ANTIPYRETIC AND ANTIINFLAMMATORY PROPERTIES OF

METHANOLIC EXTRACTS OF Kigelia africana (Lam.) Benth AND Acacia

hockii De Wild IN ANIMAL MODELS

KAMAU KIMANI JAMES (B. Ed, SCIENCE)

I56/27066/2014

A Thesis Submitted in Partial Fulfillment of the Requirements for the Award

of the Degree of Master of Science (Biotechnology) in the School of Pure and

Applied Sciences of Kenyatta University.

OCTOBER 2016

ii

iii

DEDICATION

This thesis is dedicated to my father Simon Kamau and my mother Eunice Karingi

for their sacrifices towards my education.

iv

ACKNOWLEDGEMENT

I greatly acknowledge Kenyatta University for giving me an opportunity to further

my education. Secondly, I would like to express my sincere gratitude to my

supervisors Dr. Mathew Piero Ngugi and Prof. Joseph J.N. Ngeranwa for their

patience, guidance, inspiration, invaluable constructive criticism, valuable support

and immense knowledge making completion of my research and thesis writing

possible. You have been a tremendous mentor, and I hope that one day I would

become a good advisor to my students as a supervisor.

My vote of thanks also goes to the staff of Biochemistry and Biotechnology

Department as well as Chemistry Department for their assistance. The following

people deserve special mention; Mr. Daniel Gitonga and Mr. James Adino for

offering technical support.

To my labmates, Dr. John K. Mwonjoria, Mr. Peter Nthiga, Ms. Audrey Chepkemoi,

Ms. Scholar Kibiwott, Ms. Rose Chemutai, Mr. Antony Muchori, Mr. Herman

Muraguri, Mr. Tony Onyango, Mr. Michael Musila and Mr. Shadrack Njagi thank

you for stimulating discussions, for the long hours we worked together and for all the

fun we had in the last two years.

Words cannot express how grateful I am to my father, mother, brothers and sisters,

for your financial support, inspiration, and all sacrifices that you have made on my

behalf. To my late sister Kezia Wanjiru, I will never forget your words of motivation

and support you gave me.

Above all, I sincerely thank the Almighty God for giving me strength, good health,

and sound mind to accomplish my research.

Lastly, to all who contributed to the success of my work mentioned or implied, may

the Almighty God bless you abundantly.

v

TABLE OF CONTENTS

DEDICATION……………………………………………………………………...iii

ACKNOWLEDGEMENT…………………………………………………………. iv

TABLE OF CONTENTS……………………………………………………………v

LIST OF FIGURES………………………………………………………………. viii

LIST OF TABLES…………………………………………………………………. ix

LIST OF APPENDICES……………………………………………………………. x

ABBREVIATION AND ACRONYMS…………………………………………… xi

ABSTRACT………………………………………………………………………. xii

CHAPTER ONE……………………………………………………………………. 1

INTRODUCTION………………………………………………………………….. 1

1.1 Background information ...........................................................................……1

1.2 Statement of problem and justification .............................................................4

1.3 Hypotheses ........................................................................................................5

1.4 Objectives ..........................................................................................................5

1.4.1 General objective ........................................................................................5

1.4.2 Specific objectives ......................................................................................5

CHAPTER TWO…………………………………………………………………… 6

LITERATURE REVIEW…………………………………………………………... 6

2.1 Biochemical and physiological basis of pyrexia and inflammation ..................6

2.1.1 Pyrexia ........................................................................................................6

2.1.2 Inflammation ..............................................................................................9

2.3 Experimental induction of pyrexia and inflammation.....................................12

2.3.1 Pyrexia ......................................................................................................12

2.3 Inflammation ...............................................................................................14

2.2 Modulation and conventional management of pyrexia and

inflammation ..................................................................................................16

vi

2.5 Alternative and complementary management of pyrexia and

inflammation .............................................................................................….20

2.5.1 Pyrexia ......................................................................................................20

2.5.2 Inflammation ............................................................................................22

2.6 Plants used in this study ..................................................................................24

2.6.1 Kigelia africana (Lam.) Benth .................................................................24

2.6.1.1 Plant description and geographical distribution ....................................25

2.6.2 Acacia hockii De Wild ............................................................................27

2.6.2.1 Plant description and distribution ..........................................................27

2.6.2.2 Cultural and medicinal uses ...................................................................28

CHAPTER THREE……………………………………………………………….. 29

MATERIALS AND METHODS…………………………………………………..29

3.1 Collection and preparation of plant materials .................................................29

3.2 Extraction ........................................................................................................29

3.3 Experimental design ........................................................................................30

3.3.1 Laboratory animals ...................................................................................30

3.4 Bioscreening ....................................................................................................31

3.4.1 Evaluation of antipyretic activities in Wistar rats ....................................31

3.4.2 Evaluation of anti-inflammatory activities in Swiss albino mice .............33

3.5 Qualitative phytochemical screening ..............................................................34

3.5.1 Saponins (Froth test) .................................................................................34

3.5.2 Alkaloids ...................................................................................................35

3.5.3 Terpenoids (Salkowski test) .....................................................................35

3.5.4 Flavonoids (Sodium hydroxide test) .........................................................35

3.5.5 Cardiac glycosides (Keller-Kilian test) ....................................................35

3.5.6 Steroids .....................................................................................................35

3.5.7 Phenolics ...................................................................................................36

3.6 Data management and statistical analysis .......................................................36

CHAPTER FOUR………………………………………………………………….37

vii

RESULTS…………………………………………………………………………. 37

4.1 Antipyretic activity of methanolic stem bark extract of Kigelia

africana (Lam.) Benth ....................................................................................37

4.2 Antipyretic activity of methanolic stem bark extract of Acacia

hockii De Wild ................................................................................................40

4.3 Comparison between antipyretic activities of Kigelia africana

and Acacia hockii at different doses ...............................................................43

4.4 Anti-inflammatory activity of methanolic leaf extract of Kigelia

africana (Lam.) Benth ....................................................................................45

4.6 Comparison between anti-inflammatory activities of Kigelia

africana and Acacia hockii at different doses ................................................51

4.7 Qualitative phytochemical screening ..............................................................53

CHAPTER FIVE………………………………………………………………….. 55

DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS………………..55

5.1 Discussion .......................................................................................................55

5.2 Conclusions .....................................................................................................66

5.3 Recommendations ...........................................................................................67

5.4 Suggestions for Further Studies ......................................................................67

REFERENCES……………………………………………………………………. 68

APPENDICES…………………………………………………………………….. 82

viii

LIST OF FIGURES

Figure 2. 1: Kigelia africana picture captured Embu County Kenya ......................24

Figure 2. 2: Acacia hockii picture captured Embu County, Kenya ..........................27

Figure 4. 1: Comparison of percentage reduction in rectal temperature of

methanolic stem bark extracts of Kigelia africana and Acacia

hockii on turpentine-induced pyrexia in rats at different doses……….44

Figure 4. 2: Comparison of percentage reduction in paw diameter of

methanolic leaf extract of Kigelia africana and stem bark

extract of Acacia hockii at different doses ............................................52

ix

LIST OF TABLES

Table 3.1: Treatment protocol for evaluation of antipyretic activities of

methanolic extracts of Kigelia africana (Lam) Benth and

Acacia hockii De Wild in male Wistar rats ......................................... 32

Table 3.2: Treatment protocol for evaluation of anti-inflammatory

activities of methanolic extracts of Kigelia africana (Lam)

Benth and Acacia hockii De Wild in Swiss albino mice .................... 33

Table 4.1: Effects of intraperitoneal administration of methanolic stem

bark extract of Kigelia africana (Lam) Benth on turpentine-

induced pyrexia in male Wistar rat………………....………………..39

Table 4.2: Effects of intraperitoneal administration of methanolic stem

bark extract of Acacia hockii De Wild on turpentine-induced

pyrexia in male Wistar rats ................................................................. 42

Table 4.3: Effects of intraperitoneal administration of methanolic

leaf extract of Kigelia africana (Lam) Benth on carrageenan-

induced inflammation in Swiss albino mice ....................................... 47

Table 4.4: Effects of intraperitoneal administration of methanolic stem

bark extracts of Acacia hockii De Wild on carrageenan-

induced inflammation in Swiss albino mice ....................................... 50

Table 4.5: Qualitative phytochemical composition of methanolic stem

bark and leaf extracts of Kigelia africana and methanolic

stem bark extracts of Acacia hockii .................................................... 54

x

LIST OF APPENDICES

Appendix I: Effects of intraperitoneal administration of methanolic stem

bark extract of Kigelia africana on turpentine-induced

pyrexia in male Wistar rats .................................................................82

Appendix II: Effects of intraperitoneal administration of methanolic stem

bark extract of Acacia hockii on turpentine-induced pyrexia

in male Wistar rats ..............................................................................83

Appendix III: Effects of intraperitoneal administration of methanolic leaf

extract of Kigelia africana on carrageenan-induced

inflammation in Swiss albino mice ...................................................84

Appendix IV: Effects of intraperitoneal administration of methanolic

stem bark extract of Acacia hockii on carrageenan-

induced inflammation in Swiss albino mice .....................................85

Appendix V: Figure representing antipyretic activity of methanolic stem

bark extract of Kigelia africana ........................................................86

Appendix VI: Figure representing antipyretic activity of methanolic stem

bark extract of Acacia hockii ...........................................................86

Appendix VII: Figure representing anti-inflammatory activity of

methanolic stem bark extract Kigelia africana ...............................87

Appendix VIII: Figure representing anti-inflammatory activity of

methanolic leaf extract of Acacia hockii .........................................87

Appendix IX: Analysis of the antipyretic effects of methanolic stem bark

extract of Kigelia africana ................................................................88

Appendix X: Analysis of the antipyretic effects of methanolic stem bark

extract of Acacia hockii ....................................................................90

Appendix XI: Analysis of the anti-inflammatory effects of methanolic

leaf extract of Kigelia africana .........................................................93

Appendix XII: Analysis of the anti-inflammatory effects of methanolic

stem bark extract of Acacia hockii ..................................................96

Appendix XIII: Comparison between antipyretic effects of methanolic

stem bark extracts of Kigelia africana and Acacia

hockii at various dose levels ..........................................................99

Appendix XIV: Comparison between anti-inflammatory effects of

methanolic leaf extract of Kigelia africana and stem

bark extract of Acacia hockii at various dose levels ....................100

xi

ABBREVIATION AND ACRONYMS

ANOVA Analysis of variance

COX Cyclooxygenase

DMH Dorsomedial hypothalamus

DMSO Dimethylsulphoxide

GBIF Global Biodiversity Information Facility

IFN Interferon

IL Interleukin

ILDIS International Legumes Database and Information Service

LPB Lipopolysaccharide binding protein

LPS Lipopolysaccharides

NF-κB Nuclear factor kappa B

NO Nitric oxide

NSAIDs Non-steroidal anti-inflammatory drugs

PGE2 Prostaglandin E2

PLA2 Phospholipase A2

PVN Paraventricular nucleus

ROS Reactive oxygen species

rRPa Rostral raphe pallidus nucleus

TNF Tumor necrosis factor

WHO World Health Organization

xii

ABSTRACT

Pyrexia and inflammation cause discomfort, suffering and lower productivity of the

victims. Non-steroidal anti-inflammatory drugs which are highly prescribed in

medication of pyrexia and inflammation have been reported to possess adverse

effects. Herbal medicines may possess bioactive compounds that are safer and

efficient in the management of various diseases and disorders. Kigelia africana and

Acacia hockii are traditionally used to manage pyrexia and inflammation among the

Embu and Mbeere communities in Kenya but there lacks scientific data to support

their use. The present study determined antipyretic and anti-inflammatory activities

of the two extracts in animal models to scientifically confirm their traditional use.

The plant samples were collected with the help of local herbalists in Embu County,

Kenya and transported to Kenyatta University for cleaning, air drying, milling, and

extraction in Biochemistry and Biotechnology laboratories. Animal models were

randomly divided into six groups of 5 animals each; three experimental groups (50,

100 and 150mg/kg body weight), normal control group, negative control group and

positive control group. The antipyretic effect was determined using turpentine-

induced pyrexia, while the anti-inflammatory effect was determined using

carrageenan-induced hind paw edema method. The antipyretic and anti-inflammatory

activities of the extracts were compared to reference drugs aspirin and diclofenac

respectively. The stem bark extract of K. africana reduced the elevated rectal

temperature by between 0.06 and 3.07 percent, while the stem bark extract of A.

hockii reduced the raised rectal temperature by between 0.62 and 3.88 percent. The

aspirin reduced the rectal temperature of pyretic rats by between 0.63 and 3.1 percent.

The leaf extract of K. africana reduced inflamed hind paw diameter of mice by

between 0.21 and 4.98 percent, while the stem bark extract of A. hockii reduced

inflamed hind paw diameter by between 0.6 and 5.38 percent. The diclofenac reduced

inflamed hind paw diameter by between 1.11 and 4.9 percent. The qualitative

phytochemical screening indicated the presence of flavonoid, alkaloids, steroids,

saponins, terpenoids, phenolics, and cardiac glycosides. The present study

demonstrated potent antipyretic and anti-inflammatory activities of methanolic

extracts of K. africana and A. hockii in a dose-dependent manner, which supports

their traditional use. The present study, therefore, recommends that K. africana and

A. hockii can be used as a potential candindate in development of antipyretic and

anti-inflammatory agents.

1

CHAPTER ONE

INTRODUCTION

1.1 Background information

Pyrexia, also known as fever (Axelrod and Diringeret, 2008), is a medical sign

associated with the elevation of body temperature above the normal range (36.5°C-

37.5°C) due to the cytokine-induced upward displacement of the thermoregulatory

set-point of the hypothalamus (Karakitsos and Karabinis, 2008). The elevation of

the body temperature occurs when prostaglandin E2 (PGE2) increases within the

pre-optic region and alter the firing rate of neurons that control temperature

regulation of the hypothalamus (Biren and Avinash, 2010). Symptoms of fever

include shivering, sweating, headache, dehydration, muscle aches and general

weakness (Anochie et al., 2013).

The impacts of secondary infection, tissue damage, neoplasm or other diseased

states induce fever. Infected or damaged tissue usually initiates the production of

cytokines such as interleukins 1 (β and α), tumor necrosis factor (β and α) and

interleukin-6 which stimulates the synthesis of PGE2 near the pre-optic region of

the hypothalamus. The increase in production of PGE2 stimulates the hypothalamus

to generate responses to raise body temperature (Saper and Breder, 1994).

Most of the conventional synthetic antipyretic drugs such as paracetamol, aspirin,

ibuprofen and naproxen inhibit the active sites of cyclooxygenase enzymes leading

2

to inhibition of PGE2 biosynthesis (Weissmann, 1991). However, they are toxic to

hepatic cells, glomeruli, heart muscle and cause gastrointestinal ulceration,

bleeding and perforation (Cheng et al., 2005).

Inflammation refers to body’s normal protective response to tissue injury caused

by physical trauma, toxic chemicals or microbiological agents (Calixto et al., 2004).

The classical signs of inflammation are skin redness, swelling, pain, heat, and loss

of function (Hurley, 1972). The process of inflammation involves changes in blood

flow, destruction of tissues, increased vascular permeability and the synthesis of

pro-inflammatory mediators, such as prostaglandin E2 (PGE2), leukotrienes and

platelet-activating factors induced by phospholipase A2, lipoxygenases and

cyclooxygenases (COXs) (Shah et al., 2008).

The NSAIDs (non-steroidal anti-inflammatory drugs) such as naproxen,

indomethacin, ibuprofen, diclofenac, and ketoprofen are the most commonly used

conventional drugs in the treatment of inflammation (Warden, 2010). The NSAIDs

act by inhibiting the synthesis of prostaglandins through acetylation and

consequently inactivation of cyclooxygenase 2 (COX-2) enzyme responsible for

inflammation induction (Vane and Botting, 1987). The use of NSAIDs is linked

with severe effects on the gastrointestinal tract, kidney, and cardiovascular system

(Traversa et al., 1995).

3

The World Health Organization (2008) estimates that 80% of the developing

countries populations depend on ethnomedicine for their primary health care. The

demand for herbal medicine is increasing due to the growing recognition that

natural products have few side effects, easily available, better cultural acceptability,

better compatibility with the human body and being comparatively affordable

(Kamboj et al., 2000). The value of herbal medicines depends on the presence of

various phytochemicals which brings a particular physiological effect in the human

body (Dubey et al., 2004). Herbal preparations have become the subject of

extensive recent studies regarding whether their traditional uses can be

scientifically evaluated (Hina et al., 2013).

According to Kareru et al. (2007), Kigelia africana (Lam) Benth and Acacia hockii

De Wild are used traditionally to manage pyrexia and inflammation among Embu

and Mbeere communities in Embu County Kenya, but lacks scientific data to

confirm their use. The present study was designed to determine the antipyretic and

anti-inflammatory potential of the two extracts to act as a preliminary step towards

the development of safer and more efficient plant-derived antipyretic and anti-

inflammatory agents.

4

1.2 Statement of problem and justification

Pyrexia and inflammation cause suffering, discomfort and lowers productivity of

the victims. Studies have established that NSAIDs used in the treatment of pyrexia

and inflammation are toxic to the hepatic cells, glomeruli, and the heart muscle

(Luo et al., 2005). Besides, the NSAIDs are associated with the tendency to

promote gastrointestinal bleeding, ulceration and platelets dysfunction due to

inhibition of cyclooxygenase 1 enzyme (Traversa et al., 1995; Cryer and Kimmey,

1998). Although K. africana and A. hockii are used traditionally to manage pyrexia

and inflammation among Embu and Mbeere communities in Kenya, validation to

support their ethnomedicinal use is yet to be done (Kareru et al., 2007).

Studies have established that herbal medicines are comparatively safer and efficient

in the management of various diseases and disorders and therefore, could serve as

an alternative to conventional synthetic drugs (Hassan et al., 2013). In addition,

herbal medicines are readily available, affordable and have fewer side effects

(Kamboj, 2000). The present study was carried out to scientifically confirm the

folklore use of K. africana and A. hockii. The information from the present study

may help to generate herbal formulation that is affordable, readily available and

with fewer side effects in the management of pyrexia and inflammation.

5

1.3 Hypotheses

i. Methanolic extracts of K. africana and A. hockii have no antipyretic effects

in Wistar rats.

ii. Methanolic extracts of K. africana and A. hockii have no anti-inflammatory

effects in Swiss albino mice.

1.4 Objectives

1.4.1 General objective

To determine the antipyretic and anti-inflammatory activities of methanolic

extracts of K. africana and A. hockii in animal models.

1.4.2 Specific objectives

i. To determine the antipyretic properties of methanolic extracts of K. africana

and A. hockii in Wistar rats.

ii. To determine the anti-inflammatory properties of methanolic extracts of K.

africana and A. hockii in Swiss albino mice.

iii. To determine the qualitative phytochemical composition of methanolic

extracts of K. africana and A. hockii.

6

CHAPTER TWO

LITERATURE REVIEW

2.1 Biochemical and physiological basis of pyrexia and inflammation

2.1.1 Pyrexia

Pyrexia, also known as fever and febrile response (Axelrod and Diringer, 2008),

refers to a medical sign associated with an elevation of body temperature above the

normal range (36.5°C to 37.5°C) due to increasing in body temperature regulatory

set-point (Karakitsos and Karabinis, 2008). The increase in thermoregulatory set-

point triggers increased muscle contraction and cold sensation resulting in heat

production and efforts to conserve heat. When the set-point returns to normal, a

person feels hot and may begin to sweat (Sue et al., 2014). A person is said to be

pyretic if the temperature measured in the mouth is over 37.7°C, if the temperature

in the rectum is over 38.3°C and the temperature under the arm or inside the ear is

over 37.2°C (Nordqvist, 2015a).

The general symptoms of pyrexia include sweating, lethargy, shivering and cold

sensation (Anochie et al., 2013). The infectious causes of pyrexia include viral,

bacterial, parasitic infections, common cold, malaria, and meningitis among others,

while the non-infectious causes of fever include deep vein thrombosis, cancer, and

side effects of medication among others (Anochie et al., 2013). Hyperthermia

differs from pyrexia in that body temperature elevate above 41.2°C due to failed

7

thermoregulation that occurs when the body absorbs or produces more heat than it

dissipates (Ogoina, 2011).

Pyrogens are the substance that causes pyrexia, and two types exist; endogenous

and exogenous pyrogens. The pyrogens that originate outside the body, such as

lipopolysaccharide (LPS) from gram negative bacteria are known as exogenous

pyrogen, while pyrogens that are produced by body’s cells due to an outside

stimulus are known as endogenous pyrogens (Luheshi, 1998). The endogenous

pyrogens act directly and immediately on the hypothalamic thermoregulatory

center to increase its set-point, while exogenous pyrogens act indirectly and may

require some hours to induce pyrexia. Pyrogenicity can vary, for instance, some

bacterial pyrogens are superantigens and cause rapid and dangerous pyrexia

(Anochie, 2013).

All the endogenous pyrogens are cytokines molecules produced by phagocytic

cells. Some of these endogenous pyrogens include cytokines such as interleukins 1

(β and α), interleukin 6 (IL-6), tumor necrosis factor- β (TNF-β), and interferons (β

and α) (Walter and Boron, 2003). Tumor necrosis-α also acts as a pyrogenic

cytokine and is usually mediated by interleukin 1 (IL-1) release (Stefferl et al.,

1996). Upon the release of pyrogenic cytokines into the blood circulation, they

migrate into the brain circumventricular organs and then bind to endothelial

receptors on vessel walls or interact with local microglial cells, leading to activation

8

of the arachidonic acid pathway (Dinarello, 1999). The activation of the arachidonic

acid pathway leads to the synthesis of prostaglandins E2 which is the ultimate

mediator of fever (Saper and Breder, 1994).

The exogenous pyrogens include lipopolysaccharides, which are the cell wall

components of gram-negative bacteria (Freudenberg and Galanos, 1990). An

immunological protein lipopolysaccharide-binding protein (LBP) usually binds to

lipopolysaccharide (LPS) to form LBP-LPS complex. The ensuing complex (LBP-

LPS) then binds to CD14 receptor of a neighboring macrophage. The binding of

CD14 receptor and LBP-LPS complex results in the production and release of some

endogenous pyrogenic cytokines, such as tumor necrosis factor-α, interleukin 1 (IL-

1) and interleukin 6 (IL-6) (Anochie et al., 2013).

The exogenous pyrogens, therefore, mediate the release of endogenous pyrogenic

cytokines which activate the arachidonic acid pathway leading to the production of

PGE2. The enzymes phospholipase A2 (PLA2), cyclooxygenase-2 (COX-2), and

prostaglandin E2 synthase mediate the arachidonic acid pathway (Phipps et al.,

1991), which help in synthesis and release of PGE2. The set-point temperature of

the body remains elevated until PGE2 synthesis reduces. Prostaglandins E2 usually

acts on the neurons in the pre-optic area of the hypothalamus known as

prostaglandin receptor 3 (EP3). Prostaglandin E receptor 3 expressing neurons in

the pre-optic region supply dorsomedial hypothalamus (DMH), paraventricular

9

nucleus (PVN) of the hypothalamus and rostral raphe pallidus nucleus of the

medulla oblongata (rRPa) with nerves (Anochie, 2013).

Fever signals are sent to the DMH and rRPa to stimulate sympathetic nervous

system to initiate non-shivering thermogenesis to generate body heat and

vasoconstriction of blood vessels. The surplus of nerves from the pre-optic region

of the hypothalamus to the PVN mediates the neuroendocrine effects of pyrexia

through the pathway involving pituitary and some endocrine organs (Anochie,

2013).

The endogenous pyrogens induce fever by stimulating the release of PGE2, which

stimulates the hypothalamus to generate a systemic response in the body to produce

heat. When the set-point raises, body temperature increases through active

generations of heat by shivering or retaining heat through vasoconstriction of blood

vessels. When pyrexia stops, and the set-point lowered, vasodilation and sweating

are used to cool the body to lower the set-point (Anochie, 2013).

2.1.2 Inflammation

Inflammation refers to body’s normal physiological response to tissue injury. The

causes of inflammation include physical trauma, autoimmune reactions (such as

asthma and rheumatoid arthritis), microbial agents, intense heat and toxic chemicals

(Calixto et al., 2004). Symptoms of inflammation include skin redness, heat,

10

swelling, pain and loss of function (Ravi et al., 2009). Inflammation helps body

defense chemicals and defense cells to leave the circulatory system and enter the

injured tissue or infected site. Inflammatiory response is therefore, involved in

immune surveillance, optimum tissue repair and regeneration of tissue after injury

(Vodovotz et al., 2008).

The injured cells, lymphocytes, phagocytes, mast cells and blood proteins are the

sources of inflammatory mediators. The most important inflammatory mediators

include bradykinins, serotonins, prostaglandins, histamine, and lymphokines.

These chemicals promote dilation of the small blood vessels in the area of the

injury, and more blood flows into the injured area leading to blood congestion these

accounts for the heat and skin redness of the damaged tissue (Williams and Maier,

1992).

Inflammatory process has two phases: acute and chronic. The acute inflammation

occurs a few minutes after tissue damage. It is characterized by an increase in

permeability of blood vessels, extravasation of fluid and proteins and accumulation

of white blood cells for a short period (Posadas et al., 2004). The primary mediators

of acute inflammation include histamine, serotonin, and bradykinins (Ravi et al.,

2009). Some of diseases and conditions associated with acute inflammation include

sore throat, acute bronchitis, acute appendicitis, acute dermatitis and acute infective

meningitis (Nordqvist, 2015b).

11

The failure of the management of acute inflammation and an autoimmune response

to a self-antigen lead to chronic inflammation and disease (Recio et al., 2012).

Chronic inflammation is characterized by ailments such as chronic peptic ulcers,

rheumatoid arthritis, tuberculosis, asthma, and chronic periodontitis. Moreover,

chronic inflammation can cause several diseases and conditions such as some

cancers, rheumatoid arthritis, periodontitis and hay fever (Nordqvist, 2015b).

Among the first inflammatory cells are phagocytic immune cells such as

neutrophils and macrophages. Macrophages play a significant role in the

development of chronic inflammation by stimulating overproduction of pro-

inflammatory cytokines such as TNF-α, IL-6, and IL-1β. Macrophages also help in

generation of pro-inflammatory mediators in response to microbial products, such

as reactive oxygen species (ROS), PGE2, COX-2, nitric oxide (NO) and interferon-

γ (IFN-γ) (Bosca et al., 2000; Kaplanski et al., 2003). These mediators are the

activators of components of the pro-inflammatory signal transduction cascade,

including nuclear factor kappa- light-chain- enhancers of activated B cells (NF-κB)

inducing kinase, protein kinase C (PKC) and mitogen-activated protein kinase

(MAPK) (Barnes and Karvin, 2009). The PGE2 are produced when phospholipases

release arachidonic acid, which is later metabolized by cyclooxygenases (COXs)

and specific isomerases from the plasma membrane (Kuehl and Egan, 1980).

12

During the inflammatory response, both the level and the profile of PGE2

production can change dramatically. However, PGE2 are at low levels in tissues

with no inflammation and increase immediately in acute inflammation. As immune

cells infiltrate the tissues, further increases in PGE2 levels is observed (Tilley et al.,

2001). Studies indicate that COX-2 plays a significant role in inflammation

(Oshima et al., 1996).

2.3 Experimental induction of pyrexia and inflammation

2.3.1 Pyrexia

Exogenous pyrogens such as lipopolysaccharides (LPS), steam distilled turpentine,

brewer’s yeast, muramyl dipeptide (MDP) and polyinosinic: polycytidylic acid

(poly I: C) are used to induce pyrexia in experimental animals (Soszynski et al.,

1991; Soszynski and Krajewska, 2002). Similarly, endogenous pyrogenic cytokines

such as interferon-β, tumor necrosis factor-α, interleukins (1 and 6) are also used to

induce pyrexia in animal models (Anochie et al., 2013).

Lipopolysaccharide (LPS) usually binds to an immunological protein-

lipopolysaccharide binding protein (LBP) to form LBP-LPS complex. The ensuing

complex then binds to the CD14 receptor of the neighboring macrophage, resulting

in the production and release of various endogenous pyrogenic cytokines like

interleukins (1β and 6) and the tumor necrosis factor-α. These pyrogenic cytokines

activate the arachidonic acid pathway leading to the production of PGE2. The PGE2

13

is the mediator of fever response (Cannon et al., 1990; Klir et al., 1994; Anochie et

al., 2013).

Turpentine acts directly on the brain and unlike LPS, a small dose is required to

obtain the same physiological responses (Luheshi et al., 1997). The synthesis of

interleukins (1β and 6) and tumor necrosis factor-β are associated with turpentine-

induced pyrexia. These pyrogenic cytokines enhance the production of PGE2,

which ultimately increase the body temperature (Zhu et al., 2011). The turpentine-

induced pyrexia is fast, has persistent fever pattern and experimental animals have

a high tolerance to turpentine compared to other exogenous pyrogens (Soszynski

and Krajewska, 2002; Tung et al., 2006).

Brewer’s yeast contain a lipopolysaccharide which binds to the immunological

protein LBP (Bhattacharya et al., 2014). The resultant effect of this binding is the

synthesis and release of various endogenous pyrogenic cytokines such as

interleukins (1 and 6), and TNF-α. These endogenous cytokines readily penetrate

the blood-brain barrier and act on the thermoregulatory center in the hypothalamus,

thus activating the arachidonic acid pathway resulting in the synthesis and release

of PGE2. The PGE2 are the ultimate mediators of pyrexia (Gege-Adebayo, 2013).

Endogenous pyrogenic cytokines such as interferon-β, tumor necrosis-α factor,

interleukin (1 and 6) are also used in experimental animals to induce fever (Anochie

14

et al., 2013). These cytokines activate the arachidonic acid pathway leading to the

synthesis of PGE2 which causes fever (Conti et al., 2004).

2.3 Inflammation

Carrageenan, xylene, arachidonic acid, dextran, histamine, serotonin and formalin-

induced paw edema; cotton pellet induced granuloma edema; Freund’s adjuvants

are the standard agents for producing acute, sub-acute and chronic inflammation

respectively (Ismail et al., 1997; Mujumdar and Misar, 2004).

Carrageenan-induced paw edema is widely used in experimental animals to

investigate anti-inflammatory effects (Sini et al., 2010). The development of edema

in the hind paw following the carrageenan injection is believed to be biphasic.

Serotonin, histamine, and bradykinin are the first detectable inflammatory

mediators in the early phase (one hour) of carrageenan-induced paw edema.

Prostaglandins are involved in the increased vascular permeability and are

detectable at the late phase (more than one hour) (Nantel et al., 1999). Carrageenan

also causes the production and release of nitric oxide (NO) responsible for inducing

inflammation (Handy and Moore 1998; Omote et al., 2001; Necas and Bartosikova,

2013).

Serotonin, histamine, bradykinin and arachidonic acid-induced paw edema are

other methods used to induce acute inflammation in experimental animals.

15

Serotonin, histamine, and bradykinin are important mediators of inflammation and

are potent vasodilator substances, which increase the vascular permeability and

dilate capillaries (Cuman et al., 2001). The metabolites of arachidonic acid formed

through cyclooxygenase and lipoxygenase pathways represent two classes of

inflammatory mediators. The PGE2 enhances the cardinal signs of inflammation

(Calder, 2009)).

Dextran (polysaccharide of high molecular weight) causes an anaphylactic reaction

after injection in the sub-plantar tissue of the hind paw in animal models, which is

characterized by extravasation and edema formation due to the liberation of

serotonin and histamine from mast cells (Van Wauve and Goosens, 1989). Xylene-

induced mouse ear edema is also used to induce acute inflammation. The

application of a drop of xylene to the inner surface of the ear causes irritation, fluids

accumulation, and edema occurs (Rotelli et al., 2003; Okoli et al., 2006).

Formalin-induced paw edema is used to induce sub-acute inflammation. Histamine,

serotonin, prostaglandins and bradykinin are examples of some inflammation

mediators associated with formalin-induced inflammation in animal models.

Formalin is one of the most suitable test procedures to screen inflammation and

antiarthritic agents as it closely resembles human arthritis (Lai et al., 2009).

16

Cotton pellet granuloma-induced inflammation is a method used to induce sub-

acute inflammation. The animals are anaesthetized with pentobarbitone (30mg/kg

body weight). The back skin is shaved and sterilized with 70% ethanol. An incision

is made in the lumbar region. Subcutaneous tunnels are formed by a blunted forceps

and a sterilized pre-weighed cotton pellet placed on both sides in the scapula region.

Inflammation and granuloma develop in a duration of several days and it involves

proliferation of macrophages, neutrophils, and fibroblasts which are the basic

source of granuloma formation (Kalawole et al., 2013).

Freund’s adjuvants are widely used to induce chronic inflammation in experimental

animals. The key pro-inflammatory cytokines such as interleukins (1β and 6) and

tumor necrosis factor-α are involved in the development of inflammation in

experimental animals. The expression of these cytokines is characterized by

mechanical sensitivity during the acute (4 hours), sub-acute (4 days) and chronic

(14 days) phases of complete Freund’s adjuvants-induced peripheral inflammation

(Raghavendra et al., 2004).

2.2 Modulation and conventional management of pyrexia and inflammation

The fundamental elements of pyrexia pathway are the release of endogenous

pyrogenic cytokines by the body cells in response to some exogenous pyrogens,

activation of the arachidonic acid, synthesis of COX-2, and production of PGE2 by

17

the hypothalamic vascular endothelial cells (Mackowiak, 1998). The synthesis of

PGE2 stimulates thermoregulatory neurons located in the pre-optic area of the

hypothalamus to raise the hypothalamic thermal set-point by inducing thermogenic

mechanisms to elevate core body temperature (Anochie, 2013). The antipyretic

activity of many antipyretic drugs is achieved through inhibition of COX-2 and

thereby leading to reduction in PGE2 levels within the hypothalamus (Biren and

Avinash, 2010).

On the other hand, the pro-inflammatory mediators, such as PGE2, leukotrienes,

nitric oxide (NO), COX-2 and inflammatory cytokines such as, interleukin-1β,

interleukin-6 induce inflammation (Shah et al., 2008). The anti-inflammatory

activity is achieved through reduced biosynthesis of these pro-inflammatory

mediators and inflammatory cytokines. The PGE2 are produced in the body cells

by the enzyme cyclooxygenase 2 (COX-2). The inhibition of the production of

COX-2 enzyme lead to the reduction in the biosynthesis of PGE2 and thus managing

inflammation (Mitchell et al., 1993).

The NSAIDs such as naproxen, acetaminophen, aspirin, ibuprofen, diclofenac,

indomethacin, piroxicam, ketoprofen and oxaprozin are conventionally used to

treat pyrexia and inflammation (Warden, 2010). The NSAIDs such as aspirin and

acetaminophen treat pyrexia by inhibiting COX-2 synthesis (Aronoff and Neilson,

2001). The synthesis of PGE2 depends on expression of enzyme COX-2. Inhibitors

18

of COX-2 are potent antipyretics and inhibit the transformation of arachidonic acid

to PGE2 (Chandrasekharan et al., 2002). If a bacterial infection causes pyrexia, a

physician may prescribe an antibiotic to manage pyrexia. However, NSAIDs may

be used to relieve uncomfortable symptoms (Nordqvist, 2015a).

The NSAIDs such as diclofenac and acetaminophen treat inflammation by

inhibiting enzyme cyclooxygenase (COX). There are two types of COX enzymes,

COX-1, and COX-2. The COX-2 produces prostaglandins that promote

inflammation, while COX-1 synthesizes prostaglandins that support platelets,

blood clotting and protect the stomach (Markenson, 1999).

The use of NSAIDs in the treatment of pyrexia and inflammation have shown

various side effects. The common side effects associated with NSAIDs include

vomiting, nausea, diaorrhea, dizziness, constipation, decreased appetite, rash,

headache, and drowsiness (Nordqvist, 2015b). Aspirin has a unique capacity for

causing Reye syndrome (Soumerai et al., 1992), a children's disorder associated

with hepatic failure (Rahwan and Rahwan, 1986).

Since NSAIDs block the COX enzymes to reduce the synthesis of prostaglandins

throughout the body, COX enzyme that protects the stomach and supports the

platelets and blood clotting are also reduced, and these can cause gastrointestinal

toxicity (Brooks, 1998). Gastrointestinal toxicity is a common side effect associated

19

with NSAIDs therapy. Such toxic effects are divided into three categories: mucosal

lesions, gastrointestinal discomfort (dyspepsia), and severe gastrointestinal

complications such as perforated ulcer (Wolfe et al., 1999; Plaissance, 2000). The

use of NSAIDs medications can also cause abnormalities of the skin and the

respiratory system, blood circulatory system and central nervous systems

(Plaissance, 2000).

Corticosteroids drugs are also used as anti-inflammatory agents. Corticosteroids

inhibit release of phospholipase A2, required for the synthesis of arachidonic acid

from the membrane and also inhibit transcription of pyrogenic messenger

ribonucleic acid (Hong et al., 1976). Corticosteroids are a class of steroid hormones

produced by the cortex of the adrenal gland and are usually synthesized in

laboratories and added to medications. Two types of corticosteroids exist;

glucocorticoids and mineralocorticoids. The glucocorticoids are produced in

response to stress (Nordqvist, 2015b). Synthetic glucocorticoids are used to treat

arthritis, inflammatory bowel disease, systemic lupus, and asthma.

Mineralocorticoids regulate salt and water balance in the body and are used to treat

cerebral salt wasting (Nordqvist, 2015b).

Corticosteroid side effects are more if administered orally compared to inhalers or

injections. Long-term medications employed in the treatment of asthma through

inhalation may risk the development of oral thrush. Glucocorticoids can also cause

20

Cushing's syndrome while mineralocorticoids can cause hypertension, low blood

potassium levels, high blood sodium levels, connective tissue weakness and

metabolic alkalosis (Nordqvist, 2015b).

2.5 Alternative and complementary management of pyrexia and inflammation

2.5.1 Pyrexia

The World Health Organization (WHO) defined herbal medicine as therapeutic

practices that existed before the development and spread of modern medicine and

are still in use today (WHO, 1978). In the early 19th century, when phytochemical

analysis became available, scientists began to extract and modify the active

phytochemical compounds from herbal plants. Later, chemists began to produce

their version of plant compounds and with time, the use of herbal medicines

reduced due to availability synthetic drugs (Kamboj, 2000).

According to World Health Organization (WHO), herbal medicines would be the

best alternative to replace synthetic drugs which are associated with severe effects.

The WHO (2008) estimated that about 80% of the world population relies on herbal

medicines for their primary health care needs. Demand for herbal medicine has

significantly increased in the developing countries mainly for their health care, due

to their fewer side effects, efficacy, availability and better cultural acceptability

(Kamboj, 2000).

21

Herbal preparations contain many ingredients that work in a synergistic manner to

produce a beneficial effect (Dubey et al., 2004). The herbal medicine contains

phytochemical compounds (secondary metabolites) that provide definite

physiological action on the human body. Some of these bioactive substances

include alkaloids, saponins, cardiac glycosides, terpenoids, steroids and flavonoids

(Edeoga, 2005). The levels of bioactive compounds in medicinal plants is

dependent on varous factors including soil quality, soil quality, intraspecies

variation, environment, age harvesting and processing procedures (Kamboj, 2000).

Target of Secondary metabolites include ion channels, ion pumps, neurotransmitter

enzymes which degrade neurotransmittesr or elements of cytoskeleton such as

tubulin and microtubules. Alkaloids are specific and target on receptors of

neurotransmitters, while phenolics and terpenoids are less specific and and attack a

magnitutude of proteins by building hydrogen, hydrophobic and ions bonds, thus

modulating their three dimensional structure and their bioactivies (Wink, 2015).

Different cultural systems use herbal preparations to manage pyrexia (Biren and

Avinash, 2010). A number phytochemicals such as saponins, steroids, alkaloids,

flavonoids, and terpenoids possesses inhibitory effects in production of PGE2 and,

as a result, produce an antipyretic effect (Bhaskar and Balakrishnan, 2009).

Flavonoids inhibit arachidonic acid peroxidation, resulting in the reduction of PGE2

levels (Baummann et al., 1980). Flavonoids also inhibit the synthesis of tumor

22

necrosis factor-α which stimulates the production of PGE2 necessary for fever

induction (Adesokan et al., 2008). Alkaloids and steroids inhibit the synthesis of

prostaglandins synthase which stimulates the production of PGE2 (Niazi et al.,

2010). Steroids are also reported to lower the PGE2 synthesis by preventing the

conversion of linoleic acid to arachidonic acid, a substrate required for PGE2

synthesis (Barnes et al., 1993).

The use of Peruvian cinchona bark extracts as an antipyretic agent date back to the

early 1600s (Bruce-Chhwatt, 1998). In addition, various medicinal plants like

Neem, Arjuna, Ashwagandha and Tulsi are used traditionally in the management

of fever in India (Umashanker and Shruti, 2011). According to Nagaraj and

Venkateswarlu (2013), aqueous extract of the whole plant of Fagonia cretica L.

was scientifically confirmed to possess antipyretic activity in Wistar rats. Similarly,

according to Mwonjoria et al. (2011), the root bark extract of Solanum incanum

was scientifically confirmed to possess compounds with antipyretic effects in

Wistar rats. Akatheeswaran (2013), confirmed the antipyretic activity of ethanol

and aqueous root extracts of Asparagus racemosus in Wistar rats.

2.5.2 Inflammation

Herbal preparations have been used for centuries to manage inflammation

(Reynolds et al., 1995). Convetional drugs work by inhibiting the expression of

23

COX-2 enzyme that is vital in inducing inflammation. However, many herbal

medicines acts by inhibiting nuclear factor-kB (NF-kB) inflammatory pathways

(Maroon et al., 2010). The NF-kB can detect noxious stimuli, such as infectious

agents, cellular injuries and free radicals, and then synthesize inflammatory

cytokines. Thus, their inhibition leads to magement inflammation (Frantz et al.,

1994).

The stem bark extract of the white willow tree is one of the oldest herbal remedies

for inflammation dating back to ancient Egyptian, Greek, Roman, and Indian

civilizations. The mechanism of action of white willow bark is a non-selective

inhibitor of COX-2 and COX-1, and usually blocks inflammatory PGE2

biosynthesis (Vane, 2000). Curcuma longa is traditionally used as an anti-

inflammatory agent in both Ayurvedic and Chinese medicines. Several clinical

trials have demonstrated curcumin (a yellow pigment derived from turmeric C.

longa) possessing anti-inflammatory activity (Chainani-Wu, 2003). Curcumin

inhibits inflammation by suppressing the expression of NF-kB (Bremner and

Heinrich, 2002). The stems bark of Uncaria tomentosa and Uncaria guianensis are

used to manage arthritis and intestinal disorders in Peru. The active compounds of

U. tomentosa and U. guianensis appear to be phenols, flavonoids, alkaloids, sterols

and tannins (Sandoval et al., 2002). Various studies have indicated Peruvian herb

inhibiting production of pro-inflammatory mediators such as prostaglandins,

histamine, serotonin, protease and lysosome (Sandoval et al., 2002).

24

Flavonoids have been reported to inhibit TNF-α and phospholipase necessary to

cause inflammation (Bhagyasri et al., 2015). Flavonoids also block both the

cyclooxygenase and lipoxygenase pathways of the arachidonate cascade, which are

responsible for inflammation induction (Di Carlo et al., 1999). Research findings

have revealed that triterpenoids suppress some function of macrophages,

neutrophils and also inhibit nitric oxide (NO), NF-κB signaling and PGE2

production relevant for the inflammatory response (Salminen et al., 2008).

2.6 Plants used in this study

2.6.1 Kigelia africana (Lam.) Benth

Figure 2. 1 Kigelia africana picture captured Embu County Kenya

25

2.6.1.1 Plant description and geographical distribution

Kigelia africana De Wild belong to the family bignoniaceae. Its common names

include sausage tree or cucumber (English); mvungunya, mwegea, mwicha, mranaa

(Swahili) (Grace et al., 2002); muratina (Kikuyu) and yago (Luo). It is known as

the sausage or cucumber tree due to its huge fruits (about 0.6m in length and 4kg

in weight) which hang from long fibrous stalks (Grace et al., 2002).

Kigelia africana is a tree growing up to 20m tall or more. The tree is evergreen

where rainfall occurs throughout the year, although deciduous where there is a long

dry season. The bark is grey, smooth and peeling off on older trees. The leaves are

opposite, 30-50cm long, pinnate, with six to ten oval leaflets up to 6cm broad and

20cm long. The flowers are bell shape, reddish or purplish greenish in color and

about 10cm wide. The flowers hang down from branches on long flexible stems (2

- 6 m long) (Grace et al., 2002).

Kigelia africana is mostly found on riverbanks, along streams, high-rainfall

savannah, floodplains and open woodland. It occurs on sandy loams, loamy red

clay soils, and from sea level up to 3000 m altitude with an annual rainfall of 900-

2000 mm. The plant is well distributed in the Southern, Eastern, Central and West

Africa (Burkill, 1985).

26

2.6.1.2 Cultural and medicinal uses

The slices of the baked fruit of K. africana are used to aid in fermentation of local

honey beer throughout East Africa. In times of drought, the seeds are roasted in hot

ashes and eaten. The flowers and leaves of K. africana are consumed by livestock

when they fall to the ground. The wood makes yokes, fruit boxes, and shelving.

The heartwood is usually brown and makes drums, cutlery, and utensils. Inhabitants

of areas along rivers the Zambezi and Chobe make their dugout canoes from K.

africana. Black dye is produced from the fruit and tannin can be extracted from the

roots and stem bark (Orwa et al., 2009).

Kigelia africana is traditionally used to treat diseases such as cold, flu,

inflammation, and dysentery among Embu community in Kenya (Kareru et al.,

2007). Traditional African healers also used K. africana to treat a broad range of

skin diseases from acne, boils, and fungal infections, through to more serious

illnesses, such as syphilis, skin cancer, and leprosy. It is also used effectively to

dress sores and wounds (Grace et al., 2002). According to Akah (1996), aqueous

leaves extract of K. africana has shown to possess anti-diarrheal activity. The plant

has also been reported to possess antimalarial activities (Weenen et al., 1990). The

root bark extracts of K. africana has been recommended to treat uterine cancer

(Msouthi and Mangombo, 1983). The plant has also been screened to show anti-

molluscidal activity (Kela et al., 1989).

27

2.6.2 Acacia hockii De Wild

Figure 2. 2 Acacia hockii picture captured Embu County, Kenya

2.6.2.1 Plant description and distribution

Acacia hockii also known as Acacia white thorn acacia belongs to family fabaceae.

Other common names of A. hockii in Kenya include chepnyaliliet (Kalenjin),

iguisuria (Kisii), mugaa (Kikuyu), and olerai (Maasai) (ILDIS, 2013). It is a multi-

stemmed shrub 2-4m tall or a small tree 6-7m tall with an open crown occasionally

9m wide. The bark is red-brown to greenish or greenish-brown, peeling off in

papery layers. The thorns are spinescent stipules and short (2cm long). Leaves have

2-11 pairs of pinnae; each with 9-29 pairs of leaflets, 0.5-1.2mm wide and 2.0-

6.5mm long, usually densely ciliolate. Flowers are bright yellow to orange, with

pedunculate heads 5-12mm in diameter (ILDIS, 2013).

Acacia hockii is native to many dry areas in tropical Africa, East Africa, Southern

Africa and South of Sahel (ILDIS, 2013). It is also present in Saudi Arabia (GBIF,

28

2012). In West Africa, it is well distributed in the moist savannah regions of the

Guinea zone. In East Africa, it is well distributed in wooded grassland, deciduous

and semi-evergreen bushland, thickets and scrub. A. hockii is well distributed from

sea level to at least 2,400m altitude. A. hockii is also common on sloping or rocky

ground and often associated with poor soils, where it often becomes the dominant

shrub (ILDIS, 2013).

2.6.2.2 Cultural and medicinal uses

Acacia hockii is used traditionally in the construction of thatched houses, shade for

housing cattle, source of charcoal, fencing and making cattle pens (Musinguzi et

al., 2012). Bark yields fibre used for making basket in Tanzania. Leaves, pods, and

seeds forms forage for livestock (Gwyne, 1969), and flowers are a good source of

bee forage. The inner bark yields an edible famine food, and inner bark fibers are

chewed for their sweet juice by Maasai’s community to quencher thirst. A. hockii

also produces edible reddish exudates gum (Anderson, 1984), that is also used by

the Mbeere tribe in Kenya as an adhesive.

Ethnomedically, A. hockii is used to manage pain, stomach discomfort reliever,

dropsy, swellings, malaria and gout by the Embu and Mbeere communities in

Kenya (Kareru et al., 2007). In Tanzania, the bark decoction is given to children

with fever, and a root decoction is used to treat tuberculosis-related ailments and

hookworm in Uganda (Tabuti et al., 2010).

29

CHAPTER THREE

MATERIALS AND METHODS

3.1 Collection and preparation of plant materials

Fresh stem barks and leaves of K. africana and stem bark of A. hockii were

collected in Mbeere North sub-county, Embu County, Kenya, with the help of local

herbalists (Muru wa Thika) in August 2015. The information gathered included

plant names in vernacular, plant parts used and the ailment treated. The plant

samples were identified by a taxonomist and a voucher specimen deposited at the

National Museum of Kenya herbarium. The plant samples were sorted out, cleaned

with tap water, rinsed with distilled water and transported in polyethene bags to

Biochemistry and Biotechnology laboratories at Kenyatta University. The plant

materials were separately chopped into small pieces, and air dried at room

temperature until dry. The dried sample materials were ground into fine

homogenous powder using an electric mill.

3.2 Extraction

For each sample, 400 grams of powder was soaked in 2 litres of methanol, stirred

for six hours and left standing for 48 hours for the bioactive compounds to dissolve.

The extracts were then filtered using Whatman’s No.1 filter paper and the filtrate

concentrated to dryness under reduced pressure using rotary evaporator at a

maximum temperature of 64°C. The concentrate was then put in an airtight

container and stored at 40C until use in the bioassay.

30

3.3 Experimental design

3.3.1 Laboratory animals

Male Wistar rats weighing between 130-150 grams and aged between 7-8 weeks

were used to bioscreen antipyretic activity, while Swiss albino mice weighing

between 20-25 grams and aged between 7-8 weeks were used to test anti-

inflammatory activity. The animals breeding colonies were acquired and bred in

the animal breeding and experimentation facility at the Department of Biochemistry

and Biotechnology, Kenyatta University.

The animals were allowed to acclimatize for seven days prior to experimentation.

The experimental animals were kept in the standard cages in the animal

experimentation facility maintained under standard laboratory condition of an

ambient temperature of 20°C-25°C with 12 hours daylight and 12 hours darkness

cycles. The experimental animals were fed on standard rodent pellets and provided

with water ad libitum (Vogel et al., 2002). Ethical guidelines and procedures for

handling experimental animals were followed as indicated in the animal

experimentation facility at the Department of Biochemistry and Biotechnology,

Kenyatta University.

31

3.4 Bioscreening

3.4.1 Evaluation of antipyretic activities in Wistar rats

The animals were fasted during the experiment but given water ad libitum. Before

fever induction, rats were weighed and their basal rectal temperature measured and

recorded. Steam-distilled turpentine (20ml/kg bw) was injected intraperitoneally to

induce pyrexia according to the method described by Grover et al. (1990). Rats

whose rectal temperatures rose by 0.8°C after one hour were termed pyretic and

used for studies. The extract was first dissolved in dimethylsulphoxide (DMSO)

solvent and then a vehicle normal saline (0.9% sodium chloride solution) added

before treatment. The extracts at a dose level of 50, 100 and 150mg/kg body weight

as well as aspirin (reference drug) at a dose of 100mg/kg body weight were

administered intraperitoneally one hour after fever induction.

Thirty male Wistar rats were divided randomly into six groups of five rats each and

treated as follows; Group I (normal control) was not induced with pyrexia but

received 4% DSMO. Group II (negative control) was induced with pyrexia and

received 4% DMSO. Group III (positive control) was induced with pyrexia and

received aspirin (reference drug) at a dose of 100mg/kg body weight. Groups IV,

V and VI (experimental groups) were induced with pyrexia and received extracts

at a doses of 50mg/kg, 100mg/kg, and 150mg/kg body weight. This design is

summarized in table 3.1.

32

Table 3.1: Treatment protocol for evaluation of antipyretic activities of

methanolic extracts of Kigelia africana (Lam) Benth and Acacia

hockii De Wild in male Wistar rats

Steam distilled turpentine; DMSO = 4%, bw. = body weight

The rectal temperatures were measured by inserting a well-lubricated thermistor

probe of a digital thermometer about 3 cm (Grover et al., 1990) into the rectum.

The digital thermometer was calibrated against a mercury thermometer. The mean

temperature was measured at 15 minutes intervals for one hour before injection of

turpentine, and this was termed as baseline/initial temperature. The rectal

temperatures were measured and recorded at 0, 1, 2, 3, and 4 hours after treatments.

Rectal temperature at the zero hours and after treatments was compared and their

percentage inhibition calculated using a formula described by Hukkeri (2006), as

follows;

Inhibition(%) =B − Cn

B × 100

Where,

B - Rectal temperature at 1 hour after turpentine administration

Cn - Rectal temperature after treatment

Group Status Treatment

I Control DMSO

II Negative control Turpentine + DMSO

III Positive control Turpentine + 100mg/kg bw Aspirin

IV Experimental group A Turpentine +50 mg/kg bw extract + DMSO

V Experimental group B Turpentine +100 mg/kg bw extract + DMSO

VI Experimental group C Turpentine +150 mg/kg bw extract + DMSO

33

3.4.2 Evaluation of anti-inflammatory activities in Swiss albino mice

Thirty Swiss albino mice of either sex were divided randomly into six groups of

five mice each and treated as follows; Group I (normal control) was not induced

with paw edema but received 4% dimethylsulphoxide (DMSO). Group II (negative

control) was induced with paw edema and received 4% DMSO. Group III (positive

control) was induced with paw edema and received diclofenac (reference drug) at

a dose of 15mg/kg body weight. Groups IV, V and VI (experimental groups) were

induced with paw edema and received the extracts at a dosage of 50mg/kg,

100mg/kg, and 150mg/kg body weight. This design is summarized in table 3.2.

Table 3.2: Treatment protocol for evaluation of anti-inflammatory activities

of methanolic extracts of Kigelia africana (Lam) Benth and Acacia

hockii De Wild in Swiss albino mice

Group Status Treatment

I Control DMSO

II Negative control Carrageenan + DMSO

III Positive control Carrageenan+15mg/kg/bw diclofenac

IV Experimental group A Carrageenan + DMSO + 50 mg/kg bw extract

V Experimental group B Carrageenan + DMSO +100 mg/kg bw extract

VI Experimental group C Carrageenan + DMSO +150 mg/kg bw extract

1% carrageenan; 4% DMSO, bw = body weight

The anti-inflammatory activity of the extracts was assessed using carrageenan-

induced right paw edema in mice as described by Winter et al. (1962). Acute

inflammation was induced by sub-plantar injection of 0.05ml 1% carrageenan

(sigma-type I) in normal saline 30 minutes after treatment. The change in paw

diameter was measured using a digital vernier caliper 30 minutes before injection

34

of carrageenan and at 1, 2, 3 and 4 hours after induction of inflammation

(Bamgbose and Noamesi, 1981). The percentages inhibition in inflammation was

calculated using the formula described by Ummageswari and Kudagi (2015), as

follows;

Inhibition (%) = Ct − Tt

Ct× 100

Where,

Ct = Paw diameter at 1 hour after carrageenan administration (control)

Tt = Paw diameter after Treatment

3.5 Qualitative phytochemical screening

The extracts were subjected to standard qualitative phytochemical screening to

identify the absence or the presence of various phytochemicals using methods of

analysis described by Harbone (1998) and Kotake (2000). Phytochemicals tested

include alkaloids, terpenoids, saponins, flavonoids, phenolics, cardiac glycosides,

and steroids. These phytochemicals are reported to possess antipyretic and anti-

inflammatory activity (Bhaskar and Balakrishnan, 2009; Bhagyasri et al., 2015).

3.5.1 Saponins (Froth test)

Few drops of sodium bicarbonate solution were added to 2ml of extract and shaken

vigorously. The extract was then allowed to stand for 15 minutes and classified for

saponin content as follows; no froth- negative, froth less than 1cm - weak positive,

froth 1.2cm high- positive, froth greater than 2cm high - strongly positive.

35

3.5.2 Alkaloids

A volume of 5ml of the extract was acidified with 1M hydrochloric acid. The acidic

medium was then heated and treated with few drops of Dragendroff’s reagent. The

formation of an orange or reddish brown precipitate showed the presence of

alkaloids.

3.5.3 Terpenoids (Salkowski test)

To 0.5g of the extracts, 1ml of ethylacetate was added and then mixed into 2ml of

chloroform. Concentrated sulphuric acid (3ml) was carefully added alongside to

form a layer of reddish brown coloration which indicated the presence of

terpenoids.

3.5.4 Flavonoids (Sodium hydroxide test)

To 2ml of extracts, 2ml of diluted sodium hydroxide solution was added. A golden

yellow precipitate indicated the presence of flavonoids.

3.5.5 Cardiac glycosides (Keller-Kilian test)

To 0.5g of the extract, 2ml of glacial acetic acid containing four drops of 10% ferric

chloride (FeCl3) solution was added and under-layered with 1ml of concentrated

sulphuric acid. The formation of a violet, greenish or a brown ring at the interphase

indicated the presence of deoxysugar characteristic of cardenolides.

3.5.6 Steroids

To 0.5g of the extract, 2ml of chloroform was added to dissolve the extract followed

by side addition of 3ml of concentrated sulphuric acid. The formation of a reddish

brown color layer at the interface indicates the presence of the steroidal ring.

36

3.5.7 Phenolics

To 2ml volume of the extract, 1ml of ferric chloride solution was added carefully.

The formation of blue to green color indicates the presence of phenolics.

3.6 Data management and statistical analysis

Rectal temperatures and paw edema diameter were measured, recorded and

tabulated on the spreadsheet in Microsoft excel. The data was exported to Minitab

statistical software version 17.0 (State College, Pennsylvania) for analysis. The

data was subjected to descriptive statistics and expressed as mean ± standard error

of mean (SEM). One-way analysis of variance (ANOVA) was used to determine

whether there was any significant difference between the means of different groups.

This was followed by Tukey’s tests to separate means and obtain the specific

significant differences among the various treatment groups. Unpaired student t-test

was used to compare the mean activities of the two extracts. The values of p≤0.05

were considered significant. The data was presented in tables and graphs.

37

CHAPTER FOUR

RESULTS

4.1 Antipyretic activity of methanolic stem bark extract of Kigelia africana

(Lam.) Benth

The methanolic stem bark extract of K. africana demonstrated antipyretic activity

on turpentine-induced pyrexia in male rats, which was indicated by the decrease in

rectal temperature after extract administration (Table 4.1). In the first hour after

treatment, the stem bark extract of K. africana at the dose of 150mg/kg body weight

and aspirin (reference drug) at the dose of 100mg/kg body weight decreased the

elevated rectal temperature by 0.31% and 0.63% respectively (Table 4.1). However,

the extract at the dosages of 50mg/kg and 100mg/kg body weight never showed

antipyretic activity in the first hour (Table 4.1). The antipyretic activity of the

extract at the dosages of 50mg/kg, 100mg/kg, and 150mg/kg showed no significant

difference and were comparable to reference drug aspirin (p>0.05; Table 4.1).

In the second hour after treatment, the stem bark extract of K. africana at the

dosages of 50mg/kg, 100mg/kg, and 150mg/kg body weight demonstrated

antipyretic activity by decreasing the elevated rectal temperature by 0.06%, 0.11%

and 1.20% respectively (Table 4.1). Similarly, the rats treated with aspirin

(reference drug) showed antipyretic activity by lowering the elevated rectal

temperature by 1.25% (Table 4.1). The antipyretic activity of extract at the dose of

150mg/kg body weight was significantly different from 50mg/kg and 100mg/kg

38

body weight (p<0.05; Table 4.1). However, the extract at the dose of 150mg/kg

body weight was comparable to aspirin (reference drug) (p>0.05; Table 4.1).

In the third hour after treatment, the stem bark extract of K. africana at the doses

of 50mg/kg, 100mg/kg, and 150mg/kg body weight reduced elevated rectal

temperature by 0.58%, 1.30% and 2.41% respectively (Table 4.1). Similarly, the

rats treated with aspirin (100mg/kg bw) at the dose of 100mg/kg body weight

reduced elevated rectal temperatures by 1.88% (Table 4.1). The antipyretic activity

of the extract at the doses of 100mg/kg and 150mg/kg body weight showed no

significant difference and were comparable to the aspirin (reference drug) (p>0.05;

Table 4.1). Besides, the antipyretic activity of the extract at the dosages 50mg/kg

and 100mg/kg was not significantly different (p>0.005; Table 4.1).

In the fourth hour, the extract at the dose levels of 50mg/kg, 100mg/kg and

150mg/kg body weight as well as the aspirin (reference drug) reduced elevated

rectal temperature by 1.41%, 2.09%, 3.07% and 2.40% respectively (Table 4.1).

The antipyretic activity of the extract at the dosages of 50mg/kg and 100mg/kg

body weight showed no significant difference (p>0.005; Table 4.1). In addition, the

antipyretic activity of the extract at the dose of 150mg/kg body weight was

comparable to the group of rats treated with reference drug aspirin (p>0.05; Table

4.1).

39

Table 4.1: Effects of intraperitoneal administration of methanolic stem bark extract of Kigelia africana (Lam) Benth on

turpentine-induced pyrexia in male Wistar rat

Values expressed as Mean ± SEM for five animals per group. Values with the same superscript letter are not significantly

different by one-way ANOVA followed by Tukey’s test (p>0.05). Percentage reduction in rectal temperature is given within

parentheses. Steam distilled turpentine = 20mg/kg bw; 100mg/kg bw aspirin and 4% DMSO.

Group Treatment Percentage (%) change in rectal temperature (0C) after treatment

0hr 1hr 2hr 3hr 4hr

Normal control DMSO 100±0.00

(0.00)

100.11±0.28b

(-0.11)

100.06±0.23b

(-0.06)

100.11±0.28b

(-0.11)

100.22±0.18b

(-0.22)

Negative control Turpentine + DMSO 100±0.00

(0.00)

101.47±0.20a

(-1.47)

102.25±0.13a

(-2.25)

102.94±0.16a

(-2.94)

103.04±0.14a

(-3.04)

Positive control Turpentine + Aspirin +

DMSO

100±0.00

(0.00)

99.37±0.26b

(0.63)

98.75±0.23c

(1.25)

98.12±0.13d

(1.88)

97.60±0.15de

(2.40)

Methanolic

extracts

Turpentine + 50mg/kg

bw + DMSO

100±0.00

(0.00)

100.42±0.24ab

(-0.42)

99.95±0.30b

(0.06)

99.42±0.30bc

(0.58)

98.59±0.24c

(1.41)

Turpentine +100mg/kg

bw + DMSO

100±0.00

(0.00)

100.06±0.29b

(-0.06)

99.89±0.23b

(0.11)

98.70±0.08cd

(1.30)

97.91±0.16cd

(2.09)

Turpentine + 150mg/kg

bw + DMSO

100±0.00

(0.00)

99.69±0.22b

(0.31)

98.80±0.15c

(1.20)

97.97±0.10d

(2.41)

96.93±0.10e

(3.07)

40

4.2 Antipyretic activity of methanolic stem bark extract of Acacia hockii De

Wild

The methanolic stem bark extract of A. hockii demonstrated antipyretic activity on

turpentine-induced pyrexia in rats, which was indicated by the reduction in elevated

rectal temperature after extract administration (Table 4.2). In the first hour after

treatment, only the rats treated with aspirin (reference drug) at the dosage of

100mg/kg body weight showed antipyretic activity by reducing the elevated rectal

temperature by 1.7% (Table 4.2). However, the stem bark extract of A. hockii at the

dosages of 50mg/kg, 100mg/kg, and 150mg/kg body weight never demonstrated

antipyretic activity in the first hour (Table 4.2). The antipyretic activity of the

extract at the three dose levels of 50mg/kg, 100mg/kg, and 150mg/kg body weight

showed no significant difference and were comparable to the negative control

(p>0.05; Table 4.2).

In the second hour, the stem bark extract of A. hockii at the dosages of 100mg/kg

and 150mg/kg body weight, as well as the aspirin (reference drug), demonstrated

antipyretic activity by reducing rectal temperature by 0.68%, 0.72% and 2.47%

respectively (Table 4.2). However, the extract at the dose of 50mg/kg body weight

never showed antipyretic activity at this hour (Table 4.2). The antipyretic activity

of extract at the three doses (50,100, and 150mg/kg bw) showed no significant

difference (p>0.05; Table 4.2). Besides, the antipyretic activity of the extract at the

41

dosages of 100mg/kg and 150mg/kg body weight were comparable to aspirin

(reference drug) (p>0.05; Table 4.2).

In the third hour, the extract of A. hockii at the dosages of 50mg/kg, 100mg/kg, and

150mg/kg body weight showed the antipyretic effect by lowering the elevated rectal

temperatures by 0.62%, 1.46% and 2.28% respectively (Table 4.2). In addition, the

rats treated with aspirin (reference drug) reduced rectal temperature by 3.16%

(Table 4.2). The antipyretic activity of extract at the dosages of 50mg/kg and

100mg/kg body weight showed no significant difference (p>0.05; Table 4.2).

Besides, the antipyretic activity of the extract at the doses of 100mg/kg and

150mg/kg body weight demonstrated no significant difference (p>0.05; Table 4.2).

Moreover, the antipyretic activity of the extract at the dose level of 150mg/kg was

comparable to the aspirin (reference drug) (p>0.05; Table 4.2).

In the fourth hour, the extract at the dosages of 50mg/kg, 100mg/kg and 150mg/kg

body weight as well as aspirin (reference drug) lowered the elevated rectal

temperatures by 1.61%, 2.41%, 3.88% and 3.1% respectively (Table 4.2). The

antipyretic activity of the extract at the dose of 150mg/kg body weight was

significantly different from the extract at the dose levels of 50mg/kg and 100mg/kg

body weight (p<0.05; Table 4.2). The antipyretic activity of the extract at the doses

of 100mg/kg and 150mg/kg body weight was comparable to aspirin (reference

drug) (p>0.05; Table 4.2).

42

Table 4.2: Effects of intraperitoneal administration of methanolic stem bark extract of Acacia hockii De Wild on turpentine-

induced pyrexia in male Wistar rats

Values expressed as Mean ± SEM for five animals per group. Values with the same superscript letter are not significantly different by

one-way ANOVA followed by Tukey’s test (p>0.05). Percentage reduction in rectal temperature is given within parentheses. Steam

distilled turpentine = 20mg/kg bw; 100mg/kg aspirin; 4% DMSO.

Group Treatment Percentage (%) change in rectal temperature (0C) after treatment

0hr 1hr 2hr 3hr 4hr

Normal control DMSO 100±0.00

(0.00)

99.89±0.11a

(0.11)

100.27±0.09ab

(-0.27)

100.32±0.10b

(-0.32)

100.27±0.09b

(-0.27)

Negative control Turpentine + DMSO 100±0.00

(0.00)

101.00±0.10a

(-1.00)

101.83±0.19a

(-1.83)

102.67±0.34a

(-2.67)

102.67±0.26a

(-2.67)

Positive control Turpentine + Aspirin +

DMSO

100±0.00

(0.00)

98.30±0.52b

(1.70)

97.53±0.66c

(2.47)

96.85±0.52e

(3.16)

96.90±0.32de

(3.10)

Methanolic

Extracts

Turpentine + 50mg/kg bw +

DMSO

100±0.00

(0.00)

100.58±0.40a

(-0.58)

100.22±0.60ab

(-0.22)

99.38±0.34bc

(0.62)

98.39±0.22c

(1.61)

Turpentine +100mg/kg bw

+ DMSO

100±0.00

(0.00)

100.05±0.31a

(-0.05)

99.32±0.36bc

(0.68)

98.54±0.24cd

(1.46)

97.86±0.21cd

(2.14)

Turpentine + 150mg/kg bw

+ DMSO

100±0.00

(0.00)

100.11±0.28a

(-0.11)

99.28±0.25bc

(0.73)

97.72±0.27de

(2.28)

96.12±0.34e

(3.88)

43

4.3 Comparison between antipyretic activities of Kigelia africana and Acacia

hockii at different doses

In comparison, the antipyretic activity of the two extract was not significantly

different at the dose of 50mg/kg body weight in the 1, 2, 3 and 4 hours of the test

period with p values of 0.75, 0.70, 0.93, and 0.56 respectively (p>0.05). Similarly,

the antipyretic activity of the two extracts at the dose of 100mg/kg body weight was

not significantly different in the 1, 2, 3, and 4 hours of the test period with p values

of 0.9, 0.23, 0.58, and 0.86 respectively (p>0.05). Besides, the antipyretic activity

of the two extracts was not significantly different at the dosage of 150mg/kg body

weight in the 1, 2, 3 and 4 hours of the test period with p values of 0.28, 0.16, 0.42

and 0.08 respectively (p>0.05). The antipyretic activity of both extracts was best

active at the dosage of 150mg/kg in the fourth hour after treatment (Figure 4.1).

44

Figure 4. 1: Comparison of percentage reduction in rectal temperature of

methanolic stem bark extracts of Kigelia africana and Acacia

hockii on turpentine-induced pyrexia in rats at different doses.

45

4.4 Anti-inflammatory activity of methanolic leaf extract of Kigelia africana

(Lam.) Benth

The methanolic leaf extract of K. africana showed significant anti-inflammatory

activity on carrageenan-induced paw edema, which was demonstrated by the

reduction in inflamed hind paw diameter after extract administration (Table 4.3).

In the first hour, the leaf extract of K. africana at the dose of 150mg/kg and

reference drug diclofenac at the dosage of 15mg/kg body weight showed anti-

inflammatory effect by reducing hind paw diameter by 0.21% and 1.10%

respectively (Table 4.3). However, the extract at the dosages of 50mg/kg and

100mg/kg body weight never showed anti-inflammatory activity at the first hour

(Table 4.3). The anti-inflammatory activity of the extract at the dose levels of

50mg/kg, 100mg/kg, and 150mg/kg body weight showed no significant difference

(p>0.05; Table 4.3). In addition, the anti-inflammatory activity of extract at the

dose level of 150mg/kg body weight was comparable to aspirin the reference drug

(p>0.05; Table 4.3).

In the second hour, the leaf extract of A. hockii at the doses of 100mg/kg and

150mg/kg body weight as well as the diclofenac (reference drug) reduced inflamed

paw diameter by 0.42%, 1.42% and 2.8% respectively (Table 4.3). However, the

extract at the dosage of 50mg/kg body weight never showed anti-inflammatory

activity at this hour (Table 4.3). The anti-inflammatory activity of the extract at the

dose levels of 50mg/kg and 100mg/kg showed no significant difference (p>0.05;

46

Table 4.3). Besides, the anti-inflammatory activity of the extract at the dose level

of 150mg/kg body weight was comparable to diclofenac (reference drug) (p>0.05;

Table 4.3).

In the third hour, the extract at the dose levels of 50mg/kg, 100mg/kg, and

150mg/kg body weight as well as the diclofenac (reference drug) reduced the

inflamed hind paw diameter by 0.86%, 2.25%, 3.41% and 4.02% respectively

(Table 4.3). The anti-inflammatory activity of the extract at the dosage of 50mg/kg

and 100mg/kg and 150mg/kg body weight showed no significant difference and

were comparable to diclofenac (reference drug) (p>0.05; Table 4.3).

In the fourth hour, the leaf extract of K. africana at the dose levels of 50mg/kg,

100mg/kg, and 150mg/kg body weight reduced inflamed hind paw diameter by

1.95%, 2.98% and 4.98% respectively (Table 4.3). Similarly, the reference drug

reduced the inflamed paw diameter by 4.43% at this hour (Table 4.3). The anti-

inflammatory activity of the extract at the dosages of 50mg/kg and 100mg/kg body

weight showed no significant difference (p>0.05; Table 4.3). In addition, the anti-

inflammatorty activity of the extract at the dose of 150mg/kg body weight was

comparable to reference drug diclofenac (p>0.05; Table 4.3).

47

Table 4. 3: Effects of intraperitoneal administration of methanolic leaf extract of Kigelia africana (Lam) Benth on carrageenan-

induced inflammation in Swiss albino mice

Values expressed as Mean ± SEM for five animals per group. Values with the same superscript letter are not significantly different by

one-way ANOVA followed by Tukey’s test (p>0.05). Percentage reduction in rectal temperature is given within parenthesis.

Carrageenan = 1%; 15mg/kg bw diclofenac and 4% DMSO.

Group Treatment Percentage (%) change in paw diameter (mm) after

treatment

0hr 1hr 2hr 3hr 4hr

Normal Control 10% DMSO 100±0.00

(0.00)

99.84±0.10b

(0.16)

99.84±0.10b

(0.16)

99.84±0.10b

(0.16)

99.93±0.07b

(0.07)

Negative Control Carrageenan + DMSO 100±0.00

(0.00)

102.13±0.20a

(-2.13)

103.61±0.08a

(-3.61)

104.60±0.30a

(-4.60)

104.95±0.48a

(-4.95)

Positive Control Carrageenan + diclofenac

+DMSO

100±0.00

(0.00)

98.89±0.22c

(1.11)

97.20±0.53e

(2.80)

95.98±0.44bc

(4.02)

95.57±0.47de

(4.43)

Methanolic

Extracts

Carrageenan + 50mg/kg

bw + DMSO

100±0.00

(0.00)

101.74±0.15b

(-1.74)

100.37±0.26bc

(-0.37)

99.14±0.18b

(0.86)

98.05±0.21c

(1.95)

Carrageenan+100mg/kg

bw + DMSO

100±0.00

(0.00)

101.10±0.25b

(-1.10)

99.58±0.77cd

(0.42)

97.75±0.60b

(2.25)

97.02±0.45cd

(2.98)

Carrageenan + 150mg/kg

bw + DMSO

100±0.00

(0.00)

99.79±0.14bc

(0.21)

98.59±0.25de

(1.42)

96.59±0.26b

(3.41)

95.02±0.20e

(4.98)

48

4.5 Anti-inflammatory activity of methanolic stem bark extract of Acacia

hockii De Wild

The methanolic stem bark extract of A. hockii demonstrated anti-inflammatory

activity on carrageenan-induced paw edema in mice, which was indicated by the

reduction in paw edema after extract administration (Table 4.4). In the first hour,

the stem bark extract of A. hockii at the dose levels of 100 and 150mg/kg body

weight as well as the diclofenac (reference drug) at the dosage of 15mg/kg body

weight reduced inflamed hind paw diameter by 0.6%, 0.77% and 1.48%

respectively (Table 4.4). However, the extract at the dose level of 50mg/kg body

weight never showed anti-inflammatory activity at this hour (Table 4.4). The anti-

inflammatory activity of the extract at the dosages of 100mg/kg and 150mg/kg body

weight showed no significant difference and was comparable to reference drug

diclofenac (P>0.05; Table 4.4). Besides, the anti-inflammatory activity of the

extract at the dose of 50mg/kg body weight was significantly different (p<0.05;

Table 4.4) from 100mg/kg and 150mg/kg body weight and comparable to negative

control (p<0.05; Table 4.4).

In the second hour, the extract at the dose levels of 100mg/kg and 150mg/kg body

weight, as well as the diclofenac, reduced inflamed paw diameter of mice by 1.43%,

3.03% and 3.37% respectively (Table 4.4). However, the extract at the dose level

of 50mg/kg body weight never showed any anti-inflammatory effect at this hour

(Table 4.4). The anti-inflammatory activity of the extract at the dose levels of

49

50mg/kg, 100mg/kg, and 150mg/kg were significantly different (p<0.05; Table

4.4). However, the anti-inflammatory activity of the extract at the dosage of

150mg/kg body weight was comparable to the diclofenac (reference drug) (p>0.05;

Table 4.4).

In the third hour, the extract at dose levels of 50mg/kg, 100mg/kg and 150mg/kg

body weight as well as the diclofenac (reference drug) reduced the inflamed hind

paw diameter by 0.96%, 3.11%, 4.28% and 4.5% respectively (Table 4.4). The anti-

inflammatory activity of the extract at the dosages of 100mg/kg and 150mg/kg body

weight demonstrated no significant difference and were comparable to diclofenac

(reference drug) (P>0.05; Table 4.4). However, the anti-inflammatory activity of

the extract at the dose level of 50mg/kg body weight was significantly different

from 100mg/kg and 150mg/kg body weight (p<0.05; Table 4.4).

In the fourth hour, the extract at the dose levels of 50mg/kg, 100mg/kg, and

150mg/kg body weight, as well as the diclofenac, reduced inflamed hind paw

diameter by 1.78%, 4.05%, 5.38% and 4.9% respectively (Table 4.4). The anti-

inflammatory activity of the extract at the dosage of 100mg/kg and 150mg/kg

showed no significant difference and were comparable to diclofenac (reference

drug) (p>0.05; Table 4.4).

50

Table 4. 4: Effects of intraperitoneal administration of methanolic stem bark extracts of Acacia hockii De Wild on carrageenan-

induced inflammation in Swiss albino mice

Values expressed as Mean ± SEM for five animals per group. Values with the same superscript letter are not significantly different by

one-way ANOVA followed by Tukey’s test (p>0.05). Percentage reduction in rectal temperature is given within parenthesis.

Carrageenan = 1%; 15mg/kg bw diclofenac and 4% DMSO.

Group Treatment Percentage (%) change in paw diameter (mm) after treatment

0hr 1hr 2hr 3hr 4hr

Normal Control DMSO 100±0.00

(0.00)

99.99±0.12bc

(0.01)

99.99±0.12bc

(0.01)

99.99±0.12b

(0.01)

100.08±0.08b

(-0.08)

Negative Control Carrageenan + DMSO 100±0.00

(0.00)

101.49±0.17a

(-1.49)

102.66±0.26a

(-2.66)

103.30±0.26a

(-3.30)

103.37±0.45a

(-3.37)

Positive Control Carrageenan + Diclofenac

+DMSO

100±0.00

(0.00)

98.52±0.28d

(1.48)

96.63±0.44d

(3.37)

95.50±0.36c

(4.50)

95.10±0.47c

(4.90)

Methanolic

Extracts

Carrageenan + 50mg/kg bw

+DMSO

100±0.00

(0.00)

101.24±0.24ab

(-1.24)

100.35±0.25b

(-0.35)

99.04±0.17b

(0.96)

98.22±0.29b

(1.78)

Carrageenan + 100mg/kg bw +

DMSO

100±0.00

(0.00)

99.40±0.56cd

(0.60)

98.57±0.58c

(1.43)

96.89±0.74c

(3.11)

95.93±0.63c

(4.07)

Carrageenan + 150mg/kg bw +

DMSO

100±0.00

(0.00)

99.23±0.41cd

(0.77)

96.97±0.28d

(3.03)

95.72±0.47c

(4.28)

94.62±0.41c

(5.38)

51

4.6 Comparison between anti-inflammatory activities of Kigelia africana and

Acacia hockii at different doses

In comparison, the anti-inflammatory activity of the two extracts at the dose of

50mg/kg body weight in the 1, 2, 3 and 4 hours of the test period was not

significantly different with p values of 0.12, 0.96, 0.70 and 0.64 respectively

(p>0.05). The anti-inflammatory activity of the two extracts at the dose level of

100mg/kg body weight was significantly different in the first hour after treatment

with p values of 0.04 (p<0.05). However, the anti-inflammatory activity of the two

extracts at the dosage of 100mg/kg body weight in the 2, 3, and 4 hours showed no

significant difference with p values of 0.32, 0.40, and 0.20 respectively (p>0.05).

The anti-inflammatory activity of the two extracts at the dose level of 150mg/kg

body weight in the 1, 3 and 4 hours of the test period showed no significant

difference with p values of 0.27, 0.16 and 0.42 respectively (p>0.05). However, the

anti-inflammatory activity of the two extracts at the dose level of 150mg/kg body

weight in the 2 hours of the treatment period showed significant difference with p

values of 0.004 (p<0.05). The extracts were more effective in the fourth hour at the

dose level of 150mg/kg body weight (Figure 4.2).

52

Figure 4.2: Comparison of percentage reduction in paw diameter of

methanolic leaf extract of Kigelia africana and stem bark extract

of Acacia hockii at different doses.

53

4.7 Qualitative phytochemical screening

The qualitative phytochemical screening of the methanolic stem bark extracts of K.

africana and A. hockii showed the presence of alkaloids, cardiac glycosides,

flavonoids, phenolics, saponin, steroids, and terpenoids (Table 4.5). However, the

methanolic leaf extract of K. africana demonstrated the presence of cardiac

glycosides, flavonoids, phenolics, steroids, and terpenoids (Table 4.5).

54

Table 4. 5: Qualitative phytochemical composition of methanolic stem bark and leaf extracts of Kigelia africana and methanolic

stem bark extracts of Acacia hockii

Presence of phytochemical is denoted by (+) sign; absence of phytochemical is denoted by (-) sign

Phytochemicals K. africana stem bark K. africana leaf A. hockii stem bark

Alkaloids + - +

Flavonoids + + +

Steroids + + +

Saponins + - +

Terpenoids + + +

Cardiac glycosides + + +

Phenolics + + +

55

CHAPTER FIVE

DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS

5.1 Discussion

Although there is a considerable progress in the treatment of diseases and disorders

using modern therapeutic drugs, search for alternative medication continues due to

shortcomings of synthetic drugs. The long-term use of NSAIDs (non-sterodal anti-

inflammatory drugs) in the treatment of pyrexia and inflammation may result in

severe complications such as gastric ulcers, intestinal bleeding, renal damage and

cardiac abnormalities (Wolfe et al., 1999). The natural products especially herbal

extracts can be a safe alternative for managing diseases and disorders. In addition,

herbal preparations have shown potent antipyretic and anti-inflammatory activities

in experimental animals (Shukla et al., 2010).

The present study was designed to determine the antipyretic and anti-inflammatory

activities of methanolic extracts of Kigelia africana (Lam.) Benth and Acacia

hockii De Wild in animal models. The antipyretic activity of the extracts was

evaluated on turpentine-induced pyrexia in male Wistar rats. Exogenous pyrogens

such as lipopolysaccharides (LPS), steam distilled turpentine, brewer’s yeast,

muramyl dipeptide (MDP) and polyinosinic: polycytidylic acid (poly I: C) are used

to induce pyrexia in experimental animals (Soszynski et al., 1991; Soszynski and

Krajewska, 2002).

56

The exogenous pyrogens produce fever by their ability to stimulate the synthesis

and release of endogenous pyrogens cytokines from the host phagocytic cells.

Moreover, it is the circulating endogenous pyrogens rather than circulating

exogenous pyrogens which act on the thermoregulatory center to raise the

thermostatic set-point in the hypothalamus and produce fever (Dinarello and Wolff,

1978). Fever is mediated by the release of endogenous pyrogenic cytokines such as

tumor necrosis-α, interleukins (IL-1α, IL-1β, and IL-6), and interferons (β and α)

which are synthesized and released by activated phagocytes in response to

exogenous pyrogens. These mediators stimulate the synthesis of PGE2, which is the

primary mediators of the well-coordinated fever response (Netea et al., 2000).

Turpentine is a fluid obtained by the distillation of resin from a pine tree.

Subcutaneous injection of steam distilled turpentine causes pyrexia by enhancing

the production of PGE2 which ultimately increase the body temperature (Zhu et al.,

2011). The turpentine-induced pyrexia has been established to be fast, has persistent

fever pattern and experimental animals have a high tolerance to turpentine

compared to other exogenous pyrogens (Soszynski and Krajewska, 2002; Tung et

al., 2006). Steam distilled turpentine was therefore, selected to induce pyrexia in

this study.

57

The present study demonstrated significant antipyretic activity of methanolic stem

bark extracts of K. africana and A. hockii on turpentine-induced pyrexia in rats

(Table 4.1 and 4.2). These findings were consistent with other study carried out on

herbal extracts in animal models. Gitahi et al. (2015), demonstrated antipyretic

activity of dichloromethane: methanolic leaf and root bark extracts of Carissa

edulis (Forssk.) Vahl on turpentine-induced pyrexia in rats. Similarly, according to

Mwonjoria et al. (2011), the root extract of Solanum incanum (Linneaus)

demonstrated antipyretic activity on lipopolysaccharides-induced pyrexia in male

Wistar rats. Iwalewa et al. (2003), reported antipyretic activity of methanolic leaf

extract of Vernonia cineral against brewer’s yeast-induced pyrexia in Wistar rats.

Non-steroidal anti-inflammatory drugs such as aspirin, acetaminophen and ibrufen

are commonly used in medication of fever (Warden, 2010). The NSAIDs manage

pyrexia through inhibition of cyclooxygenase (COX) enzyme that stimulates the

production of PGE2 responsible for fever induction (Aronoff and Neilson, 2001).

Studies have established that at least two COX isoenzyme exist; COX-1 and COX-

2. The COX-1 synthesize prostaglandins that protect the stomach and the kidney

from damage, while the COX-2 stimulate PGE2 production that contributes to fever

induction (Vane and Botting, 1998). It was therefore believed that the methanolic

stem bark extracts of K. africana and A. hockii reduced the elevated rectal

temperatures of rats by inhibiting the production of PGE2 responsible for pyrexia

induction.

58

The antipyretic activity of methanolic stem back extracts of K. africana and A.

hockii demonstrated a dose-dependent response after the second hour of the test

period, with the dose level of 150mg/kg body weight producing greater antipyretic

activity (Tables 4.1 and 4.2). The antipyretic activity of the extracts therefore,

increased with an increase in the dose of the extract. These findings were in

agreement with the study carried out by Srivastava et al. (2013), which showed a

dose-dependent response on methanol extracts of aerial parts of Costus speciosus

Koen in experimental animals. Similarly, a study carried out by Kumar et al.

(2015), demonstrated a dose-dependent response on the antipyretic activity of ethyl

acetate roots extracts of Ocimum sanctum in laboratory animals.

The stem bark extracts of K. africana and A. hockii were less efficient at the lower

dose levels of 50 and 100 compared to 150 mg/kg body weight (Tables 4.1 and 4.2).

The aspirin (reference drug) achieved its maximum antipyretic activity in the third

hour (Tables 4.1 and 4.2). Its activity decreased subsequently probably due to

metabolism and clearance of the drug. On the other hand, the maximum antipyretic

activity of the stem bark extracts of K. africana and A. hockii occurred in the fourth

hour, indicating slow but steady passive diffusion of the bioactive constituents

across the cell membrane in the peritoneal cavity (Hossain et al., 2011). In addition,

the stem bark extracts of K. africana and A. hockii at different dose levels did not

lower the rectal temperature in the first and second hours as in the third and fourth

59

hours (Tables 4.1 and 4.2). These could be attributed to biotransformation of

bioactive compounds in the extract conferring antipyretic activity.

The stem bark extracts of K. africana and A. hockii at the dose of 150mg/kg body

weight were more effective in the fourth hour of treatment compared to aspirin

(reference drug) (Tables 4.1 and 4.2). These findings indicated that stem bark

extracts of K. africana and A. hockii were able to inhibit the synthesis of

prostaglandins more than the reference drug (aspirin). However, the aspirin showed

a sudden decrease in elevated rectal temperatures as compared to the extracts of K.

africana and A. hockii. This could be due to the slow absorption of the extracts or

biotransformation of the extracts to become active compared to the the reference

drug aspirin (Tables 4.1 and 4.2).

The antipyretic activity of methanolic stem bark extracts of K. africana and A.

hockii could be due to the ability of herbal preparations to possess a wealth of

secondary metabolites required in management of various diseases and disorders

(Shukla et al., 2010). The qualitative phytochemical screening of methanolic

extracts of K. africana and A. hockii indicated the presence of alkaloids, flavonoids,

steroids, saponins, terpenoids, cardiac glycosides and phenolics (Table 4.5). Plants

phytochemicals including steroids, alkaloids and flavonoids have shown to exhibit

antipyretic activity in experimental animals (Niazi et al., 2010).

60

Flavonoids exhibit inhibition of arachidonic acid peroxidation, which results in the

reduction of prostaglandins levels thus managing fever (Baummann et al., 1980).

Flavonoids also inhibit the synthesis of tumor necrosis factor-α which stimulates

the production of PGE2 necessary for fever induction (Adesokan et al., 2008).

According to Niazi et al. (2010), alkaloids and steroids inhibit the synthesis of

prostaglandins synthase which stimulates the production of PGE2. Steroids have

also been reported to inhibit the conversion of linoleic acid to arachidonic acid, a

substrate required for PGE2 synthesis (Barnes et al., 1993). The antipyretic activity

exhibited by methanolic stem bark extracts of K. africana and A. hockii was

therefore believed to be associated with the presence of flavonoids, alkaloids, and

steroids.

On the other hand, the present study evaluated for the anti-inflammatory

(antiphlogistic) activity of methanolic leaf extract of Kigelia africana (Lam.) Benth

and methanolic stem bark extract of Acacia hockii De Wild on carrageenan-induced

paw edema in mice. Carrageenan, dextran, histamine, serotonin and formalin-

induced paw edema; cotton pellet induced granuloma; Freund’s adjuvants are the

standard agents for causing acute, sub-acute and chronic inflammation respectively

in animal models (Ismail et al., 1997; Mujumdar and Misar, 2004).

Carrageenan is a natural carbohydrate obtained from edible red seaweeds (Necas

and Bartosikora, 2013). It is widely used to induce acute paw edema in

61

experimental animals (Paschapur et al., 2009) and hence the choice in the present

study. A freshly prepared solution of 1% carrageenan in normal saline as an

intraplantar injection at a dose of 50-150µl is commonly used to induce

inflammation (Estakhr et al., 2011).

The carrageenan-induced inflammation is described as a biphasic event in which

various mediators operates to produce an inflammatory response (Gupta et al.,

2006). The first mediators detectable in the early phase (1 hour) include histamine,

serotonin, and cyclooxygenase. On the other hand, the late phase (over 1 hour) is

sustained by the production of PGE2 and it is mediated by bradykinin and

leukotrienes (Ravi et al., 2009; Unnisa and Parven, 2011). Inducible nitric oxide

synthase (iNOS) and COX-2 enzyme are responsible for the production of an

enormous amount of inflammatory mediators (Handy and Moore, 1998; Necas and

Bartosikova, 2013). Carrageenan-induced inflammation is also associated with

enhanced levels of the endogenous pyrogenic cytokines such as tumor necrosis

factor-α and interleukins (IL-1 and IL-6) (Cuzzocrea et al., 1999).

The evaluation of anti-inflammatory activity of methanolic leaf extracts of K.

africana and stem bark extract of A. hockii demonstrated a significant anti-

inflammatory activity on carrageenan-induced paw edema in Swiss albino mice

(Tables 4.3 and 4.4). These findings were consistent with other studies carried out

on herbal medicines using animal models. A similar study carried out by Mwangi

62

et al. (2015), demonstrated a significant anti-inflammatory activity of

dichloromethane: methanolic leaf extracts of Caesalpina volkensii and Maytemus

obscura on carrageenan-induced paw edema in mice. Similarly, Zakaria et al.

(2007), reported that aqueous leaves extract of Bauhinia purpurea in experimental

animals demonstrated anti-inflammatory activity on carrageenan-induced

inflammation. Mradu et al. (2013), demonstrated the anti-inflammatory effect of

methanolic stem bark extract of Tinospora cordifolia Wild, fruits of Emblica

officinalis and rhizomes of Cyperus rotundus Linn in rodents.

The NSAIDs such as diclofenac, ibuprofen, indomethacin, naproxen and

acetaminophen are commonly used in the treatment of inflammation (Fiorucci et

al., 2000). The NSAIDs block the enzyme cyclooxygenase (COX) which stimulate

biosynthesis PGE2. There are two types COX enzymes; COX-1, and COX-2. The

COX-2 produces prostaglandins that promote inflammation, while the COX-1

produces prostaglandins that support platelets and protect the stomach (Mitchell et

al., 1993). The NSAIDs less inhibit the initial phase of carrageenan-induced paw

edema, and this is attributed to the release of histamine, serotonin, and bradykinin.

However, the second phase is attributed to the induction of inducible COX-2 and

can be blocked using NSAIDs (Nantel et al., 1999). It is therefore believed that the

anti-inflammatory activities of leaf extract of K. africana and stem bark extract of

A. hockii, inhibited the synthesis and release of prostaglandins to manage edema.

63

The methanolic leaf extract of K. africana and stem bark extract of A. hockii

demonstrated dose-dependent response on carrageenan-induced paw edema in mice

(Tables 4.3 and 4.4). The anti-inflammatory activity therefore increased with

increase in concentration of the extracts. These findings were in agreement with the

study carried out by Mwangi et al. (2015), on anti-inflammatory properties of

dichloromethane: methanolic extracts of Caesalpiina volkensii Harms and

Maytemus obscura in mice. Similarly, another study carried by Agbaje and

Fageyinbo (2012), on the evaluation of anti-inflammatory activity of Strophanthus

hispidus in experimental animals, showed a dose-dependent response in anti-

inflammatory activity. Pourmottabed et al. (2010), also reported a dose-dependent

anti-inflammatory activity of Terbium chamaedrys in mice.

The methanolic leaf extract of K. africana and stem bark extract of A. hockii

showed minimal anti-inflammatory activities at lower dose levels of 50 and 100

mg/kg body weight compared to 150 mg/kg body weight (Tables 4.3 and 4.4). The

reference drug (diclofenac) achieved its maximum anti-inflammatory activity in the

third hour (Tables 4.3 and 4.4); its activity decreased subsequently probably due to

metabolism and excretion of the drugs. On the other hand, the maximum anti-

inflammatory activity of methanolic leaf extracts of K. africana and stem bark

extract of A. hockii occurred at the dosage of 150mg/kg body weight in the fourth

hour (Tables 4.3 and 4.4), indicating slow but steady passive diffusion of the

64

bioactive constituent’s across the cell membrane in the peritoneal cavity (Hossain

et al., 2011).

The methanolic leaf extract of K. africana and stem bark extract of A.hockii at

different dose levels did not reduce paw diameter in the first and second hours

compared to the third and fourth hours (Tables 4.4 and 4.5). This could be probably

due to biotransformation of the bioactive compounds to become active. However,

the extract of K. africana and A.hockii at the dose of 150mg/kg body weight was

more effective in the fourth hour of treatment compared to diclofenac (reference

drug) in the same hour (Tables 4.3 and Table 4.4). These findings showed that the

leaf extract of K. africana and stem bark extract A. hockii were able to inhibit

synthesis of prostaglandins more than the conventional drug diclofenac (Tables 4.3

and Table 4.4).

The antiphlogistic activity of methanolic leaf extract of K. africana and stem bark

extract of A. hockii, could be due to the presence of bioactive constituents that

exhibit an anti-inflammatory action. These bioactive constituents may inhibit

inflammatory mediators such as prostaglandins, histamine, serotonin and lysosome

(Dina et al., 2010). The qualitative phytochemical screening of methanolic leaf

extract of K. africana indicated the presence of flavonoids, steroids, terpenoids,

cardiac glycosides and phenolics, while the stem bark extract of A. hockii indicated

similar phytochemicals in addition to alkaloids and saponin (Table 4.5). The

65

presence of bioactive compounds such as alkaloids, flavonoids, terpenoids, and

steroids have previously been reported to exhibit an antiphlogistic activity in

experimental animals (Bhagyasri et al., 2015; Bhaskar and Blakrishnan, 2009).

Flavonoids have been reported to inhibit pro-inflammatory mediators such as TNF-

α and phospholipase A2 (Bhagyasri et al., 2015). Furthermore, some flavonoids

respond by blocking both the cyclooxygenase and lipoxygenase pathways of the

arachidonate cascade at relatively high concentration, while at the lower level only

the lipoxygenase pathway is blocked (Di Carlo et al., 1999). Research findings have

revealed that triterpenoids suppresses some function of macrophages, neutrophils

and also inhibit nitric oxide (NO), NF-κB signaling and PGE2 production

responsible for induction of inflammation (Salminen et al., 2008). The NF-kB can

detect noxious stimuli, such as infectious agents, cellular injuries and free radicals,

and then directs DNA to synthesize inflammatory cytokines. Thus, their inhibition

leads to management of edema (Frantz et al., 1994). Steroids also attenuate

inflammation by inhibiting phospholipase A2 which hydrolyzes arachidonic acid

from membrane phospholipids and subsequent formation of prostanoids and

leukotrienes via the cyclooxygenase and lipoxygenase pathways (Mencarelli,

2009).

66

5.2 Conclusions

The methanolic stem bark extracts of K. africana and A. hockii demonstrated

significant antipyretic effects on turpentine-induced pyrexia in rats. The antipyretic

activity of the two extracts demonstrated a dose-dependent response after the

second hour of treatment and were comparable to aspirin (reference drug). Besides,

the extracts of K. africana and A. hockii at the dose level of 150mg/kg body weight

were more effective in the fourth hour after treatment, implying possible

biotransformation of bioactive compounds.

On the other hand, the methanolic leaf extract of K. africana and stem bark extract

of A. hockii showed an anti-inflammatory effect on carrageenan-induced paw

edema in mice. The anti-inflammatory activity of leaf extract of K. africana and

stem bark extract of A. hockii demonstrated a dose-dependent response and were

comparable to diclofenac (reference drug). In addition, the extracts were best active

at the dose level of 150mg/kg body weight in the fourth hour of treatment,

suggesting absorption, distribution, metabolism and excretion of bioactive

compounds.

The extracts of K. africana and A. hockii could, therefore, be an alternative bio-

resource for generating antipyretic and anti-inflammatory agents. However, further

studies are necessary to elucidate the mechanism behind these effects. These

findings may serve as a footstep towards generating herbal formulation with

67

antipyretic and anti-inflammatory properties. The present study, therefore,

scientifically confirms and supports the traditional use of K. africana and A. hockii

in the management of pyrexia and inflammation and hence the null hypothesis

rejected.

5.3 Recommendations

1) The extracts of K. africana and A. hockii may be used as an alternative bio-

resource in development of antipyretic and anti-inflammatory agents.

2) The extracts were best active at the dose of 150mg/kg body weight

5.4 Suggestions for Further Studies

1) Identify and isolate the actual antipyretic and anti-inflammatory bioactive

compounds of the extracts.

2) Determine the possible mechanism of antipyretic and anti-inflammatory

action of the extracts.

3) Evaluation of antipyretic and anti-inflammatory activity when the extracts

are administered orally.

4) Evaluation of safety of the extracts and clinical studies.

68

REFERENCES

Adesokan, A. A., Yakubu, M. T., Owoyele, B. V., Akanji, M. A., Soladoye, A.

O., and Lawal, O. K. (2008). Effect of administration of aqueous and ethanolic

extracts of Enantia chlorantha stem bark on brewer’s yeast-induced pyresis in rats.

African Journal Biochemistry Research, 2(7): 165-169.

Agbaje, E. O., and Fageyinbo, M. S. (2012). Evaluating Anti-Inflammatory

activity of aqueous root extract of Strophanthus hispidus DC. (Apocynaceae).

International Journal of Applied Research in Natural Products, 4(4): 7-14.

Akah, P. A. (1996). Antidiarrheal activity of Kigelia africana in experimental

animals. Journal of Herbs, Spices and Medicinal Plants, 4(2): 31-38.

Akatheeswaran, V. D. (2013). Antipyretic activity of ethanol and aqueous extract

of root of Asparagus racemosus in yeast induced pyrexia. Asian Journal of

Pharmaceutical and Clinical Research, 6(3), 190-193.

Anderson, D. W., Bridgeman, M. E., and De Pinto, G. (1984). Acacia gum

exudates from species of the series Gummiferae. Phytochemistry, 23(3): 575-577.

Anochie, I. P. (2013). Mechanisms of fever in humans. International Journal of

Microbiology and Immunology Research, 2(5): 37-43.

Aronoff, D. M., and Neilson, E. G. (2001). Antipyretics: mechanisms of action

and clinical use in fever suppression. The American Journal of Medicine, 111(4):

304-315.

Axelrod, Y. K., Diringer, M. N. (2008). Temperature management in acute

neurologic disorders. Neurologic Clinics, 26(2): 585-603.

Bamgbose, S. O. A. and Noamesi, B. K. (1981). Studies on cryptolepine inhibition

of carrageenan-induced edema. Journal of Planta Medica, 41(4): 392-396.

Barnes, P. J., Adcock, I., Spedding, M., and Vanhoutte, P. M. (1993). Anti-

inflammatory actions of steroids: molecular mechanisms. Trends in

pharmacological Sciences, 14(12): 436-441.

Barnes, P. J., and Karin, M. (1997). Nuclear factor-κB a pivotal transcription

factor in chronic inflammatory diseases. New England Journal of Medicine,

336(15): 1066-1071.

69

Baumann, J., Bruchhausen, F. V., and Wurm, G. (1980). Flavonoids and related

compounds as inhibitors of arachidonic acid peroxidation. Prostaglandins, 20(4):

627-639.

Bhagyasri, Y., Lavakumar, V., DivyaSree, M. S., and Ashok Kumar, C. K.

(2015). An overview on anti-inflammatory activity of Indian herbal plants.

International Journal of Research and Pharmaceutical Nano Science, 4(1): 1-9.

Bhaskar, V. H., and Balakrishnan, N. (2009). Analgesic, anti-inflammatory and

antipyretic activities of Pergularia daemia and Carissa carandas. Journal of

Pharmaceutical Sciences, 17(3): 168-174.

Bhattacharya, A., Behera, R., Agrawal, D., Sahu, P. K., Kumar, S., and

Mishra, S. S. (2014). Antipyretic effect of ethanolic extract of Moringa oleifera

leaves on albino rats. Tanta Medical Journal, 42(2): 74-78.

Biren, N. S. and Avinash, K. S. (2010). Medicinal plants as a source of anti-pyretic

agents. India Archives of Applied Science Research, 2 (3): 188-195.

Bosca, A. A., Soler, M. A., Carrion, J. N., Alperi, A., Bernd, C. and Quintanilla

(2000). A hydroalcoholic extract of Curcuma longa lowers the abnormally high

values of human plasma fibrinogen. Mechanisms of Ageing and Development,

114(14): 207-210.

Bremner, P., and Heinrich, M. (2002). Natural products as targeted modulators

of the nuclear factor‐KB pathway. Journal of Pharmacy and Pharmacology, 54(4):

453-472.

Brooks, P. (1998). Use and benefits of nonsteroidal anti-inflammatory drugs. The

American Journal of Medicine, 104(3): 9-13.

Bruce-Chwatt, L. J. (1998). Cinchona and its alkaloids: 350 years. New York State

Journal of Medicine, 88(6): 328-332.

Burkill, H. M. (1985). The useful plants of West Africa. Royal Botanical Gardens,

Kew 2nd Edition, Families AD, 1, 479.

Calder, P. C. (2009). Polyunsaturated fatty acids and inflammatory processes: new

twists in an old tale. Biochimie, 91(6), 791-795.

70

Calixto, J.B., Campos, M.M., Otuki, M.F., and Santos, A.R.S. (2004). Anti-

inflammatory compounds from plant origin. Part II. Modulation of pro-

inflammatory cytokines, chemkines and adhesion molecules. Planta Medica, 70(2):

93-103.

Cannon, J. G., Tompkins, R. G., Gelfand, J. A., Michie, H. R., Stanford, G. G.,

Van Der Meer, J. W., and Wilmore, D. W. (1990). Circulating interleukin-1 and

tumor necrosis factor in septic shock and experimental endotoxin fever. Journal of

Infectious Diseases, 161(1): 79-84.

Chainani-Wu, N. (2003). Safety and anti-inflammatory activity of curcumin: a

component of tumeric (Curcuma longa). The Journal of Alternative and

Complementary Medicine, 9(1): 161-168.

Chandrasekharan, N. V., Dai, H., Roos, K. L. T., Evanson, N. K., Tomsik, J.,

Elton, T. S., and Simmons, D. L. (2002). COX-3, a cyclooxygenase-1 variant

inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning,

structure, and expression. Proceedings of the National Academy of Sciences,

99(21): 926-931.

Cheng, L., Ming-liang, H. and Lars, B. (2005). Is CO-2 a perpetrator or a

protector? Selective COX-2 inhibitors remain controversial. Acta Pharmacologica

Sinica, 26 (8): 926-933.

Conti, B., Tabarean, I., Andrei, C., and Bartfai, T. (2004). Cytokines and fever.

Front Bioscience, 9(12): 1433-1449.

Cryer, B. and Kimmey, M. B. (1998). Gastrointestinal side effects of non-

steroidal anti-inflammatory drugs. American Journal of Medicine, 105(1): 20-30.

Cuman, R. K. N., Bersani-Amado, C. A., and Fortes, Z. B. (2001). Influence of

type 2 diabetes on the inflammatory response in rats. Inflammation Research, 50(9):

460-465.

Cuzzocrea, S., Sautebin, L., De Sarro, G., Costantino, G., Rombolà, L.,

Mazzon, E., and Caputi, A. P. (1999). Role of IL-6 in the pleurisy and lung injury

caused by carrageenan. The Journal of Immunology, 163(9): 5094-5104.

71

Di Carlo, G., Mascolo, N., Izzo, A. A., and Capasso, F. (1999). Flavonoids: old

and new aspects of a class of natural therapeutic drugs. Life sciences, 65(4): 337-

353.

Dina, T. A., Rahman, M. A., Ahmed, N. U., and Uddin, M. N. (2010). Analgesic

and anti-inflammatory properties of Argyreia argentea methanol extract in animal

model. Journal of Taibah University for Science, 3(1): 1-7.

Dinarello, C. A. (1999). Cytokines as endogenous pyrogens. Journal of Infectious

Diseases, 179(2): 294-304.

Dinarello, C. A., and Wolff, S. M. (1978). Pathogenesis of fever in man. New

England Journal of Medicine, 298(11): 607-612.

Dubey, N. K., Kumar, R. and Tripathi, P. (2004). Global promotion of herbal

medicine: India’s opportunity. Current Science Journal, 86(1): 37-41.

Edeoga, H. O., Okwu, D. E., and Mbaebie, B. O. (2005). Phytochemical

constituents of some Nigerian medicinal plants. African Journal of Biotechnology,

4(7): 685-688.

Estakhr, J., Sanchooli, N., Najafi, S. H., and Javdan, N. (2011). Anti-

Inflammatory Activity of Ethanolic Extract of Physalis alkekengi. Research

Journal of Pharmaceutical, Biological and Chemical Sciences, 2(3): 421-425.

Fiorucci, S., Santucci, L., Cirino, G., Mencarelli, A., Familiari, L., Del Soldato,

P., and Morelli, A. (2000). IL-1β converting enzyme is a target for nitric oxide-

releasing aspirin: new insights in the antiinflammatory mechanism of nitric oxide-

releasing nonsteroidal antiinflammatory drugs. The Journal of Immunology,

165(9): 5245-5254.

Frantz, B., Nordby, E. C., Bren, G., Steffan, N., Paya, C. V., Kincaid, R. L.,

and O'neill, E. A. (1994). Calcineurin acts in synergy with PMA to inactivate I

kappa B/MAD3, an inhibitor of NF-kappa B. The European Molecular Biology

Organization Journal, 13(4): 861-869.

Freudenberg, M. A., and Galanos, C. (1990). Bacterial lipopolysaccharides:

structure, metabolism and mechanisms of action. International Reviews of

Immunology, 6(4): 207-221.

GBIF, (2012). Global Biodiversity Information Facility. Global Biodiversity

Information Facility (GBIF). http://data.gbif.org.

72

Gege-Adebayo, G. I., Bassi, A. S., Igbokwe, V. U., and Shafe, M. O. (2013). Antipyretic effect of Ocimum gratissium on brewer’s yeast induced fever in wistar

rats. Journal of Medicine Medical Science, 4(6): 247-251.

Gitahi, S. M. (2015). Antipyretic properties of dichloromethane: methanolic leaf

and root bark extracts of Carissa edulis in Rats. Asian Journal of Biomedical and

Pharmaceutical Sciences, 5(43): 12-20.

Grace, O. M., Light, M. E., Lindsey, K. L., Moholland. D. A., Staden, J. V.,

and Jager, A. K. (2002). Antibacterial activity and isolation of antibacterial

compouds from fruit of the traditional African medicinal plant, Kigelia africana.

South Africa Journal of Botany, 68(2): 220-222.

Grover, J. K. (1990). Experiments in Pharmacy and Pharmacology. CBS Publisher

and Distributor. Shahdara Delhi, India, 1(2): 155.

Gupta, M., Mazumder, U. K., Gomathi, P., and Selvan, V. T. (2006). Anti-

inflammatory evaluation of leaves of Plumeria acuminata. Biomedical central,

Complementary and Alternative Medicine, 6(1): 36-39.

Gwynne, M. D. (1969). The nutritive values of Acacia pods in relation to Acacia

seed distribution by ungulates. East African Wildlife Journal, 7: 176-178.

Handy, R. L. C., and Moore, P. K. (1998). A comparison of the effects of L‐NAME, 7‐NI and L‐NIL on carrageenan‐induced hind paw edema and NOS

activity. British Journal of Pharmacology, 123(6): 1119-1126.

Hassan, M. M., Khan, S. A., Shaikat, A. H., Hossain, M. E., Hoque, M. A.,

Ullah, M. H and Islam, S., (2013). Analgesic and anti-inflammatory effects of

ethanol extracted leaves of selected medicinal plants in animal model. Veterinary

World International Journal, 6(2): 68-71.

Harbone, J. B. (1998). Phytochemical methods: A guide to modern techniques of

plant analysis. Chapman and Hal Publishers, London, United Kingdom, 3: 60-66.

Hina, J., Shazia, E., Sobia, T. ad Farhana, A. (2013). An overview on medicinal

importance of Thymus vulgaris. Journal of Asian Scientific Research, 3(10): 974-

982.

73

Hong, S. L., and Levine, L. (1976). Inhibition of arachidonic acid release from

cells as the biochemical action of anti-inflammatory corticosteroids. Proceedings

of the National Academy of Sciences, 73(5): 1730-1734.

Hossain, E., Mandal, S. C., and Gupta, J. K. (2011). Phytochemical screening

and in-vivo antipyretic activity of the methanol leaf-extract of Bombax

malabaricum DC (Bombacaceae). Tropical Journal of Pharmaceutical Research,

10(1): 55-60.

Hukkeri, V. I., Nagathan, C. V. and Karadi, R. V. (2006). Antipyretic and

wound healing activities of Moringa oeifera in rats. Indian Journal of

Pharmaceutical Sciences, 68 (1): 124-126.

ILDIS, (2013). International Legume Database and Information Service. Reading,

United Kingdom: School of Plant Sciences, University of Reading.

http://www.ildis.org/.

Ismail, T. S., Gopalakrishnan, S., Begum, V. H., & Elango, V. (1997). Anti-

inflammatory activity of Salacia oblonga Wall. and Azima tetracantha Lam.

Journal of Ethnopharmacology, 56(2): 145-152.

Iwalewa, E. O., Iwalewa, O. J., and Adeboye, J. O. (2003). Analgesic,

antipyretic, anti-inflammatory effects of methanol, chloroform and ether extracts

of Vernonia cinerea less leaf. Journal of ethnopharmacology, 86(2): 229-234.

Kamboj, V. P. (2000). Herbal medicine. Current Science Bangalore, 78(1): 35-38.

Kaplanski, G., Marin, V., Montero-Julian, F., Mantovani, A., and Farnarier,

C. (2003). IL-6: a regulator of the transition from neutrophil to monocyte

recruitment during inflammation. Trends in Immunology, 24(1): 25-29.

Karakitsos, D. and Karabinis, A. (2008). Hypothermia therapy after traumatic

brain injury in children. New England Journal of Medicine, 359(11): 1179-80.

Kareru, P. G., Kenji, G. M., Gachanja, A. N., Keriko, J. M., and Mungai, G.

(2007). Traditional medicines among the Embu and Mbeere people of Kenya.

African Journal of Traditional, Complementary and Alternative Medicines, 4(1):

75-86.

74

Kela, S. L., Ogunsusi, R. A., Ogbogu, V. C., and Nwude, N. (1988). Screening

of some Nigerian plants for molluscicidal activity. Revue D'élevage et de Médecine

Vétérinaire des Pays Tropicaux, 42(2): 195-202.

Klir, J. J., McClellan, J. L., and Kluger, M. J. (1994). Interleukin-1 beta causes

the increase in anterior hypothalamic interleukin-6 during LPS-induced fever in

rats. American Journal of Physiology-Regulatory, Integrative and Comparative

Physiology, 266(6): 1845-1848.

Kolawole, O. T., Akiibinu, M. O., Ayankunle, A. A., and Awe, E. O. (2013). Evaluation of anti-inflammatory and antinociceptive potentials of Khaya

senegalensis A. Juss (Meliaceae) stem bark aqueous extract. British Journal of

Medicine and Medical Research, 3(2), 216-229.

Kotake, C. K. (2000). Practical Pharmacognosy. Vallabh Prakashan, New Delhi,

India, 4 (2): 107-111.

Kuehl, F. A., and Egan, R. W. (1980). Prostaglandins, arachidonic acid, and

inflammation. Science, 210(44): 978-984.

Kumar, A., Agarwal, K., Maurya, A. K., Shanker, K., Bushra, U., Tandon, S.,

and Bawankule, D. U. (2015). Pharmacological and phytochemical evaluation of

Ocimum sanctum root extracts for its anti-inflammatory, analgesic and antipyretic

activities. Pharmacognosy Magazine, 11(42): 217-224.

Lai, S. C., Peng, W. H., Huang, S. C., Ho, Y. L., Huang, T. H., Lai, Z. R., and

Chang, Y. S. (2009). Analgesic and anti-inflammatory activities of methanol

extract from Desmodium triflorum DC in mice. The American Journal of Chinese

Medicine, 37(3): 573-588.

Luheshi, G. N. (1998). Cytokines and fever: mechanisms and sites of action.

Annals of the New York Academy of Sciences, 856(1): 83-89.

Luheshi, G. N., Stefferl, A., Turnbull, A. V., Dascombe, M. J., Brouwer, S.,

Hopkins, S. J., and Rothwell, N. J. (1997). Febrile response to tissue

inflammation involves both peripheral and brain IL-1 and TNF-alpha in the rat.

American Journal of Physiology-Regulatory, Integrative and Comparative

Physiology, 272(3): 862-868.

Luo, C., and Bohlin, L. (2005). Is COX‐2 a perpetrator or a protector? Selective

COX‐2 inhibitors remain controversial. Acta Pharmacologica Sinica, 26(8): 926-

933.

75

Mackowiak, P. A. (1998). Concepts of fever. Archives of Internal Medicine,

158(17): 1870-1881.

Markenson, J. (1999). Clinical implications of cyclooxygenase enzymes: COX-

1/COX-2 role of the new non-steroidal anti-inflammatory drugs. Cancer Control,

6(3): 22-25.

Maroon, J. C., Bost, J. W., and Maroon, A. (2010). Natural anti-inflammatory

agents for pain relief. Surgical Neurology International, 1(4): 25-32.

Mencarelli, A., Renga, B., Palladino, G., Distrutti, E., and Fiorucci, S. (2009). The plant sterol attenuates inflammation and immune dysfunction in murine models

of inflammatory bowel disease. Biochemical Pharmacology, 78(9): 1214-1223.

Mitchell, J. A., Akarasereenont, P., Thiemermann, C., Flower, R. J., and Vane,

J. R. (1993). Selectivity of nonsteroidal anti-inflammatory drugs as inhibitors of

constitutive and inducible cyclooxygenase. Proceedings of the National Academy

of Sciences, 90(24): 11693-11697.

Mradu, G., Dailya, B., and Arup, M. (2013). Studies Of Anti Inflammatory,

Antipyretic And Analgesic Effects Of Aqueous Extract Of Traditional Herbal Drug

Of Rodents. International Research Journal of Pharmacy, 4(3): 113-120.

Msonthi, J. D., and Magombo, D. (1983). Medicinal herbs in Malawi and their

uses. Hamdard, 26(2): 94-100.

Mujumdar, A. M., and Misar, A. V. (2004). Anti-inflammatory activity of

Jatropha curcas roots in mice and rats. Journal of Ethnopharmacology, 90(1): 11-

15.

Musinguzi, W. B., Okure, M. A., Wang, L., Sebbit, A., and Løvås, T. (2012). Thermal characterization of Uganda's Acacia hockii, Combretum molle,

Eucalyptus grandis and Terminalia glaucescens for gasification. Biomass and

Bioenergy, 46: 402-408.

Mwangi, B. M., Gitahi, S. M., Njagi, J. M., Mworia, J. K., Aliyu, U., Njoroge,

W. A., and Mburu, N. D. (2015). Anti-inflammatory Properties of

Dichloromethane: Methanolic Leaf Extracts of Caesalpinia Volkensii and

Maytenus Obscura in Animal Models. International Journal of Current

Pharmaceutical Research, 7(3): 83-87.

76

Mwonjoria, J.K., Kariuki, H.N., and Waweru, F.N. (2011). The antinociceptive

antipyretic effects of Solanum incanum (Linneaus) in animal models. International

Journal, 2(1): 24-26.

Nagaraj, D. S., and Venkateswarlu, B. (2013). Antipyretic effect of aqueous

extract of Fagonia cretica L. whole plant on brewer's yeast induced pyrexia in

Wistar rats. International Journal of Pharmacology Toxicology, 3(1): 39-42.

Nantel, F., Denis, D., Gordon, R., Northey, A., Cirino, M., Metters, K. M., and

Chan, C. C. (1999). Distribution and regulation of cyclooxygenase‐2 in

carrageenan‐induced inflammation. British Journal of Pharmacology, 128(4): 853-

859.

Necas, J., and Bartosikova, L. (2013). Carrageenan: a review. Veterinarni

Medicina, 58(4): 187-205.

Netea, M. G., Kullberg, B. J., and Van der Meer, J. W. (2000). Circulating

cytokines as mediators of fever. Clinical Infectious Diseases, 31(5): 178-184.

Niazi, J., Gupta, V., Chakarborty, P., and Kumar, P. (2010). Antiinflammatory

and antipyretic activity of Aleuritis moluccana leaves. Asian Journal of

Pharmaceutical Clinical Research, 3(1): 35-37.

Nordqvist, C. (2015a). "Fever: Causes, Symptoms and Treatments." Medical News

Today. Retrieved from http://www.medicalnewstoday.com/articles/168266.php.

Nordqvist, C. (2015b). "Inflammation: Causes, Symptoms and Treatment."

Medical News Today. Retrieved from

http://www.medicalnewstoday.com/articles/248423.php.

Ogoina, D. (2011). Fever, fever patterns and diseases called ‘fever’ A review.

Journal of Infection and Public Health, 4(3): 108-124.

Okoli, C. O., Akah, P. A., and Ezugworie, U. (2006). Anti-inflammatory activity

of extracts of root bark of Securidaca longipedunculata Fres (Polygalaceae).

Biomedical central, Complementary and Alternative Medicines, 2(3): 54-64.

Omote, K., Hazama, K., Kawamata, T., Kawamata, M., Nakayaka, Y.,

Toriyabe, M., and Namiki, A. (2001). Peripheral nitric oxide in carrageenan-

induced inflammation. Brain Research, 912(2): 171-175.

77

Orwa C., A. Mutua, Kindt R., Jamnadass R., and S. Anthony (2009).

Agroforestree Database:a tree reference and selection guide version 4.0.

http://www.worldagroforestry.org/sites/treedbs/treedatabase.asp.

Oshima, M., Dinchuk, J. E., Kargman, S. L., Oshima, H., Hancock, B., Kwong,

E., Trzaskos, J. M., Evans, J. F., and Taketo, M. M. (1996). Suppression of

intestinal polyposis in APC 716 knockout mice by inhibition of prostaglandin

endoperoxide synthase-2 (COX-2). Journal of Cell, 87(5): 803-809.

Paschapur, M. S., Patil, M. B., Kumar, R., and Patil, S. R. (2009). Evaluation

of anti-inflammatory activity of ethanolic extract of Borassus flabellifer L. male

flowers (inflorescences) in experimental animals. Journal of Medicinal Plants

Research, 3(2): 049-054.

Phipps, R. P., Stein, S. H., and Roper, R. L. (1991). A new view of prostaglandin

E regulation of the immune response. Immunology Today, 12(10): 349-352.

Plaisance, K. I., and Mackowiak, P. A. (2000). Antipyretic therapy: physiologic

rationale, diagnostic implications, and clinical consequences. Archives of Internal

Medicine, 160(4): 449-456.

Posadas, I., Bucci, M., Roviezzo, F., Rossi, A., Parente, L., Sautebin, L., and

Cirino, G. (2004). Carrageenan‐induced mouse paw edema is biphasic, age‐weight

dependent and displays differential nitric oxide, cyclooxygenase‐2 expression.

British Journal of Pharmacology, 142(2): 331-338.

Pourmotabbed, A., Farshchi, A., Ghiasi, G., and Malek Khatabi, P. (2010). Analgesic and anti-inflammatory activity of Teucrium chamaedrys leaves aqueous

extract in male rats. Iranian Journal of Basic Medical Sciences, 13(3): 119-125.

Raghavendra, V., Tanga, F. Y., and DeLeo, J. A. (2004). Complete Freunds

adjuvant‐induced peripheral inflammation evokes glial activation and

proinflammatory cytokine expression in the CNS. European Journal of

Neuroscience, 20(2): 467-473.

Rahwan, G. L., and Rahwan, R. G. (1986). Aspirin and Reye's syndrome: the

change in prescribing habits of health professionals. Drug Intelligence and Clinical

Pharmacy, 20(2): 143-145.

78

Ravi, V., Saleem, T. M., Patel, S. S., Raamamurthy, J., and Gauthaman, K.

(2009). Anti-inflammatory effect of methanolic extract of Solanum nigrum Linn

berries. International Journal of Applied Research in Natural Products, 2(2): 33-

36.

Recio, C. M., Andujar, I., and L Rios, J. (2012). Anti-inflammatory agents from

plants: progress and potential. Current Medicinal Chemistry, 19(14): 2088-2103.

Reynolds, J. F., Noakes, T. D., Schwellnus, M. P., Windt, A., and Bowerbank,

P. (1995). Non-steroidal anti-inflammatory drugs fail to enhance healing of acute

hamstring injuries treated with physiotherapy. Medical Journal-Cape Town-

Medical Association of South Africa, 85 (6): 517-517.

Rotelli, A. E., Guardia, T., Juárez, A. O., De la Rocha, N. E., and Pelzer, L. E.

(2003). Comparative study of flavonoids in experimental models of inflammation.

Pharmacological Research, 48(6): 601-606.

Salminen, A., Lehtonen, M., Suuronen, T., Kaarniranta, K., and Huuskonen,

J. (2008). Terpenoids: natural inhibitors of NF-κB signaling with anti-

inflammatory and anticancer potential. Cellular and Molecular Life Sciences,

65(19): 2979-2999.

Sandoval, M., Okuhama, N. N., Zhang, X. J., Condezo, L. A., Lao, J., Angeles,

F. M., and Miller, M. J. S. (2002). Anti-inflammatory and antioxidant activities

of cat's claw (Uncaria tomentosa and Uncaria guianensis) are independent of their

alkaloid content. Phytomedicine, 9(4): 325-337.

Saper, C. B., and Breder, C. D. (1994). The neurologic basis of fever. New

England Journal of Medicine, 330(26): 1880-1886.

Shah, B. N., Patel, N. P., and Pandya, P. (2008). Role of leukotriene in

inflammation and antileukotriene therapy. Journal of Pharmacy Research, 1(2):

113-123.

Shukla, S., Mehta, A., Mehta, P., Vyas, S. P., Shukla, S., and Bajpai, V. K.

(2010). Studies on anti-inflammatory, antipyretic and analgesic properties of

Caesalpinia bonducella F. seed oil in experimental animal models. Food and

Chemical Toxicology, 48(1): 61-64.

Sini, J. M., Yaro, A. H., Ayanwuyi, L. O., Aiyelero, O. M., Mallum, S. M., and

Gamaniel, K. S. (2010). Antinociceptive and anti-inflammatory activities of the

79

aqueous extract of the root bark of Combretum sericeum in rodents. African Journal

of Biotechnology, 9(51): 8872-8876.

Soszynski, D., and Krajewska, M. (2002). Lack of cross tolerance between

pyrogenic effects of LPS and turpentine in rats. Journal of Thermal Biology, 27(3):

229-237.

Soszynski, D., Kozak, W., and Szewczenko, M. (1991). Course of fever response

to repeated administration of sublethal doses of lipopolysaccharides, polyinosinic:

polycytidylic acid and muramyl dipeptide to rabbits. Experientia, 47(1): 43-47.

Soumerai, S. B., Ross-Degnan, D., and Kahn, J. S. (1992). Effects of professional

and media warnings about the association between aspirin use in children and

Reye's syndrome. The Milbank Quarterly, 70(1): 155-182.

Srivastava, S., Singh, P., Jha, K. K., Mishra, G., Srivastava, S., and Khosa, R.

L. (2013). Antiinflammatory, analgesic and antipyretic activities of aerial parts of

Costus speciosus Koen. Indian Journal of Pharmaceutical Sciences, 75(1): 83-88.

Stefferl, A., Hopkins, S. J., Rothwell, N. J., and Luheshi, G. N. (1996). The role

of TNF‐α in fever: opposing actions of human and murine TNF‐α and interactions

with IL‐β in the rat. British Journal of Pharmacology, 118(8): 1919-1924.

Sue E. Huether (2014). Pathophysiology: The Biologic Basis for Disease in Adults

and Children (7 ed.). Elsevier Health Sciences. p. 498. ISBN 9780323293754.

Tabuti, J. R. S., Kukunda, C. B. and Waako, P. J. (2010). Medicinal plants used

by traditional medicine practitioner in the treatment of tuberculosis and related

ailments in Uganda. Journal of Ethnopharmacology, 127 (1): 130-136.

Tilley, S. L., Coffman, T. M., and Koller, B. H. (2001). Mixed messages:

modulation of inflammation and immune responses by prostaglandins and

thromboxanes. Journal of Clinical Investigation, 108(1): 15-24.

Traversa, G., Walker, A. M., Ippolito, F. M., Caffari, B., Capurso, L., Dezi, A.,

and Raschetti, R. (1995). Gastroduodenal toxicity of different nonsteroidal anti-

inflammatory drugs. Epidemiology, 6(1): 49-54.

80

Tung, K., Fujita, H., Yamashita, Y., and Takagi, Y. (2006). Effect of turpentine-

induced fever during the enamel formation of rat incisor. Archives of Oral Biology,

51(6): 464-470.

Umamageswari, A., and Kudagi, B. L. (2015). Anti-inflammatory and analgesic

properties of Ocimum sanctum: a comparative study using animal models.

International Journal of Basic and Clinical Pharmacology, 4(5): 981-986.

Umashanker, M., and Shruti, S. (2011). Traditional Indian herbal medicine used

as antipyretic, antiulcer, anti-diabetic and anticancer: A review. International

Journal of Research in Pharmacy and Chemistry, 1(4): 1152-1159.

Unnisa, A., and Parven, T. D. (2011). Anti-inflammatory and acute toxicity

studies of the extracts from the rhizomes of Alpinia galanga Willd. Der Pharmacia

Sinica, 2(2): 361-367.

Van Wauwe, J. P., and Goossens, J. G. (1989). Arabinogalactan-and dextran-

induced ear inflammation in mice: differential inhibition by H1-antihistamines, 5-

HT-serotonin antagonists and lipoxygenase blockers. Agents and Actions, 28(2):

78-82.

Vane, J. O. H. N., and Botting, R. (1987). Inflammation and the mechanism of

action of anti-inflammatory drugs. The Federation of American Societies for

Experimental Biology Journal, 1(2): 89-96.

Vane, J. R. (2000). The fight against rheumatism: from willow bark to COX-1

sparing drugs. Journal of Physiology and Pharmacology, 51(4): 576-583.

Vane, J. R., and Botting, R. M. (1998). Mechanism of action of nonsteroidal anti-

inflammatory drugs. The American Journal of Medicine, 104(3): 2-8.

Vodovotz, Y., Csete, M., Bartels, J., Chang, S., and Ann, G. (2008).

Translational systems biology of inflammation. The Plant Library of Science

Computational Biology, 4(4): 1-6.

Vogel, H. G. (2002). Drug discovery and evaluation pharmacological assays.

Springer-Verlag Berlin Heidelberg New York, 1408: 2-716.

Walter F. and Boron (2003). Medical Physiology: A Cellular and Molecular

Approaoch. Elsevier/Saunders. p. 1300. ISBN 1-4160-2328-3.

81

Warden, S. J. (2010). Prophylactic use of NSAIDs by athletes: a risk/benefit

assessment. Physician and Sports Medicine, 38(1): 132-138.

Weenen, H., Nkunya, M. H., Bray, D. H., Mwasumbi, L. B., Kinabo, L. S., and

Kilimali, V. A. (1990). Antimalarial activity of Tanzanian medicinal plants. Planta

Medica, 56(4): 368-370.

Weissmann G. (1991). Aspirin. Science America, 264 (1): 84-90.

Williams, J. G., and Maier, R. V. (1992). The inflammatory response. Journal of

Intensive Care Medicine, 7(2): 53-66.

Wink, M. (2015). Modes of action of herbal medicines and plant secondary

metabolites. Medicines, 2(3), 251-286.

Winter, C. A., Risley, E. A., and Nuss, G. W. (1962). Carrageenin-induced edema

in hind paw of the rat as an assay for antiinflammatory drugs. Experimental Biology

and Medicine, 111(3): 544-547.

Wolfe, M. M., Lichtenstein, D. R., and Singh, G. (1999). Gastrointestinal toxicity

of nonsteroidal anti-inflammatory drugs. New England Journal of Medicine,

340(24): 1888-1899.

World Health Organization. (1978).The promotion and development of

traditional medicine: report of a WHO meeting [held in Geneva from 28 November

to 2 December 1977].

WHO, (2008). The World health report 2008: primary health care now more than

ever.

Zakaria, Z. A., Wen, L. Y., Abdul Rahman, N. I., Abdul Ayub, A. H.,

Sulaiman, M. R., and Gopalan, H. K. (2007). Antinociceptive, anti-inflammatory

and antipyretic properties of the aqueous extract of Bauhinia purpurea leaves in

experimental animals. Medical Principles and Practice, 16(6): 443-449.

Zhu, Z. Z., Ma, K. J., Ran, X., Zhang, H., Zheng, C. J., Han, T., and Qin, L. P.

(2011). Analgesic, anti-inflammatory and antipyretic activities of the petroleum

ether fraction from the ethanol extract of Desmodium podocarpum. Journal of

Ethnopharmacology, 133(3): 1126-1131.

82

APPENDICES

Appendix I: Effects of intraperitoneal administration of methanolic stem bark extract of Kigelia africana on turpentine-

induced pyrexia in male Wistar rats

Values expressed as Mean ± SEM for five animals per group. Values with the same superscript letter are not significantly different

by one way ANOVA followed by Tukey’s test (p>0.05). Steam distilled turpentine = 20%; 100mg/kg bw aspirin and 4% DMSO.

Group Treatment Change in rectal temperature (0C) after treatment

Base line 0hr 1hr 2hr 3hr 4hr

Normal control DMSO 37.38 37.38±0.11

37.42±0.04c 37.42±0.0c

37.50±0.08c

37.50±0.08bc

Negative control Turpentine + DMSO 37.12 38.16±0.08

38.72±0.13a

39.02±0.11a

39.28±0.09a

39.32±0.07a

Positive control Turpentine + Aspirin + DMSO 37.22 38.28±0.09

38.04±0.13b

37.80±0.13bc

37.56±0.09c

37.36±0.07bc

Methanolic

extracts

Turpentine + 50mg/kg bw +

DMSO

37.28 38.30±0.11

38.46±0.18ab

38.28±0.21b

38.08±0.21b

37.76±0.18b

Turpentine +100mg/kg bw +

DMSO

37.12 38.30±0.07

38.34±0.07ab

38.26±0.14b

37.42±0.07bc

37.46±0.08bc

Turpentine + 150mg/kg bw +

DMSO

37.24 38.42±0.05

38.30±0.10ab

37.96±0.06bc

37.64±0.04bc

37.24±0.04c

83

Appendix II: Effects of intraperitoneal administration of methanolic stem bark extract of Acacia hockii on

turpentine-induced pyrexia in male Wistar rats

Values expressed as Mean ± SEM for five animals per group. Values with the same superscript letter are not significantly

different by one way ANOVA followed by Tukey’s test (p>0.05). Steam distilled turpentine = 20%; 100mg/kg bw aspirin and

4% DMSO.

Group Treatment Change in rectal temperature (0C) after treatment

Base line 0hr 1hr 2hr 3hr 4hr

Normal control DMSO 37.15 37.18±0.03

37.14±0.07d

37.28±0.02d

37.30±0.03d

37.28±0.16c

Negative control Turpentine + DMSO 37.17 38.08±0.09

38.46±0.07bc

38.76±0.07ab

39.10±0.09a

39.16±0.02a

Positive control Turpentine + Aspirin +

DMSO

37.45 38.64±0.25

37.98±0.12c

37.68±0.19cd

37.42±0.02c

37.44±0.16c

Methanolic

extracts

Turpentine + 50mg/kg

bw + DMSO

37.32 38.44±0.14

38.66±0.17ab

38.52±0.18a

38.20±0.14ab

37.82±0.17c

Turpentine +100mg/kg

bw + DMSO

37.52 39.12±0.20

39.09±0.20a

38.90±0.20ab

38.44±0.17b

38.18±0.19b

Turpentine + 150mg/kg

bw + DMSO

37.23 38.56±0.18

38.60±0.09ab

38.28±0.12bc

37.68±0.09c

37.06±0.19c

84

Appendix III: Effects of intraperitoneal administration of methanolic leaf extract of Kigelia africana on

carrageenan-induced inflammation in Swiss albino mice

Values expressed as Mean ± SEM for five animals per group. Values with the same superscript letter are not significantly

different by one way ANOVA followed by Tukey’s test (p>0.05). Carrageenan =1%; 15mg/kg bw diclofenac and 4% DMSO.

Group Treatment Change in paw diameter (mm) after treatment

Base line 0hr 1hr 2hr 3hr 4hr

Normal control DMSO 2.50 2.50±0.06

2.50±0.06b

2.50±0.07c

2.50±0.06c

2.50±0.06c

Negative control Turpentine + DMSO 2.56 2.83±0.04

2.89±0.01a

2.93±0.06a

2.96±0.01a

2.97±0.02a

Positive control Turpentine + Aspirin +

DMSO

2.55 2.75±0.06

2.72±0.06a

2.67±0.07bc

2.64±0.07bc

2.63±0.06bc

Methanolic

extracts

Carrageenan + 50mg/kg bw +

DMSO

2.49 2.76±0.03

2.81±0.02a

2.77±0.01ab

2.74±0.02b

2.71±0.02b

Carrageenan + 100mg/kg bw

+ DMSO

2.52 2.75±0.04

2.78±0.04a

2.73±0.03b

2.68±0.04b

2.66±0.04bc

Carrageenan + 150mg/kg bw

+ DMSO

2.58 2.81±0.03

2.81±0.02a

2.77±0.03ab

2.72±0.02b

2.67±0.03bc

85

Appendix IV: Effects of intraperitoneal administration of methanolic stem bark extract of Acacia hockii on

carrageenan-induced inflammation in Swiss albino mice

Values expressed as Mean ± SEM for five animals per group. Values with the same superscript letter are not significantly

different by one way ANOVA followed by Tukey’s test (p>0.05). Carrageenan 1%; 15mg/kg bw diclofenac and 4% DMSO.

Group Treatment Change in paw diameter (mm) after treatment

Base line 0hr 1hr 2hr 3hr 4hr

Normal control DMSO 26.64 2.64±0.05

2.64±0.05c

2.64±0.05c

2.64±0.05c

2.64±0.04c

Negative control Turpentine + DMSO 2.65 3.09±0.01

3.13±0.01a

3.17±0.01a

3.19±0.01a

3.19±0.01a

Positive control Turpentine + Aspirin +

DMSO

2.64 2.98±0.04

2.93±0.04ab

2.88±0.05b

2.84±0.05b

2.83±0.04b

Methanolic

extracts

Carrageenan + 50mg/kg bw

+ DMSO

2.62 2.90±0.02

2.96±0.02ab

2.93±0.02b

2.89±0.02b

2.87±0.02b

Carrageenan + 100mg/kg

bw + DMSO

2.65 2.95±0.09

2.94±0.10ab

2.91±0.07b

2.86±0.07b

2.83±0.07b

Carrageenan + 150mg/kg

bw + DMSO

2.64 2.90±0.02

2.88±0.03bc

2.83±0.04bc

2.78±0.03bc

2.75±0.02bc

86

Appendix V: Figure representing antipyretic activity of methanolic stem

bark extract of Kigelia africana

Appendix VI: Figure representing antipyretic activity of methanolic stem

bark extract of Acacia hockii

87

Appendix VII: Figure representing anti-inflammatory activity of methanolic

stem bark extract Kigelia africana

Appendix VIII: Figure representing anti-inflammatory activity of methanolic

leaf extract of Acacia hockii

88

Appendix IX: Analysis of the antipyretic effects of methanolic stem bark

extract of Kigelia africana

Descriptive Statistics: 0hr, 1hr, 2hr, 3hr, 4hr

Variable C1 Mean SE Mean

0hr 100mg/kg 100.00 0.000000

150mg/kg 100.00 0.000000

50mg/kg 100.00 0.000000

Negative control 100.00 0.000000

Normal control 100.00 0.000000

Positive control 100.00 0.000000

1hr 100mg/kg 100.06 0.293

150mg/kg 99.688 0.224

50mg/kg 100.42 0.241

Negative control 101.47 0.195

Normal control 100.11 0.277

Positive control 99.372 0.257

2hr 100mg/kg 99.894 0.228

150mg/kg 98.800 0.154

50mg/kg 99.946 0.303

Negative control 102.25 0.132

Normal control 100.06 0.231

Positive control 98.746 0.225

3hr 100mg/kg 98.696 0.0823

150mg/kg 97.970 0.0962

50mg/kg 99.424 0.303

Negative control 102.94 0.156

Normal control 100.11 0.277

Positive control 98.120 0.128

4hr 100mg/kg 97.912 0.164

150mg/kg 96.928 0.0952

50mg/kg 98.588 0.242

Negative control 103.04 0.136

Normal control 100.22 0.179

Positive control 97.598 0.149

One-way ANOVA: 1hr versus C1

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

C1 5 13.143 2.6286 8.42 0.000

Error 24 7.492 0.3122

Total 29 20.635

Tukey Pairwise Comparisons

89

Grouping Information Using the Tukey Method and 95% Confidence

C1 N Mean Grouping

Negative control 5 101.468 A

50mg/kg 5 100.418 A B

Normal control 5 100.110 B

100mg/kg 5 100.064 B

150mg/kg 5 99.688 B

Positive control 5 99.372 B

Means that do not share a letter are significantly different.

One-way ANOVA: 2hr versus C1

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

C1 5 40.429 8.0857 33.59 0.000

Error 24 5.777 0.2407

Total 29 46.206

Pooled StDev = 0.490627

Tukey Pairwise Comparisons

Grouping Information Using the Tukey Method and 95% Confidence

C1 N Mean Grouping

Negative control 5 102.252 A

Normal control 5 100.056 B

50mg/kg 5 99.946 B

100mg/kg 5 99.894 B

150mg/kg 5 98.800 C

Positive control 5 98.746 C

Means that do not share a letter are significantly different.

One-way ANOVA: 3hr versus C1

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

C1 5 85.324 17.0648 91.13 0.000

Error 24 4.494 0.1872

Total 29 89.818

Pooled StDev = 0.432722

Tukey Pairwise Comparisons

Grouping Information Using the Tukey Method and 95% Confidence

90

C1 N Mean Grouping

Negative control 5 102.936 A

Normal control 5 100.110 B

50mg/kg 5 99.424 B C

100mg/kg 5 98.6960 C D

Positive control 5 98.120 D

150mg/kg 5 97.9700 D

Means that do not share a letter are significantly different.

One-way ANOVA: 4hr versus C1

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

C1 5 126.996 25.3993 181.88 0.000

Error 24 3.352 0.1397

Total 29 130.348

Tukey Pairwise Comparisons

Grouping Information Using the Tukey Method and 95% Confidence

C1 N Mean Grouping

Negative control 5 103.040 A

Normal control 5 100.216 B

50mg/kg 5 98.588 C

100mg/kg 5 97.912 C D

Positive control 5 97.598 D E

150mg/kg 5 96.9280 E

Means that do not share a letter are significantly different.

Appendix X: Analysis of the antipyretic effects of methanolic stem bark

extract of Acacia hockii

Descriptive Statistics: 0hr, 1hr, 2hr, 3hr, 4hr

Variable C1 Mean SE Mean

0hr 100mg/kg bw 100.00 0.000000

150mg/kg bw 100.00 0.000000

50mg/kg bw 100.00 0.000000

Negative control 100.00 0.000000

Normal control 100.00 0.000000

Positive control 100.00 0.000000

1hr 100mg/kg bw 100.05 0.312

150mg/kg bw 100.11 0.279

50mg/kg bw 100.58 0.403

Negative control 101.00 0.0984

91

Normal control 99.892 0.108

Positive control 98.304 0.517

2hr 100mg/kg bw 99.324 0.355

150mg/kg bw 99.277 0.250

50mg/kg bw 100.22 0.604

Negative control 101.83 0.186

Normal control 100.27 0.0852

Positive control 97.528 0.660

3hr 100mg/kg bw 98.542 0.241

150mg/kg bw 97.722 0.267

50mg/kg bw 99.381 0.343

Negative control 102.67 0.338

Normal control 100.32 0.101

Positive control 96.851 0.524

4hr 100mg/kg bw 97.864 0.207

150mg/kg bw 96.116 0.341

50mg/kg bw 98.386 0.223

Negative control 102.67 0.257

Normal control 100.27 0.0852

Positive control 96.901 0.316

One-way ANOVA: 1hr versus C1

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

C1 5 21.13 4.2268 8.10 0.000

Error 24 12.52 0.5217

Total 29 33.65

Pooled StDev = 0.722254

Tukey Pairwise Comparisons

Grouping Information Using the Tukey Method and 95% Confidence

C1 N Mean Grouping

Negative control 5 100.996 A

50mg/kg bw 5 100.576 A

150mg/kg bw 5 100.108 A

100mg/kg bw 5 100.052 A

Normal control 5 99.892 A

Positive control 5 98.304 B

Means that do not share a letter are significantly different.

One-way ANOVA: 2hr versus C1

Analysis of Variance

92

Source DF Adj SS Adj MS F-Value P-Value

C1 5 50.82 10.1650 11.84 0.000

Error 24 20.60 0.8584

Total 29 71.43

Pooled StDev = 0.926495

Tukey Pairwise Comparisons

Grouping Information Using the Tukey Method and 95% Confidence

C1 N Mean Grouping

Negative control 5 101.832 A

Normal control 5 100.269 A B

50mg/kg bw 5 100.216 A B

100mg/kg bw 5 99.324 B C

150mg/kg bw 5 99.277 B C

Positive control 5 97.528 C

Means that do not share a letter are significantly different.

One-way ANOVA: 3hr versus C1

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

C1 5 107.34 21.4687 39.92 0.000

Error 24 12.91 0.5378

Total 29 120.25

Pooled StDev = 0.733368

Tukey Pairwise Comparisons

Grouping Information Using the Tukey Method and 95% Confidence

C1 N Mean Grouping

Negative control 5 102.672 A

Normal control 5 100.323 B

50mg/kg bw 5 99.381 B C

100mg/kg bw 5 98.542 C D

150mg/kg bw 5 97.722 D E

Positive control 5 96.851 E

Means that do not share a letter are significantly different.

One-way ANOVA: 4hr versus C1

Analysis of Variance

93

Source DF Adj SS Adj MS F-Value P-Value

C1 5 144.750 28.9499 90.94 0.000

Error 24 7.640 0.3183

Total 29 152.390

Pooled StDev = 0.564220

Tukey Pairwise Comparisons

Grouping Information Using the Tukey Method and 95% Confidence

C1 N Mean Grouping

Negative control 5 102.672 A

Normal control 5 100.269 B

50mg/kg bw 5 98.386 C

100mg/kg bw 5 97.864 C D

Positive control 5 96.901 D E

150mg/kg bw 5 96.116 E

Means that do not share a letter are significantly different.

Appendix XI: Analysis of the anti-inflammatory effects of methanolic leaf

extract of Kigelia africana

Descriptive Statistics: 0hr, 1hr, 2hr, 3hr, 4hr Variable C1 Mean SE Mean

0hr 100mg/kg bw 100.00 0.000000

150mg/kg bw 100.00 0.000000

50mg/kg bw 100.00 0.000000

Negativecontrol 100.00 0.000000

Normal control 100.00 0.000000

Positive control 100.00 0.000000

1hr 100mg/kg bw 101.10 0.247

150mg/kg bw 99.788 0.143

50mg/kg bw 101.74 0.150

Negativecontrol 102.13 0.200

Normal control 99.836 0.100

Positive control 98.894 0.220

2hr 100mg/kg bw 99.582 0.765

150mg/kg bw 98.578 0.254

50mg/kg bw 100.37 0.260

Negativecontrol 103.61 0.0762

Normal control 99.844 0.0957

Positive control 97.202 0.530

3hr 100mg/kg bw 97.754 0.604

150mg/kg bw 96.590 0.260

50mg/kg bw 99.138 0.178

94

Negativecontrol 104.60 0.299

Normal control 99.844 0.0957

Positive control 95.978 0.436

4hr 100mg/kg bw 97.018 0.445

150mg/kg bw 95.024 0.200

50mg/kg bw 98.050 0.209

Negativecontrol 104.95 0.480

Normal control 99.926 0.0740

Positive control 95.570 0.471

One-way ANOVA: 1hr versus C1

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

C1 5 40.168 8.0337 47.61 0.000

Error 24 4.050 0.1687

Total 29 44.218

Pooled StDev = 0.410790

Tukey Pairwise Comparisons

Grouping Information Using the Tukey Method and 95% Confidence

C1 N Mean Grouping

Negativecontrol 5 102.126 A

50mg/kg bw 5 101.740 A B

100mg/kg bw 5 101.104 B

Normal control 5 99.836 C

150mg/kg bw 5 99.788 C

Positive control 5 98.894 D

Means that do not share a letter are significantly different.

One-way ANOVA: 2hr versus C1

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

C1 5 115.46 23.0915 27.35 0.000

Error 24 20.27 0.8444

Total 29 135.72

Pooled StDev = 0.918933

Tukey Pairwise Comparisons

95

Grouping Information Using the Tukey Method and 95% Confidence

C1 N Mean Grouping

Negativecontrol 5 103.608 A

50mg/kg bw 5 100.368 B

Normal control 5 99.8440 B

100mg/kg bw 5 99.582 B

150mg/kg bw 5 98.578 B C

Positive control 5 97.202 C

Means that do not share a letter are significantly different.

One-way ANOVA: 3hr versus C1

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

C1 5 242.80 48.5605 77.46 0.000

Error 24 15.05 0.6269

Total 29 257.85

Pooled StDev = 0.791799

Tukey Pairwise Comparisons

Grouping Information Using the Tukey Method and 95% Confidence

C1 N Mean Grouping

Negativecontrol 5 104.598 A

Normal control 5 99.8440 B

50mg/kg bw 5 99.138 B C

100mg/kg bw 5 97.754 C D

150mg/kg bw 5 96.590 D E

Positive control 5 95.978 E

Means that do not share a letter are significantly different.

One-way ANOVA: 4hr versus C1

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

C1 5 333.33 66.6667 108.27 0.000

Error 24 14.78 0.6157

Total 29 348.11

Tukey Pairwise Comparisons

Grouping Information Using the Tukey Method and 95% Confidence

96

C1 N Mean Grouping

Negativecontrol 5 104.950 A

Normal control 5 99.9260 B

50mg/kg bw 5 98.050 C

100mg/kg bw 5 97.018 C D

Positive control 5 95.570 D E

150mg/kg bw 5 95.024 E

Means that do not share a letter are significantly different.

Appendix XII: Analysis of the anti-inflammatory effects of methanolic stem

bark extract of Acacia hockii

Descriptive Statistics: 0hr, 1hr, 2hr, 3hr, 4hr

Variable C1 Mean SE Mean

0hr 100mg/kg 100.00 0.000000

150mg/kg 100.00 0.000000

50mg/kg 100.00 0.000000

Negative control 100.00 0.000000

Normal control 100.00 0.000000

Positive control 100.00 0.000000

1hr 100mg/kg 99.402 0.561

150mg/kg 99.234 0.411

50mg/kg 101.24 0.237

Negative control 101.49 0.167

Normal control 99.994 0.119

Positive control 98.524 0.281

2hr 100mg/kg 98.566 0.577

150mg/kg 96.970 0.283

50mg/kg 100.35 0.245

Negative control 102.66 0.260

Normal control 99.994 0.119

Positive control 96.634 0.441

3hr 100mg/kg 96.894 0.743

150mg/kg 95.722 0.469

50mg/kg 99.040 0.166

Negative control 103.30 0.260

Normal control 99.994 0.119

Positive control 95.496 0.356

4hr 100mg/kg 95.930 0.634

150mg/kg 94.620 0.409

50mg/kg 98.224 0.292

Negative control 103.37 0.454

Normal control 100.08 0.0780

Positive control 95.098 0.470

97

One-way ANOVA: 1hr versus C1

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

C1 5 34.37 6.8734 12.49 0.000

Error 24 13.21 0.5504

Total 29 47.58

Tukey Pairwise Comparisons

Grouping Information Using the Tukey Method and 95% Confidence

C1 N Mean Grouping

Negative control 5 101.490 A

50mg/kg 5 101.238 A B

Normal control 5 99.994 B C

100mg/kg 5 99.402 C D

150mg/kg 5 99.234 C D

Positive control 5 98.524 D

Means that do not share a letter are significantly different.

One-way ANOVA: 3hr versus C1

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

C1 5 224.36 44.8722 53.41 0.000

Error 24 20.17 0.8402

Total 29 244.53

Tukey Pairwise Comparisons

Grouping Information Using the Tukey Method and 95% Confidence

C1 N Mean Grouping

Negative control 5 103.304 A

Normal control 5 99.994 B

50mg/kg 5 99.040 B

100mg/kg 5 96.894 C

150mg/kg 5 95.722 C

Positive control 5 95.496 C

Means that do not share a letter are significantly different.

One-way ANOVA: 4hr versus C1

Analysis of Variance

98

Source DF Adj SS Adj MS F-Value P-Value

C1 5 286.19 57.2371 63.16 0.000

Error 24 21.75 0.9063

Total 29 307.9

Pooled StDev = 0.951988

Tukey Pairwise Comparisons

Grouping Information Using the Tukey Method and 95% Confidence

C1 N Mean Grouping

Negative control 5 103.368 A

Normal control 5 100.078 B

50mg/kg 5 98.224 B

100mg/kg 5 95.930 C

Positive control 5 95.098 C

150mg/kg 5 94.620 C

Means that do not share a letter are significantly different.

One-way ANOVA: 2hr versus C1

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

C1 5 129.33 25.8652 41.46 0.000

Error 24 14.97 0.6238

Total 29 144.30

Tukey Pairwise Comparisons

C1 N Mean Grouping

Negative control 5 102.658 A

50mg/kg 5 100.348 B

Normal control 5 99.994 B C

100mg/kg 5 98.566 C

150mg/kg 5 96.970 D

Positive control 5 96.634 D

99

Appendix XIII: Comparison between antipyretic effects of methanolic

stem bark extracts of Kigelia africana and Acacia

hockii at various dose levels

Two-Sample T-Test and CI: 50mg/kg A. hockii 1hr, 50mg/kg K. africana 1hr

Difference = μ (50mg/kg A. hockii 1hr) - μ (50mg/kg K. africana

1hr)

Estimate for difference: -0.158

95% CI for difference: (-1.310, 0.994)

T-Test of difference = 0 (vs ≠): T-Value = -0.34 P-Value = 0.749

DF = 6

Two-Sample T-Test and CI: 50mg/kg kigelia2hr, 50mg/kgA. hockii 2hr Difference = μ (50mg/kg kigelia2hr) - μ (50mg/kgA. hockii 2hr)

Estimate for difference: 0.280

95% CI for difference: (-1.463, 2.023)

T-Test of difference = 0 (vs ≠): T-Value = 0.41 P-Value = 0.697

DF = 5

Two-Sample T-Test and CI: 50mg/kg K.africana 3hr, 50mg/kgA. hockii 3hr Difference = μ (50mg/kg K.africana 3hr) - μ (50mg/kgA. hockii 3hr)

Estimate for difference: -0.044

95% CI for difference: (-1.124, 1.036)

T-Test of difference = 0 (vs ≠): T-Value = -0.10 P-Value = 0.926

DF = 7

Two-Sample T-Test and CI: 50mg/kgA. hockii 4hr_, 50mg/kg K.africana 4hr Difference = μ (50mg/kgA. hockii 4hr_) - μ (50mg/kg K.africana 4hr)

Estimate for difference: 0.200

95% CI for difference: (-0.576, 0.976)

T-Test of difference = 0 (vs ≠): T-Value = 0.61 P-Value = 0.561

DF = 7

Two-Sample T-Test and CI: 50mg/kgA. hockii 4hr_, 50mg/kg K.africana 4hr Difference = μ (50mg/kgA. hockii 4hr_) - μ (50mg/kg K.africana 4hr)

Estimate for difference: 0.200

95% CI for difference: (-0.576, 0.976)

T-Test of difference = 0 (vs ≠): T-Value = 0.61 P-Value = 0.561

DF = 7

Two-Sample T-Test and CI: 100mg/kg K.africana 1hr, 100mg/kgA. hockii 1hr Difference = μ (100mg/kg K.africana 1hr) - μ (100mg/kgA. hockii

1hr)

Estimate for difference: -0.052

95% CI for difference: (-1.024, 0.920)

T-Test of difference = 0 (vs ≠): T-Value = -0.13 P-Value = 0.903

DF = 7

Two-Sample T-Test and CI: 100mg/kg K.africana 2hr, 100mg/kgA. hockii 2hr Difference = μ (100mg/kg K.africana 2hr) - μ (100mg/kgA. hockii

2hr)

Estimate for difference: -0.570

95% CI for difference: (-1.602, 0.462)

T-Test of difference = 0 (vs ≠): T-Value = -1.35 P-Value = 0.225

DF = 6

100

Two-Sample T-Test and CI: 100mg/kg K.africana 3hr, 100mg/kgA. hockii 3hr Difference = μ (100mg/kg K.africana 3hr) - μ (100mg/kgA. hockii

3hr)

Estimate for difference: -0.154

95% CI for difference: (-0.860, 0.552)

T-Test of difference = 0 (vs ≠): T-Value = -0.61 P-Value = 0.578

DF = 4

Two-Sample T-Test and CI: 100mg/kg K.africana 4hr, 100mg/kg A. hockii 4hr Difference = μ (100mg/kg K.africana 4hr) - μ (100mg/kg A. hockii

4hr)

Estimate for difference: -0.048

95% CI for difference: (-0.671, 0.575)

T-Test of difference = 0 (vs ≠): T-Value = -0.18 P-Value = 0.861

DF = 7

Two-Sample T-Test and CI: 150mg/kg K.africana 1hr, 150mg/kgA. hockii 1hr Difference = μ (150mg/kg K.africana 1hr) - μ (150mg/kgA. hockii

1hr)

Estimate for difference: 0.420

95% CI for difference: (-0.427, 1.267)

T-Test of difference = 0 (vs ≠): T-Value = 1.17 P-Value = 0.279

DF = 7

Two-Sample T-Test and CI: 150mg/kg K.africana 2hr, 150mg/kgA. hockii 2hr Difference = μ (150mg/kg K.africana 2hr) - μ (150mg/kgA. hockii

2hr)

Estimate for difference: 0.470

95% CI for difference: (-0.244, 1.184)

T-Test of difference = 0 (vs ≠): T-Value = 1.61 P-Value = 0.158

DF = 6

Two-Sample T-Test and CI: 150mg/kg K.africana 3hr, 150mg/kgA. hockii 3hr Difference = μ (150mg/kg K.africana 3hr) - μ (150mg/kgA. hockii

3hr)

Estimate for difference: -0.248

95% CI for difference: (-0.977, 0.481)

T-Test of difference = 0 (vs ≠): T-Value = -0.87 P-Value = 0.422

DF = 5

Two-Sample T-Test and CI: 150mg/kg K.africana 4hr, 150mg/kgA. hockii 4hr Difference = μ (150mg/kg K.africana 4hr) - μ (150mg/kgA. hockii

4hr)

Estimate for difference: -0.812

95% CI for difference: (-1.796, 0.172)

T-Test of difference = 0 (vs ≠): T-Value = -2.29 P-Value = 0.084

DF = 4

Appendix XIV: Comparison between anti-inflammatory effects of

methanolic leaf extract of Kigelia africana and stem bark

extract of Acacia hockii at various dose levels

Two-Sample T-Test and CI: A. hockii 50mg/kg bw 1hr, K. africana 50mg/kg bw 1hr Difference = μ (A. hockii 50mg/kg bw 1hr) - μ (K. africana 50mg/kg

bw 1hr)

101

Estimate for difference: 0.502

95% CI for difference: (-0.185, 1.189)

T-Test of difference = 0 (vs ≠): T-Value = 1.79 P-Value = 0.124

DF = 6

Two-Sample T-Test and CI: K. afiricana 50mg/kg bw 2hr, A. hockii 50mg/kg bw 2hr Difference = μ (K. afiricana 50mg/kg bw 2hr) - μ (A. hockii 50mg/kg

bw 2hr)

Estimate fr difference: -0.018

95% CI for difference: (-0.862, 0.826)

T-Test of difference = 0 (vs ≠): T-Value = -0.05 P-Value = 0.961

DF = 7

Two-Sample T-Test and CI: A. hockii 50mg/kg bw 3hr, K. africana 50mg/kg bw 3hr Difference = μ (A. hockii 50mg/kg bw 3hr) - μ (K. africana 50mg/kg

bw 3hr)

Estimate for difference: 0.098

95% CI for difference: (-0.477, 0.673)

T-Test of difference = 0 (vs ≠): T-Value = 0.40 P-Value = 0.699

DF = 7

Two-Sample T-Test and CI: A. hockii 50mg/kg bw 4hr, K. africana 50mg/kg bw 4hr Difference = μ (A. hockii 50mg/kg bw 4hr) - μ (K. africana 50mg/kg

bw 4hr)

Estimate for difference: -0.174

95% CI for difference: (-1.024, 0.676)

T-Test of difference = 0 (vs ≠): T-Value = -0.48 P-Value = 0.643

DF = 7

Two-Sample T-Test and CI: A. hockii 100mg/kg bw 1hr, K. africana 100mg/kg bw 1hr Difference = μ (A. hockii 100mg/kg bw 1hr) - μ (K. africana 100mg/kg

bw 1hr)

Estimate for difference: 1.702

95% CI for difference: (0.127, 3.277)

T-Test of difference = 0 (vs ≠): T-Value = 2.78 P-Value = 0.039

DF = 5

Two-Sample T-Test and CI: K. africana 100mg/kg bw 2hr, A. hockii 100mg/kg bw 2hr Difference = μ (K. africana 100mg/kg bw 2hr) - μ (A. hockii 100mg/kg

bw 2hr)

Estimate for difference: -1.016

95% CI for difference: (-3.282, 1.250)

T-Test of difference = 0 (vs ≠): T-Value = -1.06 P-Value = 0.324

DF = 7

Two-Sample T-Test and CI: A. hockii 100mg/kg bw 3hr, K. africana 100mg/kg bw 3hr Difference = μ (A. hockii 100mg/kg bw 3hr) - μ (K. africana 100mg/kg

bw 3hr)

Estimate for difference: 0.860

95% CI for difference: (-1.404, 3.124)

T-Test of difference = 0 (vs ≠): T-Value = 0.90 P-Value = 0.399

DF = 7

Two-Sample T-Test and CI: A. hockii 100mg/kg bw 4hr, K. africana 100mg/kg bw 4hr Difference = μ (A. hockii 100mg/kg bw 4hr) - μ (K. africana 100mg/kg

bw 4hr)

Estimate for difference: 1.088

95% CI for difference: (-0.743, 2.919)

102

T-Test of difference = 0 (vs ≠): T-Value = 1.41 P-Value = 0.203

DF = 7

Two-Sample T-Test and CI: A. hockii 150mg/kg bw 1hr, K. africana 150mg/kg bw 1hr Difference = μ (A. hockii 150mg/kg bw 1hr) - μ (K. africana 150mg/kg

bw 1hr)

Estimate for difference: 0.554

95% CI for difference: (-0.654, 1.762)

T-Test of difference = 0 (vs ≠): T-Value = 1.27 P-Value = 0.272

DF = 4

Two-Sample T-Test and CI: A. hockii 150mg/kg bw 2hr, K. africana 150mg/kg bw 2hr Difference = μ (A. hockii 150mg/kg bw 2hr) - μ (K. africana 150mg/kg

bw 2hr)

Estimate for difference: 1.608

95% CI for difference: (0.709, 2.507)

T-Test of difference = 0 (vs ≠): T-Value = 4.23 P-Value = 0.004

DF = 7

Two-Sample T-Test and CI: K. africana 150mg/kg bw 3hr, A. hockii 150mg/kg bw 3hr Difference = μ (K. africana 150mg/kg bw 3hr) - μ (A. hockii 150mg/kg

bw 3hr)

Estimate for difference: -0.867

95% CI for difference: (-2.178, 0.444)

T-Test of difference = 0 (vs ≠): T-Value = -1.62 P-Value = 0.157

DF = 6

Two-Sample T-Test and CI: A. hockii 150mg/kg bw 4hr, K. africana 150mg/kg bw 4hr Difference = μ (A. hockii 150mg/kg bw 4hr) - μ (K. africana 150mg/kg

bw 4hr)

Estimate for difference: 0.404

95% CI for difference: (-0.767, 1.575)

T-Test of difference = 0 (vs ≠): T-Value = 0.89 P-Value = 0.416

DF = 5

103