CHAPTER ONE

1.0 INTRODUCTION AND LITERATURE REVIEW

Balanites aegyptiaca Del., also known as 'Desert date' in English is a species of

tree, classified as a member of the Zygophyllaceae family, is one of the most

common but neglected wild plant species of the dry land areas of Africa and

South Asia (Chothani et al., 2012).

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.

(Chothani et al., 2012).

This plant is one of the most common trees in Senegal. It can be found in many

kinds of habitat, tolerating a wide variety of soil types, from sand to heavy clay,

and climatic levels(Chothani et al., 2012).

Taxonomic Classification Of Balanites aegyptiaca By National Plant Kingdom

Plantae – Plants Subkingdom Tracheobionta – Vascular plants Superdivision

Spermatophyta – Seed plantsnDivision Magnoliophyta – Flowering plants Class

Magnoliopsida – Dicotyledons Subclass Rosidae Order Sapindales Family

Balanitaceea – Creosote-bush family Genus Balanites Delile – Balanites

Species Balanites aegyptiacus (L.) Delile – desert date (Saed and Isam, 2018).

There are many common names for this plant. In English the fruit has been

called desert date, soap berry tree or bush, Thron tree, Egyptian myrobalan,

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Egyptian balsam or Zachum oil, in Arabic it is known as lalob, hidjihi, inteishit,

and heglig (hijlij). In Jieng it is called Thou or thau, in Hausa it is called aduwa,

in Tamasheq, the Tuareg language taboraq, in Fulfulde (Pulaar) Murtooki or

Tanni, in Swahili mchunju, in Kamba Kilului and in Amharic bedena ( Saed and

Isam, 2018).

DISTRIBUTION AND ECOLOGY

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 Rift Valley, and to the Arabian Peninsula, introduced into cultivation in

Latin America and India. It is the one of the furthermost neglected common

tree, usually found throughout in dried regions of Africa, the Middle East, India

and Burma. Azene Tesfaye, 2016. Pointed out that the plant is native to

Algeria, Angola, Benin, Burkina Faso, Burundi, Cameroon, Chad, Democratic

Republic of Congo, Djibouti, Egypt, Eritrea, Ethiopia, Libyan, Morocco,

Myanmar, Nigeria, Saudi Arabia, Somalia, Tanzania, Uganda, Yemen, Republic

of, Zambia, Zimbabwe (Azene Tesfaye, 2016). The plant can survive in various

types of soil including deep sands, sandy clay loams, sandy loams or clays but is

mainly found on level alluvial sites with deep sandy loam and free access to

water as described by Chothani et al., 2012 . The plant grows up to 2000 m

altitude with mean annual temperature of 20 to 30°C and mean annual rainfall

of 250 to 400 mm 3 According to Chothani et al., 2012. After the seedling

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stage, the plant is intolerant to shade and prefers open woodland or savannah

for natural regeneration. It is a lowland species with a full potential to produce

reliable yield under unreliable conditions. It is reported that the plant can

withstand high temperature, high light intensity, high wind velocity and severe

drought (Chothani et al., 2012).

DESCRIPTION

B. aegyptiaca belongs to the family Zygophyllaceae which composed of

about 25 genera and 240 species and predominant in tropical, subtropical and

warm temperate, often in drier areas including in Africa. It is annual and

perennial herbs (Azene Tesfaye, 2015).

CULTIVATION

Balanites aegyptiaca has been cultivated in Egypt for more than 4000

years, and stones placed in tombs as votive offerings have been found as far

back as the Twelfth Dynasty. The tree was described in 1592 by Prospero

Alpini under the name 'agihalid'. Linnaeus regarded it as a species of Ximenia,

but Michel Adanson proposed the new genus of Agialid. The genus Balanites

was founded in 1813 by Delile (Chothani et al., 2012).

The yellow, single-seeded fruit is edible, but bitte, many parts of the plant

are used as famine foods in Africa; the leaves are eaten raw or cooked, the oily

seed is boiled to make it less bitter and eaten mixed with sorghum, and the

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flowers can be eaten. The tree is considered valuable in arid regions because it

produces fruit even in dry times. The fruit can be fermented for alcoholic

beverages (Chothani et al., 2012).

MEDICINAL

Desert date fruit is mixed into porridge and eaten by nursing mothers, and

the oil is consumed for headache and to improve lactation (Eshetu et al., 2013).

Bark extracts and the fruit repel and destroy freshwater snails and

copepods, organisms act as intermediary hosts of parasites including

Schistosoma, Bilharzia, and guinea worm. Worm infections are treated with

desert date, as are liver and spleen disorders. A decoction of the bark is also

used as an abortifacient and an antidote for arrow poison in West African

traditional medicine (Eshetu et al., 2013).

The seed contains 30-48% fixed (non-volatile) oil, like the leaves, fruit

pulp, bark and roots, and contains the sapogenins diosgenin and yamogenin.

Saponins likewise occur in the roots,bark wood and fruit (Eshetu et al., 2013).

Balanites aegyptiaca (L.) Delile is an underutilized fruit yielding tree

native to Africa and distributed in tropical and subtropical regions of Africa,

from Senegal in the west to Somali in the east and Jordan in the north to

Zimbabwe in the south (Habieballa et al., 2021).

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 Balanites aegyptiaca is a widely grown desert plant with multi-use

potential, It is mainly found in arid and semi–arid areas throughout Africa, the

Middle East, and South Asia (Kabbashi et al., 2017). It is believed that the

plant is indigenous to all dry lands south of the Sahara, extending southward to

Malawi in the Rift Valley, and to the Arabian Peninsula (Kabbashi et al., 2017).

It has wide ecological distribution, but it is mainly found on level alluvial sites

with deep sandy loam and free access to water. It is a lowland species, growing

up to 1000 m altitude in areas with mean annual temperature of 20 to 30°C and

mean annual rainfall of 250 to 400 mm (Kabbashi et al., 2017).

Balanites aegyptiaca is used in African and India as a folk medicine, the

roots and bark of B. aegyptiaca are used as purgative and anthelmintic the root

of B. aegyptiaca is also used to treat malaria (Habieballa et al., 2021). Bark is

used to deworm cattle, and roots are used to treat edema and stomach pain.

Roots are also used as an emetic, the fruit of B. aegyptiaca is used to treat

jaundice in Sudan. Seed oil of B. aegyptiaca is used as a laxative and for the

treatment of hemorrhoids, stomach aches, jaundice, yellow fever, syphilis and

epilepsy. Bark extracts are used to kill freshwater snails and copepods

(Habieballa et al., 2021). The bark of B. aegyptiaca has antifungal activity

against Candida albicans infections, it is also antiviral and antibacterial. B.

aegyptiaca [L.] Delile seed oils has been used against the red flour beetle

5
(Tribolium castaneum Herbst). the fruit is also used to cure liver disease

( Habieballa et al., 2021).

In many African countries (e.g. Senegal, Nigeria, Ethiopia and Sudan) B.

aegyptiaca has been reported to be an anti-helminthic, a purgative, febrifuge,

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

wounds, colds, syphilis, liver and spleen disorders. Various parts of this plant

also have their own traditional medicinal properties. The seed is used as a

febrifuge and its oil is used to treat tumors and wounds (Kabbashi et al., 2017).

An aqueous extract of the bark is used in Sudanese folk medicine in the

treatment of jaundice, The kernel oil exhibited anticancer activity against lung,

liver, and braincarcinoma cell lines. It also has anti-mutagenic activity against

Fasciola gigantica-induced mutagenicity besides anthelmintic activity against

hepatic worms (Schistosoma mansoni), the aqueous leaf extract and saponins

isolated from its kernel cakes prove antibacterial activity. The branches are used

as tooth brush. The root extracts proved to be slightly effective against

experimental malaria. Infectious diseases which account for high proportion of

health. (Kabbashi et al., 2017).

The plant of Balanites aegyptiaca produces primary and secondary

metabolites which encompass a wide array of functions and can manufacture

many different types of secondary metabolites which have been subsequently

exploited by humans for their beneficial role in a diverse array of applications.

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Often, plant secondary metabolites may be referred to as plant natural products,

in which case they elicit effect on other organisms (Kashari et al., 2021).

Balanites aegyptiaca seed is used as expectorant, antibacterial, and antifungal,

the fruit is used in whooping cough, also in leucoderma and other skin diseases

while the bark is used as spasmolytic, in the treatment of syphilis, round worm

infections and as a fish poison(Kashari et al., 2021).

(Sedky et al., 2022) reported how Balanites aegyptiaca fruit as a wide

variety of compounds with different biological and pharmacological effects

such as anti-inflammatory, antioxidant, cytotoxic and antimicrobial activities.

Deserts date is rich in antimicrobial compounds including phenolics as simple

phenols, quinones, phenolic acids, flavonols, flavones, flavonoids, tannins,

coumarins, terpenoids, essential oils and alkaloids (Sedky et al., 2022) In

African folk medicine the bark of B. aegyptiaca is commonly used for the

treatment of wounds and skin diseases. The aqueous extract of B. aegyptiaca

bark inhibited the growth of Pseudomonas aeruginosa (P. aeruginosa) and S.

aureus isolated from wounds (Sedky et al., 2022).

Balanites aegyptica seed were produced annually in the tropical and sub

tropical countries of Asia and African. Like rest of the plant, they were highly

valued (Sabina and Fatimoh, 2021). The Balanites aegyptica also known as the

tree of life has a host of nutrient uses for both people and livestock, Balanites

seed have a unique and pleasing appearance. When firmly rooted, they produce
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a bountiful crop of one of the worlds of healthy plants. The trees itself was

rather slender with dropping branches that grow to approximately 1O-12m in

height. Balanites aegyptiaca is one of the most common but neglected wild

plant species of the dry land areas of Africa and South Asia (Sabina and

Fatimoh, 2021).

Balanites egyptiaca is use as medicinal plants for primary health care and

have increased in recent years worldwide. Various scientists are searching new

phytochemical for the treatment of infectious diseases, This plant have wide

variety of secondary metabolites that have antimicrobial properties (Sabina and

Fatimoh, 2021).

Balanites aegyptica is used to treat so many illnesses including laxative,

diarrhea, hemorrhoid, stomach aches, jaundice, yellow fever, syphilis and

epilepsy. It is used to treat liver disease and as a purgative and sucked by school

children as a confectionary in some countries even in Nigeria, The bark is used

in the treatment of syphilis, round worm infection and as a fish poison (Sabina

and Fatimoh, 2021).

The aqueous leaf extract and spooning isolated from it kernel cakes have

antibacterial activity. Africa is very rich with biodiversity resources, vegetation

and it is estimated about 40,000- 45,000 species of different flora. A very large

number of plant species are medicinally used for the treatment of various

diseases (Sabina and Fatimoh, 2021).Due to easy accessibility, cheaper than

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synthetic drugs, less toxic, and less side effects when compared to other

synthetic medicine the use of medicinal plants has increased now a days.

Balanites aegyptica is a highly valued plant distributed in many countries of the

tropic and sub tropic, desert, etc. It has impressive range of medicinal uses with

high nutritional value (Sabina and Fatimoh, 2021). Different parts of the plant

contain a profile of important minerals, and a good source of protein, vitamins,

amino acids and various phenols. The Balanites plant provides a rich and rare

combination of zeatin, quercetin, kaempferom and many other phytochemicals

(Sabina and Fatimoh, 2021).

Infectious diseases account for high proportion of health problems and are

the leading cause of death worldwide. Even though pharmaceutical industries

have produced a number of new antimicrobial drugs in the last years, resistance

to these drugs by microorganisms has increased (Sabina and Fatimoh, 2021).

The indiscriminate use of commercial antimicrobial drugs commonly used

for the treatment of infectious diseases. In general, bacteria have the ability to

acquire and transmit resistance to drugs used as therapeutic agents, Incidents of

epidemics due to such drug resistant microorganisms are now a common global

problem posing enormous public health concerns(Kabbashi et al., 2017). The

global emergence of multidrug resistant bacterial strains is increasingly limiting

the effectiveness of current drugs and significantly causing treatment failure of

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infections. The present study was conducted to investigate the antimicrobial

activity of B. aegyptiaca (leafs) in Sudan (Kabbashi et al., 2017).

The production of primary and secondary metabolites by Balanites

aegyptiaca has been subsequently exploited by humans for their beneficial role

in a diverse array of applications and this has been used in traditional ways to

treat different kinds of ailments and disorders resulting from microorganisms,

since orthodox antimicrobial agents have been engulfed with wide range of

challenges ranging from issues of resistants, side effects, allergy, high cost and

unavailability of such drugs(Kashari et al., 2021). In addition to the use of tree

in different aspects of life, also the bark, fruit, and oil of B. aegyptiaca have

been widely used to treat diseases such as cancer, tuberculosis, malaria,

diabetes, sleeping sickness wounds, colds, syphilis, liver and spleen disorders

jaundice, yellow fever and snakebite (Habieballa et al., 2021).

(Anani et al., 2015). The bark of B. aegyptiaca is used to make well water

portable, also used in rural milieu to purify freshwater, and every part of the

plant have it's own beneficial properties. It is used as abortifacient and an

antidote for arrow-poison in West African traditional. Many parts of the plant

are used as famine foods in Africa; the leaves raw or cooked, the oily seed

mixed with sorghum can be use to prepare meal. The bark, fruits, seeds, seed

oil, root and leaves are widely used for the treatment of malaria, wound,

jaundice, intestinal worm infection, syphilis, epilepsy, skin disease and

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dysentery. It also provides food, medicines, cosmetics, fodder, and pesticides

valued for subsistence living in the rid and semi-arid areas where other options

are few (Anani et al., 2015).

(Saed and Isam, 2018). Explained the medical properties of "Desert Date"

plants (Balanites aegyptiaca). which is known to have multiple uses and

multiple users arid land tree with a wide range products and values such as

food, fodder, shade, oil and traditional medicine and potential shelterbelts and

agroforestry species. However, the most important part of the tree is its fruits.

The fruit is known as Desert date (common name) and lalobe (Arabic name) is a

drupe, pubscent when green becoming yellowish and glabous after ripening. It

contains four layers, the outer skin is called epicarp, the fleshy pulp is called

mesocarp, the wooden shell called endocarp and the inner seed called kernel.

All of this four layers is utilized for different industrial and pharmaceutical

product (Saed and Isam, 2018). The seed contains high amount of oil, the

oil is consumed in human food and can also be converted into biodiese. The oil

can be use medicinally, the cake remains after oil extraction, is a good source

for animal feed supplement (Saed and Isam, 2018).

However, the most important product obtains from different parts of the

tree is the sapontes. This compund proved to have wide range of industrial and

pharmaceutical applications. Some of this different part of the plant have

medicinal property in many ethnobotanical studies as antihelmethic, a

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purgative, leukoderma and emetic. Also used as anticancer, antivirus as well as

antimicrobial and act as a good antidiabetic and antioxidant agent (Saed and

Isam, 2018).

Edible uses of Balanites aegyptiaca which is the fleshy pulp of both unripe

and ripe fruit which can be eaten dried or fresh. It has a slightly astringent taste

and can be purgative in larger quantities. The fruit is processed into a drink and

sweetmeats in Ghana, an alcoholic liquor in Nigeria, a soup ingredient in Sudan

(Ajna and Richard, 2014). The ellipsoid fruit is up to 4cm long. Brown or pale

brown when fully ripe, with a brittle coat enclosing a brown or brown-green

sticky pulp and a hard stone seed (Ajna and Richard, 2014). Young leaves and

tender shoots are used as a vegetable. They are prepared by boiling and

pounding, and are then fried or mixed with fat. The flowers are a supplementary

food in West Africa and an ingredient of ‘dawa dawa’ flavouring in Nigeria.

They are sucked in order to obtain their nectar. A greenish-yellow to orange-red

resin is produced from the stems. It is sucked and chewed when fresh (Ajna and

Richard, 2014). The kernels produce an edible oil used for cooking. The oil

remains stable when heated and has a high smoking point, and therefore its free

fatty acid content is low (Ajna and Richard, 2014).

The use of medicinal plants as antibacterial, antiviral and anticancer

therapeutic agent has progress in the past decades. However, the emergence of

resistant strains for the antimicrobial and antiviral agents and the non-specific
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effect of the anticancer drugs, in addition to their severe side effects, limited

absorption and poor bioavailability reduce the clinical efficacy of currently used

therapies. Thus, the need to discover and to develop new, alternative, or

synergistic drugs from natural or synthetic origin remains urgent(Manal et al.,

2013). Another study's proved that the mixture of balanitin-6 (28%) and

balanitin-7 (72%), saponins from B. aegyptiaca kernels significantly increase

the survival time of mice bearing murine L1210 leukemia grafts to the same

extent reported for vincristine ( Manal et al., 2013).

Shashi et al., 2011. Describe antimicrobial principle from the tissue

cultures of B. aegyptiaca which is a multipurpose plant used as fodder,

medicines, charcoal and pesticides. The plant has antidiabetic, antihelmintic,

antibacterial and antiviral activities mainly attributed due to the production of

various types of secondary metabolites like flavonoids, steroids and alkaloids.

Antimicrobial activity of the various plants has been reviewed by many

workers. Callus tissues maintained as static cultures and suspension cultures

have also been screened for their antimicrobial substances. However, there have

been only a few attempts, to isolate the active antimicrobial principles from

tissue cultures and even less has been done on the identification of these

substances. The present work deals with the screening of B. aegyptiaca cultures

for their antimicrobial activity against eight microorganisms and isolating,

13
identifying and testing of the possible antimicrobial substances produced by it

(Shashi et al., 2011).

(Jahan et al., 2013). Balanites aegyptiaca Del. (Zygophyllaceae), also

known as ‘desert date’ in English, ‘Hingoli’ in Hindi and ‘Angavriksha’ in

Sanskrit, is a medicinal plant that grows in Sahel-Savanna regions and drier

parts of middle-belt zones of Nigeria, Ghana, and Ivory Coast, and is cultivated

as a fruit tree in semisavanna parts of the continent. (Jahan et al., 2013). This

plant is a small evergreen thorny treefound in drier parts of India. It grows to 6

to 10 m in height, is highly resistant to stresses such as sandstorms and heat

waves, and grows with minimal available moisture. The trees produce date-

like fruits between March and October. In ethnomedicinal studies, it has been

found that various parts of the Balanites tree have been used as folk medicine in

many regions of Africa and Asia (Jahan et al., 2013). Literature has revealed

antifeedant, antidiabetic, molluscicide, anthelminthic, and contraceptive

acteported to have anthelminthic, antifertility, purgative and antidysenteric

properties. Aqueous suspensions of dried fruits of this plant are being used as

abortifacient by local healers (Jahan et al., 2013). The root has been indicated

for the treatment of malaria, herpes zoster, and venereal diseases (Irvine, But

unfortunately, these plants are disappearing at an alarming rate due to

indiscriminate deforestation and uncontrolled collection of plant materials

(Jahan et al., 2013). Through in vitro cultivation it would be possible to

14
preserve and conserve these endangered plants and obtain phytotherapeutic

compounds especially at places where the plant does not grow naturally due to

adverse atmospheric conditions. (Jahan et al., 2013).

Despite the medicinal importance of Balanites aegyptiaca (L.) Del, the

antimicrobial potential of these plant has not been explored extensively, Various

parts of the Balanites tree have been used in folk medicine in many regions of

Africa and Asia. Literature has revealed antifeedant, antidiabetic, molluscicide,

anthelminthic, and contraceptive activities in various Balanites extract (Shahid

et al., 2012). The bark, unripe fruits, and leaves of this plant are reported to

have anthelminthic,antifertility, purgative and antidysenteric properties.

Aqueous suspensions of dried fruits of this plant are being used as abortifacient

by local healers. The root has been indicated for the treatment of malaria, herpes

zoster, andvenereal diseases ( Shahid et al., 2012).

Although there are preliminary reports on antimicrobial activities of

Balanites aegyptiaca, Moreover, Balanites aegyptiaca is reported as

endangered plant species in India. Therefore, an attempt was made to optimize

the in vitro callus induction from various parts of the plant, and then to find out

the antimicrobial activity from the extracts of the various parts (leaf, fruit and

stem) of parent plant ( Shahid et al., 2012).According to Azene et al., 2015.

multipurpose tree, B. aegyptiaca provide food, medicinal products and fuel-

wood valued for subsistence living in arid and semi-arid areas where other
15
options are few. The potential of B. aegyptiaca under management remains

unexplored and it is a priority to construct a picture of variation within the

natural range and to generate the capacity to raise plants with desirable features.

Thus, the present paper point out the overall potential of B. aegyptiaca and its

nutraceutical application ( Azene et al., 2015).

Nutritionally, Balanites leaves, flowers and fruit pulp are good sources of

protein, K, Fe, Mn, Zn and Cu. The fleshy pulp of both unripe and ripe fruit is

edible and eaten dried or fresh, the fruit is used as sweetmeats in Ghana,

alcoholic liquor in Nigeria, a soup ingredient in Sudan. Young leaves and tender

shoots are used as a vegetable, which is boiled, pounded, then fried or fat added

to prepare it. The flowers are a supplementary food in western part of Africa.

The kernels produce edible oil used for cooking. The oil remains stable when

heated and has a high smoking point, in which the free fatty acid content is low.

Its scent and taste are good. The leaves are eaten raw or cooked, the oily seed is

boiled to make it less bitter and eaten mixed with sorghum, and the flowers can

be eaten. The fruit can be fermented for alcoholic beverages. The seed contains

seed oil used as cooking oil. The seed cake remaining after the oil is extracted is

commonly used as animal fodder (Azene et al., 2015).

1.1 AIM AND OBJECTIVE

To Determine The Bacteriocidal Activity Effect Of Balanites aegyptiaca

On Spp Of Escherichia coli and Klebsiella.

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 CHAPTER TWO

2.0 MATERIALS AND METHODS

2.1 MATERIALS USED: Mueller Hinton agar, Stove, Pressure pot, Hot air

oven, Mortar and Pestle, Conical flask, Cotton wool, Wire loop, Test tube,

Weighing balance, Measuring cylinder, Aluminum foil, Petri dishes, Incubator,

Dropper pipette, Soxhlet extractor, (apparatus), Separating funnel, Cork borer,

Spirit lamp, Beaker, Handkerchief.

2.2 REAGENT USED: Distilled water, Petroleum ether, Dimethyl sulfoxide

(dmso) and Balanites oil extract.

2.3 MEDIA USED: Meuller Hinton agar and Nutrient agar.

2.4 STERILIZATION OF MATERALS

The materials such as ( Test tube, pipette, measuring cylinder, conical flask,

beaker were washed with soapy water and rinsed with distilled water, and were

allowed to dry and wrapped with foil paper and further sterilized in hot air oven

at 180C for 2hours, then allowed to cool and stored at 4C of which the media

used was also sterilized.

2.5 SAMPLE COLLECTION

Sample of Balanites aegyptiaca seed were collected from Gambari market

in Ilorin, kwara-state. These samples were put into a sterile polythene bag and

was taken to the laboratory for analysis.

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2.6 SAMPLE PREPARATION AND EXTRACTION

The Balanite aegyptiaca sample was sun dried for easy removal of the seed

from the shell, it was washed to remove unwanted particles and was dried off.

Then pounded fine powder using mortar and pestle.

Extraction was carried out according to the method adopted by (Prince, 2000).

 137g of the powder crushed from the seed was weighted and then poured

into a handkerchief and steepled properly.

 The handkerchief containing the powder was into the extraction chamber

of soxhlet extractor.

 250ml of petroleum ether was poured into the round button flask of the

extractor.

 Heating mantle was switched on at 40 degree Celsius and allowed to heat

the solvent petroleum ether.

 Oil flows through the tube of the extraction chamber into the boiling flask

and was filtered and poured into a beaker.

 It was inserted into hot air oven at 30 degree Celsius for the remaining

solvent in the oil evaporate

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2.7 SEPARATION METHOD

After the sample was removed out of the hot air oven, it was allowed to

cool. Then it was poured into a separating funnel and allowed to settle

completely. Different layers were observed, the button was opened and allowed

to drop off, when the residue layer finished, it was discarded and the oil was

collected and ready for use.

2.8 PREPARATIONS OF EXTRACT STOCK SAMPLES

 1ml of extract was measured using a sterile pipette and poured into a

clean and sterile test tube.

 9ml of dimethyl sulfoxide (dmso) was measured and added on the

extract test-tube, shaked and was labelled as “stock sample” test-tube

at 100,00

 Another five test-tubes were arranged in a row and labelled as test-

tube 1,2,3,4 and 5.

 The 1st test-tube: 0.1ml of extract sample was measured and poured

into the first test-tube and 0.9ml of dmso was measured and added on

it, which made it up to 1ml (100g/ml).

 The 2nd test-tube: using a sterile pipette, 0.2ml of extract sample was

poured into the 2nd test-tube and 0.8ml dmso was added on it, making

1ml sample (200g/ml).

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  The 3rd test-tube:0.3ml of extract sample was poured into the 3rd test-

tube and 0.7ml of dmso was added on it, also making 1ml sample

(300g/ml).

 The above procedure was also done for test-tube where 4ml and 5ml

sample and 6ml and 5ml of dmso was diluted respectively (where

made up of 400g/ml and 500g/ml.

2.9 PREPARATION OF MEDIA

 9.5g of Mueller Hinton agar was weighed and was dissolved in 250g

of distilled water in a conical flask.

 The conical flask was closed with a cotton and foil paper then sealed

with a masking tape.

 The conical flask was placed into an autoclave and sterilized at 121C

for 15minutes.

 The autoclave was turned off and the conical flask was removed and

allowed to cool for sometimes.

 An amount of the prepared media was aseptically poured into six

petri-dishes covered and allowed to solidify.

2.10 INOCULATION OF BACTERIA

 The six petri-dishes containing the solidified Mueller Hinton agar

were arranged and labelled according to the species of bacterial used

i.e Escherichia coli and Klebsiella.

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  Three labelled as E.coli and three as Klebsiella.

 Using strict plating method

 Using a sterile wire loop small amount of E.coli was inoculated in an

aseptic condition into the three petri-dishes and was strictly robbed on

the surface of the media and the petri-dishes were closed

 After sterilizing the wire loop again, calories of Klebsiella was also

inoculated throughout the surface of the three (3) plates and was

closed.

2.11 WELL DIFFUSION METHOD

After inoculating, well diffusion method was used for the experiments.

Cork borer was sterilized and well was dugged on the three media plates, two

E.coli plates were divided into two and the first was labelled 100mg, the second

200mg. the second plate divided and was also labelled 300mg and 40omg

respectively. Then the third plate of E.coli was labelled 500mg. this same

method was done to the Klebsiella plates. Then little amount of the diluent

extract of the five test-tubes were poured into each hole of the six plates which

were labelled as 100mg/ml, 200mg/ml, 300mg/l, 400mg/ml and 500mg/ml

respectively. The plates were the incubated.

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2.12 PROXIMATE ANALYSIS

The proximate analysis of soymilk samples was carried out quantitatively

to determine the moisture, crude protein, fat, crude fibre, ash and carbonhydrate

content of the samples.

Moisture Content Determination

This was carried out using the conventional method (A.O.CA.C; 1990).

Two moisture cans were dreid in the oven and then put into desiccation to cool.

5g of the sample was put in each of the moisture can and placed in the oven and

dried at 105 for 3hours. It was brought out and transferred into desiccator t cool

before weighing. The cycle of heating, cooling and weighing was repeated until

a constant weight was obtained which was determined by weight difference and

expressed as a percentage of the sample weighed.

It was given by:

% moisture = / W2-W1×100: where:

W1= weighed of empty can : W2=weight of can + sample before drying : W3=

weight of can + sample at constant weight.

Ash Content Determination

The method recommended by A.O.A.C. (1990) was used. The crucibles

were dried and cooled in a desiccator before use. 5g of the sample was weighed

22
into the crucibles, covered and placed in a muffle furnace at temperature of

55C. This temperature was maintained for 2hours until a whitish ash is

maintained. The muffle furnace was switched off and the crucible were

removed and placed in a desiccator to cool. The crucible containing the sample

were weighed and the percentage ash content as determined.

% Ash=W2-W3/W2-W1×100

Where W1= weight of crucible; W2=weight of sample + crucible; W3+weight

of crucible + ash.

Fibre Determination

Crude fibre was determined by the method described by (Josyln,1990).

Exactly 2g of the sample was treated with 20ml of 1.5M H2SO4 and boiled for

30minutes. The resultant was filtered under suction, washed with hot distilled

water and boiled again for another 30 minutes with 1.25M NaOH. The digested

sample was then washed severally with hot distilled water. The washed sample

was scrapped into a crucible, dried at 100 for 1hour, cooled and weighed. The

loss in weight on incinerator was taken as the weight of the crude fibre.

Thus % crude fibre = loss in weight on incinerator/weight of original sample ×

100.

23
Fat and determination (BY SOXHLET EXTRACTIION METHOD)

About 2g of sample was placed into soxhlet extractor. The extractor was

placed into a pre-weighed dried distillation flask. Then the solvent (acetone)

was introduced into the distillation flask via the condenser and attached to the

soxhlet extractor. The setup was held in place with a retort stand clamp. Cooled

water jet was allowed to flow into the condenser and the heated solvent was

refluxed as a result, the lipid was observably extracted to concentrate the lipid;

the flask was then dried with the air oven at 600 to constant weight and re-

weighed to obtain the weight of lipid. This was done by continuous extraction in

a soxhlet reflux apparatus (Pearson, 1976).

%Ash = W1-W3/W2-W1×100

Where W1=weight of empty flask; W2=weight of sample; W3=weight of flask

+ oil extract.

Distillation

About 20ml diluted digest was measured into a distillation flask and the

flask was held in place on an electro-thermal heater hot plate. To the distillation

was attached condenser, 40% sodium hydroxide is injected into the digest via a

syringe at the head to the micro arm steel head until the digest becomes strongly

alkaline. The mixture was heated to boil and distill the ammonia gas vi the

24
condenser into the receiver beaker. The color of the acid changed from purple to

greenish Ammonia distillate was introduces into the acid.

Titration

The distillate was titrated with 0.1N hydrochloric acid back to purple color from

greenish. The volume of hydrochloric acid added to effect the change was

recorded as titre value.

CALCULATION: %N = titre value × 1.4×100

Carbohydrate Determination:

Carbohydrate was determined by weight difference. Thus % carbohydrate =

100- (%moisture + %ash + %fat + %fibre + %crude protein).

25
 CHAPTER THREE

3.0 RESULT ANALYSIS AND TABLES

The table below shows the Antibacterial activity of Balanites aegyptiaca

seed oil extract against Klebsiella using well diffusion method:

TABLE-1:

CONCENTRATION OF ZONE OF INHIBITION

EXTRACTION
100mg/ml No visible zone

200mg/ml 0.2mm/dm

300mg/ml No visible zone

400mg/ml No visible zone

500mg/ml 0.1mm/dm

Antibacterial activity of Balanites aegyptiaca seed oil extract against

Escherichia coli using well diffusion method.

TABLE-2:

CONCENTRATION OF EXTRACT ZONE OF INHIBITION

100mg/ml No visible zone

200mg/ml No visible zone

26
300mg/ml No visible zone

400mg/ml 0.1mm/dm

500mg/ml 0.3mm/dm

3.1PROXIMATE ANALYSIS RESULT

Sample %Moisture %Ash %Fibre %Lipid %Protein %Cho %Dry

Matter
Balanites 4.68 0.79689 0.760719 0.615 4.375 88.8123 95.36

aegytiaca

27
 CHAPTER FOUR

4.1 DISCUSSION

Antibacterial activity of the Balanites aegyptiaca oil at different concentrations

on Klebsiella and Escherichia coli bacteria and different meters in diameter of

the zones was observed. In some cases the higher the concentration the higher

the zone of inhibition found in the experiment of Klebsiella where 0.2mm/dm,

0.1mm/dm was recorded for 200mg/ml and 500mg/ml and no of visible zone

was recorded for 10mg/ml. 30mg/ml and 400mg/ml. Also in the case of

Escherichia coli the highest zone of inhibition where 0.3ml/dm and 0.1mm/dm

was observed and recorded for 500mg/ml and 400mg/ml concentration and no

visible zone was recorded for 100mg/ml, 200mg/ml and 300mg/ml respectively.

The results obtained shows that Balanites aegyptiaca has an antibacterial agent

that acted on bacterial. However my own experiment did not meet the general

rule, because it is stated that seed extract concentration should be considered

active against bacteria when the zone of inhibition is greater than 6mm. After

the inoculation and incubation of the cultured plates, the observation was done

after 24hours, and it was found out that there were clear spaces around the cup

borrer well also known as zone of inhibition and it was measured using a metre

ruler, whereas the other part of the plate there were presence of bacteria growth,

this indicates the effect of the oil on the bacteria. The inhibition zone was

supposed to increase gradually with increase in concentration.

28
4.2 CONCLUSION

The findings from the bacterocidal activity effect of Balanites aegyptiaca

extract on the spp of Eschrichia coli and Klebsiella studies shows that Balanites

aegyptiaca has an antibacterial agent that acted on bacterial. It is stated that seed

extract concentration should be considered active against bacteria when the

zone of inhibition is greater than 6mm.

4.3 RECOMMENDATION

29
 REFERENCES

Ajna Fern and Richard Morris (2014). Useful Tropical Plant Balanites

aegyptica.

https://tropical.theferns.info/viewtropical.php?id=Balanites+aegyptiaca.

Anani Kokou , Yao Adjrah, Yaovi Améyapoh, Simplice Damintoti Karou,

Amegnona Agbonon, Comlan de Souza, Messanvi Gbeassor (2015).

Antimicrobial activities of Balanites aegyptiaca (L.) Delile

(Balanitaceae) on bacteria isolated from water well. Journal of

Applied Pharmaceutical Science Vol. 5 (10), pp. 052-058, Available

online at http://www.japsonline.com DOI:

10.7324/JAPS.2015.501010 ISSN 2231-3354.

Azene Tesfaye (2015). Balanites (Balanite aegyptiaca) Del., Multipurpose Tree

a Prospective Review. International Journal of Modern Chemistry and

Applied Science 2015, 2(3), 189-194.

Chothani, H. Daya L. U. Vaghasiya (2012). A review on Balanites aegyptiaca

Del (desert date): phytochemical constituents, traditional uses, and

pharmacological activity.

Eshetu Molla; Mirutse Giday; Berhanu Erko (2013). "Laboratory assessment of

the molluscicidal andcercariacidal activities of Balanites aegyptiaca.

30
 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3703561) . Asian

Pacific Journal of Tropical Biomedicine.

Habieballa Abdalla Gobara, Halima Elfadel Alebead, Madena Komi Koko,

Awad Salim Ibrahim and Asha Fadllallah Wady ( 2021).

Antimicrobial activity and physicochemical properties of Balanites

aegyptiaca seed oil. European Journal of Chemistry Pp 450-453.

Jahan Noor, Razia Khatoon, Anwar Shahzad and Mohammad Shahid (2013).

Antimicrobial activity of medicinal plant Balanites aegyptiaca Del.

and its in vitro raised calli against resistant organisms especially those

harbouring blagenes .Vol. 7(25), pp. 1692-1698, DOI:

10.5897/JMPR2013.5109 ISSN 1996-0875 Academic Journals

http://www.academicjournals.org/JMPR Journal of Medicinal Plants

Research.

Kabbashi S. Ahmed, Shams Eldien Koko, Talib M. A., Suliman I. Suliman and

Muddathir S. Alhassan (2017). Antimicrobial activity of Balanites

aegyptiaca leafs. pp 43-46 ISSN 2455-3301 WORLD JOURNAL OF

PHARMACEUTICAL AND MEDICAL RESEARCH

www.wjpmr.com.

Kashari, O., Anyekema, M., Malik, A.I, Henshaw, E and Udeme, A. M (2021).

Antibacterial Activity of Balanites aegyptiaca Leaf and Stem bark

31
 Extracts against Some Clinical Test Bacteria. International Journal of

Advanced Academic Research | ISSN: 2488-9849Vol. 7, Issue 8.

Manal A. Awad, Rabab A. El Dib, Nawal Almusayeib, Shaza Al-massarani,

Khalid M. O. Ortashi, Awatif A. Hendi (2013). Novel Balanites

aegyptiaca mesocarp synthesized silver nanoparticles: formation,

characterization, antimicrobial, cytotoxicity and ANTIVIRAL

EFFECTS. Vol. 8, No. 4, p. 1665-1677, Digest Journal of

Nanomaterials and Biostructures.

Sabina Khanam and Fatima Zakari’yau Galadima(2021). Antibacterial activity

of Balanites aegyptiaca Oil Extract on Staphylococcus aureus and

Escherichia coli.uthor/funder. All rights reserved. No reuse allowed

without permission.

https://doi.org/10.1101/2021.03.23.436600 ;Department of Biological

Sciences.

Saed A. Al-Thobaiti and Isam M. Abu Zeid(2018). Medicinal Properties of

Desert Date Plants (Balanites aegyptiaca). Global Journal of

Pharmacology.

Shahid Mohd, Noor Jahan, Anwar Shahzad, Aastha Sahai, Shivali Sharma and

Shahina Parveen (2012). Antimicrobial Potential of Balanites

Aegyptiaca (L.) Del, Stevia Rebaudiana you(Bert.) Bertoni, Tylophora

32
 Indica (Burm.f.) Merrill, and Cassia Sophera(Linn.).The Open

Conference Proccedings Journal, 3, Pp 63-69.

Shashi Bidawat, Ruchi Nag And T. N. Nag (2011). Antimicrobial principles

from tissue cultures of Balanites aegyptiaca. Vol. 16, Romanian

Biotechnological Letters.

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