Effect of Aqueous Extract of Balanite Aegyptiaca Seed on Multidrug Resistant

Bacteria

By

Egbule Christian Chibuike

ID. Number: 17/01/05/091

Supervisor: Prof. S.T. Balogun

A PROJECT SUMMITED TO THE DEPARTMENT OF MEDICAL

LABORATORY SCIENCES, UNIVERSITY OF MAIDUGURI, IN PARTIAL
FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF
BACHELOR OF MEDICAL LABORATORY SCIENCE. (BMLS)

SEPTEMBER, 2023
 CHAPTER ONE

1.0 INTRODUCTION

1.1 Background Study

The effectiveness of improved santitation, antibiotics, and vaccination programs

created a confidence in the 1960s that infectious diseases would soon be
eliminated. Consequently, chronic diseases such as cardiovascular disease and
cancer received more attention in the United States and industralised countries.
But infectious diseases have continued to be the major cause of suffering and
mortality in developing countries, moreover, disease agents adapt and evolve so
that new infectious disease have emerged and some existing diseases have
reemerged. It is clear that human and animal invasions of new ecosystems,
global warming, environmental degradation, increased international travel and
changes in economic patterns will continue to provide opportunity for new and
existing infectious diseases. The emerging and reemerging diseases has led to
the revival interest in infectious diseases. Bacterial resistance traditional to
antimicrobial treatment has been linked to about 20,000 resistant genes and
numerous mechanism has been proposed to explain it (Aslam et al, 2018). Some
bacteria are naturally antibiotic resistant, whereas others develop resistance
primarily as a result of antibiotic abuse and the introduction of new resistant
strains (Vargiu et al., 2016). Antibacterial agents inhibits the growth of bacteria
and may rapidly kill them by disrupting one or more of their essential cellular
functions. Each time an antibacterial agent is used to treat an infection, there is a
risk the agent will select in the population infecting bacteria, for bacteria that
are resistant to it, thus resulting in colonisation by resistant bacteria which may
subsequently be responsible for another infection at the same or another body
site. Eventhough pharmacological industries have produced a number of new
antibiotics in the last three decades, resistance to these drugs by microorganisms
has increased. In general bacteria have the genetic ability to transmit and
acquire resistance to drugs, which are utilized as therapeutic agents. Large
amount of antibiotics used for human therapy, as well as for farm animals and
even for fish aquaculture, resulted in the seletion of pathogenic bacteria resistant
to multiple drugs.

Multidrug resistance in bacteria may be generated by one of two mechanisms.

Firstly, these bacteria may accumulate multiple genes, each coding for
resistance to a single drug, within a single cell. This accumulation occurs
typically on resistance (R) plasmids. Secondly, multidrug resistance may also
occur by the increased expression of genes that codes for multidrug efflux
pumps. Multidrug resistant bacteria represent a major threat to the success of
almost all branches of medical practice. Such a fact, is a cause for concern
because the number of patients in the hospital who have suppresssed immunity
due to bacterial strains, such patients are especially vulnerable to acquring
multidrug resistant bacterial infections as a consequence for treatments for
underlying illness such as organ transplant patients, haemodialysis patients and
those with various type of cancers. Bacterial resistance potentially complicates
the management of every infection, no matter how mild it may be at the time of
first presentation. Multidrug resistance among bacteria is a global probem and
organism are easily carried across international boundaries thus all regions of
the world are already experiencing the effects of multidrug resistance on clinical
practice. The growing gap between the increasing frequency of infections
caused by multidrug resistant bacteria and the decline in research and
development of new antibiotics is now threatening to take us back to
preantibiotic era strategies to curtail the the spread of multidrug resistant
bacteria. Since ancient times, people have been exploring the nature particularly
plants in search of new drugs, this resulted in the use of large numbers of
medicinal plants with curative properties to treat various diseases[5]. Nearly
80% of the world’s population relies on traditional medicine for primary
healthcare, most of which involve the use of plant extracts [9]. High cost of
convectional drugs particularly in resource poor communities of the African
continent has led to the increased use of plants as an alternative for the
treatment of diseases (Sibanda and Okoh, 2018). Medicinal plants have been
used for centuries as remedies for human diseases and offer a new source of
biologically active chemical compounds as antimicrobial agent. Medicinal
plants are the richest bio-resources of drugs of traditional medicinal systems,
modern medicines, nutraceuticals, food supplements, folk medicines,
pharmaceuticals, intermediate and chemical entitled for synthetic drugs
(Hammer et al., 1999). It has been estimated that 14-28% of higher plant
species are used medicinally and that 74% of pharmacologically active plant
derived components were discovered after following up on ethno-medicinal use
of the plants (Ncube et al., 2008). Also some plant parts have been used as
antimicrobial agents, especially their extract as decoctions, infusions, or oral
administration (Okemo et al., 2001). Importantly, plant products have been
known to have medicinal effects on the organs of animals. If the toxic effect
after administration is low, there is a possible chance of introduction of such
drugs for therapeutic purpose (Ibeh, 1998).

Important subclasses in this group of compounds include phenols,

phenolic acids, quinones, flavones, flavonoids, flavonols, tannins and
coumarins. These groups of compounds have antimicrobial effects and serve as
plant defense against pathogenic microorganisms. Plants that contain substances
which can be used for therapeutic purposes or which can be used as precursors
for the synthesis of useful drugs is a medicinal plant (WHO, 1997; Sofowora,
1982). In spite of the millions of chemical structures currently available for
screening of therapeutic agents, natural products particularly of plant origin
remain the most important source of new drugs (Odugbemi and Akinsulire,
2006). Plant based natural constituents can be derived from any part of the plant
including bark, leaves, flowers, roots, fruits and seeds. The systematic screening
of plant species with the purpose of discovering new bioactive compounds is a
routine activity in many laboratories. Scientific analysis of plant components
follows a logical pathway. Plants are collected either randomly or by following
leads supplied by local healers in geographical areas where the plants are found
(Parekh et al., 2006). Fresh or dried plant materials can be used as a source for
the extraction of secondary plant components. Many authors had reported about
plant extract preparation from the fresh plant tissues. The logic behind this came
from the ethno medicinal use of fresh plant materials among the traditional and
tribal people.

Balanite aegyptiaca (L.) Del., known as Hegleig is a plant that belongs to

the Zygophyllaceae family. The tree was figured and described in 1592 by
Prosper Alpinus under the name ‘Agihalid’ but the genus was founded by Delile
in 1813. It is an evergreen savanna tree, 4.5 to 6 m high, woody and with a
small spine scents (Koko et al., 2000). A few of its numerous names are aduwa
in Hausa, tanni in Fulfulde, heglig in Arabic, eduwa in Yoruba and thorn
tree/desert date (fruit) in English. It is widely grown in the Sudano-Sahielian
region of Africa, the Middle East and South Asia. It has a multiplicity of uses
and almost every part of the plant is useful including leaves, thorns, bark of the
root, fruits and seed. Balanite aegyptiaca (L) Del has a long history of usage in
Africa folk medicine to treat so many illness including laxative, diarrhoea,
hermorrhoid, stomach aches, jaundice, yellow fever, syphilis and epilepsy (Ojo
et al., 2007). The bark is used in the treatment of syphilis, round worm
infections, and as a fish poison. The aqueous leaf extract and saponins isolated
from its kernel cakes have antibacterial activity (Bashir et al., 1984 and
Doughari et al., 2007) and potent larvicidal activity (Zarroug et al., 1988)
respectiviely.
 Balanites aegyptiaca of various origins based on geographical and
environmental conditions according to studies conducted has indicated the
presence of many flavanoids, saponins, and other important phytochemicals
(Maksoud and Al-Hadidi, 1988). Differences in phytochemical composition,
including oil, protein, and some minerals, together with the antioxidant activity
of a wide range of biological activity has been confirmed for B. aegyptiaca
extracts including antioxidant, anticancer, antidiabetic, anti-inflammatory,
antimicrobial, hepatoprotective and molluscicidal activities. Recent studies have
confirmed the medicinal use of different extracts of B. aegyptiaca fruits in the
treatment of hyperactive gut disorders, diabetes mellitus, hyperglycemia, and
dermatophytes. The antibacterial activity of B. aegyptiaca fruit extract has been
reported by several previous studies against a number of fungal and bacterial
species. These effects are ascribed to phytochemicals detected in the extract
including palmitic acid and hexadecanoic acid, 2,3-dihydroxypropyl ester.

In Nigeria, the seed oil obtained from B. aegyptiaca has been used
especially in the northern part, as substitute to groundnut oil which is relatively
expensive. The oil is used for frying food and adding flavor to tea. This is in
addition to medicinal uses such as treatment of skin disease and rheumatism.
Despite such wide spread use, there is limited literature on the possible effects
of B. aegyptiaca seed extract on multi drug bacteria. This study attempts to
evaluate the acute toxicity of the aqueous seed extracts of B. aegyptiaca on rats
and the determination of phytochemical biactive antimicrobial activity of the
seed extract.

1.2 Statement of the Problem

Antimicrobial resistance is now a major challange to clinicians for treating

patients. Although continued effort toward the development of new antibiotics,
particularly those with novel mechanisms of action, remains crucial, this alone
is not enough due to diminishing efficacy of our current armamentarium of
antibacterial drugs, no constant supply of new drugs to replace those rendered
obsolete and without means of prolonging the life span of current antibiotics.
From an economic standpoint, new antibiotics have a poor projected return on
investment compared to drugs targeted at chronic diseases such as heart disease
and high blood pressure because antibiotic regimens are typically much shorter
in duration and are often curative. Most of the antimicrobial agents have
considerable drawback in terms of limited antimicrobial spectrum. Antibiotic
resistance among bacteria is becoming more and more serious problem
throughout the world, it is a serious health concern in Nigeria being a
developing country, which has conditions and practices that promote the
development and spread of pathogens that are resistant to antimicrobials. Hence,
the identification of new medicinal plant with impressive antimicrobial activity,
hopefully new mode of action is one of the ways of tackling the problem.

1.3 AIMS AND OBJECTIVES

1. To screen and ascertain the phytochemical constituents of the seed extract

of Balanites aegyptiaca.
2. To ascertain the associated acute toxicity of the seed extract.
3. To determine minimum inhibitory concentration( MIC) and minimum
bacteriocidal concentration (MBC) of the extract against MDR bacteria
isolates by antimicrobial assay.

1.4 SIGNIFICANCE OF THE STUDY

1. The result of this study explored the toxicological and antibacterial
effects of the aqueous seed extract of Balanites aegytiaca and data
obtained from this study could serve as baseline data upon which further
research could be concluded.
 2. To ensure the effectiveness of traditional use of the plant in management
disease.

1.5 Scope of the Study

The study will include the toxicological, preliminary phytochemical screening

and the antibacterial activities of aqueous seed extract of Balanites aegyptiaca
on MDR bacteria in an attempt to improve the global public health.

1.5 Research Questions

1. Does the seed extract of Balanites aegyptiaca contains bioactive

substances of pharmacological interest?
2. Does the prolong use of seed extracts of Balanites aegyptiaca have
adverse effect?
3. Does the aqueous seed extract of Balanites aegyptiaca have antibacterial
activity.
 CHAPTER TWO

2.0 LITERATURE REVIEW

Balanite aegyptiaca Del, (Zygophyllacea) known as desert date is spiny shrub or

tree up to 10m tall, idely distributed in dry land areas of africa and south Asia. It
is traditionally used in treatment of various ailments i.e Jaundice, intestinal
worm infection, wound, malaria, syphilis, epilepsy, dysentery, constipation,
diarrhoea, hemorrhoid, stomach aches, asthma and fever. It contains protein,
lipid, carbohydrates,alkaloid, saponins, flavonoids and organic acid. Present
review summarizes the traditional claims, phytochemical and pharmacology of
B. aegyptiaca Del reported in scientific literature. It is also known as desert date
in English, a member of the family Zygophyllaceae, is one of the most common
but neglected wide plants species of the dry land areas of africa and south asia.
This tree is native to much of Africa and parts of the middle East. In india it is
particularly found in Rajasthan, Gujarat, Madhya, Pradesh and Deccan. This is
one of most common tree in senegal. It can be found in many kinds of habitat
tolerating a wide variety of soil types from sand to heavy clay and climate
moisture levels.

2.1 PLANT TAXONOMIC HIERARCHY

Kingdom: Plantae

Division: Magnoliophyta

Class: Magnoliopsida

Order: Saplindales

Family: Zygophyllaceacae
Genus: Balanite Delile

Specie: Balanite aegyptiaca (L.,) Delile

2.2 SYNONYMNS: Ximenia aegyptiaca L. (excl. Balanites romburghii

planch), Agialiaa senegalensis van Tiegh., Agialida barteri van Tiegh.,

Agialida tombuctensis van Tiegh., Balanites ziziphoides Milbr, Et Schlechter,

Balanites latifolia (van Tiegh.) Chiov.

2.3 LOCAL NAMES

Ayurvedic: Ingudi, Angaar Vrksha, Taapasadrum, Taapasa

Vrksha Dirghkantaka.

Unani: Hingan, Hanguul.

Siddha: Nanjunda.

Folk: Hingol Hingota, Hingothaa.

English: Desert date, Soapberry tree, Thorn tree, Egyptian balsam

French: Dattier du desert, Hagueleg balanite

Arab: Helglig
Spanish: Corona di jesus

Hausa: Aduwa

Fulfulde: Tanni

Yoruba: Teji

Igbo: Utazi

2.4 Botanical Description

It is multibranched spiny shrub or tree up to 10m tall crown spherical, in one or

several distinct masses, trunk short and often branching from near the base, bark

dark brown to grey, deeply fissured branches armed with stout yellow to green

thorns up to 8cm long. Leaves with two seperate leaflets obovate, asymmetric

2.5 to 6cm long bright green leathery with fine hairs when young flowers in

fascicles in the leaf axils and are fragrant yellowish green.

2.5 Seeds and Fruit Description

The seeds of B. aegyptiaca yielded four new cytoststic saponins, namely

balanitins 4, 5, 6 and 7. Seeds of B. aegyptiaca also contains deltonin and

isodeltonin (steroidal spirostanol glycosides ) which are used as mulluscicidal

agents (Gnoula et al., 2007).

Fruit is a rather long, narrow drupe 2.5 to 7cm long, 1.5 – 4cm in diameter.

Young fruits are green and tormentose turning yellow and glabrous when

mature. Pulp is bitter sweet and edible, seed is the pyrene (stone ) 1.5 - 3cm

long light brown, fibrous and extremely hard. It takes up to 50 to 60% of the

fruits, there are 500 to 1500 dry clean seeds per kg. The fruit of B.aegyptiaca

consists of an epicarp, a mesocarp, an endocarp and a kernel. The total saponin

content has been found to be 7.2% in the mesocarp and 6.7% in the kernel,

Balanitin A, B, C, D, E and Balanitin F and G have been isolated from pulp and

kernel, respectively. The oil extracted from the the kernel constituted

44-55%w/w and is composed of mainly triglycerides, with small quantities of

diglycerides, phytosterols, sterol, esters and tocopherols(Sarker et al., 2000).

Besides, a known spirostanol glycoside, balanitin-3, and a new sapogenol,

6-methyldiosgenin, new furostanol saponin, balanitoside and two pregnane

glycosides have been isolated from the fruits (mesocarp) of B. aegyptiaca

(Maksoud et al., 1988).

2.6 Distribution and Habitat

Natural distribution is obscured by cultivation and naturalization. It is believed

indigenous to all dry lands south of the sahara extending southward to malawi in

the the rift valley and to the Arabian Peninsula, introduced into cultivation in

latin America and india, it has wide ecological distribution but is mainly found

on level alluvial sites with deep sandy loam and free access to water. After the

seedling stage it is intolerate to shade and prefers open woodland or savannah

for natural regeneration. It is a lowland specie growing up to 1000m altitude in

area with mean annual teperature of 20 to 300c and mean annual rainfall of 250

to 400mm (Hamid et al., 2001)

2.7 Traditional Uses

Various parts of B.aegyptiaca have their own traditional medicinal properties.

This plant has been reported to be an antihelminthic, a purgative , vermifuge

febrifuge, emetic and can also cure other types of ailments like skin boils,

leucoderma, malaria, wounds, colds, syphilis, liver and spleen disorders, and

aches (Hamid et al., 2001). The bark of the plant is useful in curing mental

diseases, epilepsy, yellow fever, jaundice and syphils and also act as a fumigant

to heal circumcision wounds (Hamid et al., 2001). The boiled root of the plant

can be used as a soup against stomach pain, anthrax, and the infusion of root

bark has been used in diarrhoea, in haemorrhoid,and also acts as a fish poison

(Bukar et al., 2004). The paste of shoot has been used for dressing of wounds

and as tooth brushes when frayed. The thorns are used in the treatment of

leprosy. Plant leaves are used in curing anthrax, for the antihelminthic activities

and to clean malignant wounds (Hamid et al., 2001). The fruit can cure mouth

ulcer, whooping cough, sleeping sicknes and sick diseases. Fruit kernal has been

found as a mild laxative, an antidote to narrow poison, and also act as a

vermifuge. Kernel oil helps in curing skin disease. The seeds are useful as
oilments, to cure cough, colic pain and also have magico- religious properties

(Ojo et al., 2006).

2.7.1 Pharmacological Activity

2.7.2 Insecticidal Activity

B. aegyptiaca acts a potential natural larvicidal agent against mosquitoe larvae

due to the larvicidal activities present in the saponin rich extracts in the various

tissues such as fruit pulp, kernel, root, bark and leaf (Zarroug et al., 1990). The

water extracts of the B. aegyptiaca were found to be effective against the larvae

of Aedes arabiensis, Culex quinquefasciatus and Aedes aegypti and it was

concluded that A. arabiensis larvae were the most susceptible, followed by C.

Quinquefasciatus and the A. aegpti. The root extract of the plant was found to

be most lethal, followed by the the bark among the various parts tested (fruit

pulp, seed kernel, root bark and leaves ) (Chapagain and Wiesman, 2005 ). On

that basis the very low adult emergence ( only 18%) on using only 50 ppm of
the root derived callus, saponins of B. aegyptiaca showed a great possibility for

drastically reducing the A. aegypti population in the concerned areas. The main

reason behind the larvicidal activity of plant extract may be the interaction of

saponin molecules with cuticle membrane of the larva, ultimately these

(Morrissey et al., 1999).

2.7.3 Antibacterial Activity

The leaf extracts of B. aegyptiaca, prepared in aqueous and organic solvents

(acetone and ethanol), were tested for their actibacterial activity against

Salmonella more details typhi, by using the disc diffusion method. Ethanolic

extracts demonstrated higher antibacterial activity (16 mm zone of inhibition )

at 100mg/ml. The preliminary phytochemical analysis revealed the presence of

saponins, tannins, pherols, anthraquinones inthe extracts and these were

considerd for antibacterial activity (Douglas et al., 2007).

Methanolic and aqueous extracts of whole plant extract showed 4 mm

inhibition zone in Staphylococcus aureus and 11 mm zone of inhibiton in case

of staphylococcus epidermidis (Parekh and Chanda., 2007 ). The extract of B.

aegyptiaca supplemented with a 60-100 mg mineral (kadosero) revealed 100%

reduction in bacterial kadosero revealed the presence of SO4 (0.0038 mg/g), Fe2

(0.0027 mg/g ), Cl-(232.683 mg/g) and Na+ (151.25 mg) (Otieno et al.,2007).

2.7.4 Antifungal Activity

Aqueous and methanolic (80%) extrats of root bark were screened for

anticandidal activity by bio-authography agar overlay method, using a standard

strain of candida albicans (ATCC 90028). These extracts reveal strong anti

candidal activity. The identification of compounds responsible for the activity

was not done (Runyero et al., 2006). The stem of the extracts isolated in various

solvents were sxreened for their antifugal effects against Aspergillus niger and

C, albicans (MFC 250mg/ml) (Maregesi et al., 2008). The fruit msocarp saponin

rich extract has been tested against common phytopathogenic fungal ( Pythium

ultimun, Fusarium oxysporum, Alternaria solani, Colletotrichum coccodes and

Verticillium dahlia). The inhibitory effects of these extacts were measured in

vitro and the concentrations that reduced the colony diameter of fungus to 50%
of the control were determined. At 4% concentration, growth inhibitions were

reported against P. Ultimum (81,1%) and A. solani (34.7%). The antifungal

activity may be due presence of several triterpene saponins and saponins in B.

aegyptiaca (Chapagain et al., 2007).

2.7.5 Hepatoprotective Activity

The extracts of leaf, stem, stem bark and root of B.aegyptiaca were screened

for hepatoprotective activity in Wistar albino rats. The stem bark extracts of the

plant showed significant (P< 0.05) hepatoprotective effects as revealed by a

decrease in the activity of serum transaminase and alkaline phosphatase

enzymes as compared on control rats. The effect of lyophilised extracts of B.

aegyptiaca (1 g/kg) and llymarin (0.1 g/kg), a standard hepatoprotective agent ,

given for 5 consecutive days, was tested on liver damage induced by

paracetamol (0.6 g/kg) in the mice. B. aegyptiaca had a relatively modest

hepatoprotective activity (27%) while silymarin protected about 92% of the

treated mice (Ali et al., 2001). These results suggest that the extract could

protect the paracetamol- induced liver damages perhaps by eliminating the

delecterious effects of metabolite from the drug.

2.7.6 Anticancerous And Antioxidant Activity

The mixture of balanitin-6 (28%) and balanitin-7 (72%) was evaluated in vitro
for anticancer activity against six different human cancer cell lines, using the
[3-(4, 5- dimethylthiazol-2yl)-diphenyltetrazolium bromide] colorimetric assay
and in vivo in the murine L1210 leukaemia model. The mixture has
demonstrated appreciable amticancer effects in human cancer cell lines in vitro
as it displayed higher antiproliferative activity than etoposide and oxaliplatin
but markedly lesser activity than taxol. The in vitro anticancer activities resulted
in atleast partially from depletion of ATP, leading in turn to major
gisorganisation of actin cytoskeleton, ultimately resulting in the impairment of
cancer cell proliferation and migretion. In vivo, bal 6/7 increased the survival
time of mice bearing murine L1210 leukaemia grafts to the same extent as that
reported for vincristine. These preliminary in vivo data suggest that it may be
possible to generate novel semi- synthetic derivatives of balanitin-6 and -7 with
potentially improved in vitro and in vivo anticancer activity and reduced in vivo
toxicity, thus markedly improving the therapeutic ratio (Gnoula et al., 2008).

Balatinin B1 and B2, isolated from mrthonol and butanol extracts of B.

aegyptiaca bark, have been evaluated in vitro and in vivo using amethod based
on the Briggs-Rauscher (BR) oscillating reaction and this revealed the
antioxidantn activity (Speroni et al.,2005 ).
2.7.7 Anthelminthic And Molluscicidal Activity

The root, bark seed kernel, fruit and whole plant extracts were found to be lethal
to snails, miracidia and cereariac of schistosomes in various studies (Guyatt and
Evens, 1992). A mixture of deltonin and 25-isodeltonin extracted from seeds
was found to be molluscicidal against snail species Biomphalaria glabrata
(Brimer et al .,2007). The anthelminthic properties of extract of B. aegyptiaca
were compared with those of albendazole and praziquantel (Koko et al., 2005).
The efficacy of therapeutics of aqueous mesocarp extract against Fasciola
gigantica in goat was 93.2 - 97.7%. the characteristic lesions of liver fasciolosis,
egg count per gram of faeces (EPG), packed cell volume (PCV) and total blood
count were significantly different from those of control and treated groups
(P<0.05) (Koko et al., 2005 ). The efficacay of B. aegyptiaca fruit mesocarp
(200 mg/kg) was compared with that of praziquantel (200mg/kg) in mice
infected with Sudanese strain of schistosoma mansoni. A significant reduction
was observed in EPG, egg burden in tissue and recovery of adult worms
(P<0.05) for both the extract and the drug treated animals (Koko et al., 2005).

2.7.8 Antiparasitic Activity

The crude methanolic extract has been found to have a moderate biological
activity on leishmania major in an in vitro study (Fatima et al., 2005).

2.7.9 Antidiabetic Activity

The bark of B. aegyptiaca has been also shown to have a moderate effect on the

amylase which is responsible for the degradation of oligosaccharides (Funke

etal., 2005). B. aegyptiaca fruit extracts (1.5 g/kg bw) reduced the blood
glucose-6- phophatase activity in diabetic rats (Mohammed et al., 2006 ). The

water and methanolic extacts of B. aegyptiaca fruit extract induced significant

reduction in serum glucose, glucagon, total lipids, total cholesterol, triglycerides

level and transaminases [aspartate aminotransferase (AST), alanine

aminotransferase (ALT) and gGT (gamma aminotransferase)] activities. An

aqueous extract of mesocarps of the fruits of B. aegyptiaca exhibited a

prominent antidiabetic activity of oral administration in streptozotocin-induced

diabetic mice. It is believed that the antidiabetic activity was due to the presence

of steroidal saponins in the extracts (Kamal et al., 1991).

2.7.10 Anti-Inflammatory Activity

The ethanolic extract of aerial parts of B. aegyptiaca, when given orally as a

suspension at 300mg/kg bw per day, reduced the paw volume by 55.03%,

whereas in the case of adminstration of 600 mg/kg per day it was 65.54%,

indicating that the effect was dose dependant. The significant anti-inflammatory

activity was evaluated in methanolic and ethanol extract of the bark in two

different animal models, the carregeenan-induced oedama in the rat, and acetic

acid-induced writhing test in mice (Speroni et al., 2005). Ethanolic extract of

fruit of B.aegyptiaca also exhibited a proinflammatory activity. The

pytochemicals responsible for these activities were found to be flavonoids,

saponins B1 and B2 isolated from bark and aerial parts of the plant (Gaur et al.,

2008).

2.8 History Of The Test Organism

2.8.1 Escherichia coli (E.coli)

Scientific Classfication

Domain: Bacteria

Phylum: Proteobacteria

Class: Gamma Proteobacteria

Order: Enterobacteriales

Family: Enterobacteriaceae

Genus: Escherichia

Specie: E.coli

Binomial name Escherichia coli

This organism is a gram-negative, rod-shaped bacterium which belongs

to the family enterobacteraceae(Cheesbrough, 2005). They are naturally found

in the intestinal tract, soil, water. Enterobacteraceae comprises of bacteria that

are gram negative, mostly motile by petrichous flagella. It is a facultative

anaerobe. E. coli is a member of the normal flora of the human body and is not
highly pathogenic. It is still of great importance because of its frequency of

potentially serious infectious disease that it causes. The organism colonizes the

large intestine without causing apparent harm. However, its presence in faeces

makes it possible for it to cause disease in other parts of the body especially the

urinary tract and peritoneum (Prescott et al., 2005).

Pathogenicity: strains of Escherichia coli possses a range of diffferent

pathogenic mechanisms. This organism produces three different types of

antigens; K, O and H, out of which the O and K antigens protect the organism

from bactericidal effect of complement and phagocytes. Mannose sensitive and

mannose resistant pilli are the types of pilli produced by Escherichia coli which

dertermine pathogenicity (Brooks et al., 2001).

Pathogenesis: The clinical manifestation of infections with E. coli and other

enteric bacteria depend on the site of infection and cannot be differentiated by

signs and symptoms f rom processes caussed by other bacteria. E. coli are not

the most common cause of urinary tract infection in young women. The

symptoms and sign includes polyuria, hematuria, pyuria and dysuria. Urinary

tract infection can result in bacteraemia with clinical signs of sepsis (Brooks et

al., 2001).

2.8.2 Staphylococcus aureus (Staph. Aureus)

Scientific classification
Domain: Bacteria

Kingdom: Eubacteria

Phylum: Firmicutes

Class: Bacilli

Order: Bacillales

Family: Staphylococcaceae

Genus: Staphylococcus

Specie: Staph. Aureus

Binomial name Staphylococcus aureus

Staphylococcus aureus is a facultative anaerobic gram-positive coccal

bacterium. It is known as “golden grape-cluster berry” , and also known as

golden staph and Oro staphira. It is frequently found as part of the normal flora

on the skin and nasal passages (Kluytmans et al., 1997). The reason S. aureus is

sucessful pathogen is a combination of the host and bacterial immune-evasive

strategies. One of these strategies is the production of carotenoid pigment

staphyloxanthin which is responsible for the characteristic golden color of S.

aureus colonies. This pigment acts as a virulence factor, primarily being a

bacterial antioxidant which helps the microbe evades the host’s immune system

in the form of reactive ovygen spicies which the host uses to kill pathogens.
Pathogenicity: S. aureus strains possess some extracellular factors, some of

which contribute to the ability of thr organism to overcome the body defence, to

invade, survive and colonize tissues (Prescott, 2005). These extracellular

substances may be enzymes or toxins. These includes catalase (can convert

hydrogen peroxide to water), coagulase (this clots oxalate or citrated plasma in

the presence of a factor contained in sera), hyaluonidase (spreading factor) and

staphylokinase (Brooks et al., 2001).

Pathogenesis: nasal carriage of S. aureus occurs in 40-50% of humans. The

pathogenic capacity of a given strain of S.aureus is the combined effect of

extracellular factors and toxins together with the invasive properties of the

strain. At one end of the diseasespectrum is staphylococcal food poisoning

attributed solely to the ingestion of preformed enterotoxins at the other end as

staphylococcal bacteraemia and dissemination abscess in all organs. The

potential contribution of the various extracelluar substances in pathogenesis is

evident from the nayure of their individual action (Brooks et al., 2001).

2.8.3 Klebsiella pneumonia (K.spp)

Domain: Bacteria

Phylum: Pseudomonadota

Class: Gammaproteobacteria
Order: Enterobacteriales

Family: Enterobacteriaceae

Genus: Klebsiella

Species: K. Pneumonia

Klebsiella pneumonia is a genus of gram-negative rod like non motile

facultative anaerobic bacteria mostly lactose fermenting found in the respiratory

and urinoligical tracts of animals and humans. It is rod-shaped and measure 2m

by 0.5m. it is 160nm thick fine fibers that protrude out from the outer membrane

at right angels (Amako et al., 1988; lawler et al., 2008). Its habitat is not limited

to humans butis ubiquitous to the ecological environ ment. This includes

surfacewater, sewage and soil (Brissel et al., 2000). It can affect the liver,

urinary tract, lungs to name but a few (Wen-chen et al., 2002).

Pathogenicity: In 1882, Friedlander C. Uber first discovered Klebsiella to be a

pathogen that caused pneumonia. It is found in the gastrointestinal tract,

nasopharynx and hands of hospital personnel. The reason for its pathogenicity is

the thick capsule layer surroumding the bacterium. Community Acquired K.

pneumonia has been responsible for the increased number of bacteremic liver

abscess and results in complications like pulmonary emboli or abscess, brain

abscess, pyogenic meningitis, endophthalmitis, prostatic abscess, asteomyelitis,

septic arthritis, or psoas abscess and meningitis (Lee CC et al., 1998; Yarng SS
et al., 1997). The bacteria also produces 10% of all hospital-acquired infextions,

including pneumonia and urinary tracts infections, billary tract and surgical

wounds. The predisposing factors are catheters and endotracheal tubes, old age,

alcoholism, diabetes, immunosuppression, congestive heart failure, chronic

obstructive pulmonary disease and other debilitating diseases. Patients with

Klebsiella pneumonia tend up a property to cough up sputum it rank second to

E. coli for urinary tract infections in the elderly. Symptoms include: toxic

presentation with sudden onset, high fever and haemoptysis.

Treatment is done by antibiotics such as clindafloxacin however, Brissel et al

(2000) and Sylvian et al (1999) have documented that Ciprofloxacin and

cephalosporins are antibiotics that are becoming less effective.

2.9 Multidrug Resistance In Bacteria

The discovery of penicillin in 1928 was followed by the discovery and

commercial production of many other antibiotics. We now take for granted that

infectious diseases is curable by antibiotics therapy. Antibiotics are

manufactured at an estimated scale of about 100,000 tons annually worldwide,

and their use had a profound impact on the life of bacteria on earth. More strains

of pathogens have become antibiotic resistant, and some have become resistant
to many antibiotic and chemotherapeutic agents, the phenomenon of multidrug

resistance.

Multidrug resistance in bacteria occurs by the accumulation of resistance (R)

plasmids or transposons of genes with the coding for resistance agent, and/or by

the action of multidrug efflux pump, each of which can jump out more than one

drug type.

2.10 BIOCHEMICAL MECHANISM OF RESISTANCE

.10.1 Mutational Alteration Of The Target Protein: Man-made compounds,

such as fluoroquinolones, are unlikely to become inactivated by the enzymatic

mechanism. However, bacteria can still become resistant through mutations that

make the target protein less susceptible to the agent. Fluoroquinolones

resistance is mainly (but not exclusively) due to mutations inthe tsrget enzymes,

DNA topoisomerases, whether resistance of this type is easily transfered to

other cells on plasmids depends on the mode of drug action. High level

production of drug resistant target enzymes from plasmids can make bacteria

resistant and these resistant genes can spread widely on plasmids.

2.10.1 Enzymatic Inactivation Of The Drug: This is a common resistance

mechanism for antibiotics of natural origin such as aminoglycosides

(kanamycin, tobramycin and amikacin). Which are inactivated by enzymatic

phosphorylation[by aminoglycoside phosphoryltransferase (APH)], acetylation

(by aminoglycoside acetylatransferasea (AAC), adenylation (by aminoglycoside

adenyltransferase or nucleotidyltransferase) and β-lactams (penicillins,

cephalosporins and carbapenem such as imepenem), which are inactivated by

enzymatic hydrolysis by β-lactamases, usually in the periplasm. Genes coding

for these inactivating enzymes can easily produce resistance as additional

genetic components on plasmids.

2.10.3 Acquisition Of Genes For Less Susceptible Target Proteins For

Other Species: Sequencing of the genes coding for the target of penicillin,

DD-transpeptidase or penicillin binding proteins (PBPs) revealed that penicillin

resistance among streptococcus pneumoniae was due to the production of

mosiac proteins, part of which came from other organisms. We note that s.

pneumoniae is an organism capable of natural transformation and may import

foreign DNA. Interestingly, a similar mechanism of penicillin resistance was

also found in another organism capable of natural transformation, Neisseria

meningitidis. An extreme case of these can be seen in the generation of MRSA

strains which contain a new methicillin-resistant PBP called PBP-2A or 2’

whose expression is often induced by methicillin and other β-lactams. The gene

for this new PBP is located in a large segment of DNA, ehich apparently came

from an organism other than S. aureus and also contains other antibiotic
resistance genes. S. aureus is not naturally transformable and it is unclear how

the horizontal transfer of a large DNA segment occurred.

2.10.4 Bypassing The Target: vancomycin, a fermentation product from

streptomycetes, has an unusual mode of action, instead of inhibiting an enzyme,

it binds to substrate the lipid-linked-disaccharidepentapeptide, a precussor of

cell wall peptidoglycan. Because of this mechanism many assume that it would

be impossible to generate resistance against vancomycin. However, vancomycin

resistance is now prevalent among enterococci, normal inhabitants of our

intestinal tract because enterococci are naturally resistant to β-lactams,

aminoglycosides,macrolides, and tetracycline. These vancomycin resistant

strains of enterococci became prevalent in the hospital environment, colonize

the patients and cause infections that are difficult to treat. Study of the

resistance mechanism showed that the enf of the pentapeptide, D-Ala-D-Ala,

where vancomycin binds was replaced in the resistant strain by an ester

structure, -Ala-D-lactic acid, which is not bound by vancomycin (160).

D

Production of altered structure requires the participation of several imported

genes.

2.10.5 Preventing Drug Access To Targets: Drug acess to the target can be

reduced locally. It can also be reduced by an active refluxed process in gram-

negative bacteria, the access can be reduced generally by decreasing the reflux

across the outer membrane barrier.

 Local Inhibition Of Drug Acess: Tet(M) or Tet(S) proteins, produced by

plasmid-coded genes in gram-positive bacteria bind to ribosomes with high

affinity and apperently change the ribosomal conformation , thereby preventing

the association of tetracyclines to ribosomes. Plasmid-coded-Qnr protein s

which have become more prevalent in recent years are thought to protect DNA

topoisomerases from (Fluoro)quinolones.

Drug Specific Efflux Pumps: Drug resistance owing to active efflux was

discovered with the common teetracycline resistance protein TetA in

gram-negative bacteria which catalyze a protein-motive-force dependent

outward pumping of a tetracycine-Mg complex.

2.11 SOURCES OF THE RESISTANCE GENES

.11.1 Producing Organisms: Some of the aminoglycoside - resistant genes

appear to be derived from streptomycetes producing these antibiotics. The genes

coding for vancomycin resistance appear to originate in a similar way.

Resistance here requires the production of several new enzymes, and it is

unlikely that the genes coding for these enzymes evolved in the few decades

after vancomycin was introduced. Indeed, the genes in the vancomycin-

resistant clinical isolates of enterococci were found to be homologs of those

found in the vancomycin-producing streptomycetes, organized in exactly the

same manner, an observation that leaves no doubt on the origin of the resistance

genes.
.11.2 Microorganisms In The Environment, Especially Soil: Some resistance

genes are found in the chromosome of environmental bacteria. A classical case

is the ampC gene in the environmental genera of Enterobacteriaceae, such as

Enterobacter, Serratia, and Proteus, and in the soil organism P. aeruginosa.

These genes do not show signs that they have been imported in the recent past,

and it is revealing that the ampC gene of the exclusive animal symbiont E. coli

lacks the induction mechanism and that the pathogen Salmonella spp. Lacks the

ampC gene entirely. In this connection, examination of a random collection of

soil-dwelling strains of streptomyces and their relatives showed that 6o% to

100% of them were resistant to several antibiotics teasted, suggesting that

antibiotic-resistant genes are abundantly present in this habitat. Although most

antibiotics may be present in soil only at very low concentrations, the recent

discovery of microoganisms that utilize antibiotics as nutrients is suggestive of

the evolutionary origin of some antibiotic degradation (resistance) genes.

 CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Glass wares and apparatus

Conical flask, Petri dishes, Bijou bottles, Water bath, Syringe and needle, Wire

loop, Hand gloves, Rotary evaporator, Analytical weighing balance, Autoclave,

Incubator, Oven, Glass funnels, Large size cylinder, Glass stirrer, Cotton wool,

Spatula, Test tube, Stainless steel tray, Bunsen burner.

3.2 Solvents and reagents:

Distilled water, Ethanol, 1% aqueous hydrochloric acid, Dragendorff’s reagent,

Mayer’s reagent, Sodium chloride solution, Glacial acetic acid, concentrated

sulpuric acid, 10% ferric chloride solution, Molisch’s reagent, Fehling solution
A and B, Aqueous extract were obtained from the Department of Chemistry,

University of Maiduguri.

3.3 SOURCES OF THE MICROORGANISMS:

Clinical isolates of the the tests organisms (Staphylococcus aureus, Escherichia

coli, Klebsiella specie) were obtained from the Department of Microbiology,

University of Maiduguri Teaching Hospital (UMTH).

3.4 PLANT COLLECTION, IDENTIFICATION AND PREPARATION

3.4.1 PLANT COLLECTION AND IDENTIFICATION

The fruits of balanites eyptiaca ( desert date) were purchased from Gamboru

market in Borno state, Nigeria and was brought to the department of Biological

Sciences, University of Maiduguri, Borno, for identification by a plant

taxonomist. The plant was identified and authenticated by Prof. S. S. Sanusi.

3.4.2 PLANT PREPARATION

The fruits of balanites aegyptiaca were soaked in water for four (4) hours,

washed thoroughly to remove the unwanted pulp of the fruit to expose the

kernel shell before sun drying for seven days. The shell was cracked using a

steel hammer to obtain the seed kernels, the seed kernels were then air dried in a
room temperwture for three (3) weeks before it was then grinded to form

powder using mortar and pestle.

3.4.3 PLANT EXTRACTION

The powdered sample (500g) was added to a flask containing distilled water

(300ml) and refluxed for 2 hours. Then the resulting mixture was filtered and

dried using a hot air oven at 400c (Dhanani et al., 2017). An Aqueous extract

was obtained, weighed and kept in the dessicator until required for use.

3.5 PHYTOCHEMICAL SCREENINNG

Basic phytochemical screening test consist of performing simple chemical tests

to detect the presence of an alkaloids, tannins, saponins, carbohydrates,

glycosides flavonoids (Sofowara 2008).

3.5.1 TEST FOR CARBOHYDRATES

General Test (Molish’s Test)

Few drops of molish’s reagent were added to 0.5g of Balanites aegyptiaca

aqueous seed extract disolved in distilled water. This was followed by addition

of 1ml of concentrated tetraoxosulphate vi acid (H2SO4) by the side of the test

tube; so that the acid will form a layer beneath the aqueous layer. The mixture
was then allowed to stand for two minutes and then diluted with 5ml of distilled

water, formation of a red or dull colour at the interphase of the two layers was

observed indicating the presence of carbohydrates (Farnsworth, 1989, Trease

and Evans 2002).

Salivanoff’s Test (Standars Test For Ketones)

To the half gram (0.5g) of the aqueous extract, few crystals of resorcinol and

2ml of Hydrochloric acid was added and the mixture was boiled for 5minutes.

The appearance of red colouration indicates the presence of ketones (Vishnor,

1979).

3.5.2 TEST FOR FLAVONOIDS

Sodium Hydroxide

To the half gram (0.5g) of the aqueous extract was disolved in water and

filtered. To the filtrate, 2ml of 10% aqueous sodium hydroxide was added to

produce a yellow colouration. A change in the colour from yellow to colourless

on addition of dilute hydrochloric acid was an indication of the presence of

flavonoids (Trease and Evans, 1989).

3.5.3 TEST FOR TERPENOID

Half gram (0.5g) of the aqueous extract was disolved in ethanol. 1ml of acetic

anhydride was added, followed by the addition of concentrated H2SO4. A

colour change from pink to violet was observed showing the presence of

terpenoids (Silva et al, 1998).

3.5.4 TEST FOR GLYCOSIDES

Salkowski’s Test

To the half gram (0.5g) of the aqqueous extract, 2ml of chloroform was added,

then tetraoxosulphate (vi) acid was added carefullyby the side of the tube to

form a lower layer. Appearance of a reddish brown colour or yellow at the

interphase indicates the presence of steriodal ring (i.e aglycone portion of

cardiac glycoside) or methylated sterols (Silva et al, 1998).

3.5.5 TEST FOR CARDIAC GLYCOSIDES

Keller- Killiani’s Test

Glacial acetic acid (1ml) was added to about 3mlof the solution containing 2mg

of the aqueous extract in atest tube held at 450c then two drops of concentrated

tetraoxosulphate (vi) was added along the side of the test tube carefully.

Formation of purple ring colour at the interphase indicates the presence of

cardiac glycosides (Trease and Evans, 1998).

3.5.6 TEST FOR SAPONIN GLYCOSIDE

One gram of each extract was boiled with 5ml of distilledwater, filtered and the

filtrate was divided into two portions.To the first porrtion, about 3ml of distilled

water wasadded and then shaken for about 2minutes. Frothing which persist on

warming is the evidence for the presence of saponins (Sofowara, 2008). To the

second portion 2.5ml of the mixture of equal volume of fehling’s solution A and

B was addded. Appearance of a brick-red precipitate taken shows the presence

of saponin glycosides (Vishnoi, 1979).

3.5.7 TEST FOR ALKALOID

Zero point five grams (0.5g) of the aqueous extract was stirred wuth 5ml of 1%

aqueous hydrochloric acid on a water bath and the filtered. Of the filtrate, 3ml

was taken and divided equally into 2 portions in a test tube.

To the first portion few drops of dragendorff’s reagent was added indicating the

occurrence of orange-red precipitate which was taken as an indication for the

presence of alkaloids.

To the second, 1ml of mayer’s reagent was added and the appearance of

buff-coloured precipitate was an indication for the presence of alkaloids.

3.5.8 TEST FOR TANNINS

Zero point two grams (0.5g) of the aqueous extract was stirred with abouut

10ml of distilled wateerand then filtered. Then filtrate was used for the

following test:

Ferric Chloride Test

To 2ml of the filtrate, few drops of the 1% ferric chloride solution was added

occurrence of a blue-black, green or blue-green precipitate was not observed

showing the absence of tannins.

Lead Ethanoate Test

A mixture of equal volume of 10% lead ethanoate was added to 2ml of the

filtrate. Formation of awhite precipitate was not observed indicating the absence

of tannins (Sofowora 1993 Trease and Evans, 2002).

3.6 PREPARATION OF MEDIA FOR ANTIBACTERIAL SCREENING

3.6.1 Sterilization Of The Equipment And Disinfection

All bench work surface were mopped with moist rag and was disinfected with

cotton wool soaked in

3.6.2 Moist Heat Sterilization

All the materials used in the course of this project that is not sensitive to moist

heat sterilization were adequately sterilized using autoclave. Materials such as

glasss wares, conical flask were properly waashed with detergent and water to
remove dirt and contaminants and were allowed to dry prior to usage. These

materials were then sterilized in a digital laboratory autoclave at 1200c for

15minutes.

The media preparation were carried out based on the manufacturer’s instruction

as follows:

3.6.3 PREPARATION OF THE NUTRIENT AGAR PLATE

Nutrient Agar: This was prepared by disolving 50g of the agar in one litre of

distilled water and then covered with aluminium foil. The media was boiled to

dissolution and then sterilized at 1210c for 15minutes. The media was allowed

to cool to 450c and 20ml of the sterilized medium was poured into the sterile

petri dish which was allow to cool and solidify. The plates were labelled and

dried at 370c for 30minutes and are to be used for well diffusion method. The

plates were labelled with the test microorganism ( each plate with a test

microbe). The microbes were spread evenly over the surface the medium before

boring a hole using a cork borer.

3.6.3 Antibacterial Screening Using Cup Plate Method

Prepared agar plate was evenly spread with test organism after which 4 cups of

holes were bored on the surface of the prepared plates using standard cup borer
of 8mm in diameter into which concentrated aqueous extract of balanites

aegyptiaca and gentamicin were poured into holes, the centre being the control.

Twenty grams (20g) each of the extract was disolved in 30mls sterile distilled

water to make an initial concentration of 667mg/ml. These were then serially

diluted using 1:1 dilution of the initial concentration and distilled water to

produce a concentration of 334mg/ml and 167mg/ml. The bacteria plates were

incubated at 370c for 24hours and observation for the zone of inhibition were

made. The zone of inhibition were measured with a transparent ruler and the

result recorded in millimeters. Gentamycin was used as a positive control.

3.6.4 Minimum Inhibitory Concerntration (Serial Dilution Method)

Mininum inhibitory concentration (mic) of balanites aegyptiaca aqueous seed

extract was carried out using serial dilution method. Three grams (3g) of the

extract was dissolved in 30ml of distilled water. A set of 9 test tubes each for

specific test organism were arranged serially and filled wih 5ml of peptone

water, 5ml of the extract was added into the first test tube mixed thoroughly and

5ml of the extract was withdrawn and poured into the second test tube. This was

continued serially until the ninth (9 ) tube, and then 5ml was withdrawn from

the tube number nine (9 ) and discarded. The tube number ten (10) was used as

the negative control containing only peptone water while the tube number
eleven (11) was used as the positive control containing peptone water and the

test organisms; (Staphylococcus aureus, Escherichia coli and Klebsiella spp).

All the test tubes were incubated at 370c in the incubator for 24 hours. The

lowest concentration which showed no turbidity in the bottle was recorded as

the Minimum Inhibitory Concerntration (MIC).

3.6.5 Minimum Bacteriocidal Concentration

All the test tubes content showing no tuebidity in the minimum Inhibitory

concentration (MIC) assay was subcultured into a nutrient agar plate. The

content of the MIC in the serial dilution without turbidity were subcultured in a

nutrient agar plates and incubsted at 370c for 24 hours in the incubator and

observed for colony growth. The Minimum Bacteriocidal Concentration (MIC)

was observed with colonial growth of bacteria

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