CHAPTER 1

INTRODUCTION

1.0 Study Background

In modern days, traditional medicine has been brought into focus for meeting the goals of a wider
coverage of primary healthcare delivery not only in Africa, but also, to various extents, in all countries of
the world and is the first choice healthcare treatment for at least 80% of Africans who suffer from high
fever and other common ailments (Treben, N.,1998). Newbouldia laevis is found in tropical Africa and it
grows to a height of about 10 meters with a cauliferous habit.

Newbouldia laevis is popularly known as the tree of life or fertility tree in Nigeria (Ainooson, et al, 2009).
Newbouldia laevis is commonly called African Border tree or boundary tree (Gbile and Adesina, 1986). It
is called "Aduruku" in Hausa; "Ogirisi" in Igbo; *Kkhimi" in Edo and "Akoko" in Yoruba languages
(Ogunlana and Ogunlana, 2008) .

Newbouldia laevis bark is chewed and swallowed to heal stomach pain, diarrhea and toothache. (Lewis
and manony, 1977, Machina. 2003). The extracts of this plant are used for the treatment of
elephantiasis, dysentery, rheumatic swellings, syphilis, constipation, pile and as a vermifuge for round
worms. It is also useful for the treatment of ear ace, sore foot, chest pain, epilepsy and convulsion
(Akunvili. 2000). The leaf, stem and fruits have been used for febrifuge, wound dressing and for

stomach ache (lwu, 2000). The plant is a multipurpose one and its leaves, fruits and flowers, bark

and roots have various medicinal value. Studies by (Oliver-Bever, 1986) revealed that the leaf

and root bark contain flavonoids, saponins, quinines, terpenes and steroids. Recent studies have

revealed the presence of alkaloids, quinoids, phenyl propanoids amongst other chemicals.

The aqueous and ethanol leaf extracts of Newbouldia laevis displayed uterine contractile effects (Bafor
and Sanni, 2009). Further, like many medicinal plants, many of the uses and possible adverse effects of
the plant by traditional medicine practitioners have not been investigated. Hence, this research
investigated possible effect of the leaf extract on ocular inflammation, corneal ulcer and intra-ocular
pressure.

1.1 Background Information

1.1.1 Cornea and Vision

The cornea is a vascular transparent structure that occupies the anterior 1/6th of the outer

fibrous coat of the eyeball. The cornea is horizontally oval measuring 11-12mm horizontally

and 9-11mm vertically. It is responsible for two-third of the eye's total refractive power of

43D with a refractive index of 1.376. It helps to focus rays of light on the retina. The cornea
has a lot unmyelinated nerve endings that are sensitive to touch, temperature, and chemicals stimulus.
The cornea transparency is very important as it is how it carries out its refractive purpose. There are
different mechanisms that the cornea uses to maintain its transparency. It does not have or need blood
vessels within it. Nutrients also are transported to the cornea by diffusion from the tear film outside and
the aqueous humor inside the eye.

1.1.2 Cornea Histology

Histologically, the cornea has five distinct layers (Khurana 2003 pg 89). From anterio- posterior these
are;

 Epithelium
 Bowman's membrane
 Substantia propria (Stroma)
 Descemet's membrane
 Endothelium

 Corneal Epithelium: This is a very thin multicellular stratified squamous cell that

becomes continuous with the epithelium of the bulbar conjunctiva at the limbus. It

consists of 5-6 layers which are shed constantly on the exposed layer and regenerate

by multiplication of the basal layer.it is a fast growing and easily regenerated cell type

kept moist with tears. It serves as a barrier to chemicals, water and also to microbes. It

also possesses some immunological functions due to the presence of the Langerhan's

cell.

 Bowman's Layer: it is a tough layer composed of collagen, laminin, perlecan that

protects the stroma. It is not a true elastic membrane but simply a condensed

superficial part of the stroma. It is considerably resistant to infection but once destroyed, does not
regenerate. The Bowman's layer helps in maintaining corneal

shape.

 Corneal Stroma (Substantia Propria) : This layer is about 0.5mm in thickness and

consists about 90% of the total thickness of the cornea. It consists of collagen fibrils

(lamellae) embedded in hydrated matrix of proteoglycans. The cornea stroma helps

give mechanical strength to the cornea, aids in corneal transparency and is also the

main refracting lens of the cornea.

  Descemet's membrane: The Descemet's membrane is the resting layer for the

endothelia cells. It consists of collagen and glycoproteins. It binds the stroma

posteriorly and is very resistant to chemical agents, trauma, and pathological

processes. Therefore, descemetocoele can maintain the integrity of the eyeball for

long. Unlike the Bowman's layer, it can regenerate.

 Endothelium: it consists of a single layer of flat polygonal cells that helps maintain

corneal clarity by removing water from the corneal stroma. It is a simple squamous or

low cuboidal monolayer approximately micrometer. Thick of mitochondria rich

cells.

1.1.5 Corneal Inflammation

Corneal inflammation (keratitis ) is a condition where the corneal is inflamed and shows the following
characteristics of corneal oedema, cellular infiltration and cilliary congestion. It may be superficial or
deep and can lead to corneal ulcer when not properly treated. The five cardinal signs of inflammation
are;

 Pain
 Redness
 Immobility
 Swelling and
 Heat

Keratitis has many potential causes which includes; infections (such as bacteria, virus, fungi,

contact lens induced acanthamoeba keratitis), physical or chemical trauma, dry eyes, etc. The

symptoms include; eye pain, blurred vision, photophobia, tearing and red eye.

Once the corneal epithelium has been invaded by the inflammatory agents, the sequences of

pathological changes that occur include the following;

Infiltration

Active ulceration

Regression

Cicatrization

1.1.10 Intra ocular pressure

Intra ocular pressure is the measurement of the amount of fluid pressure ( aqueous humor ) inside the
eye. It is determined by the production

and drainage of the aqueous humour by the cilliary body and drainage via the trabecular

meshwork and uveosclera outflow. The normal IOP of humans is between 11-21mmHg. There is diurnal

variation of IOP of about 3-6mmHg for normal eyes and even more for the eyes of those with glaucoma.

1.3.1 Measurement

Intraocular pressure is determined by the production and drainage of aqueous humour by the

ciliary body and its drainage via the trabecular meshwork and uveoscleral outflow. The reason

for this is because the vitreous humour in the posterior segment has a relatively fixed volume and

thus does not affect intraocular pressure regulation.

An important quantitative relationship is provided by Aptel, et al. (2016) below:

Po=F-UV/C+Pv

Where:

Po is the IOP in millimeters of mercury (mmHg)

F the rate of aqueous humour formation in microliters per minute (uL/min)

U the resorption of aqueous humour through the uveoscleral route (in uL/min)

C is the facility of outflow in microliters per minute per millimeter of mercury

(uL/min/mmHg)

P the episcleral venous pressure in millimeters of mercury (mmHg).

The above factors are those that drive IOP.

1.3.2 Significance of the Intraocular Pressure

A prominent and consistent increased pressure can result in a number of ocular complications. One of
such complications is glaucoma. Glaucoma is not a single disease process but a group of disorders

characterized by a progressive optic neuropathy resulting in a characteristic appearance of the

optic disc and a specific pattern of irreversible visual field defects that are associated frequently

but not invariably with raised intraocular pressure (IOP). Raised intraocular pressure causes

mechanical stretch on the lamina cribosa, leading to axonal deformation and ischemia by altering
capillary blood flow. As a result of this, neurotrophins (growth factors) are not able to reach the

retinal ganglion cell bodies in sufficient amount needed for their survival (this is the mechanical

theory of glaucoma). Thus, intraocular pressure is the most common risk factor, but not the only

risk factor for the development of glaucoma.

Consequently, the term 'ocular hypertension' is used for cases having constantly raised

intraocular pressure without any associated glaucoma damage. Conversely, the term 'normal' or

*low' tension glaucoma (NTG/LTG) is suggested for the typical cupping of the disc and/or visual

field defects associated with a normal or low intraocular pressure.

1.3.3 Variation of IOP

A healthy eye should have roughly consistent intraocular pressure at all times. This demonstrates how
distinctive and efficient the pressure controlling system is. This is due to the fact that the rate of
aqueous generation and drainage is roughly similar. Therefore, a change in the equilibrium between
aqueous production and outflow is typically to blame for an unexpected increase in intraocular pressure.
The intraocular pressure changes throughout the day (diurnal fluctuation), and is often highest just after
awakening and lowest in the late afternoon. In glaucomatous eyes, where it can reach up to 40mmHg in
fluctuation, this diurnal variation can range from 3-4mmHg and considerably higher. Other differences
include changes in blood pressure, pulse, and breathing that have an impact on the arterial pulse might
vary by up to 2-3 mmHg due to the intraocular pressure expanding the ocular coatings and vessel walls.
The systolic phase of the pulse causes the arteries in the eyes to dilate, which raises intraocular
pressure.

1.3.4 Factors that Influence Intraocular Pressure

 Age
 Heredity
 Sex
 Diurnal variation
 Postural variation
 Blood pressure:
 Osmotic pressure of blood

l
CHAPTER 2

LITERATURE REVIEW

2.1.1 Anti-inflammatory effect of Newbouldia laevis leaf extract

Various study done with Newbouldia laevis suggest a fair amount of potency in its pharmacological
properties. A study was carried out by Udeozo et al., (2014) to deteremine determine the
phytochemical, anti-inflammatory and acute toxicity properties of Newbouldia laevis flower. Acetic acid-
induced writhing in mice and formalin test in rats were used to carry out this study. The ethanolic
extract of the Newbouldia laevis flower caused a significant decrease (p<0.05) in the induced conditions
which was not a dose dependent inhibition on acetic acid induced writhing and the neurogenic pains
which was induced by formalin. The Newbouldia laevis flower extract at the doses of 25, 50 and 100
mg/kg showed 59. 71 and 47% inhibitions of the abdominal construct in mice respectively.

Kolawole et al., (2014) carried out a study to investigate the effect of the leaf extract of Newbouldia
laevis on some enzymes of hepatic glucose metabolism in induced diabetic rats. Intravenous injection of
streptozotoan in the rat was used to induce diabetes and for four weeks the rats were orally treated
with Newbouldia laevis leaf extract. The activities of hepatic glucokinase and glucose 6-phospate

were evaluated at the end of the study and it was deduced that the Newbouldia laevis leaf

extract stimulates the activity of hepatic glucokinase and inhibits the activity of glucose 6-

phosphate in induced diabetic rats.

Usman et al, (2008) did a study on the analgesic and anti-inflammatory effects of the ethanolic flower
extract of Newbouldia laevis in fifty rodents. They were induced with acetic acid and carrageenan-
induced hind paw edema in rats. The results showed that the ethanol extract possessed significant
(p<0.001) anti-nociceptive activity between 50 and 200 mgkg-' intraperitoneally in mice and also dose
dependent anti-inflammatory activity between 50 and 200mgkg' in rats ( the extract exhibited highest
toxicity at this value ) .

Although Agbafor et al., (2015) found similar results in the phytochemical analysis of the leaf and root
extracts of Newbouldia, during their investigation on the cardio-protective effect of the leaf and root
extracts of Newbouldia laevis against carbon tetrachloride induced-cardiotoxicity in albino rats, the
analysis also revealed the presence of Tannins in the leaf extract and Glycosides in the root extract.

The presence of these phytochemicals gives the nudge to further investigations on the plant's

ability in dealing with specific pathological cases. Investigations seem to be geared towards its

effect on inflammatory conditions and infective conditions, usually bacterial and fungal infections.

Chukwujekwu, et al. (2005) did an investigation the antibacterial, anti-inflammatory and antimalarial
activity of some Nigerian medicinal plants, in which Newbouldia laevis was one of the plant species
investigated. Cyclooxygenase (COX-1 and COX-2) assays were used to test for inflammatory activity. All
the plant species, except for two, showed anti-inflammatory activity with Newbouldia laevis root extract
having one of the highest activity (86‡1.9% with petroleum ether root extract, and 781.4% with
dichloromethane root extract) of prostaglandin synthesis inhibition, thereby treating inflammation.

2..1.2 Effect of Newbouldia laevis on Intraocular Pressure

Though Newbouldia lavis's leaves extracts has been used to combat myriad of ailments, from
antiinflamatory, antibacterial and antimalarial activity it effect on the intraocular pressure has not been
investigated. Hence, this study will be one of the first to the best of our knowledge to investigate its
effect on the intraocular pressure of laboratory models

(rabbits).
CHAPTER 3

RESEARCH DESIGN

This study was an experimental study that was performed on the eyes of 30 live
New Zealand rabbits.

STUDY POPULATION

This study included 30 clinically normal adult New Zealand rabbits, which
includes 15 males and 15 females.

STUDY LOCATION

This study was done in the Animal House of the Department of Animal and
Environmental biology, Faculty of Life Science, in the University of Benin, Edo
State.

STUDY DURATION

This study was carried out within a time frame of four (4) weeks.

SAMPLE SIZE

A total number of thirty (30) subjects was used for this study.

SAMPLING TECHNIQUE

This study made use of a simple random sampling technique. Simple random
sampling, in that 30 rabbits was pooled together and one rabbit was selected at
random as a member of a group.
INCLUSION CRITERIA

● Healthy new Zealand rabbits

● Rabbits within the weight range (2.0kg -3.0kg)
● Rabbits without ocular pathology
EXCLUSION CRITERIA
● Sick rabbits
● Rabbits with any ocular pathology
● Rabbits outside the stimulated weight range
● Rabbits of different species

ETHICAL CONSIDERATIONS
Ethical consideration for this study was obtained from the ethical committees of
the Departments of Optometry, University of Benin, Benin City, Edo State.

RESEARCH INSTRUMENTS AND MATERIALS

The materials, instruments and drugs used for this research include the following;
● Newbouldia laevis leaf, stem and root
● Soxhlet Extractor
● Applanation tonometer
● 30 adult New Zealand rabbits
● Rabbit feed, anticoccidal drugs, antifilarial drugs , multivitamins
● Metal cages
● Weighing balance
● Sterile syringes(2ml and 5ml)
● Topical Anaesthetics
● Slit knife
● Dexamethasone tablets
● Dexamethasone eyedrop
● Atropine 1%
● Sterile Fluorescein sodium strips
● Placebo (saline)
● Distilled Water
● Sterile Filter paper
● Beakers, Petri dish, conical flasks
● Sterile drop containers
● Cotton wool
● Methylated Spirit
● Sterile Latex gloves
● Handheld magnifier(8.00-10.00D)
● Penlight
● Direct Ophthalmoscope
● Iphone 12 Pro Camera
● Refrigerator
● Writing materials for documentation

● Blue filter button lamp

DESCRIPTION OF PROCEDURE

COLLECTION OF LEAF, STEM BARK, AND ROOT BARK OF

NEWBOULDIA LAEVIS PLANT AND PREPARATION OF THEIR
EXTRACT

Fresh leaf, stem bark, and root bark of Newbouldia laevis plants was obtained from
a garden in the Senior Staff Quarters in the University of Benin. After which they
were taken to the Department of Plant Biology and Biotechnology, in the Faculty
of Life Sciences for appropriate identification. After which they were washed, the
stem and root bark will be cut into pieces and air dried for fourteen days before
being grounded into powder using a milling machine. Ethanolic Extraction of the
leaf, stem bark and root bark will be carried out in the Department of
Pharmacognosy, Faculty of Pharmacy in the University of Benin. Two kilograms
of the grinded powder was subjected to Soxhlet extraction with methanol.

ANIMALS AND HANDLING

This study was a controlled experimental study which was divided in two parts: the
Adaptive Phase (Phase 1) where the animals was made to adapt to their
environment for a period of two week and then the Experimental Phase (Phase
2&3) during which the conditions of the experiment was administered and the
treatments was initiated. 30 healthy adult New Zealand rabbits of either sex, with
an average weight ranging from 2.0kg -3.0kg, was obtained from an animal farm in
Benin City. The Rabbits was kept in metal cages in the Animal House in the
Department of Animal and Environmental biology and was given two weeks to
adapt to their new environment (acclimatization), with appropriate antibacterial
(Keproceryl) and antifiliarial (Ivermectin) and anticoccidal (Embazin) treatments that was
administered in the rabbit feed along with multivitamins and nutritional
rations .The end of the acclimatization phase (Phase 1) begins the experimental
phase (Phase 2) in which the inflammation was inflicted on the rabbits eyes and
also the IOP was increased with the administration of dexamethasone eyedrops and
tablet , once the pressure was well increased and the inflammation has set in, the
various plants extracts was administered three times daily and the rate of healing of
the rabbits was divided into three groups .

Grouping and Handling of Animals

Six rabbits were separated as control for the test. This control group was tagged
group G. The remaining 30 rabbits were grouped into seven groups, Groups A, B,
C, D, E, F and G. One group was tested for the effect of the extract on one ocular
condition. Each group had the respective conditions of Inflammation and intra
ocular pressure induced in their eyes.

GROUP A: This group comprise of four Rabbits that was inflicted with inflammation on their
right eyes and this inflammation was attained by the use of ophthalmic slit knife to physically
induce injury on the corneo-conjuctival of the rabbits. After twenty four hours, excess tearing
along with other classical signs of uveitis (red eye/cilliary injection, photophopia). (Villena eya
love 1999). Baseline data of their schrimmer test was taken and also was taken daily after
inflammation was induced . After which they were treated with the ethanolic leaf extracts of
newbouldia Leavis plant three times daily. Until the inflammation was healed and the schrimmer
test value returned to baseline data.

GROUP B: This group comprise of four Rabbits that was inflicted with inflammation on their
right eyes and this inflammation was attained by the use of ophthalmic slit knife to physically
induce injury on the corneo-conjuctival of the rabbits. After twenty four hours, excess tearing
along with other classical signs of uveitis (red eye/cilliary injection, photophopia). (Villena eya
love 1999). Baseline data of their schrimmer test was taken and also was taken daily after
inflammation was induced . After which they were treated with the ethanolic Stem bark extracts
of newbouldia Leavis plant three times daily. Until the inflammation was healed and the
schrimmer test value returned to baseline data.

GROUP C: This group comprise of four Rabbits that was inflicted with inflammation on their
right eyes and this inflammation was attained by the use of ophthalmic slit knife to physically
induce injury on the corneo-conjuctival of the rabbits. After twenty four hours, excess tearing
along with other classical signs of uveitis (red eye/cilliary injection, photophopia). (Villena eya
love 1999). Baseline data of their schrimmer test was taken and also was taken daily after
inflammation was induced . After which they were treated with the ethanolic root bark extracts
of newbouldia Leavis plant three times daily.

GROUP D: Group D were tested on induced increase IOP, baseline data of the rabbits' intraocular
pressure were collected using the Perkins Tonometer. The rabbits were given one tablet of
Dexamethasone orally and had one drop of Dexamethasone 0.1%, then one drop Atropine 1% instilled
in one eye (right eye) four times daily for two weeks . After 2 weeks the steroid had artificially increased
the intraocular pressure of the rabbits' eyes. After which they were treated with the ethanolic Stem
bark extracts of newbouldia Leavis plant three times daily

GROUP E: This group of rabbit were tested on induced increase IOP, baseline data of the
rabbits' intraocular pressure were collected using the Perkins Tonometer. The rabbits were given one
tablet of Dexamethasone orally and had one drop of Dexamethasone 0.1%, then one drop Atropine 1%
instilled in one eye (right eye) four times daily for two weeks . After 2 weeks the steroid had artificially
increased the intraocular pressure of the rabbits' eyes. After which they were treated with the
ethanolic root bark extracts of newbouldia Leavis plant three times daily

GROUP F: This group of rabbit were tested on induced increase IOP, baseline data of the
rabbits' intraocular pressure were collected using the Perkins Tonometer. The rabbits were given one
tablet of Dexamethasone orally and had one drop of Dexamethasone 0.1%, then one drop Atropine 1%
instilled in one eye (right eye) four times daily for two weeks . After 2 weeks the steroid had artificially
increased the intraocular pressure of the rabbits' eyes. After which they were treated with the
ethanolic leaf bark extracts of newbouldia Leavis plant three times daily.

GROUP G
This group will serve as the control. This group of animals won't have any kind of
physically caused eye inflammation or elevated intraocular pressure. Additionally,
no ethanolic extracts of the Newbouldia Leavis plant may be given to them. There
will be six rabbits in it

LIMITATIONS OF STUDY

● The cost to carry out the study might be very high due to the current state of
recession in the country
● It can really take a long time for meaningful results to be gotten.
● The number of the test subjects might be reduced due to unexpected death
thereby causing a reduction in sample size
● The laboratory environment and other variables might have an effect on
study outcomes.

DATA ANALYSIS
Data collected was analyzed using analysis of variance (ANOVA) as processed by
Statistical Package for social sciences (version 22.0). IOP data was expressed as
the mean and was analyzed by a two-tailed paired t-test for ratios compared to 1.0
or for differences compared with 0.0.