CHAPTER ONE

1.0 INTRODUCTION
Higher animals are always at risk of been injured or infected.The body has a
central element of natural immunity which acts as a protective mechanism in
other to defend them,which is in the form of inflammation.This occurs by the
activation and release of chemical signals which include monocytes, eosinophils
and neutrophils from the venous system to the target sites of the injury.The
inflammatory mechanism involves a four step Viz;firstly, adhesion molecules
such as E,L and P selectin are activated then followed by triggering chemical
signals modulated by leukocytes activating molecules and cytokines which then
activate the upregulation of leucocyte integrins.Next, neutrophils are
immobilized on the surface of the vascular endothelium by the action of alpha
4 beta 7 and alpha 4 beta 1 integri s binding to MadCAM-1 and VCAM-1
accordingly.Lastly, inflammatory cells are then transmitted to the affected
sites via the endothelium by extracellular proteases like matrix
metalloproteinases(MMPs)(Chettibi et al.,1999). Inflammation is of two types;
acute and chronic inflammation. Acute inflammation is the immediate and
early response to injury designed to deliver leucocytes to sites of injury.once
there, leucocytes clear any invading microbes and begin the process of
breaking down necrotic tissues. This process has two major components;
vascular changes which includes alterations in vessel caliber resulting in
increased blood flow (vasodilation) and structural changes that permit plasma
proteins to leave the circulation(increased vascular permeability) and the
second is cellular events which involves emigration of the leucocytes from the
microcirculation and accumulation in the focus of injury (cellular recruitment
and activation). The cascade of events in acute inflammation is integrated by
local release of chemical mediators. The vascular changes and cell recruitment
account for three of the five classic local signs of acute
inflammation:heat(calor),redness(rubor),and swelling (tumor). The two
additional cardinal features of acute inflammation are pain(dalor) and loss of
function (functiolaesa) ,occur as consequences of mediator elaboration and
leukocyte-mediated damage.Acute inflammation generally has one of three
outcomes:

Resolution:when the injury is limited or short-lived ,when there has been no or

minimal tissue damage ,and when the tissue is capable of replacing any
irreversibly injured cells,the usual outcome is restoration to histologic and
functional normalcy.This involves neutralization or removal of the various
chemical mediators, normalization of vascular permeability,and cessation of
leukocyte emigration with subsequent death(by apoptosis) of extravasated
neutrophils. Eventually,the combined efforts of lymphatic drainage and
macrophage ingestion of necrotic debris lead to the clearance of the edema
fluid, inflammatory cells,and detritus from the battlefield.
Scarring results after substantial tissue destruction or when inflammation
occurs in tissues that do not regenerate. In addition,extensive fibrinous
exudates(due to increases vascular permeability) may not be completely
absorbed and are organized by ingrowth of connective tissue with resultant
fibrosis. Abscess formation may occur in the setting of extensive neutrophilic
infiltrates or in certain bacterial or fungal infections. Due to the extensive
underlying tissue destruction,the only outcome of abscess formation is
scarring.

Progression to chronic inflammation may follow acute inflammation, although

signs of chronic inflammation may be present at the onset of injury. Depending
on the extent of the initial and ongoing tissue injury,as well as the capacity of
the affected tissues to regrow , chronic inflammation may be followed by
regeneration of normal structure and function or may lead to scarring.(Feghali
and Wright., 1997)

The second type is chronic inflammation which is considered to be

inflammation of prolonged duration (weeks to months to years) in which active
inflammation, tissue injury and healing proceed simultaneously. In contrast to
acute inflammation,which is largely neutrophilic infiltrate, chronic
inflammation is characterized by the following::: infiltration with mononuclear
cells, including macrophage, lymphocytes and plasma cells. Tissue destruction,
largely directed by the inflammatory cells and lastly repair,involving new
vessel proliferation and fibrosis.
Chronic inflammation arises in the following settings:

•viral infections, intracellular infections of any kind typically require

lymphocytes to identify and eradicate infected cells.

•persistent microbial infections,most characteristically by a selected set of

microorganisms including mycobacteria,treponema pallidum and certain
fungi. These organisms are of low direct pathogenicity,but typically they evoke
an immune response called delayed hypersensitivity, which may culminate in a
granulomatous reaction.

•prolonged exposure to potentially toxic agents, examples include no

degradable exogenous material such as inhaled particulate silica, which can
induce a chronic inflammatory response in the lungs and endogenous agents
such as chronically elevated plasma lipid components ,which may contribute to
atherosclerosis.

• autoimmune diseases,in which an individual develops an immune response to

self-antigens and tissues.Because the responsible antigens are in most instances
constantly renewed, a self-perpectuating immune reaction results (e.g
rheumatoid arthritis or multiple sclerosis).(Feghali and Wright., 1997).
Anti-inflammatory drugs (non steroidal anti-inflammatory drugs NSAIDs and
corticosteroids) are used to treat the inflammation,even though they cause
renal toxicity and gastric ulcerations over a long period of time. However,oral
administration of such drugs is limited by the first pass effect, pH if the GIT
and the intestinal barrier to drug absorption which reduce the bioavailability
of the drug(Bowman,K et al .,. 2006).

1.1 BACKGROUND INFORMATION

Annona muricata is a lowland tropical fruit bearing tree in the annonaceae

family,which is a family of flowering plants. Annonaceae is the largest family
in the magnoliales order and comprises approximately 130 genera and 2,300
species. (Mishra S et al. 2013). Annona muricata is a species of the genus
annona,derived from the Latin word 'anon' which means'yearly produce' or
'annual harvest'. As the name suggests, Various species in this genus generally
have annual fruit production.(Oyekachukwu AR, et al., 2017).
Annona muricata is an evergreen,small and upright tree that grows up to 9.1m
tall (Jillian F. 1987). It has an edible whitish aromatic fruit that is pricky and
green or dark green externally with about 30cm long and a slightly firm
texture. ( Walker JW 1971). The peduncles are usually woody and are
opposite to the leaves while the petals are thick and yellowish. The young
branches are hairy and are oblong to oval.(Walker JW 1971). In Nigeria, the
plant is called soursop,choo-choo and sapi sapi (Ana v. et al., 2018).
Traditional medicine uses of annona muricata include analgesic,
antipyretic ,anti-inflammation management of hypertension, respiratory
diseases, management of diabetes as well as cancer. (Ana. V et al., 2018).

1.2 STATEMENT OF RESEARCH PROBLEMS

According to Russell (2021) non steroidal anti-inflammatory drugs (NSAIDs)
are the most extensively used drugs to alleviate inflammation and pain, over
20 million prescriptions are written every year in the UK and over 70 millions
in the USA. Robert L. Barking(2015) stated that the use of NSAIDs is limited
by the dose dependent adverse effects like renal toxicity, cardiovascular events
and gastrointestinal disturbances. It is high time to involve in research using
nanoparticle technology which provides particles with smaller sizes,large
surface areas and even potentially modifiable surfaces to curb with such
adverse effects and enhance bioavailability of the drug at the site of action.

1.3 SIGNIFICANCE OF THE STUDY

Data obtained from this study could serve as baseline data upon which further
research could be conducted.

To expand the studies made on the leaves and fruits of annona muricata to
include the stem bark.

To ensure the effectiveness of the traditional uses of the plant in the

management of inflammations.
1.4 SCOPE OF THIS STUDY

This study will include the preliminary phytochemical studies and th in-vivo
anti-inflammatory activity of the alcohol extract of annona muricata and
comparing it with chitosan loaded annona muricata alcoholic extract of the
stem bark on whit albino rat.

1.5 RESEARCH HYPOTHESIS

1. Does annona muricata stem bark alcoholic extract contain phytochemicals

of pharmacology importance?

2. Does annona muricata stem bark alcoholic extract has anti-inflammatory

properties?

3. Does the chitosan loaded annona muricata stem bark alcoholic extract
enhance the concentration of the drug at the site of action when compared with
conventional annona muricata stem bark alcoholic extract?

1.6 AIMS AND OBJECTIVES OF THE STUDY

1. Determination of the phytochemical content of annona muricata stem bark
extract.

2. Determination of the anti-inflammatory property of annona muricata stem

bark extract.

3. Compare the anti-inflammatory property of annona muricata stem bark

extract alone and with chitosan loaded annona muricata.

2.0 CHAPTER TWO

2.1.1 Taxonomical classification of annona muricata

Kingdom plantae

Clade tracheophytes

Subphylum. angiosperm

Order annonales

Family annoanceae

Genus. annona

Specie. annona muricata L

Synonyms : A. macrocarpa Werkle

A. crassiflora Mart

A. bonplandiana kunth

A. cearensis Barb. Radr

M. gomez

Guanabanus muricatus L

Common name : Soursop

English name: Graviola, prickly custard apple

2.2 Botanical distributions of A. muricata

Soursop is an evergreen tree that is small,upright which usually grow up to 9.1m

high(30feet) tall. (JuliaF.Morton(1987).). The leaves are oblong to

oval bearing dark green colouration with no hairs above.

The flowers are arranged opposite from the leaves,each with one or more

flowers. The fruits are prickly with mild to moderate firm texture which

when flesh it is juicy,whitish and aromatic.

Picture of annona muricata leaves and fruit.

2.3 Geographical destibution of annona muricata

The plant is widely distributed In many countries in the world such as

Nigeria,Ghana,Tanzania,China,India,Indonesia,Malaysia,Pakistan,Singapore,Taiw

an,Veitnam,Grenada,Jamaica,Mexico,Panama,Brazil.Bolivia,Ecuador,Colombia,Pa

raguay,Peruand Venezuela.

2.4 Ethnobotanical uses of annona muricata

The plant is used tradionally in Indian medicine as well as Brazil and Haitio to cure

kidney diseases,fever,nervousness,ulcers,wounds, as well as

antispasmodic and antidysentric and parasiticidal acitivity. The flowers are

used as cough suppressant . The seeds are used as insecticidal astringent

and also a fish poison. Annona muricata has also been used as a bitter

tonic,febrifuge abortifacient,antidote for scorpion stings,respiratory

stimulant and for blood pressure.

2.5 Pharmacological activities of a. muricata

2.5.1 Anti-malarialactivity

Malaria is a popular illness caused by a plasmodium species after a

mosquito bite. Annona muricata aqueous leaf extract was reported to

signinficantly reduce parasitimia of PbANKA in an in vivo study in mice

which results in the potential anti-malarial property. In vivo study display

potential anti-malaria activity and lack of toxic effect of A . muricata used

for bioassay to build a new lead compound to develop a safe potential and

abortable drug against malaria in humans. (Somsaketal.,2016).

2.5.2 Cytotoxicity and antileishmanial activity of Annona muricata

When the hexane, ethyl acetate and methanol extracts of Annona muricata pericarp were tested in

vitro against Leishmania braziliensis and L. panamensis promastigotes and also against the cell line

U 937, ethyl acetate extract was found to be more active than GlucantimeW which was used as the

reference substance and the other extracts. Further fractionation of the extract has resulted in the

isolation of three acetogenins namely annonacin, annonacin A and annomuricin.

2.5.3 Anti-viral activity

Annona muricata extract was screened against Herpes simplex virus-1 (HSV-1) and clinical isolate

(obtained from the human keratitis lesion) in order to check whether they inhibit the cytopathic effect

of HSV-1 on vero cells which is the indicative of anti-HSV-1 potential. The minimum inhibitory
concentration of ethanolic extract of A. muricata was found to be 1 mg/ml which shows that the A.

muricata could be used as the potential antiherptic drugs.

2.5.4 Anticarcinogenic and genotoxic effects

Acetogenins (Ace) are the chemicals whish possess various biological properties
including the cytotoxic effect against the neoplasic cells which suggests their
potential usage as the antitumoral agents. Acetogenins also possess the capacity to
reduce the mouse colon crypts that is induced by azoxymethane (Azo) and was
found that 50% reduction in the amount of crypts in the animals treated with
acetogenin when compared with the level determined in mice treated with Azo .

2.5.5 Wound Healing Activity

Wound is the first medical problem that is faced by the human race. The knowledge
about wounds and their management remains still in the primitive and stunted stage .
A wound is a disrupted state of tissue that is caused by the physical, chemical,
microbial or immunological insult which heals either by the regeneration or
fibroplasias finally. The wound healing activity of alcoholic extract of stem and
bark of Annona muricata was found to show the marked reduction in area of the
wound which was tested in the albino rats which proves their possible use in the
healing the wound.

2.5.6 Anti-microbial activity

The antibacterial effect of the methanolic and aqueous extract of the leaves of
Annona muricata was tested against various bacterial strains such as
Staphylococcus aureus ATCC29213, Escherichia coli ATCC8739, Proteus
vulgaris ATCC13315,Streptococcus pyogenes ATCC8668, Bacillus subtilis
ATCC12432, Salmonella typhimurium ATCC23564, Klebsiella pneumonia
NCIM No.2719 and Enterobacter aerogenes NCIM No. 2340.Among the above
organisms tested, B. subtalis and S.aureus was found to be the most susceptible
Grampositive bacteria while K. pneumoniae and P. vulgaris was found to be the
most susceptible Gram-negative bacteria . Leaf extract of Annnona muricata is used
in the treatment of various bacterial infectious diseases such as pneumonia ,
diarrohea, urinary tract infection and even some skin disease. Annona muricata
extract contains a wide spectrum of activity against a group of bacteria that are
responsible for the most common bacterial diseases. Thus, the plant possesses an
abundant of the antibacterial compounds.

2.5.7 Hypoglycemic activity

A. muricata leaf extracts showed hypoglycemic activity in murine models

(Adewole and Caxton-Martins, 2006). In these studies, the effect of aqueous and
methanolic extracts of A. muricata leaves on reducing the concentration of blood
glucose in rats with diabetes induced with streptozotocin (STZ) was evaluated , and
the histology and biochemistry of the pancreas were observed. Pancreatic b-cells in
rats that were administered with extracts of A. muricata did not show the alterations
that are normally found in diabetic rats. An increase in the antioxidant enzymatic
activity and insulin content in pancreatic serum was reported. Near normal blood
glucose levels, body weight, food and water intake, lipid profile and oxidative
defense were achieved after a month of daily treatment with A.muricata extract ,
which could prevent the deleterious effect of STZ by its antioxidant and protective
effect of pancreatic bcells (Florence et al., 2014). It has also been reported that
there is a positive correlation between tannins, flavonoids and triterpenoids content
and the inhibition of a-glucosidase. Flavonoids inhibit a-glucosidase through
hydroxylation bonding and substitution at b ring (Hardoko et al., 2015). This
inhibition decreases carbohydrate hydrolysis and glucose absorption, and inhibits
carbohydrates metabolism into glucose. Additionally, glycemic index (GI) and
glycemic load (GL) have been reported for A. muricata fruit. GI indicates the effect
of the content and type of carbohydrates of a food on blood glucose content, while
GL estimates how much the food will raise blood glucose level after eating it. GI
and GL are considered low for A. muricata, which agrees with its hypoglycemic
potential.

2.5.8 Anxiolytic and anti-stress activities

The anxiolytic and the anti-stress effects were more effective in the alkaloid fraction
than in the crude hydroalcoholic extracts (Oviedo et al., 2009). It is possible to
attribute this bioactivity to the alkaloid compounds; especially because two of the
isolated alkaloids (anonaine and asimilobine) have relaxing activity . These
compounds can influence the central nervous system via the 5HT1A receptor. The
5HT1A receptor binds with the endogenous neurotransmitter serotonin and is
involved in the modulation of emotion (Hasrat et al., 1997a, 1997b). This
bioactivity can validate the reason for the traditional use of A. muricata as sedative .

2.5.9 Hypotensive activity

Leaf extract of A. muricata caused a dose-dependent reduction in mean arterial

pressure (MAP) in normotensive rats(Nwokocha et al., 2012). The suggested
hypotensive mechanism of action of aqueous extract of A. muricata did not involve
the endothelial or nitric oxide-dependent pathways.Studies suggested that plant
extracts lower blood pressure through the blockage of calcium ion channel, and this
Ca+ antagonism is further demonstrated by its ability to relax high K+ induced
contractions (Nwokocha et al.,2012). The hypotensive effect has been attributed to
alkaloids such as coreximine, anomurine, and reticulin, and some essential oil
components such as b-caryophyllene (Nwokocha et al., 2012).

2.6 Drug Delivery Using Chitosan Nanoparticles

Chitosan is a biodegradable, biocompatible polymer regarded as safe for human dietary use and

approved for wound dressing applications. Chitosan has been used as a carrier in polymeric

nanoparticles for drug delivery through various routes of administration. Chitosan has chemical
functional groups that can be modified to achieve specific goals, making it a polymer with a

tremendous range of potential applications. Chitosan is the most important derivative of chitin,

derived from crustacean shells such as those from prawns or crabs, as well as from the cell walls

of fungi. It is produced by removing the acetate moiety from chitin. (Munawar et al., 2017)

2.6.1 Cancer-Targeted Multidrug Resistance Small Interfering RNA (Mdr-siRNA) Delivery

Using Self-Cross-Linked Glycol Chitosan Nanoparticles to Overcome Drug Resistance

Yhee et al. (2015) delivered small interfering RNA (siRNA) for p-glycoprtein (pgp) down

regulation to tumors to overcome multidrug resistance in cancer and the resulting nanoparticles

protected siRNA molecules from enzymatic degradation. The formed stable nanoparticles, p-

glycoprotein targeted poly-siRNA thiolated glycol chitosan nanoparticles (psi-Pgp-tGC NPs)

showed great potential as a supplementary therapeutic agent for drug-resistant cancer.

2.6.2 Application of Fungal Chitosan/Ceftriaxone Nano-composite to Strengthen and

Sustain their Antimicrobial Potentiality Against Drug Resistant Bacteria.

Alshubaily and Al-Zahrani, (2019) reported that Nano-biopolymers could be employed for the

delivery of active compounds to increase their stability, bioavailability, efficacy and

sustainability. The ceftriaxone/chitosan nanoparticles antibacterial activity was confirmed

against 3 strains of Staphylococcus aureus (methicillin resistant), which elucidate that CFT/NCT

Nano-composite had a vigorous action toward bacterial cells; most cells were ruptured and

exploded after 6 hours of exposure and entirely lysed after 9 hours.

2.6.3 Antibacterial and Mechanical Properties of Bone Cement Impregnated with Chitosan

Nanoparticles.

Schito et tal., (2006) investigated the use of chitosan nanoparticles (CS NP) and quaternary

ammonium chitosan derivative nanoparticles (QCS NP) as bactericidal agents in poly(methyl

methacrylate) (PMMA) bone cement with and without gentamicin. The antibacterial activity was

tested againstS. aureus andS. epidermidis. A 103-fold reduction in the number of viable bacterial

cells upon contact with the surface was achievable.

CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 MATERIALS

The following materials, reagents and apparatus were used:

Weighing balance

Beakers

Spatula

Whatman filter paper

Stainless steel tray

Pestle and mortar

Evaporator

Soxhlet evaporator

Test tubes

Test tube racks

Test tube holder

Conical flasks

Syringes

Needles

Test tube holder

Conical flask

Chitosan nanoparticles

Conc. H2SO4

Ethanol

Acetic anhydride

Lead acetate

Ferric chloride

Aqueous sodium chloride

Rats