ANALGESIC, ANTIPYRETIC AND ANTI-INFLAMMATORY …

ANALGESIC, ANTIPYRETIC AND ANTI-INFLAMMATORY

POTENTIAL OF DICHLOROMETHANOLIC ROOT EXTRACT OF

CLUTIA ABYSSINICA JAUB AND SPACH IN RATS AND MICE

MODELS

KOECH SAMSON CHERUIYOT (BSc Biochem)

I56/28505/2014

A Research Thesis Submitted in Partial Fulfillment of the Requirements

for the Award of the Degree of Master of Science (Biochemistry) in the

School of Pure and Applied Sciences of Kenyatta University

July, 2017

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DEDICATION

I dedicate this thesis to my family for their immense support towards my

education.

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ACKNOWLEDGEMENT

I greatly acknowledge Kenyatta University for giving me an opportunity to

further my studies. Much of my appreciation goes to my supervisor‟s Prof Eliud

N.M Njagi and Dr Mathew Piero Ngugi for their inspiration, mentorship and

tireless effort in guiding me to complete my research study. May God bless them

abundantly.

I owe special thanks to the Department of Biochemistry and Biotechnology and

her staff for the close companionship and cooperation during the entire period of

carrying thank out my research study. The following people deserve to be

mentioned, Mrs Lel, James Adino, Daniel Gitonga and James Ngunjiri for their

technical support. To Veronica Sindani, Peter Nthiga, James Kimani, John

Mwonjoria, Jane Maoga, Wylcliffe Arika, Alex Cheruiyot, Herbet Cheruiyot and

Robert Ouko you for your motivation and encouragement.

I also owe a big thanks to my parents, brothers, sister and friends for the

encouragement, motivation and support throughout the entire period. Above all I

thank the almighty GOD for giving me good health and strength to complete this

project.

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TABLE OF CONTENT

DECLARATION ............................................................................................. ii

DEDICATION ................................................................................................ iii

ACKNOWLEDGEMENT .............................................................................. iv

TABLE OF CONTENT ................................................................................... v

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

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

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

ABBREVIATIONS AND ACRONYMS ....................................................... xi

ABSTRACT ................................................................................................... xii

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

INTRODUCTION ........................................................................................... 1

1.1 Background of the Study ........................................................................... 1

1.2 Problem Statement and Justification .......................................................... 7

1.3 Hypotheses ................................................................................................. 8

1.4 General Objective ...................................................................................... 9

1.4.1 Specific Objectives ................................................................................9

CHAPTER TWO ........................................................................................... 10

LITERATURE REVIEW .............................................................................. 10

2.1 Biochemical and Physiological Basis of Pain, Fever and

inflammation ............................................................................................ 10

2.2 Screening Models for Pain, Pyrexia and Inflammation ........................... 15

2.2.1 Screening Models for Pain ..................................................................15

2.2.1.1 Test Based on Thermal Stimuli..................................................15

2.2.1.1.1 Tail flick test using radiant heat ................................................15

2.2.1.1.2 Hot plate test ..............................................................................16

2.2.1.2 Test Based on Mechanical Stimuli ...............................................16

2.2.1.3 Test Based on Electrical Stimuli ..................................................16

2.2.1.3.1 Electrical stimulation of the tooth-pulp .....................................16

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2.2.1.3.2 Electrical stimulation of the tail .................................................17

2.2.1.4 Test Based on Chemical Stimuli ..................................................17

2.2.1.4.1 Formalin-Induced pain test ........................................................17

2.2.1.4.2 Acetic acid-Induced pain test ....................................................17

2.2.2 Screening Models for Pyrexia .............................................................18

2.2.2.1 Turpentine-Induced test ................................................................18

2.2.1.2 DNP-Induced test .........................................................................18

2.2.2.3 Brewer‟s Yeast-Induced test .........................................................19

2.3.3 Screening Models for Inflammation ...................................................19

2.3.3.1 Carragenaan-Induced Hind Paw Edema test ................................19

2.2.3.2 Xylene-Induced Ear Edema test ...................................................20

2.2.3.3 Formalin-Induced Paw Edema Test .............................................21

2.2.3.4 Cotton Pellet Granuloma Test ......................................................21

2.3 Conventional Management of Pain, Pyrexia and Inflammation .............. 21

2.4 Use of Medicinal Plants in Management of Diseases .............................. 25

2.5 Herbal Management of Pain, Fever and Inflammation ............................ 26

2.5.1 Pain ......................................................................................................26

2.5.2 Pyrexia .................................................................................................28

2.5.3 Anti-inflammatory ...............................................................................29

2.6 Clutia abyssinica ...................................................................................... 31

2.6.1 Description and Distribution ...............................................................31

2.6.2 Medicinal uses .....................................................................................32

CHAPTER THREE ....................................................................................... 34

MATERIALS AND METHODS ................................................................... 34

3.1 Collection and Preparation of Plant Materials ......................................... 34

3.2 Extraction ................................................................................................. 34

3.3 Experimental Design ................................................................................ 35

3.3.1 Laboratory Animals.............................................................................35

3.4 Determination of Analgesic Effect .......................................................... 35

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3.5 Evaluation of Antipyretic Effect .............................................................. 37

3.6 Determination of Anti-inflammatory Effect ............................................ 39

3.7 Qualitative Phytochemical Screening ...................................................... 41

3.7.1 Flavonoids ...........................................................................................41

3.7.2 Terpenoids (Salkowski test) ................................................................41

3.7.3 Steroids ................................................................................................42

3.7.4 Cardiac glycosides...............................................................................42

3.7.5 Phenolics .............................................................................................42

3.7.6 Alkaloids- Mayer‟s test .......................................................................42

3.7.7 Saponins (Frothing test) ......................................................................43

3.8 Data Management and Statistical Analysis .............................................. 43

CHAPTER FOUR .......................................................................................... 44

RESULTS ...................................................................................................... 44

4.1 Analgesic Activity of DCM Root Extracts of C. abyssinicaon acetic acid-

. induced pain in Swiss albino mice .......................................................... 44

4.2 Antipyretic Activity of DCM Root Extract of C. abyssinica on turpentine-

. Induced pyrexia in Wistar albino rats. ..................................................... 46

4.3 Anti-inflammatory Activity of DCM Root Extract of C. abyssinica....... 50

on Carrageenan-Induced Inflammation in Swiss Albino Mice................ 50

4.4 Qualitative Phytochemical Screening ...................................................... 55

CHAPTER FIVE ........................................................................................... 56

DISCUSSION, CONCLUSION, RECOMMENDATIONS AND

SUGGESTIONS FOR FURTHER STUDIES ............................................... 56

5.1 Discussion ................................................................................................ 56

5.2 Conclusion ............................................................................................... 67

5.3 Recommendations .................................................................................... 68

5.4 Suggestions for Further Studies ............................................................... 69

REFERENCES .............................................................................................. 70

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LIST OF FIGURES

Figure 2. 1: Clutia abyssinica…………………………………………………..32

Figure 4. 1: Percentage change in rectal temperature of DCM root

extract of Clutia abyssinica on turpentine-induced

pyrexia in Wistar albino rats..............................................................49

Figure 4. 2: Percentage change in hind paw diameter of DCM root

extract of Clutia abyssinica on carragenaan induced

inflammation in Swiss albino mice .................................................. 54

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LIST OF TABLES

Table 3. 1: Treatment protocol for assessment of analgesic activity of

DCM root extract of C. abyssinica in Swiss albino mice ................. 36

Table 3. 2: Treatment protocol for antipyretic activity of DCM root

extract of C. abyssinica in wistar albino rats ..................................... 38

Table 3. 3: Treatment protocol for evaluation of anti-inflammatory

activity of DCM root extract of C. abyssinica in swiss

albino mice. ....................................................................................... 40

Table 4. 1: Analgesic activities of DCM root extracts of C. abyssinica

on acetic acid-induced pain in Swiss albino mice……..……………45

Table 4.2: Antipyretic activities of DCM root extracts of

C. abyssinica on Turpentine-induced pyrexia in Wistar

albino rats………………………………………………………….48

Table 4. 3: Anti-inflammatory activities of DCM root extracts of

C. abyssinica on carragenaan-induced inflammation in

Swiss albino mice .............................................................................. 53

Table 4. 4: Phytochemical composition of DCM root extract of

....................C. abyssinica ..................................................................................... 55

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LIST OF APPENDICES

Appendix I: Mean percentage change in the number of abdominal

writhes after administration of DCM root extract in

Swiss.albino mice………………………………………………..87

Appendix II: Effects of DCM root extract of Clutia abyssinica on

turpentine-induced pyrexia in Wistar albino rats…………….88

Appendix III: Effects of DCM root extract of Cluta abyssinica on

carrageenan-induced inflammation in Swiss albino mice…….....89

Appendix IV: A map of the location where the plant was

collected Courtesy of Google Earth maps………………………90

Appendix V: Analysis of analgesic activity of DCM root extract of .

………………Clutia abyssinica in Swiss albino mice………………………….91

Appendix VI: Analysis of antipyretic activity of DCM root extract

of Clutia abyssinica in Wistar albino rats……………………….92

Appendix VII: Analysis of anti-inflammatory activity of DCM root

extract of Clutia abyssinica in Swiss albino mice……………...95

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ABBREVIATIONS AND ACRONYMS

5-HT Serotonin (5-hydroxytryptamine receptors)

A δ A delta fibers

ANOVA Analysis of variance

Bw Body weight

CAM Complemetary and alternative medicine

COX 1 Cyco-oxygenase 1

COX 2 Cyclo-oxygenase 2

COX cyclo-oxygenase

DAMPs Damaged associated molecular patterns

DCM Dichloromethane

DMSO Dimethyl sulfoxide

DNP 2,4-Dinitrophenol

IASP International association for the study of pain

IL-1RA Interleukin 1 RA

IL-1α Interleukin 1α

IL-1β Interleukin 1β

IL-6 Interleukin 6

Ip Intraperitoneally

KG Kilograms

M Meters

M/L Milliliters

M/S Meters per second

MG Milligrams

Ms Microsoft

NFκB Nuclear factor kappa activated Bcells

NLRS Nord-like receptors

NSAIDs Non-steroidal anti-inflammatory drugs

OVLT Organum vasculum of the laminae terminalis

PGE2 Prostaglandin E2

PGs prostaglandins

SEM Standard error mean

TLR-4 Toll-like receptor 4

TLRs Toll like receptors

TNFα Tumour necrosis factor α

UL Microliters

WHO World health organization

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ABSTRACT

Various diseases and injuries are always presented with pain, fever and

inflammation. These are considered as symptoms associated with various

pathological processes in an animal body. Drugs that are used to alleviate pain,

fever and inflammation such as non-steroidal anti-inflammatory drugs exhibit

adverse effects for example cardiac abnormalities, peptic ulcers, liver toxicity and

kidney failure. Therefore, there is need to come up with alternative remedies.

Herbal medicines are deemed to be safe, have good efficacy and have fewer side

effects. Clutia abyssinica is a shrub that is found in East, Central, and South

Africa and it has been used traditionally to cure several ailments including

malaria, chest pain, gonorrhea, fever, infertility, pain, inflammation, skin diseases

and cancer. Roots of this medicinal plant have been used traditionally to prepare

decoctions. The aim of the project was to evaluate the analgesic, antipyretic and

anti-inflammatory potential of dichloromethanolic root extract of Clutia

abyssinica in animal models. Plant sample material was collected from Kaptebee

village, Turbo sub-county in Uasin Gishu County Kenya, and the active

components extracted using dichloromethane. Pain, fever and inflammation were

induced Swiss albino mice and Wistar albino rats using acetic acid, turpentine and

carrageenan respectively. Swiss albino mice and Wistar albino rats were grouped

into normal control, negative control, positive control and 3 experimental groups.

Extracted root extracts were administered intraperitoneally to Swiss albino mice

and Wistar albino rats at predetermined doses (50, 100, and 150 mg/kg body

weight). The analgesic and anti-inflammatory activities of the plant root extract

were compared to diclofenac the (reference drug) while the antipyretic activity

was compared to aspirin. The dichloromethanolic root extract of C. abyssinica

demonstrated significant analgesic, antipyretic and anti-inflammatory activities.

Number of abdominal writhing was reduced between 33.95-49.51% by

dichloromethanolic root extract while the diclofenac (reference drug) reduced

abdominal writhing by 46.51%. Reduction in number of abdominal writhings by

the extract indicates the plant posse‟s analgesic properties. The elevated

temperature was reduced between 0.68-3.34% by the dichloromethanolic root

extract while Aspirin the (reference drug) reduced elevated temperature between

3.32-4.96%. Edema was reduced between 0.88-5.34% by the plant extract while

diclofenac reduced edema between 2.21-5.35% respectively. Rectal temperature

and the size of the edema was reduced more in the third and fourth hours

signifying better blockage of mediators responsible for fever and inflammation.

Data was analyzed using one way analysis of variance followed by turkey‟s test.

Qualitative phytochemical analysis revealed the presence of alkaloids, flavonoids,

terpenoids, steroids, saponins and cardiac glycosides. The extract from Clutia

abyssinica may be used as an alternative bioresource in development of analgesic,

antipyretic and anti-inflammatory agent. The study therefore, confirms the

folklore use of the medicinal plant by Kalenjin community of Kenya to manage

pain, fever and inflammation.

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CHAPTER ONE

INTRODUCTION

1.1 Background of the Study

Pain is defined as multidimensional, subjective and unpleasant experience that is

allied to tissue damage comprising sensory experiences that include; time,

intensity, space, emotion, cognition and motivation (Maze et al., 2000). Pain

contains both sensory and psychological mechanisms. However, pain is beyond

sensation, it comprises of perception and subjective interpretation of the

discomfort (Maze et al., 2000). Major mediators of pain include; Bradykinin,

histamine, serotonin and prostaglandins. Pain in its real sense lack a way to

define it, but in general term, occurs whenever the body tissues are damaged

(Arome et al., 2016). Sensation of pain is a sign that something in the body is

wrong. Pain plays an important role in drawing attention to tissue injury from

harmful stimuli and reflexes are elicited to protect the injured part of the body

(Arome et al., 2016).

Damage caused by mechanical, thermal, chemical and electrical stimuli through

peripheral receptors triggers pain sensation to nociceptors in an organism (Guyton

and Hall, 2006). Perception of pain is a normal physiological response that is

mediated by nervous system and is used for diagnosing various diseases such as

diabetes, arthritis and cancer that are normally associated with chronic pain

(Apkarian et al., 2005).

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Fever or pyrexia is a common clinical sign that is characterized by rise in body

temperature beyond the normal range of (36.5°C -37.5

°C) (Axelrod and Diringer,

2008). Pyrexia depicts an increase in body temperature due to cytokine-induced

upward displacement of the set point in the hypothalamic thermoregulatory center

(Karakitsos and Karabinis, 2008). Fever is considered a normal reaction in a

number of health problems. It is even considered as an alarming sign when fever

itself is absent than when it is present. Symptoms of fever include; sweating,

chills, sensation of cold and other subjective sensations (Guyton and Hall, 2000).

High temperature with absence of these symptoms may indicate a sign of a

serious illness (Saper and Breder, 1994).

A number of different microorganisms and other substances can cause fever and

are collectively termed as pyrogens (Shalini and Donna, 2006). Pyrogens are

classified into either exogenous or endogenous pyrogens. Exogenous pyrogens are

those that originate outside the body for example bacteria toxins on the other hand

endogenous pyrogens are derived from immune cells in the body responding to

stimuli from an external environment (Shalini and Donna, 2006). Products

released by bacterial cell membranes such as lipopolysaccharide, toxins and

breakdown of protein products in an organism body can cause the set point of the

hypothalamic thermostat to increase (Guyton and Hall, 2000). Under normal

health conditions the range for oral temperature is between 33.2 – 38.2°C, for the

armpit 35.5 – 37.0°C, the rectal 34.4 – 37.8°C, while for tympanic membrane

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35.4-37.8°C (Sund-Levander et al., 2002). Fever is associated with sickness

conditions, such as depression, anorexia, sleepiness, lethargy, inability to

concentrate and hyperalgesia (Kelly et al., 2003).

Inflammation on the other hand is a complex response in the vascularised

connective tissue that may be triggered by either exogenous or endogenous

stimuli (Rang et al., 2003). Inflammation is considered a normal process that

provide protective role to tissue injury brought about by microbiological agents,

physical trauma and noxious chemicals. Inflammation is utilized by the immune

system as a means of defence. During the process of inflammation, foreign

material responsible for inflammation are destroyed as a means of tissue repair

and regeneration (Rang et al., 2003).

Inflammation is characterised by a number of components that result from tissue

injury and this include; leukocyte infiltration, edema formation and granuloma

formation (Guyton and Hall, 2000; Mitchell and Cotran, 2000). For edema to be

formed, synergism between various inflammatory mediators are involved that

increase vascular permeability and blood flow into the damaged sides (Lalenti et

al., 1995). Inflammation can either be acute or chronic. Acute inflammation is

considered as that initial response to harmful stimuli by the body while chronic

inflammation on the hand is considered inflammatory response that is out of

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proportion resulting in damage to the body tissue or organs (Palladino et al.,

2003).

Cardinal signs of inflammation include; redness, increased temperature, swelling,

pain, and loss of function (Craig and Stitzel, 2003). A diverse number of

substances can provoke inflammation for example toxins, infections, frostbite,

chemicals, noxious agents, physical injuries, foreign materials, pathogens,

antibodies, immune reaction and necrosis (Young et al., 2002).

Inflammation can also be triggered by a number of diverse inflammatory

mediators for example prostaglandins, leukotrienes, neuropeptides, eicosanoids,

histamine, serotonin, kinins and platelet activating factors (Burke et al., 2006).

Different immune cells like neutrophils, monocytes, lymphocytes, basophils, mast

cells, resident macrophages and eosinophils are also involved in pathogenesis of

inflammation (Richardson, 1971).

Pain, fever and inflammation are beneficial to the immune system. However, they

cause a lot of suffering and discomfort to the victims lowering the quality of life

and therefore need to be managed. Non-steroidal anti-inflammatory drugs

(NSAIDs) are commonly used to manage inflammation, fever and pain (Barar,

2009). Opoid analgesics are choice drugs for severe or chronic malignant pain

(Richard et al., 2008). Their mechanism of action involves inhibition of cyclo-

5

oxygenase (COX) enzyme which results in disruption of prostaglandins synthesis

(Burke et al., 2006). However, non-steroidal anti-inflammatory drugs and opioid

analgesics that are normally used to treat inflammation, fever and pain manifest a

great number of adverse effects (Beg et al., 2011).

Despite numerous progress in medical science and in the production of new

synthetic conventional drugs for management of pain, fever and inflammation,

there is still need for development of more cost-effective and improved remedies

with lesser side effects. Recent studies by world health organisation (WHO)

indicate that these compounds/drugs used in the management of pain,

inflammation and fever manifest a lot of side effects after long term use that

include gastric irritation, ulceration, prolonged bleeding, renal failure, interstitial

corrosion, and pruritis (Beg et al., 2011). Reduction in ligament formation,

tendon, cartilage healing and delay in muscle regeneration in many studies has

been associated with NSAIDs (Almekinders, 1999). Conventional drugs used to

manage pain, inflammation and fever only provide asymptomatic relief and the

greatest disadvantage lies in their toxicity to the liver, kidney and reappearance of

symptoms after discontinuation (Shah et al., 2006).

In this regard, herbal medicines have been employed in complementary and

alternative medicine (CAM) for treatment of pain, fever and inflammation as well

as diseases related to these conditions. Many traditionally used medicinal plants

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are known to contain antipyretic, analgesic and anti-inflammatory properties but

only a small percentage are included in health care systems after clinical research

to manage these conditions (Singh et al., 2008).

In general, natural products and in particular, medicinal plants, are believed to be

an important source of novel chemical substances with potential therapeutic

capabilities. Considering that most of anti-inflammatory, analgesic, anti-malarial

and antipyretic synthetic drugs such as aspirin, morphine chloroquine and

artemisinin were derived from plant products, the search for plant species with

anti-inflammatory, antipyretic and analgesic properties should be viewed as a

fruitful strategy in search of new drugs (Gupta et al., 2006).

Clutia abyssinica has been used traditionally by the kalenjin community in

Kaptebee village, Turbo sub-county in Uasin Gishu County Kenya in

management of pain, malaria, kidney problem, fever and inflammation (Jeruto et

al., 2014). Though there are some ethnobotanical studies conducted on this

medicinal plant, there has been no scientifically evaluated data about its potential.

It is against this background that bioscreening of analgesic, antipyretic and anti-

inflammatory potential of dichloromethanolic (DCM) root extracts of C.

abyssinica in animal models was investigated. The study aimed at providing

preliminary information for production of plant derived analgesic, antipyretic and

anti-inflammatory drugs.

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1.2 Problem Statement and Justification

Recently, there has been a remarkable development in medical science. However,

treatment and management of many serious indicators of ill health including pain,

fever and inflammation is still problematic and complex (Adedapo et al., 2009).

Pain, fever and inflammation cause suffering and discomfort among the victims

(Kariuki et al., 2012). Recent studies by WHO indicates that the NSAIDs used in

management of these conditions manifest a lot of side effects (Robotin, 2006; Beg

et al., 2011).

This is therefore, a challenge to the research sector to find alternative approaches

of managing pain, fever and inflammation. Herbal medicines are deemed to be

safe, have good efficacy, are culturally accepted and have lesser side effects than

the synthetic drugs (Sen et al., 2010).

Although C. abyssinica is widely used in Kaptebee village, Turbo sub-county in

Uasin Gishu County Kenya by the Kalenjin community to manage pain, fever,

inflammation, malaria and other ailments in the traditional system of medicine, an

extensive search on the literature reveals that no data has been documented about

the medicinal use of the plant against pain, fever and inflammation.

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1.3 Hypotheses

i. The DCM root extracts of C. abyssinica do not have analgesic effects in

mice models.

ii. The DCM root extracts of C. abyssinica do not have antipyretic effects in

rat models.

iii. The DCM root extracts of C. abyssinica do not have anti-inflammatory

effects in mice models.

iv. The DCM root extracts of C.abyssinica do not have phytochemical

compounds associated with analgesic, antipyretic and anti-inflammatory

activities.

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1.4 General Objective

To determine analgesic, antipyretic and anti-inflammatory potentials of DCM root

extracts of C. abyssinica in rats and mice models.

1.4.1 Specific Objectives

i. To determine effects of DCM root extracts of C. abyssinica on acetic acid

induced-pain in Swiss albino mice models.

ii. To determine the effects of DCM root extracts of C. abyssinica on

turpentine-induced pyrexia in Wistar albino rat models.

iii. To determine the effects of DCM root extracts of C. abyssinica on

carragenaan-induced inflammation in Swiss albino mice models.

iv. To determine the qualitative phytochemical composition of DCM root

extracts of C. abyssinica.

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CHAPTER TWO

LITERATURE REVIEW

2.1 Biochemical and Physiological Basis of Pain, Fever and Inflammation

The international association for the study of pain (IASP) defines pain as

“unpleasant sensory and emotional experience that is caused by actual or potential

tissue damage”. The emotional component differ from one person to the other

and in the same individual from time to time and it can be classified in several

ways, but in therapeutic application into; nociceptive and neuropathic (Rajagopal,

2006). In the body, Sensory nerve endings are generally found in every part of the

body such as the blood vessels, internal organs, muscles, joints, and the skin.

Damage caused by the chemical, mechanical, and thermal stimuli sensitize

nociceptors. When cells are damaged a number of chemical mediators are

released which then activate and sensitize nociceptors to other mediators of pain

(Thorp, 2008). Sensation of pain due to mechanical, thermal and electrical

stimuli is initiated by peripheral receptors (Guyton and Hall, 2006). In the brain

pain stimulus are processed and generated impulses are send down the spinal cord

following the appropriate nerves and instructs the body to respond, for instance

withdrawing your hand from fire (Rang et al., 2006).

Peripheral nerves transmit pain stimulus to the spinal cord which then links to the

brain. Two types of nerve fibers are involved in this process; slow pain fibers and

Fast pain fibers. Transmission of fast pain is through the A delta fibers (Aδ fibers)

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to the spinal cord. The activity of fast pain fibers is terminated at luminal in the

spinal cord. A second neuron is excited which follows the neospinothalamic

pathway and terminates its transmission in the brainstem (Arome et al., 2016).

Fast pain nerve endings secrete a neurotransmitter called glutamate, which

transmits fast pain impulses to the brain in the cortex. Therefore localization of

pain in certain part of the body becomes relatively precise (Rang et al., 2006).

Bradykinin, histamine, serotonins and prostaglandins are the major mediators of

pain (Craig and Stitzel, 2003). It is a sensory modality that is essential for survival

of an organism from harmful stimuli. It provides a warning signal to the nervous

system to initiate a response that would otherwise minimize injury to the tissues

(Arome et al., 2016).

Pain can also be classified into fast and slow pain. A second after application of

pain stimulus such as pricking of hand or touching a hot object fast pain is felt at

that moment. This kind of pain is shallow, felt within underneath of the skin but

not felt in most internal tissues of the body. Its transmission is through the A delta

fibers at a velocity of about 5-30 m/s and due to its high speed in conduction of

pain stimulus it allows the body to immediately withdraw from the stimuli

(Guyton and Hall, 2006). Slow pain on the other hand is throbbing and diffused.

It is felt immediately after pain stimulus is applied lasting for few minutes, weeks

or even months and if not properly processed by the body, may result in chronic

12

pain. Slow pain is felt in internal tissues of the body and its transmission is

through C-fibers at a speed of 0.5-2 m/s to the brain. Other types of pain include

the visceral, somatic and neuropathic pain (Guyton and Hall, 2006).

Fever means the body temperature is above normal and fever is a symptom, not a

disease. It is a mechanism used by the immune system to fight or defend against

infections. Fever turns on the body‟s immune system and helps fight infections

(Blatteis, 2007). Regulation of temperature is in the hypothalamus. Fever is

triggered by substances called „‟pyrogen‟‟ and this pyrogens causes release of

prostaglandin E2 (PGE2). Prostaglandin E2 acts on the hypothalamus and a

systemic response is generated which act on the rest of the body, causing heat-

creating effects to match the new temperature. The hypothalamus works like a

thermostat in many situations (Anochie and Philip, 2013).

In essence, endogenous pyrogens are cytokines which are associated with innate

immune system. Phagocytic cells produce this molecules that causes increase in

thermoregulatory set-point and this includes; interleukin 1α and interleukin 1β

(Walter, 2003). When the set-point is raised, the body develop‟s mechanism of

heat generation and retention. Vasoconstriction decreases loss of heat through the

skin and makes an individual to feel cold (Anochie and Philip, 2013). This differs

with hyperthermia, in which the body overheats due to undesirable retention of

heat or overproduction of heat under the normal conditions (Fauci, 2008).

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Febrile response involves innate immune system activation via Toll-like receptor

4 (TLR-4) leading to production of pyrogenic cytokines such as; (IL)-1β, IL-6,

and tumor necrosis factor (TNF-α) that act on an area of the brain known as the

OVLT and eventually leading to the release of PGE2 via activation of COX-2

enzyme (Young and Sexana, 2014). In the hypothalamus, PGE2 binds to receptors

leading to an increase in heat generation mechanism and reduction in heat loss

until a new elevated set-point in the hypothalamus is reached (Young and Sexana,

2014).

Inflammation is defined as a biological response to a disrupted tissue homeostasis

(Medzhitov, 2008). Inflammation simply involves tissue-destroying process in

which blood-derived products such as plasma proteins, fluids and leukocytes are

recruited into perturbed tissues (Medzhitov, 2008). A number of processes are

involved in inflammation for example mediator release, cell migration, tissue

breakdown, enzyme activation, regeneration and repair (Vane and Bolting, 1995).

There are two kinds of inflammation, acute and chronic inflammation. Acute

inflammation begins immediately after the injury of tissues and is usually marked

by cardinal signs such as redness, heat, pain and loss of function. On the other

hand chronic inflammation is marked by continuation of acute inflammation, new

connective tissue formation, persistent and is prolonged (Bhagyasri et al., 2015).

14

Germ-line encoded receptors such as intracellular nucleotide binding domains

(NOD) and transmembrane toll-like receptors (TLRs) recognize damaged signals

(Lange et al., 2001; Proell et al., 2008). Once ligands are recognized, TLRs

activate ordinary signaling pathways and these results in the activation of NF-κB

(Ghosh et al., 1998). When the signal is transducted, NF-κB is released from IκB

and is translocated to the nucleus. In the nucleus transcription is upregulated by

binding to target genes (Friendman and Hughes, 2002). Increasing numbers of

damaged associated molecular patterns (DAMPs) trigger the intracellular nod-like

receptors that alert the immune system to cell injury and provide ways in which

possible exposure to toxins or pollutants in environment can be detected (Nathan,

2002). Transcription and translation of genes lead to expression of inducible

cytokines that promote inflammation that include TNF-α, IL- 1β, IL-6, and others.

Neutrophils create an environment in which the toxins or the micro-organism

cannot survivor by releasing noxious chemicals from cytoplasmic granules such

as reactive oxygen species and nitrogen species. Both oxygen and glucose are

consumed when these noxious chemicals are released from the cytoplasmic

granules a process termed as respiratory burst (Nathan, 2002).

Prostaglandins act as short-lived localized hormones that are released when any

cell in the body is exposed to traumatic injury or any other form harmful stimuli.

Once present in the intracellular space prostaglandins can induce fever, pain and

15

inflammation (Rehman and Sack, 1999). Thromboxane a hormone activator

responsible for generating inflammatory response plays an active role in

regulation of blood vessel tone, clot formation and platelet aggregation (Rehman

and Sack, 1999). The inflammatory process is considered a complex process in

which once stimulated by injury or harmful stimuli leads to production of pro-

inflammatory mediators in a sequential manner whose early effect is pain, tissue

destruction then followed by healing and recovery (Fitzgerald, 2004).

2.2 Screening Models for Pain, Pyrexia and Inflammation

2.2.1 Screening Models for Pain

Pain models have been classified into: Thermal, electrical, mechanical and

chemical stimuli according to the kind of stimuli applied. The neuronal basis of

this models is not clearly known, however they are used to predict analgesic

activity of new substances (Parle and Yadav, 2013).

2.2.1.1 Test Based on Thermal Stimuli

2.2.1.1.1 Tail flick test using radiant heat

It is a model used for screening analgesic agents response in animals. When

thermal radiation is applied to the tail/paw of the animal it triggers the animal to

withdraw its tail/paw from the thermal source (Smith et al., 1943). Tail

withdrawal from the heat source is referred to as “tail flick latency‟‟. Time taken

for the animal to withdraw its paw/tail from the heat source in this model is timed

and recorded.

16

2.2.1.1.2 Hot plate test

This involves placing an animal in an open-ended cylindrical space with the

surface consisting of hot metal or boiling liquid by a thermode. The time taken

during paw licking and jumping from the heat source is timed and recorded, this

is called reaction time. Both licking of the paw and jumping from heat source are

considered as supra-spinally integrated response. The model is used for assessing

new analgesics agents (Parle and Yadav, 2013).

2.2.1.2 Test Based on Mechanical Stimuli

The hind paw and the tail are ideal sites for applying nociceptive mechanical

stimuli. In this model the tail or paw is jammed between two plane surfaces and

the pressure of increasing intensity is applied until the animal begins a response

behavior of withdrawing its tail or hind paw from the two planes. The vocal

reaction taken by the animal to withdraw tail or hind paw from two the plane

surfaces is timed and recorded (Green et al., 1951).

2.2.1.3 Test Based on Electrical Stimuli

2.2.1.3.1 Electrical stimulation of the tooth-pulp

Electrical current is applied to the tooth-pulp of the laboratory animal in this

model. This produces behavioral characteristic reaction such as head flick, biting,

chewing, and licking of the tooth pulp due to induction of pain. Time taken for the

above observation is timed and recorded (Parle and Yadav, 2013).

17

2.2.1.3.2 Electrical stimulation of the tail

In this model electrical current of increasing intensity is applied to the tail of the

rat or mice. This will generate some observed reflex movement in the tail with

increasing electrical currents. Time taken for the animal to withdraw its tail from

electrical stimuli is timed and recorded. Morphine or drugs falling in the same

class as morphine are effective in this model (Parle and Yadav, 2013).

2.2.1.4 Test Based on Chemical Stimuli

2.2.1.4.1 Formalin-Induced pain test

This model is sensitive to peripherally acting analgesics agents. Nociceptive

effect of formalin is considered biphasic. The initial phase is mediated by

serotonin (5-HT), histamine and kinin. The second phase is mediated by

prostaglandins (Turner, 1965). When formalin is injected into the hind paw of the

animal it elicits a painful behavior such as licking of the paw, bitting and lifting.

The time taken by the animal to lick, lift and bit the paw is timed and recorded

(Parle and Yadav, 2013).

2.2.1.4.2 Acetic acid-Induced pain test

In this model acetic acid or phenylquinone is used to induce pain in mice or rats

by injecting these irritants into the peritoneal cavity. The animal responds by

characteristics such as stretching of hind paw, turning of trunk, abdominal

musculature contraction and stomach touching the floor. This model is sensitive

to peripherally acting analgesics. This agent irritates the serous membrane and

18

therefore eliciting a stereotype behavior. The number of abdominal writhes within

a given period is timed and recorded (Parle and Yadav, 2013).

2.2.2 Screening Models for Pyrexia

2.2.2.1 Turpentine-Induced test

Turpentine induces fever slowly reaching its peak at 11th

hour after its injection.

Macrophages release protein signals such as interleukin-1 and interleukin-6 to

counteract the pyrogen (Leon, 2002). Interleukin acts on the temperature

regulating hypothalamus to increase body temperature. Stimulating the

hepatocystes to secrete acute phase proteins and increasing number of circulating

eosinophils and neutrophils begin to neutralize turpentine (Cartmell et al., 2000).

Turpentine injection into experimental animals induces a persistently high fever

pattern (Kuochung et al., 2006).

2.2.1.2 2,4-Dinitrophenol (DNP)-Induced test

2,4-Dinitrophenol (DNP) is known to induce febrile response through uncoupling

oxidative phosphorylation. This then would result in fast consumption of energy

without generating adenosine triphosphate causing the release of calcium from its

mitochondrial stores and subsequently prevent the uptake of calcium. This leads

to free intracellular calcium, muscle contraction and hyperthermia and Energy

proton gradient would then be lost as heat (Kumar et al., 2002).

19

2.2.2.3 Brewer’s Yeast-Induced test

Brewer‟s yeast induces pyrexia after injection subcutaneously into the

experimental animal. When subcutaneously injected into the experimental animal;

brewer‟s yeast binds to an immunological protein called lipopolysaccharide-

binding protein and these results to release of various endogeneous cytokine

factors such as phagocyte and interleukin-1; interleukin will act on T lymphocytes

and this will in turn result in hypothalamus producing prostaglandins (Dubey and

Maheswari, 2005). Brewer‟s yeast is also known to induce TNFα and

prostaglandins (Kluger, 1991).

2.3.3 Screening Models for Inflammation

2.3.3.1 Carragenaan-Induced Hind Paw Edema test

Carrageenan has widely been used as a harmful agent for inducing inflammation

in laboratory animals and for screening compounds possessing anti-inflammatory

activities. When injected into experimental animal this phlogistic agent produces

a severe inflammatory reaction (Marzouk et al., 2010). Freshly prepared solution

of carrageenan of between 1-3% is commonly used and is injected into

experimental animal at a dose of 50-150 ul (Naude et al., 2010). Carrageenan is

known to induce inflammation in two phases and is dependent upon age and

weight. It is known to induce edema in mice models in two phases and is

dependent on age and weight of the experimental animal (Vinegar et al., 1969).

The first phase (0-2 hours) is due to release of inflammatory mediators such as

20

serotonin and histamine, resulting in sensitization of central nociceptor neurons.

The lysosome, protease, prostaglandins and bradykinin are released majorly in the

second phase (D‟Amour et al., 1965). The second phase (2.5-5 hours) of edema is

sensitive to clinically used anti-inflammatory drugs. During the second phase

prostaglandins play a major role in inflammatory reaction and can stimulate the

nociceptors and thus induce pain (Dacie, 1958). The initial phase is due to release

of inflammatory mediators such as serotonin and histamine while the second

phase is mediated by lysosome, protease, bradykinin and prostaglandins (Vinegar

et al., 1969).

2.2.3.2 Xylene-Induced Ear Edema test

Application of xylene to the ear of laboratory animal induces a neurogeneous

edema that is partly related with substance P. Substance P is released from

neurons in the midbrain in response to stress and is an undecapeptide of central

and peripheral nervous system. When substance P is released from sensory

neurons in the periphery, it causes plasma extravasations and vasodilatation

resulting into inflammation (Kou et al., 2005).

21

2.2.3.3 Formalin-Induced Paw Edema Test

This technique is based on the ability of the drug to hinder inflammation produced

in the hind paw of the mice or rat after injection with formalin. Nociceptive effect

of formalin is biphasic, consisting of two phases. The first phase is mediated by

release of histamine, serotonin (5-HT), and kinin while the second phase is

majorly mediated by prostaglandins. The size of edema is measured using string

or a vernier caliper and recorded (Turner, 1965).

2.2.3.4 Cotton Pellet Granuloma Test

In this model, laboratory animal is subcutaneously implanted with cotton pellets

in the dorsal region to induce granulomas lesion. This model is used to asses

proliferative phase of inflammation. Inflammation involves the proliferation of

macrophages, neutrolphils and fibroblast which are responsible for granuloma

formation (Winter and Porter, 1957).

2.3 Conventional Management of Pain, Pyrexia and Inflammation

Non-steroidal anti-inflammatory drugs (NSAIDs) belong to an important class of

therapeutic agents that are prescribed all over the world for treatment of

orthopaedics conditions such as fractures, soft-tissue injuries and osteoarthritis

(Boursinos et al., 2009). The NSAIDs posses antipyretic, anti-inflammatory and

analgesic activities (Lascelles et al., 2007; Sparkes et al., 2010). These NSAIDs

for instant aspirin is known to acetylate the active site of COX enzyme called

Tyro 385 and Ser 530. Aspirin and arachidonic acid compete for the active

22

binding site in the COX enzyme. This competition result in displacement of

arachidonic acid from binding to the active site leading to inhibition of

prostaglandins synthesis (Rao and Knaus, 2008). Generally NSAIDs exhibit three

modes of inhibiting COX;

i. Competitive reversible inhibition that competes with arachidonic acid for

binding to the COX site for example ibuprofen and piroxicam.

ii. Competitive, reversible inhibitors, time dependent that bind COX active

site in the early phase to form a reversible enzyme inhibitor complex for

example diclofenac and flurbiprofen.

iii. Competitive, time independent irreversible inhibitors that form an enzyme

inhibitor complex for example aspirin.

During the early 18th

century, salicin a compound responsible for the synthesis of

acetylsalicylclic acid was discovered and isolated. In the 1990s there was the

emergence of selective COX-2 inhibitors and development of small molecule

therapies to manage pain, pyrexia and inflammation (Marnett, 2009). NSAIDs for

example indomethacin, diclofenac, ibuprofen and aspirin that show non-selective

COX inhibition, represents various types of NSAIDs prescribed to relieve short

term fever, pain and inflammation (Inotai et al., 2010). Analgesic drugs such as

diclofenac relieve pain peripherally/centrally by inhibiting cyclooxygenase

enzyme (COX-1 and COX-2). Inhibition of cyclooxygenase enzyme reduces the

production of pain mediators such as prostaglandins, substance P, histamine,

23

serotonin, and Bradykinin. Pain sensation is eventually reduced in the nociceptors

(Davies et al., 1984).

Antipyretic drugs such as aspirin, exhibit their antipyretic activity by inhibiting

cyclooxygenase enzyme (COX). Inhibition of cyclooxygenase enzyme results in

blockage of synthesis of prostaglandins consequently the levels of PGE2 in the

hypothalamic region is also reduced. Hence fever is reduced to normal by

resetting the hypothalamic regulatory center (Paya and Katzung, 1995).

Acetylsalicylic acid is known for its ability to irreversibly disable COX enzymes

and therefore blocking formation of prostaglandins (Maroon et al., 2010). They

also act by suppressing the production of pyrogenic cytokines such as TNF-α and

IL-β (Aronoff and Neilson, 2001). Mechanism of action of NSAIDs is mainly

through interaction with pro-inflammatory cytokines and interleukin such as IL-

1b, IL-1a, TNF-α and IL-6 (Ghosh et al., 1998). New generation of NSAIDs

which are selective in the inhibition of cyclooxygenase enzyme exhibit analgesic

and anti-inflammatory activities greater than non-selective NSAIDs and have

reduced side effects (Boursinos et al., 2009). Ability of NSAIDS to disrupt the

synthesis of prostaglandins during the inflammatory process makes them have

anti-inflammatory activity (Talalay, 2001).

Glucocorticoid is an important class of drugs belonging to corticosteroids and

over a number of years its therapeutic application in the management of allergy,

24

inflammation and pain has spread (Hawkey and Rampton, 1985). Conventional

NSAIDs that work as analgesic, antipyretic and anti-inflammatory agents are non-

specific cyclooxygenase enzyme blockers on the other hand selective NSAIDs are

specific in their inhibition by inhibiting COX-2 only (Vane et al., 1998). Non-

selective conventional NSAIDs block both constitutive COX-1 and inducible

COX-2 (Vane et al., 1998). COX 1 enzyme plays an important role in renal and

gastrointestinal blood flow as well platelet aggregation while COX 2, on the hand

plays a key role in pain and inflammation process (Rao and Knaus, 2008).

Eventually disrupting the synthesis of PGs. Prostaglandins (PGs) generally acts in

an autocrine or paracrine manner (Hawkey and Rampton, 1985).

Nonsteroidal anti-inflammatory drugs (NSAIDs) like aspirin, indomethacin,

ibuprofen, and naproxen are usually used for management of pyrexia, pain and

inflammation (Paya and Katzung, 1995). NSAIDs represent a class of drugs that

are used to prevent and treat postoperative pain (Luna et al., 2007). However,

NSAIDs manifest adverse effects for example impairment in bone/fracture

healing, prolonged healing and causing deterioration in the quality of bone

formed. Its negative effects are linked to the inhibition of prostaglandin which has

an effect on oesteoblasts (Boursinos et al., 2009). It has been shown that long

term use of predinisolone in experimental rabbits caused delayed fracture healing

in ulnar osteotomy (Waters et al., 2000). Recent studies by WHO indicates that

these conventional drugs used in the management of pain, inflammation and fever

25

manifest a lot of side effects after long term use for example gastric irritation,

ulceration, prolonged bleeding, renal failure, interstitial corrosion, and pruritis

(Robotin, 2006; Beg et al., 2011). At typical doses acetaminophen is effective in

management of fever and pain. It is a weak anti-inflammatory drug but appears to

have fewer side effects (Burke et al., 2006).

2.4 Use of Medicinal Plants in Management of Diseases

Traditional medicinal herbs for over centuries have served as potential source for

alternative medicine and the knowledge of herbal medicine has been passed on

from generation to generation. From ancient time, Indian, Chinese, Egyptian,

Greek, Syrian, African and Roman medicinal practices documented the use herbal

medicine for curing different diseases (Komboj, 2000). Many communities all

over the world have utilized Phytopharmaceuticals for centuries in curing an array

of ailments (Semwal et al., 2010). Herbal medicines from medicinal plants have

been utilized by societies as the principal source of curing a number of diseases

(Shinwari, 2010).

Therefore, medicinal plants are an assuming important role in people‟s wellbeing

(Vasundra and divya, 2013). Nature is endowed with complete store of remedies

to cure all diseases affecting the human being (Kokate et al., 2002). Nature is

also endowed with natural remedies inform of herbs, animal products and algae

capable of curing diseases without any side effects (Trease and Evans 1983).

26

Herbal medicine is an important source of discovering new therapeutic agents

with potential therapeutic capabilities. Investigation into plants used traditionally

to relieve inflammation, pain and fever should be seen as a step towards discovery

of novel analgesic, antipyretic and anti-inflammatory agents (Gupta et al., 2006).

According to WHO, a world population of between 75-80% still depends on

herbal medicines (Gupta et al., 2006). Secondary active constituents from plant

sources are directly used as therapeutic agent and can serve as lead molecule in

discovery and synthesis of new drugs (Komboj, 2000). In a study conducted in

2002 by the National Center for complementary and alternative medicine (CAM)

surveyed a total of 31,044 adults (Barnes et al., 2004). Found out that during the

last 12 months, 36% of respondents used some form of complementary and

alternative medicine (CAM) therapy (Barnes et al., 2004).

2.5 Herbal Management of Pain, Fever and Inflammation

2.5.1 Pain

Throughout history man has used different forms of therapy to relief pain.

Morphine for example was isolated from a medicinal plant Papaver somniferium

(De souza, 2011). The search of herbal plants with analgesic activities, used as

pain relievers should be viewed as a successful search for new pain relieving

drugs (Elisabetskey et al., 1995). Considering that most of anti-inflammatory,

analgesic, anti-malarial and anti-pyretic synthetic drugs such as aspirin, morphine,

27

artemisinin, atrophine and chloroquine were derived from the plant products

(Gupta et al., 2006).

White Willow Bark (Salix alba) has been used traditionally in management of

inflammation, mild feverish colds, influenza, headache, arthritic conditions and

muscle spasm (Vadivelu et al., 2011). Its anti-inflammatory, antipyretic and

antiuricosuric activities have been attributed to flavonoids, tannins and salicylates.

Salicin extract when taken at a dose level of 120-240 mg on daily basis can

reduce back pain in some patients (Vadivelu et al., 2011). Harpagophytum

procumbens (devil's Claw) has traditionally been used to manage symptoms

associated with pain such as, low back pain, osteoarthritis, rheumatoid arthritis,

gastrointestinal upset gout, myalgia, chronic low back pain and lumbago.

Inhibition of both cyclooxygenase and lypoxygenase inflammatory pathways

according to current research have been linked to herpagoside (Chrubasik et al.,

2004; Vadivelu et al., 2011).

The analgesic activities of ginger and ibuprofen showed no significant difference

in management of pain indicating that herbal extract do have antinociceptive

activity (Bliddal et al., 2000). The antinociceptive activity of ginger is assumed to

be through inhibition of COX 2 and lipooxygenase (Srivastava and Mustafa,

1989). Likewise, Mworia et al.,(2015) demonstrated antinociceptive activities

of leaf extract of Carissa spinarum on acetic acid-induced pain test in Swiss

28

albino mice models. The extract showed dose dependent response with 100 mg/kg

body weight having the highest inhibition percentage compared to 50 mg/kg body

weight. A similar study conducted by Kariuki et al. (2012) on root extract of

Toddalia asiatica exhibited antinociceptive activity in adult Swiss albino mice

when pain was induced using acetic acid, hot plate and tail flick test in laboratory

animals.

2.5.2 Pyrexia

Though medicinal plants from time immemorial have been used as a source of

antipyretic agents to manage fever, emergence of synthetic drugs however,

resulted in neglect of their use. But due to its availability, low cost and fewer side

effects herbal medicine is gaining its popularity (Sharma et al., 2010). Treatment

of fever dates back to 400 B.C years ago when Greek Hippocrates prescribed an

extract from the willow bark and leaves (Rao and Knaus, 2008). Tumeric

(Curcuma longa) is an ancient spice that has been used traditionally as condiment,

medicine, and flavouring agent. Its medicinal properties is attributed to the

compound curcuminoid. Curcuminoids, a major phytochemical compound in the

plant is believed to exert it antipyretic activity by inhibiting both 5-lipooxygenase

and cyclooxygenase enzymes (Chandra and Gupta, 1972).

According to Vadivelu et al. (2011), white Willow Bark (Salix alba) contains

heavy concentration of salicin and glycoside a precursor for aspirin. The

therapeutic application of Salix alba in traditional system of medicine has

29

spanned over centuries in management of headache, mild feverish colds, arthritic

conditions, influenza and inflammation. The antipyretic activities of Salix alba

has been atributed to flavonoids, tannins and salicylates present in the extract

(Vadivelu et al., 2011).

In a study conducted by Lapah et al. (2014) on aqueous extract of phragmanthera

capitata, the extract exhibited antipyretic activity in sprague dawley rats. Dose

level of 100 mg/kg and 200 mg/kg body weight extract reduced body temperature

significantly when compared to refference drug. In a similar study conducted by

Mwonjoria et al. (2011) on antinociceptive and antipyretic activities of

methanolic root extract of Solanum incanum (linneaus) in animal model using

brewer‟s yeast induced-pyrexia demonstrated significant antipyretic activities.

The antipyretic activities of 50 mg/kg and 100mg/kg body weight were

comparable to reference drug (Mwonjoria et al., 2011).

2.5.3 Anti-inflammatory

For centuries, mankind has used herbal medicine for relieving inflammation and

pain (Sen et al., 2010). Boswellia serrata is among many herbal plants used in

ayurvedic medicine and it has been used traditionally in management of pain

associated conditions such as osteoarthritis, tendonitis and rheumatoid arthritis.

Boswellic acids in Boswelia serrata in a number of laboratory studies about its

anti-inflammatory activity have shown to exert its effects by inhibiting

30

leukotrienes an inflammatory mediator (Vadivelu et al., 2011). The principle

components that have been associated with anti-inflammatory activities are

boswellic acid, α-boswellic and β-boswellic (Vadivelu et al., 2011). Tumeric

(Curcuma longa) is an ancient spice and condiment that has been used as herbal

medicine in India and china. Its medicinal value is attributed to curcuminoid.

Curcuminoids exert is activity by inhibiting 5-lipooxygenase and cyclooxygenase

enzymes resulting in a well established anti-inflammatory activity (Chandra and

Gupta, 1972).

Camellia sinensis commonly known as tea plant is an important herbal plant. The

leaves and buds produce tea, the most consumed beverage in the world. The anti-

inflammatory activity of green tea has been attributed to high content of

polyphenols/catechins and mainly epigallocatechin-3-gallate. Potential of green

tea in management of arthritis on collagen type-II-induced arthritis in mice has

been reported (Curtis et al., 2004). Likewise, study carried out by Mwangi et al.

(2014) on leaf extract of Caesalpinia volkensii and Maytenus obscura exhibited

anti-inflammatory activities in animal models. In addition, a study by Onasanwo

et al. (2012) on methanolic, hexane, dichloromethane and chloroform leaf extracts

of Anacardium occidentalis demonstrated strong anti-inflammatory and analgesic

activity in animal models. Furthermore, a study conducted by Ravi et al. (2009)

on methanolic bark extract of solanum nigrum berries demonstrated a dose

dependent response in reducing the inflamed hind paws of rats.

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2.6 Clutia abyssinica

2.6.1 Description and Distribution

Clutia abyssinica (Figure 2.1) belongs to the genus Clutia and family of

Euphorbiaceae. It is dioecious, erect, lax shrub that grows up to 4–5 m tall, with

brittle branches and glabrous to evenly hairy. Leaves alternate, simple and entire.

Stem is green tinged red-brown; leaves are green but turn red when old.

Inflorescence an auxiliary white-yellow flower, fruits are green. The plant is

usually harvested from the wild for its local medicinal use (Matu, 2008).

Clutia abyssinica (Figure 2.1) is found in Central Africa to Eritrea, Ethiopia and

through eastern Africa south to Zambia, Angola, Mozambique and South Africa.

The vernacular names used by the local communities include; Muhende

(shambaa), Turmanyat (Nandi), Kurbanyat (Kipsigis), Mrukuru (Pare),

Muthimamburi (Kikuyu) and Sambukwe by the Luhya (Kokwaro, 2009). Clutia

abyssinica is commonly found in dry forest, forest remnants, secondary forest and

wooded grassland on rocky hillsides, and riverine, evergreen thickets, at an

altitude level of between 700–3700 m above the sea level (Matu, 2008).

32

Figure 2. 1 Clutia abyssinica

Source: Turbo Subdivision in Uasin Gishu County

2.6.2 Medicinal uses

Methanolic leave extract of Clutia abyssinica contains saponins, anthraquinone,

phenolics, terpenoids, flavanoids and alkaloids (Jeruto et al., 2011). It is

extensively used as herbal medicine in South Nandi District Kenya and it has

been used traditionally to treat several ailments that include; malaria, colds, fever,

skin diseases, chest problems, cancer and infertility in humans (Jeruto et al.,

2011). The root and leaf extracts are drunk or rubbed on the head to treat

headache. Sap from the leafy twigs is used to treat chest pain, side pain and

33

shortness of breath. To cure malaria, influenza, colds, indigestion, intestinal

worms, and fever, root extract from the plant is drunk as a remedy (Matu, 2008).

The maceration of the crushed leaves of Clutia abyssinica given orally has been

traditionally used for the treatment of animal trypanosomosis (Mergia et al.,

2014). In Eastern Africa, soup from boiled roots is taken as a remedy for

headache, stomach-ache, enlarged spleen and kidney problems (Matu, 2008).

Clutia abyssinica is used to treat yellow fever, malaria and infections of the ear,

nose and throat (Njoroge and Bushman, 2006). In Marakwet community, boiled

root extract is used to treat erectile dysfunction and works synergistically with

other herbs (Kipkore et al., 2014).

34

CHAPTER THREE

MATERIALS AND METHODS

3.1 Collection and Preparation of Plant Materials

Fresh roots of Clutia abyssinica were collected randomly from Kaptebee village,

Turbo sub-county in Uasin Gishu county Kenya with the help of local herbalist

under accepted bio-conservation methods from its natural habitat (Appendix IV).

This process was conducted during the months of January-March 2016, a season

in which the local herbalist believed that the medicinal plant had its maximum

medicinal activity. A sample of fresh twigs with leaves was presented to a

taxonomist for botanical authentication and a sample voucher was deposited at the

National Museums of Kenya herbarium Nairobi. Sample roots were then cleaned

with tap water to remove any dirt and chopped into smaller pieces. The root

samples of Clutia abyssinica were completely air dried under shade for two

weeks. The samples were then packed into burlap sacks and transported to

Kenyatta University Biochemistry laboratory. The samples were then pulverized

into fine powder using laboratory electrical mill.

3.2 Extraction

A 400 g weight of powdered root sample was soaked in 1.3 litres of

dichloromethane (DCM) with regular agitation at an interval of 1 hour to

uniformly mix the sample within the first 10 hours and left to stand for 48 hours.

Filtration was performed using Whatman‟s filter paper No.1 (sigma-aldrich). The

35

filtrate was concentrated using rotary evaporator (Buchi India) at a temperature of

41°C under reduced pressure. The concentrate was kept in sealed containers at

low temperature 4°C until use in bioscreening experiments.

3.3 Experimental Design

3.3.1 Laboratory Animals

Swiss albino mice aged between 5-6 weeks, weighing between 18-25 g and

Wistar albino rats aged 8-9 weeks and weighing 120-140 g were used in this

study. All the experimental animals were housed in the animal house at Kenyatta

University in standard laboratory cages. The experimental animals were kept at

room temperature for 12 hours in darkness followed by 12 hours light cycles

throughout the entire experimental period. The animals were fed on rodent pellet

diet and water ad libitum. Wistar albino rats were utilized in testing for antipyretic

activity assay while for analgesic and anti-inflammatory activities assays, Swiss

albino mice were used. In this study ethical guidelines and procedures were

followed while handling the laboratory animals (Lapah et al., 2014).

3.4 Determination of Analgesic Effect

Analgesic activity of DCM root extract of Clutia abyssinica was determined

through acetic acid-induced pain in experimental animals following the procedure

described by Singh and Majumdar (1995) and Akuodor et al. (2011). The

experiment animals were grouped into 6 groups of five animals each. Prior to pain

induction and administration of the experimental doses, all the experimental

36

animals were fasted for 12 hours but were allowed access to water ad libitium.

Pain was induced by injecting 3% acetic acid solution at a dose of 20 ml/kg body

weight into the left side of the abdomen intraperitoneally. Immediately after

injection with acetic-acid, abdominal muscle constriction in the abdomen and

turning of body trunk of the laboratory animal was seen as an indication of pain.

The different groups were treated as follows; Group I (Normal control) was

administered with 10% dimethyl sulphoxide (DMSO) only but pain was not

induced. All the animals in group II (negative control) were induced with pain and

administered with 10% dimethyl sulphoxide (DMSO). Those in group III

(positive control) were induced with pain and administered with the reference

drug (diclofenac 15 mg/kg body weight). Group IV-VI were induced with pain

and administered with DCM root extract of C. abyssinica at dose levels of 50, 100

and 150 mg/kg body weight respectively. This design is summarized in table 3.1.

Table 3. 1: Treatment protocol for assessment of analgesic activity of

DCM root extract of C. abyssinica in Swiss albino mice

Group Treatment

I (Normal control) 10% DMSO only

II (Negative control) 3 % Acetic acid

III (Positive control) 3 % Acetic acid + diclofenac (15

mg/kg bw)

IV (Experimental group A) 3 % Acetic acid + C.abyssinica (50

mg/kg bw)

V (Experimental group B) 3 % Acetic acid + C.abyssinica (100

mg/kg bw)

VI (Experimental group C) 3 % Acetic acid + C.abyssinica (150

mg/kg bw)

37

Thirty minutes after different treatments were administered, each mouse in groups

II−VI was intraperitoneally (ip) injected with 3% acetic acid into the left side of

the abdomen at a dose of 20 ml/kg body weight to induce pain sensation except

those in group I, which were administered with the vehicle (10% DMSO) only.

Each mouse was then placed in a transparent observation box and the number of

abdominal constrictions (writhes) for each mouse was counted for 30 minutes

commencing 5minutes after intraperitoneal injection of acetic acid. The

percentage writhing inhibition was then calculated using the formula described by

Ezeja et al. (2011);

Where;

C- The vehicle- treated control group

T - Treated group value

3.5 Evaluation of Antipyretic Effect

Male Wistar albino rats were grouped into 6 groups of five rats each. Prior to

fever induction, rectal temperature of all the experimental animals were measured

using digital thermometer and recorded. All the experimental animals were fasted

for 12 hours before induction of fever and administration of the experimental

doses but were allowed access to water ad libitium. Turpentine solution was used

to induce fever and it was injected intraperitoneally at a dose of 20 ml/kg body

38

weight and the animals left for one hour (Kuochung et al., 2006). A raise in rectal

temperature of Wistar albino rats by 0.8°C after one hour was termed pyretic and

proceeded to be used in the assay.

One hour after fever induction, pyretic animals in groups IV-VI were

administered intraperitoneally with the three experimental doses of 50, 100 and

150 mg/kg body weight respectively. Pyretic animals in group III (Positive

control) were administered with aspirin (100 mg/kg body weight). Pyretic animals

in group II (negative control) were administered with 10% DMSO only.

Experimental animals in group I (normal control) were not induced with fever but

administered 10% DMSO only. This design is summarized in table 3.2.

Table 3. 2: Treatment protocol for antipyretic activity of DCM root extract

of C. abyssinica in wistar albino rats

Plant extract doses were prepared on the same day of experiment. All the

treatments were administered intraperitoneally. The rectal temperatures of rats

Group Treatment

I (Normal control) DMSO (10%)

II (Negative control) Turpentine (20%)

III (Positive control) Turpentine (20%) + Aspirin (100 mg/kg

bw)

IV (Experimental group A) Turpentine (20%) + C.abyssinica (50

mg/kg bw)

V (Experimental group B) Turpentine (20%) + C.abyssinica (100

mg/kg bw)

VI (Experimental group C) Turpentine (20%) + C.abyssinica (150

mg/kg bw)

39

were measured by inserting a well lubricated digital thermometer about 3cm into

the anus of the rats. The mean body temperature of Wistar albino rats was

recorded at 20 minutes intervals over 1 hour before turpentine injection and was

recorded as the baseline/initial temperature. Rectal temperature was measured and

recorded at an interval of one hour continuously for four hours after treatments.

Percentage inhibition of anal temperature was calculated by formula described by

Maria et al. (2014);

Where,

B - Rectal temperature at 1 hour after turpentine injection.

Cn - Rectal temperature after dose administration

3.6 Determination of Anti-inflammatory Effect

Edema was induced by injecting 0.05 ml of 1% (w/v) carragenaan solution into

the sub-plantar region of right hind paw of the Swiss albino mice according to

procedure described by Winter et al. (1962). Swiss albino mice (male) were

grouped into 6 groups of 5 animals (n=5) and treated as follows; each mouse in

group I (normal group) was administered with the vehicle (10% DMSO) only but

inflammation was not induced. All the mice in Group II (negative control) were

induced with inflammation and administered with the vehicle (10% DMSO) while

40

those in group III (positive control) were induced with inflammation and

administered with diclofenac (15 mg/kg body weight) one hour prior to

administration of 1% carragenaan solution into the sub-plantar region of right

hind paw of Swiss albino mice. Animals in groups IV-VI were induced with

inflammation and administered with the plant extract at 50 , 100 and 150 mg/kg

body weight respectively, one hour prior to administration of 1% carragenaan

solution. This design is summarized in table 3.3.

Table 3. 3: Treatment protocol for evaluation of anti-inflammatory activity

of DCM root extract of C. abyssinica in swiss albino mice.

Measurement of the hind paw diameter was carried out using a vernier calliper in

order to find out the diameter of right hind paw immediately before and after 1, 2,

3 and 4 hours following carragenaan injection. Percentage inflammation

inhibition was calculated according to the formula described by Jia et al. (2003);

Group Treatment

I (Normal control) DMSO (10%)

II (Negative control) Carragenaan (1%)

III (Positive control) Carrageenan (1%) + Diclofenac (15

mg/kg bw)

IV (Experimental group A) Carrageenan (1%) + C. abyssinica (50

mg/kg bw)

V (Experimental group B) Carrageenan (1%) + C.abyssinica (100

mg/kg bw)

VI (Experimental group C) Carrageenan (1%) + C.abyssinica (150

mg/kg bw)

41

Where;

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

Tt = Paw diameter after Treatment

3.7 Qualitative Phytochemical Screening

The dichloromethanolic (DCM) root extract of C. abyssinica was subjected to

qualitative phytochemical screening to determine the presence/absence of

different active secondary metabolites following the methodology described by

Trease and Evans (1983). The phytochemicals tested included flavonoids,

phenolics, saponins, alkaloids, cardiac glycosides, steroids and terpenoids. This

phytochemicals compounds are associated with analgesic, antipyretic and anti-

inflammatory activities.

3.7.1 Flavonoids

A quantity of 1 ml of the plant root extract was put in a test tube followed by

additional few drops of dilute NaOH solution. A golden intense yellow

precipitate indicated the presence of flavonoids.

3.7.2 Terpenoids (Salkowski test)

Dry crude extract of the plant (5 mg) was dissolved in 2 ml chloroform and 1 ml

of acetic acid added to it. Concentrated (1ml) sulphuric acid was carefully added

42

to the solution alongside the test tube. Formation of a reddish violet colour

indicated the presence of terpenoids.

3.7.3 Steroids

A volume of 2 ml of acetic anhydride was added to 5ml of the extract followed by

2 ml of concentrated sulphuric acid (H2SO4). A formation of violet to blue or

green confirmed the presence of steroids.

3.7.4 Cardiac glycosides

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

indicated the presence of cardiac glycosides.

3.7.5 Phenolics

A solution of 2ml of the extract was put in a test tube and 1ml of ferric chloride

solution added carefully. Formation of blue to the green color indicated the

presence of phenolics.

3.7.6 Alkaloids- Mayer’s test

A volume of 1 ml of plant sample extract, two drops of Mayer‟s reagents were

added along the sides of test tube. Appearance of white creamy precipitate

indicated the presence of alkaloids.

43

3.7.7 Saponins (Frothing test)

A volume of 1 ml of the root extract was dissolved in 10 ml of distilled water and

shaken vigorously for 15 minutes. A foam layer was formed at the top of the

mixture indicated the presence of saponins.

3.8 Data Management and Statistical Analysis

In this study, quantitative and qualitative data was obtained and recorded.

Quantitative data on the number of abdominal constrictions, the paw edema

diameter and the rectal temperature were obtained from experimental animals,

recorded and tabulated on a broad sheet using Ms Excel program. Data obtained

was then exported to statistical software (Minitab version 17.0) for analysis. Data

was subjected to descriptive statistics and results were expressed as Mean ±

Standard Error of the Mean (SEM). Results among the groups was analysed for

statistical significance using one way ANOVA followed Tukey‟s post hoc test for

pairwise comparison and separation of means. Data on percentage inhibition on

the number of abdominal constrictions was presented in form of a table while

percentage inhibition on rectal temperature and edema paw diameter was

presented in form of tables and graphs. A value of P ≤0.05 was considered

significant.

44

CHAPTER FOUR

RESULTS

4.1 Analgesic Activity of DCM Root Extracts of C. abyssinicaon acetic

acid-induced pain in Swiss albino mice

The DCM root extract of C. abyssinica demonstrated an analgesic activity

in acetic acid-induced pain in Swiss albino male mice by reducing the

number of abdominal writhes 30 minutes after the administration of the

plant extract (Table 4.1). The intraperitoneal administration of DCM root

extracts of C. abyssinica into Swiss albino mice at all the three dose levels

(50, 100 and 150 mg/kg body weights), reduced the number of abdominal

writhing by 49.3%, 38.6% and 33.95% respectively (Table 4.1). However,

diclofenac (reference drug) reduced the number of abdominal writhes by

46.51% (Table 4.1).

The analgesic effects of DCM root extract of C. abyssinica showed no

significant difference at the three doses and were comparable to diclofenac

(P> 0.05; Table 4.1). The DCM root extract of C. abyssinica, at the three

dose levels (50, 100 and 150 mg/kg body weight), reduced the number of

abdominal writhings in a reverse dose dependent manner (Table 4.1).

45

Table 4. 1: Analgesic activities of DCM root extracts of C. abyssinica on acetic

Acid-Induced pain in Swiss albino mice

Values are expressed as Mean ± SEM for five animals per group. Values with the

same superscript letter are not significantly different (one way ANOVA followed

by Tukey‟s test) (P>0.05).

Group Treatment % inhibition

Normal Control 10% DMSO 100.00±0.0a

Negative Control Acetic acid + DMSO 0.00±0.0c

Positive Control Acetic acid + Diclofenac

(15 mg/kg bw)

46.51±4.22b

Experimental group

A

Experimental group

B

Experimental group

C

Acetic acid + C.abyssinica

(50 mg/kg bw)

49.77±4.69b


(100 mg/kg bw)

38.60±4.33b


(150 mg/kg bw)

33.95±8.01b

46

4.2 Antipyretic Activity of DCM Root Extract of C. abyssinica on turpentine-

. Induced pyrexia in Wistar albino rats.

The DCM root extract of C. abyssinica showed antipyretic effects against

turpentine-Induced pyrexia in rats, which was indicated by reduction in rectal

temperature (Table 4.2; Figure 4.1). In the first hour after treatment with the

experimental doses, all the three doses (50, 100 and 150 mg/kg body weight),

lowered the elevated anal temperature by 0.81%, 0.68% and 1.14% respectively

(Table 4.2; Figure 4.1). On the other hand, aspirin lowered the anal temperature

significantly by 3.32%, indicating a stronger antipyretic activity than the all the

three experimental doses (P˂ 0.05; Table 4.2; Figure 4.1). However, the

antipyretic effects of all the three doses of the DCM root extract of C.abyssinica

were not significantly different (P>0.05; Table 4.3).

In the 2nd

hour, experimental animals administered with DCM root extract of C.

abyssinica at 50, 100 and 150 mg/kg body weight, reduced the anal temperature

by 2.54%, 1.84% and 2.48% respectively. The reference drug, at this hour,

reduced rectal temperature by 3.53% (Table 4.3; Figure 4.1). At this hour, a dose

of 50 mg/kg body weight demonstrated the highest fever reducing activity. Even

though the three experimental doses of C. abyssinica and the reference drug

reduced the rectal temperature at different percentages, their antipyretic effects

were not significantly different (P>0.05; Table 4.2; Figure 4.1).

47

In the third hour, the DCM root extract of C. abyssinica, at the three doses

exhibited antipyretic effects by reducing the elevated anal temperature by 2.38%,

2.88% and 3.34% respectively in a dose dependent manner. Reference drug

(aspirin) reduced elevated rectal temperature by 4.56% (Table 4.2; Figure 4.1).

The antipyretic effects of 150 mg/kg body weight and 100 mg/kg body weight

were comparable to the reference drug (aspirin) (P> 0.5; Table 4.2).

At the end of four hours of the test period, the DCM root extract of C. abyssinica

at all three dose levels, reduced the rectal temperature by 2.44%, 2.88% and

3.21% respectively in a dose dependent manner. The reference drug reduced

rectal temperature by 4.96% (Table 4.2; Figure 4.1). Administration of extract to

the experimental groups at all the three dose levels (50, 100 and 150 mg/kg body

weight), exhibited strong antipyretic activity but showed no significant difference

(P> 0.5; Table 4.2; Figure 4.1). The antipyretic effects of 100 mg/kg body weight

and 150 mg/kg body weight were comparable to the reference drug (aspirin) (P>

0.5; Table 4.2; Figure 4.1).

48

Table 4. 2: Antipyretic activities of DCM root extracts of C. abyssinica on Turpentine-Induced pyrexia in Wistar albino rats

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

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

Group Treatment % change in anal temperature in (°C) after dose

administration

0hr 1hr 2hr 3hr 4hr

Normal Control 10% DMSO 100.00±0.00

(0.00)

99.79±0.16bc

(0.21)

99.79±0.16bc

(0.21)

99.94±0.12c

(0.06)

99.95±0.31c

(0.05)

Negative Control Turpentine 100.00±0.00

(0.00)

101.08±0.51c

(-1.08)

101.39±0.5c

(-1.39)

100.82±0.2c

(-0.82)

100.61±0.4c

(-0.61)

Positive Control Turpentine + Aspirin (100

mg/kg bw)

100.00±0.00

(0.00)

96.68±0.19a

(3.32)

96.47±0.12a

(3.53)

95.44±0.29a

(4.56)

95.04±0.23a

(4.96)

Experimental

group A

Experimental

group B

Experimental

group C

Turpentine + C.abyssinica

(50 mg/kg bw)

100.00±0.00

(0.00)

99.19±0.56b

(0.81)

97.46±0.48a

(2.54)

97.62±0.53b

(2.38)

97.56±0.46b

(2.44)


(100 mg/kg bw)

100.00±0.00

(0.00)

99.32±0.54b

(0.68)

98.17±0.68ab

(1.83)

97.12±0.80ab

(2.88)

97.12±0.79ab

(2.88)


(150 mg/kg bw)

100.00±0.00

(0.00)

98.86±0.16b

(1.14)

97.52±0.34a

(2.48)

96.64±0.33ab

(3.34)

96.79±0.45ab

(3.21)

49

Figure 4. 1: Percentage change in rectal temperature of C. abyssinica on

.....................turpentine-Induced pyrexia in Wistar albino rats

50

4.3 Anti-inflammatory Activity of DCM Root Extract of C. abyssinica

on Carrageenan-Induced Inflammation in Swiss Albino Mice

Generally, the DCM root extract, at the three doses demonstrated an anti-

inflammatory activity on carragenaan-induced paw edema in Swiss albino

mice. This was indicated by reduction in the diameter of the hind paw

after administration of the three extract doses (Table 4.3 and Figure 4.2).

In the first hour, the DCM root extract of C. abyssinica, at 50, 100 and 150

mg/kg body weight, reduced the inflamed hind paw diameter by 0.88%,

1.88% and 3.30% respectively while diclofenac reduced by 2.21% (Table

4.3; Figure 4.2).

At this hour, the anti-inflammatory activities of DCM root extract of C.

abyssinica, at all the three extract doses, were significantly different

(P<0.05; Table 4.3). However, the anti-inflammatory activities of DCM

root extract of C. abyssinica, at 100 mg/kg body weight and diclofenac

were not significantly difference (P>0.05; Table 4.3). The anti-

inflammatory effects of 50, 150 mg/kg body weight and diclofenac were

statistically significant (P<0.05; Table 4.3).

In the 2nd

hour, the DCM root extract of C. abyssinica, at 50, 100 and 150

mg/kg body weight and diclofenac reduced the inflamed hind paw

diameter by 1.47%, 3.04%, 3.76% and 3.57% respectively thereby

51

demonstrating anti-inflammatory activities (Table 4.3; Figure 4.2). The

anti-inflammatory effects of 100 mg/kg and 150 mg/kg body weight were

not significantly different (P>0.05; Table 4.3). However, the anti-

inflammatory activity of DCM root extract of C. abysssinica at 50 mg/kg

body weight was significantly different from the anti-inflammatory

activities of the other two dose levels (100 mg/kg body weight and 150

mg/kg body weight) and diclofenac (P< 0.05; Table 4.3).

In the 3rd

hour, the DCM root extract at 50, 100 and 150 mg/kg body

weight and diclofenac reduced the inflamed hind paw diameter by 1.61%,

3.59%, 4.64% and 5.01% respectively (Table 4.3; Figure 4.2). The anti-

inflammatory effects of 100 mg/kg body weight and 150 mg/kg body

weight were not significantly different (P>0.05; Table 4.3; Figure 4.2).

The anti-inflammatory effects of 50 mg/kg body weight was significantly

different from diclofenac (P<0.05; Table 4.3). The anti-inflammatory

activity of 150 mg/kg body weight and diclofenac showed no significant

differences (P>0.05; Table 4.3; Figure 4.2).

In the fourth hour, the DCM root extract at 50, 100 and 150 mg/kg body

weight reduced inflamed hind paw diameter by 1.84%, 4.30% and 5.34%

respectively while diclofenac (reference drug) reduced inflamed hind paw

edema diameter by 5.35% (Table 4.3; Figure 4.2). The anti-inflammatory

52

activities of 150 mg/kg body weight and 100 mg/kg body weight were not

significantly different and were comparable to diclofenac (P> 0.05; Table

4.3; Figure 4.2). However, the anti-inflammatory effect of 50 mg/kg body

weight was significantly different from the other two doses and diclofenac

(P< 0.05; Table 4.3; Figure 4.2).

53

Table 4. 3: Anti-inflammatory activities of DCM root extracts of C. abyssinica on carragenaan-induced inflammation in Swiss albino

mice

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

(one way ANOVA followed by Tukey‟s test) (p>0.05). Percentage reduction in the size of the edema is given within parenthesis.

Group Treatment % change in paw diameter in (mm) after dose administration

0hr 1hr 2hr 3hr 4hr

Normal Control 10% DMSO 100.00±0.00

(0.00)

99.92±0.08d

(0.08)

99.92±0.08c

(0.08)

99.84±0.16d

(0.09)

99.92±0.08c

(0.08)

Negative Control Carrageenan 100.00±0.00

(0.00)

102.40±0.24e

(-2.4)

103.46±0.42d

(-3.46)

104.34±0.34e

(-4.35)

104.73±0.32d

(-4.74)

Positive Control Carrageenan + Diclofenac (15

mg/kg bw)

100.00±0.00

(0.00)

97.79±0.09b

(2.21)

96.43±0.24a

(3.57)

94.98±0.30a

(5.01)

94.64±0.35a

(5.35)

Experimental

group A

Experrimental

group B

Experimental

group C

Carrageenan + C.abyssinica

(50 mg/kg bw)

100.00±0.00

(0.00)

99.12±0.18c

(0.88)

98.41±0.28b

(1.47)

98.39±0.56c

(1.61)

98.16±0.6b

(1.84)

Carrageenan+ C. abyssinica

(100 mg/kg bw)

100.00±0.00

(0.00)

98.12±0.05b

(1.88)

96.96±0.22a

(3.04)

96.41±0.23b

(3.59)

95.70±0.22a

(4.30)

Carrageenan + C.abyssinica

(150 mg/kg bw)

100.00±0.00

(0.00)

96.70±0.14a

(3.30)

96.24±0.29a

(3.76)

95.35±0.21ab

(4.64)

94.64±0.18a

(5.34)

54

Figure 4. 2: Percentage change in hind paw diameter of C. abyssinica on

…………….carragenaan induced inflammation in Swiss albino mice

55

4.4 Qualitative Phytochemical Screening

The DCM root extract of C. abyssinica upon phytochemicals test revealed the

presence of the following compounds; terpenoids, alkaloids, flavonoids, steroids,

cardiac glycosides and saponins. However, Phenolics compounds were absent

(Table 4.4).

Table 4. 4: Phytochemical composition of DCM root extract of C. abyssinica

Present phytochemicals denoted by (+), absent phytochemicals denoted by (-)

Phytochemicals DCM root extract of C. abyssinica

Alkaloids +

Flavonoids +

Steroids +

Saponins +

Cardiac glycosides +

Phenolics -

Terpenoids +

56

CHAPTER FIVE

DISCUSSION, CONCLUSION AND RECOMMENDATIONS

5.1 Discussion

Various injuries and diseases are most often presented with fever, pain and

inflammation among other clinical signs. Although during the past years there has

been remarkable progress in medical science in management of pain, fever and

inflammation using conventional methods (Adedapo et al., 2009), there is need to

seek alternative methods to manage these indicators of ill health. Conventionally

used analgesic, antipyretic and anti-inflammatory drugs such as NSAIDs, opiods

analgesics and glucocorticoids pose a lot of side effects after long term use like

cardiac abnormalities, peptic ulcers, prolonged bleeding, hepatic failure and renal

failure among others effects leading to their limited use in clinical settings

(Castellsague et al., 2012).

Therefore, due to these limitations and other associated problems of these

conventional drugs, search for newer drugs from medicinal plants with analgesic,

antipyretic and anti-inflammatory activities in traditional system of medicine is

essential. In this regard, alternative medicines from natural sources such as

medicinal plants are an important option into discovery of novel drugs because

currently available conventional drugs are derived from traditionally used

medicinal plants (Robinon and Zhang, 2011).

57

The present study was designed to evaluate the analgesic, antipyretic and anti-

inflammatory potential of DCM root extract of C. abyssinica in animal models.

To evaluate the analgesic activity of the DCM root extract, acetic acid-induced

pain test was used to induce abdominal writhings in Swiss albino mice. Acetic

acid-induced pain test has widely been used for screening new analgesic agents

and it majorly involves cholinergic, histaminic peritoneal receptors, acetylcholine

and histamine mediators. It is also used to asses peripherally acting analgesics

(Collier et al., 1968; Fujiyoshi et al., 1989).

The DCM root extract of C. abyssinica in this study, exhibited analgesic activities

by reducing the number of abdominal writhes in acetic acid-induced pain in male

Swiss albino mice after treatment with the extract. After thirty minutes of test

period, the DCM root extract at 50 mg/kg body weight demonstrated the highest

analgesic activity by reducing the number of writhes by 49.77%, while 100 mg/kg

body weight decreased the number of writhes by 38.6% and the dose of 150

mg/kg body weight decreased the number of writhes by 33.95% in a reverse dose

dependent manner (Table 4.1).

These findings strongly suggest that the DCM root extract of C. abyssinica posses

peripherally or centrally analgesic property. Perhaps acting in a similar manner as

conventionally used therapeutic drugs that reduce the pain perception in

nociceptors by inhibiting production of prostaglandins. These results concur with

58

other research studies on the evaluation of analgesic activity of herbal plant

extracts using Swiss albino mice. Reduction in the number of abdominal

writhings in this study is in agreement with a study carried out by Mworia et al.

(2015) on analgesic properties of acetone leaf extracts of Carissa spinarum in

mice. The findings are also in line with studies by Kariuki et al. (2012) on

antinociceptive activity of Toddalia asiatica (L) Lam in models of central and

peripheral pain. The same effect in reduction of number of abdominal writhes was

observed by Elson et al. (2007) while examining the analgesic and anti-

inflammatory effects of DCM root bark extract of Cheiloclinium cognanum in

mice models.

The non-dose dependent analgesic activities of DCM root extract of C.

abyssinica, are in agreement with a study carried out by Gitahi et al. (2015) on

analgesic activities of root and leaf extract of Carissa edulis in Wistar albino rats.

The higher analgesic activity of a dose level of 50 mg/kg body weight than 150

mg/kg body weight (Table 4.1) might be attributed to the fact that when certain

limits in dose ranges are exceeded, the activity of that particular agent is reduced.

Also, high dose concentration of the extract could be taking longer time to diffuse

across peritoneum cavity in that compounds that are more concentrated takes

longer period to diffuse while those with lower concentration takes a shorter time.

Analgesic effects produced by a lower dose were comparable to the reference

59

drug, suggesting that the lower dose was more effective in managing pain induced

by acetic acid.

The dose range of (50, 100 and 150 mg/kg body weight) applied in bioscreening

of analgesic, anti-inflammatory and antipyretic activities of DCM root extract in

the assays were comparable to dose ranges used by Tayyaba et al. (2015) while

evaluating the antipyretic, anti-inflammatory and analgesic activity of methanolic

leaf extract of Acacia hydaspica (R) parker in laboratory animals. Similarly,

Kamau et al. (2016a) used these doses while evaluating anti-inflammatory

activity of methanolic leaf extract of Kigelia africana (lam) benth and stem bark

extract of Acacia hockii de wild in Swiss albino mice. Likewise, Elson et al.

(2007) used a similar dose range while studying the analgesic and anti-

inflammatory effects of DCM root bark extract of Cheiloclinium cognanum using

acetic acid-induced test, tail flick test and croton oil-induced ear edema test.

The analgesic activities of DCM root extract of C.abyssinica could be attributed

to one or more of phytochemical compounds present in the extract (Table 4.4).

Studies conducted on herbal plants by many researchers have linked presence of

secondary active metabolites such as flavonoids, saponins and alkaloids to

analgesic, antipyretic and anti-inflammatory activities among other properties

(Asfar et al., 2015; Kumar et al., 2015). An agent that lowers the number of

abdominal writhes is considered analgesic by inhibiting prostaglandin synthesis

60

(Alam et al., 2009), a peripheral mechanism of pain inhibition (Alam et al.,

2009). Flavonoids have the ability to disrupt synthesis of eicosanoids (Robak and

Gryglewski, 1996). Flavonoids also have the ability to reduce production of

arachidonic acid through inhibition of neutrophils degranulation (Tordera et al.,

1994). Besides flavonoids, alkaloids also have been associated with the ability to

inhibit pain perception (Uche et al., 2008). Alkaloids, for example berberine from

berberies in a skeletal based pyridine ring have shown strong anti-inflammatory

and antinociceptive activities (Kupeli et al., 2002). These mentioned

phytochemicals were confirmed in preliminary phytochemical screening of DCM

root extract of C. abyssinica (Table 4.4). Therefore, the phytochemicals found in

the extract might have antagonized peripheral mediators of pain and thereby

blocking transmission of pain.

Evaluation of antipyretic potential of DCM root extract of C. abyssinica was

tested using turpentine-induced pyrexia in Wistar albino rats. Fever can be

induced in laboratory animals using several agents collectively termed as

pyrogens such as lipopolysacharides (LPS), E-coli, amphetamines, sulphur,

brewer‟s yeast and turpentine. These agents are considered exogenous pyrogens

(Petrova et al., 1978; Vasundra and Divya, 2013). Turpentine is a clear flammable

liquid with pungent odour and bitter taste, refined from resin pine. Turpentine

causes tissue damage and induces acute phase response as well as fever (Wieslaw

et al., 1998). Turpentine injection into experimental animals induces a persistently

61

high fever pattern (Kuochung et al., 2006). Based on this information, turpentine

in the present study was chosen as an appropriate pyrogen.

The DCM root extract of C. abyssinica, in this study demonstrated antipyretic

activities against turpentine-induced pyrexia in Wistar albino rats (Table 4.2;

Figure 4.1). Findings from this study were comparable to other earlier research

studies on evaluation of antipyretic effects of extracts of herbal plants using

animal models. Similar study carried out by Nthiga et al. (2016) showed that the

methanolic stem extracts of Harrisonia abyssinica and Ladolphia buchananiii

staphf posses antipyretic activities against turpentine-induced pyrexia in Wistar

albino rats. Likewise, a study carried out by Kamau et al. (2016b) on antipyretic

properties methanolic leaf extracts of Kigelia africana (Lam) and Acacia hockii

de wild showed significant antipyretic activities in Wistar albino rats. In addition,

a study by Vasundra and Divya. (2013) observed similar trend in reduction of

elevated temperature while examining the antipyretic effects of ethanolic extract

of Asparagus racemosus using laboratory animals.

The DCM root extract of C. abyssinica, lowered the elevated rectal temperature

more in the fourth hour than (in the third hour, the second and the first hour)

(Table 4.2; Figure 4.1). This might be attributed to the fact that most of the

bioactive compounds may have not been completely absorbed across the

peritoneum cavity while in the third and fourth hours most of the bioactive

62

compounds may have been absorbed across the peritoneum cavity thereby causing

higher antipyretic activities. Lower percentage inhibition in first and the second

hours could also be attributed to the fact that the drug needed time to be

biotransformed into an antipyretic agent for example zidovudine used as an

antiviral drug (Jarko et al., 2008) . Another factor is that at certain dose ranges,

the active antipyretic phytochemical compounds in the extract were not sufficient

enough to lower the rectal temperature. However, the plant extract at 50 mg/kg

body weight lowered the rectal temperature in the second hour, then reducing

antipyretic activity in the third and fourth hours respectively (Table 4.2; Figure

4.1). This scenario could be ascribed to the fact that most of the bioactive

compounds might have been quickly metabolized and excreted because of the

small concentrations of active phytochemical compounds in the extract.

In the fourth and the third hours, the DCM root extract of C. abyssinica, showed a

dose dependent response in reducing the elevated rectal temperature in turpentine-

induced pyrexia in Wistar albino rats (Table 4.2; Figure 4.1). The dose dependent

response observed in this study are in agreement with studies by Mbiri et al.

(2016b), who observed a similar trend while examining antipyretic activities of

methanolic bark extract of T. brownii in rats. Likewise, Nthiga et al. (2016)

observed similar dose dependent response while evaluating the antipyretic

properties of methanolic stem bark extracts of H. abyssinica oliv and L.

buchananiii staphf against turpentine-induced pyrexia in Wistar albino rats. In

63

another related study carried out by Tosan et al. (2014) on antipyretic activities of

ethanolic roots extract of Abultilon mauritianum (jacq) on brewer‟s yeast-induced

pyrexia and lipopolysaccharide-induced pyrexia in Wistar albino rats, a similar

trend was observed.

From the data obtained in this study (table 2), the DCM root extract of C.

abyssinica reduced the rectal temperature significantly when compared to the

reference drug. The extract might have inhibited cyclooxygenase enzyme, a key

enzyme necessary for synthesis of prostaglandins, thereby reducing fever.

Moreover, the extract may have reduced the concentration of PGE2 in the

hypothalamus through its action on cyclooxygenase enzyme or by enhancing of

body‟s own antipyretic substances like vasopressin, IL 10 and arginine (Okokon

and Nwafor, 2010). However, other mechanism for blocking fever for example

blockage of voltage-independent sodium channels cannot be ignored.

Qualitative phytochemical screening of the extract revealed the presences of

bioactive compounds such as terpenoids, alkaloids, flavonoids, steroids, cardiac

glycosides and saponins (Table 4.4). Flavonoids have been found to inhibit

prostaglandin E synthase production and its transcription, resulting in blockage of

prostaglandin synthesis (Hamalainen et al., 2011). Flavonoids such as baicalin

have been shown to exert antipyretic activity by inhibiting TNF-α (Adesokan et

al., 2008). This study correlates with a study by Gitahi et al. (2015), that bioactive

64

compounds such as alkaloids, saponins and flavonoids could act synergistically to

produce an observed pharmacogical activity. Therefore alkaloids, flavonoids and

saponins present in the extract might have contributed to antipyretic activities.

The anti-inflammatory activity of DCM root extract of C. abyssinica was

evaluated using carragenaan induced-inflammation in male Swiss albino mice. It

is the most used primary test for screening new anti-inflammatory agents and

constitute a simple and routine animal model for evaluation of inflammation (Jain

et al., 2001; Paschapur et al., 2009). Carragenaan is obtained from a sea weed

known as carrageen moss. It is a sulphated polysaccharide (Necas and

Bartosikova, 2013). Carragenaan induces severe inflammatory reaction when

injected into the hind paw leg of the rat/mice (John and Nodine, 1999). It is also

used to access anti-inflammatory effect of natural product (Dirosa et al., 1971).

The DCM root extract of C. abyssinica in this study demonstrated strong anti-

inflammatory activities against carragenaan-induced edema in Swiss albino mice

by reducing the diameter of the edema (Table 4.3; Figure 4.2). There are some

correlations, between results obtained from this study and other studies on

evaluation of anti-inflammatory activity of medicinal plants using animal models.

Similar study conducted by Tukappa et al. (2015), on in vitro and in vivo anti-

inflammatory and toxicity studies of methanolic bark extract of Rumex vesicarius

linn using carragenaan-induced edema in Wistar albino rats exhibited anti-

65

inflammatory activities. In addition, a study conducted by Mwangi et al. (2015),

on anti-inflammatory properties of dichloromethane:methanolic leaf extract of

Caesalpinia volkensi and mytenus obscura in animal models demonstrated strong

anti-inflammatory activities. Therefore, it is possible that DCM root extract of C.

abyssinica inhibited production of prostaglandins that are responsible for

initiating inflammation, signifying that the extract may have acted on cyclo-

oxygenase enzyme, a key enzyme required for catalyzing production of

prostaglandins from arachidonic acid.

The DCM root extract of C. abyssinica, demonstrated a dose dependent response

on carragenaan-induced inflammation (Table 4.3; Figure 4.2). The dose

dependent response was observed from the first hour to the fourth hour.

Reduction in the size of the hind paw edema was more in the fourth hour than in

the third, second and the first hours respectively. These differences in percentage

inhibition in the fourth hour and the first hour might be attributed to the fact that

absorption within first hour was slow but consistent. In the third and fourth there

were sufficient quantity of active phytochemical compounds necessary to reduce

inflammation more than in the first and the second hours. Lower percentage

inhibition in the first and the second hours might be ascribed to the fact that the

plant extract needed time to be biotransformed into an anti-inflammatory agent

for example 5-flurouracil a conventional drug used as an anticancer agent (Jarko

et al., 2008). The plant extract at 150 mg/kg body weight exhibited higher anti-

66

inflammatory effects than the other two dose levels in the entire experimental

period. This can be explained in terms of sufficient quantity of bioactive

compounds in the dose level than the other two doses. Another factor is small

concentration of active phytochemicals in the other two doses might have been

quickly metabolized and excreted.

This observed anti-inflammatory activity, is in concurrence with a study carried

out by Mwangi et al. (2015), while evaluating anti-inflammatory properties of

dichloromethane:methanolic leaf extracts of Caesalpinia volkensi and Mytenus

obscura in animal models. Likewise, a study carried out by Mbiri et al. (2016a)

on anti-inflammatory activities of methanolic stem bark extract of T. brownii in

rats observed similar dose dependent response.

Preliminary phytochemical screening, of the DCM root extract of C. abyssinica,

showed the presence of phytochemical compounds such as terpenoids, alkaloids,

flavonoids, steroids and saponins (Table 4.4). Alkaloids, flavonoids, cardiac

glycosides and sterols have been reported to inhibit prostaglandin pathway

(Kumar et al., 2013). Moreover, flavonoids can inhibit cyclo-oxygenase,

phospholipase, TNF α and lipo-oxygenase enzyme of arachidonic acid

metabolism (Chi et al., 2001; Jang et al., 2002). According to Vasudevan et al.

(2007), saponins have been shown to inhibit inflammation and therefore acting as

an anti-inflammatory agent. Therefore flavonoids, alkaloids, terpenoids and

67

saponins present in the extract might have acted synergistically to bring forth anti-

inflammatory activities.

5.2 Conclusions

The following conclusions can be made from the study;

i. The DCM root extracts of C. abyssinica posse‟s analgesic activities.

Significant reduction in the number of abdominal writhings after treatment

with the extract and the reference drug indicates that extract inhibited pain

mediators.

ii. The DCM root extracts of C. abyssinica posse‟s antipyretic properties.

Reduction in elevated rectal temperature after treatment with the extract

and the reference drug indicates that the extract reduced fever.

iii. The DCM root extract of C.abyssinica posses anti-inflammatory

activities. Reduction in the diameter of the inflamed hind paw after

treatment with the extract and the reference drug indicates that the extract

disrupted synthesis of pro-inflammatory mediators.

iv. Phytochemical compounds that were confirmed in preliminary

screening of the DCM root extract of C.abyssinica have previously been

reported to posses analgesic, antipyretic and anti-inflammatory properties.

The DCM root extract of C. abyssinica, might prove useful in obtaining

agents capable of managing condition related to pain, fever and inflammation.

68

However, there is still need of identification of molecular targets of the

extract. Therefore, the present study scientifically confirmed the traditional

uses of C. abyssinica in management of pain, fever and inflammation hence

the null hypothesis was rejected.

5.3 Recommendations

i. The extract of Clutia abyssinica may be used as an alternative bio-

resource in development of analgesic, antipyretic and anti-inflammatory

agent.

69

5.4 Recommendations for Further Studies

i. Isolation and characterization of the bioactive constituent responsible

for the activity which may lead to discovery of compounds that might be

used as lead compounds in discovery of analgesic, antipyretic and anti-

inflammatory agents.

ii. Use of a different route in administration of the extract other than

intraperitoneally. The data from both routes of administration can be

compared in management of pan, fever and inflammation.

iii. Determining the expression levels of biomarkers cytokines for pain,

fever and inflammation. This will ensure that biomarkers responsible for

pain, fever and inflammation that are released during this process of pain,

fever and inflammation are quantified.

iv. Evaluation of acute and chronic toxicity to determine the safety of the

extract in animal models.

70

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APPENDICES

APPENDIX I: Mean percentage change in the number of abdominal writhes

. after administration of DCM root extract in Swiss albino mice

Doses Mean abdominal

withings

C. abyssinica root

extract

50 mg/kg bw 21.6±2.01

100 mg/kg bw 26.4±1.86

150 mg/kg bw 28.4±3.44

86

APPENDIX II: Effects of DCM root extract of C. abyssinica on turpentine-Induced pyrexia in Wistar albino rats

Group Treatment % change in rectal temperature in (°C) after dose

administration

0hr 1hr 2hr 3hr 4hr

Normal Control 10% DMSO 37.18±0.03b

37.1±0.29b

37.1±0.32b

37.18±0.35b

37.16±0.35b

Negative Control Turpentine 38.94±0.26a

39.36±0.34a

39.48±0.27a

39.26±0.24a

39.18±0.39a

Positive Control Turpentine + Aspirin (100 mg/kg

bw)

39.14±0.35a

37.84±0.34b

37.76±0.34b

37.36±0.41b

37.2±0.42b

Experimental

group A

Experimental

group B

Experimental

group C

Turpentine + C.abyssinica (50

mg/kg bw)

38.52±0.48ab

38.2±0.37ab

37.54±0.34b

37.6±0.49b

37.58±0.48ab


mg/kg bw)

38.08±0.48ab

37.82±0.32b

37.38±0.29b

36.98±0.31b

36.98±0.33b


mg/kg bw)

38.72±0.08a

38.28±0.11ab

37.76±0.16b

37.42±0.18b

37.48±0.20b

87

APPENDIX III: Effects of DCM root extract of C. abyssinica on carrageenan-Induced inflammation in Swiss albino

………………….mice

Group Treatment % change in rectal temperature in (°C) after dose

administration

0hr 1hr 2hr 3hr 4hr

Normal Control 10% DMSO 2.49±0.03b

2.47±0.03b

2.49±0.3c

2.48±0.03c

99.95±0.03c

Negative Control Turpentine 3.60±0.03a

3.69±0.05a

3.73±0.04a

3.76±0.05a

3.77±0.04a

Positive Control Turpentine + Diclofenac

(15 mg/kg)

3.53±0.09a

3.45±0.09ab

3.40±0.08b

3.35±0.09b

3.34±0.09b

Experimental

group A

Experimental

group B

Experimental

group C


(50 mg/kg bw)

3.47±0.08a

3.43±0.08ab

3.40±0.09b

3.40±0.09b

3.39±0.09b


(100 mg/kg bw)

3.62±0.04a

3.55±00.04ab

3.51±0.04ab

3.49±0.03ab

3.47±0.04ab


(150 mg/kg bw)

3.44±0.13a

3.33±0.12b

3.31±0.12b

3.28±0.12b

3.26±0.12b

88

APPENDIX IV: Source of the plant Turbo division. Courtesy of Google Earth maps

89

APPENDIX V: Analysis of analgesic activity of DCM root extract of .

. C.abyssinica in Swiss albino mice

Descriptive Statistics: pain

Variable C1 Mean SE Mean StDev

% inhibition 100 mg/kg 38.60 4.33 9.67

150 mg/kg 33.95 8.01 17.91

50 mg/kg 49.77 4.69 10.48

Negative control 0.000000 0.000000 0.000000

Normal control 100.00 0.000000 0.000000

Positive control 46.51 4.22 9.45

One-way ANOVA: writhings versus treatments Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

C1 5 26189 5237.7 51.24 0.000

Error 24 2453 102.2

Total 29 28642

Model Summary

S R-sq R-sq(adj) R-sq(pred)

10.1103 91.43% 89.65% 86.62%

Means

C1 N Mean StDev 95% CI

100 mg/kg 5 38.60 9.67 ( 29.27, 47.94)

150 mg/kg 5 33.95 17.91 ( 24.62, 43.29)

50 mg/kg 5 49.77 10.48 ( 40.44, 59.10)

Negative control 5 0.000000 0.000000 (-9.331808, 9.331808)

Normal control 5 100.0 0.0 ( 90.7, 109.3)

Positive control 5 46.51 9.45 ( 37.18, 55.84)

Pooled StDev = 10.1103

Tukey Pairwise Comparisons

Grouping Information Using the Tukey Method and 95% Confidence

C1 N Mean Grouping

Normal control 5 100.0 A

50 mg/kg 5 49.77 B

Positive control 5 46.51 B

100 mg/kg 5 38.60 B

150 mg/kg 5 33.95 B

Negative control 5 0.000000 C

90

Means that do not share a letter are significantly different.

APPENDIX VI: Analysis of antipyretic activity of DCM root extract of C.

………………….abyssinica in Wistar albino rats

Descriptive Statistics: 0 hour, 1 hour, 2 hour, 3 hour, 4 hour

Variable C1 Mean SE Mean StDev

0 hour 100 mg/kg 0.000000 0.000000 0.000000

150 mg/kg 0.000000 0.000000 0.000000

50 mg/kg 0.000000 0.000000 0.000000


Normal control 0.000000 0.000000 0.000000


1 hour 100 mg/kg 0.680 0.539 1.206

150 mg/kg 1.137 0.156 0.348

50 mg/kg 0.812 0.561 1.254

Negative control -1.077 0.513 1.146

Normal control 0.211 0.157 0.351


2 hour 100 mg/kg 1.829 0.676 1.512

150 mg/kg 2.480 0.343 0.768

50 mg/kg 2.525 0.476 1.065




3 hour 100 mg/kg 2.876 0.799 1.786

150 mg/kg 3.359 0.331 0.740

50 mg/kg 2.384 0.532 1.190




4 hour 100 mg/kg 2.879 0.788 1.761

150 mg/kg 3.203 0.446 0.997

50 mg/kg 2.436 0.463 1.036




One-way ANOVA: 1 hour versus C1 Analysis of Variance


C1 5 51.69 10.3373 13.00 0.000

Error 24 19.09 0.7954

Total 29 70.78

91

Model Summary


0.891872 73.03% 67.41% 57.86%

Means


100 mg/kg 5 0.680 1.206 (-0.144, 1.503)

150 mg/kg 5 1.137 0.348 ( 0.313, 1.960)

50 mg/kg 5 0.812 1.254 (-0.011, 1.635)

Negative control 5 -1.077 1.146 (-1.901, -0.254)

Normal control 5 0.211 0.351 (-0.612, 1.035)





C1 N Mean Grouping

Positive control 5 3.320 A

150 mg/kg 5 1.137 B

50 mg/kg 5 0.812 B

100 mg/kg 5 0.680 B

Normal control 5 0.211 B C

Negative control 5 -1.077 C


One-way ANOVA: 2 hour versus C1

Analysis of Variance


C1 5 81.16 16.2313 17.47 0.000

Error 24 22.30 0.9290

Total 29 103.45

Model Summary


0.963863 78.45% 73.96% 66.32%

Means


100 mg/kg 5 1.829 1.512 ( 0.940, 2.719)

150 mg/kg 5 2.480 0.768 ( 1.590, 3.369)

92

50 mg/kg 5 2.525 1.065 ( 1.635, 3.415)

Negative control 5 -1.392 1.170 (-2.281, -0.502)

Normal control 5 0.215 0.347 (-0.675, 1.104)





C1 N Mean Grouping


50 mg/kg 5 2.525 A

150 mg/kg 5 2.480 A

100 mg/kg 5 1.829 A B

Normal control 5 0.215 B C





C1 5 106.15 21.2295 21.25 0.000

Error 24 23.97 0.9989

Total 29 130.12

Model Summary


0.999464 81.58% 77.74% 71.21%

Means


100 mg/kg 5 2.876 1.786 ( 1.954, 3.799)

150 mg/kg 5 3.359 0.740 ( 2.437, 4.282)

50 mg/kg 5 2.384 1.190 ( 1.461, 3.306)

Negative control 5 -0.824 0.592 (-1.747, 0.098)

Normal control 5 0.002 0.270 (-0.920, 0.925)





C1 N Mean Grouping


93

150 mg/kg 5 3.359 A B

100 mg/kg 5 2.876 A B

50 mg/kg 5 2.384 B

Normal control 5 0.002 C





C1 5 108.24 21.647 18.34 0.000

Error 24 28.32 1.180

Total 29 136.56

Model Summary


1.08637 79.26% 74.94% 67.59%

Means


100 mg/kg 5 2.879 1.761 ( 1.876, 3.881)

150 mg/kg 5 3.203 0.997 ( 2.200, 4.206)

50 mg/kg 5 2.436 1.036 ( 1.433, 3.439)

Negative control 5 -0.610 1.084 (-1.613, 0.393)

Normal control 5 0.054 0.690 (-0.948, 1.057)





C1 N Mean Grouping


150 mg/kg 5 3.203 A B

100 mg/kg 5 2.879 A B

50 mg/kg 5 2.436 B




APPENDIX VII: Analysis of anti-inflammatory activity of DCM root

……………………extract of C.abyssinica in Swiss albino mice

Descriptive Statistics: 0 hour, 1 hour, 2 hour, 3 hour, 4 hour Variable C1 Mean SE Mean StDev

0 hour 100 mg/kg 0.000000 0.000000 0.000000

150 mg/kg 0.000000 0.000000 0.000000

50 mg/kg 0.000000 0.000000 0.000000


Normal control 0.000000 0.000000 0.000000

94


1 hour 100 mg/kg 1.8774 0.0518 0.1159

150 mg/kg 3.300 0.143 0.320

50 mg/kg 0.884 0.184 0.412


Normal control 0.0816 0.0816 0.1825


2 hour 100 mg/kg 3.036 0.223 0.498

150 mg/kg 3.761 0.287 0.641

50 mg/kg 1.470 0.276 0.616


Normal control 0.0778 0.0778 0.1740


3 hour 100 mg/kg 3.586 0.232 0.519

150 mg/kg 4.642 0.206 0.460

50 mg/kg 1.608 0.560 1.252


Normal control 0.1621 0.0993 0.2221


4 hour 100 mg/kg 4.305 0.221 0.494

150 mg/kg 5.359 0.182 0.408

50 mg/kg 1.839 0.602 1.347


Normal control 0.0806 0.0806 0.1803



Method

Null hypothesis All means are equal

Alternative hypothesis At least one mean is different

Significance level α = 0.05

Equal variances were assumed for the analysis.

Factor Information

Factor Levels Values

C1 6 100 mg/kg, 150 mg/kg, 50 mg/kg, Negative control, Normal

control, Positive

control



C1 5 99.726 19.9452 186.10 0.000

Error 24 2.572 0.1072

Total 29 102.298

Model Summary

95


0.327375 97.49% 96.96% 96.07%

Means


100 mg/kg 5 1.8774 0.1159 ( 1.5752, 2.1796)

150 mg/kg 5 3.300 0.320 ( 2.997, 3.602)

50 mg/kg 5 0.884 0.412 ( 0.582, 1.186)

Negative control 5 -2.401 0.537 ( -2.704, -2.099)

Normal control 5 0.0816 0.1825 (-0.2205, 0.3838)





C1 N Mean Grouping

150 mg/kg 5 3.300 A

Positive control 5 2.2090 B

100 mg/kg 5 1.8774 B

50 mg/kg 5 0.884 C

Normal control 5 0.0816 D

Negative control 5 -2.401 E

Means that do not share a letter are significantly different



C1 5 191.784 38.3568 101.56 0.000

Error 24 9.064 0.3777

Total 29 200.848

Model Summary


0.614545 95.49% 94.55% 92.95%

Means


100 mg/kg 5 3.036 0.498 ( 2.468, 3.603)

150 mg/kg 5 3.761 0.641 ( 3.194, 4.328)

50 mg/kg 5 1.470 0.616 ( 0.902, 2.037)


Normal control 5 0.0778 0.1740 (-0.4894, 0.6450)



96



C1 N Mean Grouping

150 mg/kg 5 3.761 A


100 mg/kg 5 3.036 A

50 mg/kg 5 1.470 B


Negative control 5 -3.464 D




C1 5 310.45 62.0897 118.64 0.000

Error 24 12.56 0.5234

Total 29 323.01

Model Summary


0.723431 96.11% 95.30% 93.92%

Means


100 mg/kg 5 3.586 0.519 ( 2.919, 4.254)

150 mg/kg 5 4.642 0.460 ( 3.975, 5.310)

50 mg/kg 5 1.608 1.252 ( 0.940, 2.276)


Normal control 5 0.1621 0.2221 (-0.5057, 0.8298)





C1 N Mean Grouping


150 mg/kg 5 4.642 A B

100 mg/kg 5 3.586 B

50 mg/kg 5 1.608 C

Normal control 5 0.1621 D

Negative control 5 -4.348 E

97





C1 5 384.61 76.9215 136.11 0.000

Error 24 13.56 0.5652

Total 29 398.17

Model Summary


0.751771 96.59% 95.88% 94.68%

Means


100 mg/kg 5 4.305 0.494 ( 3.611, 4.998)

150 mg/kg 5 5.359 0.408 ( 4.665, 6.052)

50 mg/kg 5 1.839 1.347 ( 1.145, 2.533)


Normal control 5 0.0806 0.1803 (-0.6132, 0.7745)





C1 N Mean Grouping

150 mg/kg 5 5.359 A


100 mg/kg 5 4.305 A

50 mg/kg 5 1.839 B


Negative control 5 -4.735 D


ANALGESIC, ANTIPYRETIC AND ANTI-INFLAMMATORY …

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