MUSA, UNEKWU-OJO.pdf - University Of Nigeria Nsukka

1

MUSA, UNEKWU-OJO

(PG/MSc/05/39544)

COMPARATIVE EFFECTS OF ESCULETIN AND SCOPOLETIN

ON PARACETAMOL- INDUCED LIVER DAMAGE IN RATS

Biochemistry

A DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR AWARD OF THE DEGREE OF MASTER OF SCIENCE (M.Sc) IN

PHARMACOLOGICAL BIOCHEMISTRY, UNIVERSITY OF NIGERIA, NSUKKA

Webmaster

2010

UNIVERSITY OF NIGERIA

2

COMPARATIVE EFFECTS OF ESCULETIN AND

SCOPOLETIN ON PARACETAMOL- INDUCED

LIVER DAMAGE IN RATS

BY

MUSA, UNEKWU-OJO

(PG/MSc/05/39544)

DEPARTMENT OF BIOCHEMISTRY


NSUKKA

AUGUST, 2010

3

TITLE

COMPARATIVE EFFECTS OF ESCULETIN AND

SCOPOLETIN ON PARACETAMOL-INDUCED

LIVER DAMAGE IN RATS

A DISSERTATION SUBMITTED IN PARTIAL

FULFILMENT OF THE REQUIREMENTS FOR AWARD

OF THE DEGREE OF MASTER OF SCIENCE (M.Sc) IN

PHARMACOLOGICAL BIOCHEMISTRY, UNIVERSITY

OF NIGERIA,

NSUKKA

BY

MUSA, UNEKWU-OJO

(PG/MSc/05/39544)

DEPARTMENT OF BIOCHEMISTRY


NSUKKA

SUPERVISOR: PROF. L. U. S. EZEANYIKA

SEPTEMBER, 2010

4

CERTIFICATION

Musa, Unekwu-ojo, a postgraduate student with registration number

PG/M.Sc/05/39544 in the Department of Biochemistry has satisfactorily completed the

requirements for course work and research for the degree of Master in Pharmacological

Biochemistry (M.Sc). The work embodied in this report is original and has not been

submitted in part or in full for any other diploma or degree of this or another university.

______________________ ____________________

Professor L.U.S. Ezeanyika Professor L.U.S. Ezeanyika

(Supervisor) (Head of Department)

____________________

External Examiner

5

DEDICATION

This work is dedicated to my beloved son, Enyo-ojo Daniel Oruma.

6

ACKNOWLEDGEMENTS

I would like to express my profound gratitude to God Almighty, for his mercy and

compassion during the course of this work.

I wish to express my sincere gratitude to my supervisor, Professor L.U.S.

Ezeanyika, for his guidance during this study. His ideas and advice have been most

valuable to me.

I am especially grateful to Dr. Joshua Parker Elijah whose patience and productive

criticism made it possible for the successful completion of this work.

I am also greatly indebted to Mr. Felix, Dr. Ode, Ginika, Geofree, Francis Egwu

and Abiola for their various contributions to make this work a success.

I express my sincere thanks to all my colleagues and the staff of the department of

Biochemistry, University of Nigeria, Nsukka, I wish to thank the Federal Polytechnic

Idah, for giving me this opportunity for further studies.

Finally, my warmest thanks are accorded to my family – Parents Dr. & Mrs. C. S.

Musa; brother, Achumugu Musa; sister, Ojone Musa; my dearest and beloved, Enyo-ojo

D. Oruma for their support, understanding and never-failing support during all these

years. Loving and humorous atmosphere is most refreshing. And to my husband Yahaya

Musa Oruma, I say a big thank you for all you have been.

7

ABSTRACT

A wide spectrum of pharmacological activities is displayed by coumarins and their

derivatives. Coumarins are bonzo-α-pyrones. They are active components of herbs used

for the treatment of various diseases such as cancers, hypertension, etc. Esculetin (6,7-

dihydroxy coumarin) is a phenolic antioxidant and scopoletin (7-hydroxy-6-

methoxycoumarin) is one of the most widespread coumarins in nature. The comparative

effects of esculetin and scopoletin on paracetamol induced liver damage in rats were

investigated. Aspartate aminotransferase, alanine aminotransferase, alkaline

aminotransferase and superoxide dismutase activities were assayed and total and

conjugated bilirubin level, malondialdehyde, albumin and globulin levels were analyzed.

The results showed that the enzyme levels of aspartate aminotransferase (AST), alanine

aminotransferase (ALT) and alkaline phosphatase (ALP) in the serum of the rats treated

with paracetamol (positive control) increased significantly (P<0.05) as compared with the

group administered distilled water (negative control). The result also showed that total

bilirubin and conjugated bilirubin level increased significantly (P<0.05) as compared with

the negative control. No significant difference (P>0.05) was observed in the activities of

the enzyme markers assayed between the test groups administered esculetin plus

paracetamol and scopoletin plus paracetamol. Significant difference (P<0.05) was

observed between the test groups and the positive control. No significant difference

(P>0.05) was observed between the rats of the test groups and the negative control. . No

significant difference (P>0.05) was observed between the level of serum proteins of the

test groups and the positive and negative controls. Significant difference (P>0.05) was

observed between the test groups and the positive control for the activity of superoxide

dismutase and malondialdehyde levels. No significant difference (P<0.05) was observed

between the test groups and the negative control for both the activity of superoxide

dismutase and malondialdehyde level. There was a significant difference (P<0.05)

between the ALP activities of the positive control and negative control. No significant

differences (P<0.05) were observed between the ALT of the esculetin plus paracetamol

and scopoletin plus paracetamol and the positive control. A significant difference

(P<0.05) in the level of AST was observed between the positive control and the test

groups administered esculetin plus paracetamol and scopoletin plus paracetamol. The

results from this study suggest that esculetin and scopoletin have similar antioxidant

effect on paracetamol induced liver damage in rats.

8

TABLE OF CONTENTS

Title Page .. .. .. .. .. .. .. .... .. i

Certification .. .. .. .. .. .. .... .. ii

Dedication .. .. .. .. .. .. .... .. iii

Acknowledgements .. .. .. .. .. .... .. iv

Abstract .. .. .. .. .. .. .. .... .. v

Table of Contents .. .. .. .. .. .. .... .. vi

List of Figures .. .. .. .. .. .. .... .. ix

List of Tables .. .. .. .. .. .. .... .. xi

CHAPTER ONE: INTRODUCTION

1.1 Coumarins --- --- --- --- --- ----- --- 1

1.1.1 Synthesis of Coumarins --- --- --- --- ----- --- 4

1.1.2 Antioxidant Property of Coumarins --- --- ---- --- --- 6

1.1.3 Esculetin --- --- --- --- --- --- ------ --- 8

1.1.3.1 Pharmacological Action of Esculetin --- --- ------ --- 9

1.1.4 Scopoletin --- --- --- --- --- --- ------ --- 10

1.1.4.1 Pharmacological Action of Scopoletin --- ------ --- 11

1.1.4.2 Uses of Scopoletin --- --- --- --- --- ------ --- 11

1.2 Acetaminophen (Paracetamol) --- --- --- ------ --- 12

1.2.1 Mechanism of Action --- --- --- --- --- ------ --- 15

1.2.2 Metabolism of Paracetamol --- --- --- --- ------ --- 15

1.2.3 Toxicity of Paracetamol --- --- --- ------ --- 17

1.3 The Liver --- --- --- --- --- ------ --- 18

1.3.1 Anatomy of the Liver --- --- --- --- --- ------ --- 18

1.3.2 Physiology/Function of the Liver --- --- --- ------ --- 18

1.3.3 Liver Intoxication/Damage --- --- --- --- ------ --- 20

1.3.4 Causes of Liver Damage --- --- --- ------ --- 21

1.3.5 Hepatotoxicity --- --- --- --- --- ------ --- 22

1.3.6 Mechanism of Liver Damage --- --- --- ------ --- 23

1.4 Liver Function Tests --- --- --- --- --- ------ --- 23

1.4.1 Aminotransferases --- --- --- --- --- ------ --- 25

1.4.2 Alanine Aminotransferase (ALT) --- --- --- ------ --- 26

9

1.4.3 Aspartate Aminotransferase (AST) --- --- --- ------ --- 26

1.4.4 Alkaline Phosphatase (ALP) --- --- --- ------ --- 27

1.5 Reactive Oxygen Species --- --- --- --- ------ --- 28

1.6 Antioxidants --- --- --- --- --- --- ------ --- 32

1.6.1 Glutathione --- --- --- --- --- --- ------ --- 34

1.6.1.1 Pharmacology and Mechanism of Action of Glutathione ------ --- 35

1.6.1.2 Pharmacokinetics of Glutathione --- --- --- ------ --- 36

1.7 Oxidative Stress and Radical Reactions --- ------ --- 36

1.7.1 Lipid Peroxidation and Free Radical --- --- ------ --- 37

1.7.2 Lipid Peroxidation and Tissue Damage --- --- ------ --- 39

1.7.3 Malondialdehyde --- --- --- --- --- ------ --- 41

1.7.4 Superoxide Dismutase --- --- --- ------ --- 42

1.7.5 Albumin --- --- --- --- --- --- ------ --- 42

1.7.6 Serum Proteins --- --- --- --- ------ --- 43

1.9 Aim and Objectives of Study --- --- --- ------ --- 44

CHAPTER TWO: MATERIALS AND METHODS

2.1 Materials --- --- --- --- --- --- ------ --- 45

2.1.1 Animals --- --- --- --- --- --- ------ --- 45

2.1.2 Chemicals/Reagent/Samples --- --- --- --- ------ --- 45

2.1.3 Equipment/Instrument--- --- --- --- --- ------ --- 45

2.2 Methods --- --- --- --- --- --- ------ --- 45

2.2.1 Experimental Design --- --- --- --- --- ------ --- 46

2.2.2 Preparation of Scopoletin Solution --- --- --- ------ --- 46

2.2.3 Preparation of Esculetin Solution --- --- --- ------ --- 47

2.2.4 Preparation of Paracetamol Solution --- --- --- ------ --- 47

2.2.5 Preparation of Liver Samples --- --- --- --- ------ .--- 47

2.2.6 Preparation of Serum Samples --- --- ------ --- 47

2.2.7 Assay Methods --- --- --- --- --- ------ --- 47

2.2.5.1 Assay of Aspartate Aminotransferase --- ------ --- 47

2.2.5.2 Assay of Alanine Aminotransferase Activity ------ --- 49

2.2.5.3 Assay of Alkaline Phosphatase Activity --- --- ------ --- 51

2.2.5.4 Assay of Bilirubin --- --- --- --- ------ --- 52

2.2.6.5 Superoxide Dismutase Activity --- --- --- ------ --- 54

2.2.6.6 Serum Protein --- --- --- --- ------ --- 55

10

2.2.6.7 Lipid Peroxidation Assay --- --- --- --- ------ --- 56

2.4 Statistical Analysis --- --- --- ------ --- 57

CHAPTER THREE: RESULTS

3.1 Comparative Effects of Esculetin and Scopoletin on

Alkaline Phosphatase (ALP) Activity --- --- ------ --- 58


Alanine Aminotransferase (ALT) Activity --- --- ------ --- 58


Aspartate Aminotransferase Activity (AST) Activity ------ --- 59


on Alanine Aminotransferase (ALT) Activity --- ------ --- 59

3.5 Comparative Effects of Esculetin and Scopoletin on Total

Bilirubin Level --- --- --- --- --- ------ --- 60

3.6 Comparative Effects of Esculetin and Scopoletin on Super-

oxide Dismutase Activity --- --- --- --- ------ --- 60


Malondialdehyde Level --- --- --- ------ --- 61

3.8 Comparative Effects of Esculetin and Scopoletin on Albumin

Level --- --- --- --- --- ------ --- 61

3.9 Comparative Effects of Esculetin and Scopoletin on Globulin

Level --- --- --- --- --- ------ --- 62

CHAPTER FOUR: DISCUSSION

4.1 Discussion --- --- --- --- --- ------ --- 72

4.2 Conclusion --- --- --- --- --- ------ --- 78

4.3 Indications for Further Studies --- --- ------ --- 79

REFERENCES --- --- --- --- --- ------ --- 80

APPENDICES --- --- --- --- --- ------ --- 91

11

LIST OF FIGURES

Fig. 1 Structures of Parent Coumarin, Scopoletin and Esculetin --- ------ 3

Fig. 2 Retrosynthesis of Tetrasubstituted Coumarins by RCM --- ------ 4

Fig. 3 Synthesis of a Coumarin by Ring Closure Metathesis (RCM) ------ 5

Fig. 4 Pechmann Coumarin Synthesis --- --- --- --- ------ 5

Fig. 5 Synthesis of Coumarin from Cinnamic Acid --- ------ 6

Fig. 6 Structure of Esculetin --- --- --- --- --- ------ 8

Fig. 7 Structure of Scopoletin --- --- --- --- ------ 10

Fig. 8 Structure of N-Acetyl-p-aminophenol --- --- --- ------ 13

Fig. 9 Metabolism of Paracetamol --- --- --- --- ------ 16

Fig. 10 Implication of ROS and RSD and of the Oxidative Stress

(OS) in the Cell Damage --- --- --- --- --- ------ 30

Fig. 11 Overview of Formation of Reactive Oxygen Species (ROS) ------ 30

Fig. 12 Reduction of Glutathione --- --- --- --- --- ------ 34

Fig. 13 Structure of Glutathione --- --- --- --- --- ------ 35

Fig. 14 Mechanism of Non-enzymatic Lipid Peroxidation --- ------ 38

Fig. 15 Comparative Effects of Esculetin and Scopoletin on

Alkaline Phosphatase (ALP) Activity --- --- --- ------ 63


Alanine Aminotransferase (ALT) Activity --- --- --- ------ 64


Aspartate Aminotransferase (AST) Activity --- --- ------ 65


the Conjugated Bilirubin Level --- --- --- --- ------ 66

Fig. 19 Comparative Effects of Esculetin and Scopoletin

on Bilirubin Level --- --- --- --- --- --- ------ 67

Fig. 20 Comparative Effects of Esculetin and Scopoletin on Super-

oxide Dismutase Activity Level --- --- --- --- ------ 68


Malondialdehyde Level --- --- --- --- --- ------ 69

12


Albumin Level --- --- --- --- --- --- ------ 70


Globulin Level --- --- --- --- --- --- ------ 71

13

LIST OF TABLES

Table 1 Structure of Coumarins used as Antioxidants --- --- ------ 7

Table 2 Several Characteristics of Isolated Substances --- --- ------ 8

Table 3 Reactive Oxygen Species --- --- --- --- ------ 29

Table 4 Serum Normal Values of AST Activity in Humans --- ------ 49

Table 5 Serum Normal Values of ALT Activity in Humans --- ------ 51

Table 6 Lipid Peroxidation Assay Procedure --- --- --- --- ------ 57

14

CHAPTER ONE

INTRODUCTION

From the beginning of human existence, man has familiarized himself with plants

and used them in a variety of ways throughout the ages. In search of food and to cope

successfully with human suffering, primitive man began to distinguish those plants

suitable for nutritional purpose from others with definitive pharmacological action. This

relationship has grown between plants and man such that many plants came to be used as

drugs (Abbasi et al., 2009).

A wide spectrum of pharmacological activities is displayed by coumarins and

their derivatives. Coumarin derivatives such as warfarin are used widely as anticoagulants

(Marcias et al., 1999). They are also used as rodenticides due to their ability to cause fatal

haemorrhaging (Basaran et al., 2007).

Natural products are typically secondary metabolites, produced by organisms in

response to external stimuli such as nutritional changes, infection and competition (Ojala,

2001). Natural products produced by plants, fungi, bacteria, insects and animals have

been isolated as biologically active pharmacophores. Approximately one-third of the top-

selling drugs in the world are natural products or their derivatives often with

ethnopharmacological background (Ojala, 2001). Moreover, natural products are widely

recognized in the pharmaceutical industry for their broad structural diversity as well as

their wide range of pharmacological activities.

Secondary metabolites, in natural products chemistry are considered as waste

products of primary metabolic processes (biosynthesis, biodegradation and other energy

conversions of intermediary metabolism) in biological systems (Arogba, 2008).

Plants are able to synthesize and accumulate a vast array of primary and

secondary chemicals useful for the plant itself for protecting against environmental stress

factors due to their specialized biochemical capabilities. These compounds have made

many plants useful also for humans for instance as spices, medicines, etc (Kostova,

2005).

1.1 Coumarins

Natural coumarins, like other unsaturated lactones, may exert various effects on

living organisms, both in plants and animals. Coumarins owe their class name to

coumaron the vernacular name of the tonka bean, from which coumarin itself was

15

isolated in 1820. Coumarin and the other members of the coumarin family are benzo--

pyrones (Daud et al., 2008). They may also be found in nature in combination with

sugars, as glycosides (Wikipedia, 2008). Coumarin (Fig. 1) (anhydride of O- coumaric

acid) is a white, crystalline lactone, obtained naturally from several plants such as tonka

bean, lavender, sweet clover grass, strawberries, citrus and cinnamon or produced

synthetically from the amino acid, phenylalanine (SCCP, 2006; Arogba, 2008).

It has a characteristic odour like that of vanilla beans and is used for the

preparation of flavours and fragrances. The coumarin nucleus (benzo-2-pyrone) is derived

from cinnamic acid (phenylacrylic skeleton) in the biosynthesis of coumarin . The

hydroxyl group attached to coumarin structure at 7 position is important in the

biosynthetic pathway (Iscan et al., 1994). Umbelliferone (7-hydroxy coumarin), esculetin

(6,7– dihydroxy coumarin, scopoletin (7-hydroxy-6-methoxycoumarin), are the

widespread coumarins in nature (Ojala, 2001).

Coumarin derivatives are used as therapeutic anticoagulants, UV absorbers

(synthetic 7–hydroxyl coumarins) (Table 2) and in the synthesis of certain drugs. They

are widely used as fragrance ingredients (SCCP, 2006).

The coumarins can be roughly categorized as follows;

Simple – these are hydroxylated, alkoxylated and alkylated derivates of the parent

compound, coumarin, along with their glycosides.

Furano coumarins – these compounds consist of a five-membered furan ring

attached to the coumarin nucleus, divided to linear and angular types with

substituent at one or both of the remaining benzenoid positions.

Pyranocoumarins – members of this group are analogous to the furanocoumarins,

but contain a six-membered ring.

Coumarins substituted in the pyrone ring (Kostova, 2005).

Like other phenylpropanoids, coumarins arise from the metabolism of

phenylalanine via cinnamic acid, (Fig. 5) p–coumaric acid. The specificity of the process

resides in the 2-hydroxylation, followed by photocatalysed isomerisation of the double

bond and spontaneous lactonisation. In some cases, glycosylation of cinnamic acid

occurs, precluding lactonisation (Kostova, 2005). The primary site of synthesis of

coumarins is suggested to be the young, actively growing leaves, with stems and roots

playing a comparatively minor role. They occur at the highest levels in the fruits,

followed by the roots and stems (Ojala, 2001).

16

Fig. 1: Structures of Parent Coumarin, Scopoletin and Esculetin

The distribution of biologically active coumarins in a wide range of plants seems

to correlate with their ability to act as phytoalexins, i.e they are formed as a response to

traumatic injury (Wikipedia, 2008).

Coumarins have a variety of bioactivities including anticoagulant, estrogenic,

dermal, photosensitizing, antimicrobial, vasodilatory, molluscidal, antihelmintic, sedative

and hypnotic, analgesic and hypothermic activity (Ojala, 2001).

Coumarin has a wide variety of uses in industry, mainly due to its strong fragrant

odour (Tkach and Lukyanets, 1992). Its uses include that of a sweetener and fixative of

perfumes (e.g. 3,4-dihydrocoumarin), an enhancer of natural oils, such as lavender, a food

additive in combination with vanillin, a flavour/odour stabilizer in tobaccos, an odour

masker in paints and rubbers and In addition, it is also used in electroplating to reduce the

porosity and increase the brightness of various deposits such as nickels. Coumarin

compounds are suggested to be beneficial for the plants themselves as natural

biocontrolling antipathogenic compounds, and for human beings as dietary supplements

on the basis of their mild antimicrobial and anti-inflammatory effects (SCCP, 2006).

Coumarins are active components of herbs used for the treatment of various

diseases (Moini et al., 2002).

H

Coumarin

O HO

H3CO

H

O

Scopoletin

O HO

HO

O

Esculetin

O H

H

O

17

1.1.1 Synthesis of Coumarins

Coumarins were first synthesized via the Perkin reaction in 1868. In the early

1900s, the Knoerengel reaction emerged as an important synthetic method to synthesize

coumarin derivatives with carboxylic acid at the 3 position. Other synthetic methods for

coumarins have been reported including the Pechmann synthesis (Fig. 4), Reformatsky

and Witting reactions (Shaabani et al., 2009).

Coumarins represent an important class of compounds due to their importance in

biological systems. There have been a wide variety of non catalytic methods to synthesize

coumarins, such as Pechmann condensation of -ketoesters. This method generally

requires strong acids and high reactions temperatures. Pechmann reaction (Fig. 4) is the

most widely applied method for coumarin synthesis since it proceeds from simple starting

materials (phenols and a -ketone sugar) and gives good yields of coumarins with

substitution in either pyrone or benzene ring or both (Joel et al.,2005). Coumarins can be

obtained in high yields upon reaction of ethyl acetoacetone with 1,3-dihydroxy benzene

(resorcinn) with sulphuric acid as solvent and condensing agent. Aluminum chloride and

trifluoroacetic acid also act as condensing agents for the synthesis of coumarins (Joshi

and Chudasama, 2008).

3-Subsituted coumarins can be synthesized by a rhodium-catalyzed carbonlylation

of alkynyl phenols. Nickel-catalyzed cross-coupling of coumarin 4-phosphonates is an

alternative method to prepare a variety of 4-substituted coumarins. The ring closure

metathesis substrate could be easily prepared by acylation of the corresponding phenol

(Figs. 2 and 3). The required alkynyl phenols may be accessible from a ketone starting

material such as acetophenone (Arnab et al., 2003).

Fig. 2: Retrosynthesis of Tetrasubstituted Coumarins by Ring Closure Metathesis

(RCM)

O

RCM

R`

O O

O

Acylation

OH OH

R R R R

18

The synthesis began with commercially available 2-propenylphenol. The acylation

step proceeds smoothly with acrolyl chloride (Arnab et al., 2003).

Fig. 3: Synthesis of a Coumarin by Ring Closure Metathesis (RCM)

Laboratory Synthesis of Coumarins

Fig. 4: Pechmann Coumarin Synthesis

Benzo-2-pyrone nucleus of the simple coumarins is derived from the

phenylacrylic skeleton of cinnamic acids (Fig. 1).

AlCl3

R

O

OH

+

O

Eto

O O

R

OH O OH O O

CH3

Et3N/CH2Cl2 CH2Cl2

(10mM)

O O

19

Fig. 5: Synthesis of Coumarin from Cinnamic Acid (Organic Chemistry.org 2008)

Keys:

a = Orthohydroxylation

b and c = trans-cis isomerization of the side reaction double bond

d = Lactonisation

1.1.2 Antioxidant Property of Coumarins

Dihydroxycoumarins are better antioxidants than monohydroxy-coumarins. The 6,

7-dihyroxy-4-methylcoumarin is a free radicals scavenger too. 7, 8-dihydroxycoumarin

showed the most promising antioxidant properties. If the two OH groups are in ortho-

positions, the corresponding dihydroxycoumarin can decrease the lipid peroxidation by

scavenging O2 and ROO. Coumarins with antioxidant properties are shown in Table 1.

Coumarin and its derivatives are widely used in medical practice (Traykova and

Kostova, 2005). The oxidative behaviour of these compounds is strongly determined by

all aspects of their molecular structure, and on the source of oxidative stress. Coumarin

b

COOH

Cinnamic acid

a

COOH

OH

COOH

O

-glucose

O

-glucose

COOH

O-coumaric acid

hv or enzyme

c

O-coumaric acid-ß-D-Glucoside O

-coumaric acid-ß-D-Glucoside

O

Coumarin

d

O

20

itself has a low absolute availability in the human body due to first-pass hepatic catalytic

conversion into 7-hydroxycoumarin followed by glycoronidation. The hydroxycoumarins

are typical phenolic compounds and therefore act as potent metal chelators and/or free

radical scavengers which may result in a powerful antioxidant effect (Traykova and

Kostova, 2005).

Table 1: Structures of Coumarins used as Antioxidants

Compound R3 R4 R5 R6 R7 R8

1 Coumarin H H H H H H

2 7-hydroxycoumarin H H H H OH H

3 7-methylcoumarin H H H H Me H

4 4-methylcoumarin H Me H H H H

5. 4-methyl-7-

hydroxycoumarin

H Me H H OH H

6 3-hydroxyscopoletin OH H H OMe OH H

7 3,7-dihydroxycoumarin OH H H H OH H

8 4-methyl-6,7-

dihydroxycoumarin

H Me H OH OH H

9 7,8-dihydroxy-4-

methylcoumarin

H Me H H OH OH

10 6,7-dihydroxycoumarin

(Esculetin)

H H H OH OH H

11 Esculin H H H OGI OH H

12 4-hydroxycoumarin H OH H H H H

13 7,8-diacetoxy-4-

methylcoumarin

H Me H H Acetoxy Acetoxy

14 7,8-diacetoxy-6-

methylcoumarin

H H H Me OH OH

15 3-hydroxycoumarin OH H H H H H

16 3-aminocoumarin NH2 H H H H H

17 3-acetylaminocoumarin Acetylamino H H H H H

18 Coumarin-3-carboxylic

acid

COOH H H H H H

Traykova and Kostova, 2005

21

Table 2: Several Characteristics of Isolated Substances Fluorescence in UV-Light

Name At

366nm

+NH3 +KOH Colour of

crystals

Good in Bad in

Umbelliferone Blue blue bright

blue

colourless alcohols Benzene,

ethers

Esculetin Blue-

violet

Green bright

blue

colourless alcohols Benzene,

ethers

Esculin Blue Blue blue colourless alcohols Benzene,

ethers

Scopoletin Blue Blue blue yellowish alcohols

CHCl3

Benzene,

ethers

Scopolin Blue Blue blue yellowish alcohols Benzene,

ethers

Traykova and Kostova, 2005

1.1.3 Esculetin

Fig. 6: Structure of Esculetin

Esculetin also known as 6, 7-dihydroxycoumarin (Fig. 6) is a phenolic antioxidant

compound found in cassava (Manihot esculenta Crantz), Cichorium intybus and

Bongavilra spectabillis (Gilani et al., 1998). Esculetin can also be isolated from many

plants such as Artemisia spp, Citrus limona and Euphobia lathyns (Leung et al., 2005). It

appears in its physical state as a yellow crystalline powder. It is slightly soluble in water

and weakly acidic. Its melting point is between 265–270oC. Esculetin is stable under

normal conditions. It is a 7-hydroxycoumarin and can be used to absorb ultraviolet rays

in sunscreen cosmetics and also used in the synthesis of drugs especially anticancer drugs

(Kostova, 2005). Esculetin has been reported to protect cells from injury by linoleic acid

hydroperoxide in cell incubated in medium supplemented with linoleic acid

hydroperoxide (Kaneko et al., 2003).

Synergistic protection against cytotoxicity induced by linoleic acid hydroperoxide

with alpha-tocopherol was provided by esculetin and 4-methyl esculetin.

HO

HO

O O

22

Esculetin has been shown to have a protective effect on paracetamol induced liver

damage (Gilani et al., 1998). Oral administration of paracetamol (640mgkg-1

) produced

liver damage in rats as manifested by the rise in serum levels of alkaline phosphatase,

alanine aminotransferase and aspartate aminotransferase (Gilani et al., 1998).

Pretreatment of rats with esculetin (6mgkg-1

) prevented the paracetamol–induced rise in

serum enzymes indicating that esculetin possesses anti-hepatotoxic activity (Gilani et al.,

1998).

Esculetin plays a role in regulating pentose phosphate pathway activity in

Artemisia, Herba alba during seed germination. Presence of esculetin during germination

of seeds and seedlings of Artemisia, Herba alba have influenced both activities and

isoenzymes patterns of Glucose–6–phosphate dehydrogenase (G6PDH) and 6-phospho-

gluconate dehydrogenase (6PGDH) (Farouk and Al-Charchatchi, 2006).

Esculetin was reported to inhibit the lipoxygenase more strongly than the

cyclooxygenase (Traykova and Kostova, 2005). In mature adipocytes, esculetin caused a

time and dose-related increase in adipocyte apoptosis and a decrease in viability (Yang et

al., 2006). Esculetin also inhibited adipogenesis of preadipocytes (Yang et al., 2006).

Also esculetin has been reported to inhibit adipocyte differentiation during the early,

intermediate and late stages of differentiation process. It induced apoptosis during the late

stage of differentiation. These findings suggest that esculetin can alter fat cell number by

direct effects on cell viability, adipogenesis and apoptosis in 313-L1 cells (Yang et al.,

2006).

1.1.3.1 Pharmacological Action of Esculetin

Several workers have demonstrated the pharmacological effect of esculetin.

Sagrario et al. (1998), examined the effect of esculetin (6,7-dihydroxycoumarin) on the

glutathione system and lipid peroxidation in mice. The GSSG/GSH ratio was

significantly lower in esculetin groups compared to the control. Also increases in

glutathione reductase (GR) activity were observed. The extent of decline in the

GSSG/GSH ratio was correlated with the increase in GR activity.

Esculetin is also shown to exert immunomodulatory effects on murine

macrophages and lymphocytes, both in vitro and in vivo and might be one of the possible

23

mechanisms by which coumarins can exert their chemopreventive and anti-tumor

activities in vivo (Kwok-nam et al., 2005).

Elliot et al. (2001) reported that esculetin inhibits degradation of bovine nasal

cartilage and human articular cartilage.

1.1.4 Scopoletin

Fig. 7: Structure of Scopoletin

It is a yellow to beige crystalline powder. It is distributed in Noni, manaca,

passion flower and steva. Scopoletin is a fluorescent coumarin compound (Wikipedia,

2008).

Scopoletin (6-methoxy-7-hydroxycoumarin)(Fig. 7) is one of the most widespread

coumarins in nature (Basaran et al., 2007). Scopoletin is a naturally occurring compound

in cotton leaf and citrus peel (Abdulrahman et al., 2005). It is a dye that can be used to

detect the release of reactive oxygen species during the oxidative burst, peroxynitrite

scavenger and as acetylcholinesterase inhibitor. Scopoletin and other coumarin

compounds have been isolated from cassava, a shrubby tropical plant (Manihot

esculenta) widely grown for its large tuberous, starchy roots (Obidoa and Obasi, 1991;

Ezeanyika et al., 1999). This root is eaten as a stable food in the tropics only after

leaching and drying to remove cyanide. The consumption of this plant has been linked to

some diseases which include endemic goiter, cretinism and mental retardation

(Abdulrahman et al., 2005).

Experimental evidence strongly suggests a biochemical and/or toxicological role

for scopoletin. Scopoletin has been reported to inhibit superoxide anion production more

effectively than hydrogen peroxide production in conjunction with the fact that it has

O HO

H3CO

H

O

24

more inhibitory effect on potassium ferricyanide compared to oxygen (Abdulrahman et

al., 2005).

Scopoletin is a progressive inhibitor for substrate oxidation and also inhibits

prostatic cell proliferation (Liu et al., 2001).

1.1.4.1 Pharmacological Action of Scopoletin

The pharmacological effects of scopoletin have been demonstrated by several

workers. Ezeanyika et al. (1999) reported that scopoletin is involved in the pathogenesis

of the neuropathy seen in cassava consuming populations. Corbett (1989) demonstrated

that scopoletin is used for hydrogen peroxide activity determination while Ojewole and

Adesina (1983) reported that scopoletin has a depressor effect on anaesthetized rats. It

induces negative chromotropic and ionotropic responses on guinea pig isolated atria;

inhibits acetylcholine–induced contradiction of toad rectus abdomens muscle. They also

reported that scopoletin possesses neuromuscular and hypotensive effects, inhibits

electrically induced contraction of the vascular and extravascular smooth muscles. They

suggested that scopoletin acts as a non-specific spasmolytic agent on smooth muscles.

Obidoa et al. (1999) showed the effect of scopoletin on male guinea pig reproductive

organs and reported that scopoletin may induce testicular failure at the level of sperm

maintenance.

Ezeanyika et al. (2002) also investigated the effects of scopoletin on serum

electrolytes, urea, creatinine and some haematological parameters of rats and reported

that among the groups administered scopoletin, serum levels of K+ and creatinine

increased with increasing concentration while Na+ and urea level decreased with

increasing concentration. Scopoletin was also found to increase bleeding time and RBC

count.

1.1.4.2 Uses of Scopoletin

Scopoletin is used as a peroxide or calcium signal during the oxidative burst. It is

an acetylcholinesterase inhibitor (Wikipedia, 2007). Scopoletin seems to regulate the

blood pressure; when the blood pressure is high, scopoletin helps to lower it and when it

is too low, it helps to raise it (Corbett, 1989).

25

Evidence has shown that it has bacteriostatic activity against various species of

bacteria including Escherichia coli, Staphylococcus aureus, Streptococcus sp, Klebsiella

pneumoniae and Pseudomonas aeruginosa (Wikipedia, 2008). Scopoletin has anti-

inflammatory activity and can be used to treat bronchial illness and asthma (Kostova,

2005). Scopoletin regulates the hormone serotonin which helps to reduce anxiety and

depression (Wikipedia, 2008).

Scopoletin also relaxed the smooth muscles and inhibited the spasmogenic

activities of a wide variety of agonists on guinea pig isolated ileum (Ojewole and

Adesina, 1983). Scopoletin inhibited proliferation of certain cancer cells by inducing

apoptosis (Liu et al., 2001).

Scopoletin was found to inhibit hepatic lipid peroxidation and increased the

activity of antioxidants, superoxide dismutase and catalase (Panda and Kar, 2006).

Synthetic 7-hydroxy coumarins are used to absorb ultraviolet rays in sunscreen

cosmetics and are also used in the synthesis of drugs especially against cancer (Iscan et

al., 1994). Ezeanyika et al. (1999) investigated the effect of scopoletin and cyanide on rat

brain.

1.2 Acetaminophen (Paracetamol)

Acetaminophen is the active metabolite of phenacetin responsible for its analgesic

effect (Venkatesha and Kala, 2000). It is a weak prostaglandin inhibitor in peripheral

tissues and possesses no significant anti-inflammatory effects (Betram, 2001).

Acetaminophen is one of the most important drugs used for the treatment of mild

to moderate pain when an anti-inflammatory effect is not necessary (Nwachukwu, 2006).

Pharmacokinetics

Acetaminophen (Fig. 8) is administered orally. Absorption is related to the rate of

gastric emptying and peak blood concentrations are usually reached in 30–60 minutes

(Australian Rheumatology Association, 2007). Acetaminophen is slightly bound to

plasma proteins and is primarily metabolized by hepatic microsomal enzymes and

converted to acetaminophen sulfate and glucoronide, which are pharmacologically

inactive. Less than 5% is excreted unchanged. A minor but highly active metabolite (N-

26

acetyl-p-benzoquinone) is important in large doses because of its toxicity to both liver

and kidney (Prescott et al., 2006). The half-life of acetaminophen is 2–3 hours and is

relatively unaffected by renal function.

Fig. 8: Structure of N-acetyl-p-aminophenol

Indications

Acetaminophen differs from aspirin in that it lacks anti-inflammatory properties.

It lacks platelet-inhibiting properties and does not affect uric acid levels (Betram, 2001).

Acetaminophen alone is inadequate therapy for inflammatory conditions such as

rheumatoid arthritis. For mild analgesia, acetaminophen is the preferred drug in patients

allergic to aspirin. It does not antagonize the effects of uricosuric agents (Australian

Rheumatology Association, 2007).

Adverse Effects

In therapeutic doses, a mild increase in hepatic enzymes may occasionally occur

and with large doses, dizziness, excitement and disorientation are seen (Chan et al.,

1996). Early symptoms of hepatic damage include nausea, vomiting, diarrhoea and

abdominal pain.

For the great majority of people, paracetamol is a safe analgesic/antipyretic

provided it is taken in the recommended doses. It is widely available under several brand

names and in many proprietary combinations with other drugs. A major problem caused

by paracetamol is hepatotoxicity (Chan et al., 1996).

Paracetamol or acetaminophen is a common analgesic and antipyretic drug that is

used for the relief of fever, headaches and other minor aches and pains (Janbaz and

Gilani, 2002). It is a major ingredient in numerous cold and flu medications and many

HO

H O

Fig. 1.7: N – Acetyl-p-aminophenol

(acetaminophen)

N – C – CH3

27

prescription analgesics. It is remarkably safe in standard doses but because of its wide

availability, deliberate or accidental overdoses are fairly common (Wikipedia, 2008).

Paracetamol is the most widely used over-the-counter analgesic agent in the world

(Janbaz and Gilani, 2002). It is involved in a large proportion of accidental paediatric

exposures and deliberate self poisoning cases. It is also the single most commonly taken

drug in overdoes that lead to hospital presentation and admission (Daly et al., 2008).

Paracetamol (acetaminophen, N – acetyl – p – aminophenol, APAP, NAPA, 4 –

hydroxyl – acetanilide) was first introduced into clinical medicine towards the end of the

20th century but it attracted little attention and was soon forgotten. There was a

resurgence of interest in paracetamol when it was found to be the major metabolite of

acetanilide and phenacetin and was commonly assumed to be responsible for the

therapeutic effects of these two drugs. It has since been used increasingly as a substitute

for other analgesic such as aspirin and phenacetin (Prescott et al., 2006).

Paracetamol, unlike other common analgesics such as aspirin and ibuprofen has

no anti-inflammatory properties and so it is not a member of the class of drugs known as

non-steroidal anti-inflammatory drugs or NSAIDs (Murray et al., 2003). In normal doses,

paracetamol does not irritate the lining of the stomach or affect blood coagulation, the

kidney or the fetal ductus arteriosus (as NSAIDs can) like NSAIDs and unlike opiod

analgesics paracetamol has not been found to cause euphoria or alter mood in any way.

Since this molecule is achiral, it does not have a specific rotation (Prescott et al., 2006).

The words acetaminophen and paracetamol are both derived from the chemical names for

the compounds N acetyl-para-aminophenol and para-acetyl amino-phenol, respectively.

Paracetamol has the following properties: it has a chemical formula C8H9NO2, a

molecular weight of 151.17. Bioavailabity is almost 100%. It is metabolised in the liver

cells and has an elimination half-life of 1 – 4 hours. The melting point of paracetamol is

169oC, its density is 1.263g/cm

3 (Wikipedia, 2007a).

1.2.1 Mechanism of Action

Paracetamol has long been suspected of having a similar mechanism of action to

aspirin because of the similarity in structure. That is, it has been assumed that

paracetamol acts by reducing production of prostaglandins, which are involved in the

28

pain and fever processes by inhibiting cyclooxygenase (COX) (Murray et al., 2003).

There are however, important differences between the effects of aspirin and those of

paracetamol. Prostaglandins participate in the inflammatory response but paracetamol has

no appreciable anti-inflammatory action. Also, COX produces thromboxanes, which aid

blood clotting – aspirin reduces blood clotting, but paracetamol does not (Wikipedia,

2008). Finally, aspirin and the other NSAIDs commonly have detrimental effects on the

stomach lining, where prostaglandins serve a protective role, but paracetamol is safe.

While aspirin acts as an irreversible inhibitor of COX and directly blocks the active site

of the enzymes, paracetamol indirectly blocks COX and this blockade is ineffective in the

presence of peroxide (Ho and Lam, 1996). It is reported that paracetamol selectively

blocks a variant of COX. This enzyme which is only expressed in the brain and the spinal

cord is referred to as COX–3 (Wikipedia, 2008).

1.2.2 Metabolism of Paracetamol

Paracetamol (or acetaminophen) is metabolised in the liver via three pathways –

glucuronidation, sulphation (both account for 95% of metabolism) or via the cytochrome

P450 system (5%). In this pathway, acetaminophen is converted to a toxic metabolite, N-

acetyl-p- benzoquinone imine(NAPQI) (Fig. 9). Glutathione a tripeptide (y-glu-cys-gly)

then binds to this toxic metabolite resulting in a non-toxic compound. Hepatotoxicity

occurs when glutathione stores are depleted faster than they can be regenerated and the

toxic metabolite is left to accumulate (Kumar et al., 2005).

29

Paracetamol Metabolism

Ac – glucoronide Ac Ac-sulfate

Cytochrome P450

Reactive electrophillic compound

(Ac*)

GSH Cell macromolecules(protein)

GS – Ac * Ac* - protein

Ac – mercapturate Hepatic cell death

Fig. 9: Pathway of Metabolism of acetaminophen (Ac) to hepatotoxic metabolites

(GSH=Glutathione (Reduced), GS=glutathione moeity, Ac*=Active intermediate)

(Betram, 2001)

Non-toxic

metabolite

Cell death

Glutathione O

Sulphate

HNCOCH3

OH

Acetaminophen

HNCOCH3

Toxic

metabolite

HNCOCH3

Glucuronide

HNCOCH3

30

Paracetamol is rapidly absorbed from the small intestine. Peak serum

concnetrations occur within 1 – 2 hours for standard tablet or capsule formulations and

within 30 minutes for liquid preparations. Peak serum concentrations after therapeutic

doses do not usually exceed 130nmol/l (20gm/l) (Leshna et al., 1976).

Twenty percent of the ingested dose undergoes first-pass metabolism in the gut

wall (sulphation). Distribution is usually within 4 hours of ingestion for standard

preparations and 2 hours for liquid preparation. Volume of distribution is 0.9l/kg2.

Further elimination occurs by hepatic biotransformation. After therapeutic doses, the

elimination half-life is 1.5 – 3 hours (Nahid et al., 2005). Over 90.6% is metabolised to

inactive sulphate and glucuronide conjugates that are excreted in the urine. Metabolism

of the remainder is via cytochrone P450 and results in the highly reactive intermediary

compound N – acetyl-p–benzoquinone imine (NAPQI) (Prescott and Mathew, 1974). In

normal conditions, NAPQI is immediately bound by intracellular glutathione and

eliminated in the urine as mercapturic adducts (Daly et al., 2008). With increased

paracetamol doses, greater production of NAPQI may deplete glutathione stores. When

glutathione depletion reaches a critical level (about 30% of normal stores), NAPQI binds

to other proteins, causing damage to the hepatocyte. Glutathione depletion itself may be

injurious (Kupeli et al., 2006).

The commonest target organ in paracetamol poisoning is the liver and the primary

lesion is acute centrilobular hepatic threshold. In adults, the single acute threshold dose

for severe liver damage is 150 to 250 mg/kg though there is marked individuals variation

in susceptibility (Prescott et al., 2006).

1.2.3 Toxicity of Paracetamol

The mechanism of paracetamol hepatotoxicity shows that a minor route of

paracetamol metabolism involves its conversion by cytochrome P-450-dependent mixed

function oxidase to a reactive acrylating metabolite, N–acetyl–p–benzoquinone imine

(NAPQI). This is produced by a two electron oxidation of paracetamol to NAPQI by

cytochrome P-450 or alternatively, a one electron oxidation to N–acetyl-p-

benzosemiquinone imine by peroxidase prostaglandin H synthetase or cytochrome P-450

(Tanaka and Misawa, 2000). NAPQI causes a depletion of both the mitochondrial and

31

cytosolic pools of reduced glutathione (GSH). Once GSH is depleted, cellular proteins

are directly acrylated and oxidized by the reactive metabolite, resulting in inhibition of

enzyme activities (Prescott et al., 2006). Two of the enzymes that have been shown to be

inhibited in paracetamol treated animals are glutathione peroxidase and thiol transferase.

Inhibition of these enzymes renders the cell vulnerable to endogenous activated oxygen

species with further oxidation of protein thiols (Prescott et al., 2006). Paracetamol

hepatotoxicity depends on the metabolic balance between the rate of formation of the

toxic acrylating metabolite and the rate of glutathione conjugation (Guzy et al., 2004)

Treatment for paracetamol overdose is gastro-intestinal decontamination (Prescott

et al., 2006). In addition, N-acetyl cysteine (NAC) administration plays an important role

(Prescott et al., 2006). The maintenance of hepatic glutathione concentrations by

administration of N-acetyl cysteine was first suggested as a treatment for paracetamol

poisoning by Prescott and Mathew (1974). GSH itself, due to its inability to cross the

plasma membrane, cannot be used as an antidote. However, GSH precursors such as N-

acetyl cysteine have been found to be effective both in experimental animals and in

humans (Dong et al., 2000).

N-acetyl cysteine may reduce the severity of the liver necrosis by directly

conjugating with or reducing the reactive metabolite NAPQI. In addition, N-acetyl

cysteine forms other nucleophiles such as cysteine and GSH that are capable of

detoxifying NAPQI (Prescott et al., 2006).

1.3 The Liver

The liver is an organ present in vertebrates and some other animals. This organ is

also the largest glandular organ in the human body. It lies below the diaphragm in the

thoracic region of the abdomen. Medical terms related to the liver often start in hepato or

hepatic from the Greek word for liver, ‘hepa’. The adult human liver normally weighs

between 1.4 – 1.6 kilograms and it is a soft, pinkish-brown boomerang shaped organ

(Medicinenet.com, 2007b).

32

1.3.1 Anatomy of the Liver

The liver is a multi-lobed organ that is located at the most forward part of the

abdomen. Flow of blood in the liver is from the splenic vein which joins the inferior

mesenteric vein, which together joins with the superior mesenteric vein to form the

hepatic portal vein, bringing venous blood from the spleen, pancreas, stomach, small

intestine, so that the liver can process the nutrients and by products of food digestion. The

role of the liver is to keep the complex internal chemistry of the body in balance

(Wikipedia, 2007c).

The hepatic veins drain directly into the inferior vena cava. The liver is among the

few internal human organs capable of natural regeneration of lost tissue – as little as 25%

of remaining liver can regenerate into a whole liver again. This is predominantly due to

the hepatocytes acting as unipotential stem cells (i.e a single hepatocyte can divide into

two hepatocyte daughter cells) (Wikipedia, 2007c).

There is also some evidence of bipotential stem cells, called ovalocytes, which

exist in the canals of Hering. These cells can differentiate into either hepatocytes or

cholangiocytes (cells that line the bile ducts) (Wikipedia, 2007c). The liver is covered

entirely by visceral peritoneum, a thin double layered membrane that reduces friction

against other organs. Ligaments connect the upper surface of the liver to the diaphragm

and the abdominal wall and the under surface to the stomach and duodenum. The gall

bladder is located on the under-surface of the right lobe of the liver. Neighbouring organs

include the colon, the intestines and the right kidney. The liver is a large network of units

called hepatic lobules. The hepatic lobule is very small and looks like a six-sided

cylinder.

1.3.2 Physiology and Functions of the Liver

The liver plays a major role in metabolism. Every second, the liver cell

undertakes thousands of complex biochemical interactions that influence all the organs in

the body. The liver has as many as 500 vital functions. It is involved in glycogen storage,

decomposition of red blood cells, plasma protein synthesis and detoxification.

33

The liver produces bile, an alkaline compound which aids in digestion, via the

emulsification of lipids. It also performs and regulates a wide variety of high-volume

biochemical reactions requiring very specialized tissues (Wikipedia, 2007c).

The liver takes raw nutrients from the digestive system and processes them, so

they can be stored and also sent to different parts of the body in the right form and

quantity. The liver regulates the level of sugar in the human body and helps to remove

toxins, drugs and hormones from the blood stream.

The liver performs various functions which are carried out by liver cells or

heptocytes.

The functions performed by the liver are

Produces and excretes bile (a greenish liquid required for emulsification of fats).

Performs several roles in carbohydrate metabolism: Gluconeogenesis,

Glycogenolysis, Glycogenesis; the breakdown of insulin and is responsible for the

mainstay of protein metabolism.

Performs several roles in lipid metabolism, cholesterol synthesis, the production

of triacylglycerols.

The liver produces coagulation factors I (fibrinogen), II (prothrombin), V, VII,

IX, X and XI as well as protein C, protein S and antithrombin.

The liver breaks down haemoglobin.

It converts ammonia to urea.

It stores a multitude of substances including glucose, vitamin B12, iron and

copper.

The liver breaks down toxic substances and most medicinal products in a process

called drug metabolism.

The liver also produces albumin, the major osmolar component of blood serum.

1.3.3 Liver Intoxication/Damage

Common liver disorders include hepatitis and cirrhosis. Symptoms of liver

damage are often vague and may not be noticed until damage is severe since the liver can

still function with up to 80 percent deterioration. Symptoms and signs of liver damage

34

include yellowing of the white of the eye and skin, jaundice, pale stools, sleep

disturbance, abdominal pain, loss of appetite, drowsiness after meals, intolerance of fatty

food and energy loss among others.(Atreyee et al., 1998)

Liver function is a dominant factor in the development of fatty degeneration

diseases because one of the functions of the liver is managing fats and cholesterol. Such

deranged fat metabolism contributes to many diseases including cardiovascular disease,

cancer, obesity and diabetes. High blood pressure, anaemia and infertility, may also arise

from poor use of fats caused by liver dysfunction and insufficient bile production (Muriel

et al., 1992).

Environmental and dietary toxins place a great deal of stress on the liver as it

works to detoxify air and food. Dietary fat further increases the liver workload because

toxins are concentrated in animal fats. Also dietary fats of any kind can be damaging

when rancidity, overheating and processing cause them to oxidize and create free radical

damage in the body (Wikipedia, 2007c).

1.3.4 Causes of Liver Damage

Causes of liver damage are:

Inflammation – drugs, viruses, bacteria, bile and toxins.

Anaemia – haemolytic anaemia can decrease the oxygen available to liver cells

and lead to their death.

Infection – bacteria, viruses and fungi can cause liver disease.

Toxins – thousands of chemicals are available which are toxic to the liver.

Examples include

- acetaminophen

- Phenobarbital

- Parasiticides

- Ketacimazole (fungal treatment)

- Glucocorticoids (cortisone)

- Antihelmintics (worming medication) (Song et al., 2001).

35

Liver Repair

The liver has a capacity to regenerate and optimum nutrition can help produce

quick improvements. Antioxidants also facilitate liver healing. Supplementing the diet

with vitamins C and E and the minerals, zinc and selenium can help protect the liver from

free radical damage. The B complex vitamins may also support regeneration of the liver.

Methionine is a source of the major lipotropic compound, S–adenosylmethionine

of the body. It contributes to the formation of glutathione, a molecule that aids

detoxification by making toxins water-soluble. Excessive exposure of the liver to drugs

and other chemical toxins tend to deplete the supply of glutathione to the liver.

Supplemental N-acetylcysteine (NAC), another sulphur – containing amino acid and a

powerful antioxidant also stimulate glutathione synthesis (Prescott et al., 1999).

Phosphatidylcholine also called lecithin is a phospholipid composed largely of choline.

Cell membranes are made largely of lecithin. Lecithin not only maintains the integrity of

liver cells but may also help regenerate damaged tissue and normalize bile function (Ken,

2004).

1.3.5 Hepatotoxicity

This implies chemical-driven liver damage. The liver plays a central role in

transforming and cleaning chemicals and is susceptible to the toxicity from these agents.

Certain mechanical agents when taken in overdoses and sometimes even when introduced

within therapeutic ranges may injure the organ (Pablo et al., 1992). Other chemical

agents such as those used in laboratories and industries, natural chemicals and herbal

remedies can also induce hepatotoxicity. Chemicals that cause liver injury are called

hepatotoxins. The liver is a major organ for metabolism of foreign substances and also

functionally interposed between the site of resorption and the systemic circulation. These

conditions render the liver not only the most important organ for detoxification of foreign

substances but also a major target of their toxicity. More than 1000 drugs have been

associated with idiosyncratic hepatotoxicity and drug-induced liver injury is the main

reason for removing approved medications from the market (Chau, 2008).

Moreover, drug-induced hepatotoxicity contribute more than half of the cases of

acute liver failure with paracetamol being the principal contributing agent in Western

36

countries (Chau, 2008). Hepatotoxicity may be predictable or unpredictable. Predictable

reactions typical are dose-related and occur with short latency (within a few days) after

some threshold toxicity is reached. Paracetamol (acetaminophen) is a classic example.

Conversely, idiosyncratic reactions occur with variable, sometimes prolonged latency (1

week to 1 year), with low incidence and may not be dose-related (Chau, 2008).

1.3.6 Mechanism of Liver Damage

Drugs continue to be taken off the market due to late discovery of hepatotoxicity.

Due to its unique metabolism and close relationship with the gastro-intestinal tract, the

liver is susceptible to injury from drugs and other substances. Seventy five percent (75%)

of blood coming to the liver arrives directly from gastrointestinal organs and the spleen

via portal veins which bring drugs and xenobiotics in concentrated form. Several

mechanisms are responsible for either inducing hepatic injury or worsening the damage

process. Many chemicals damage mitochondria, an intracellular organelle that produce

energy. Its dysfunction releases excessive amount of oxidants which in turn injures

hepatic cells. Activation of some enzymes in the cytochrome P- 450 system such as

CYP2EI also leads to oxidative stress (Wikipedia, 2007a). Injury to hepatocytes and bile

duct cells lead to accumulation of bile acids in the liver. This promotes further liver

damage.

1.4 Liver Function Tests

Liver function tests represent a broad range of normal functions performed by the

liver. The diagnosis of liver disease depends upon a complete history, complete physical

examination and evaluation of liver (Jensen, 2007).

The liver performs different kinds of biochemical, synthetic and excretory

functions, and so no single biochemical test can detect the global functions of the liver.

The various aspects/uses of liver function tests include:

1. Screening – they are a noninvasive yet sensitive screening modality for liver

dysfunction,/ pattern of diseases – they are helpful to recognize the pattern of

liver disease like being helpful in differentiating between acute viral hepatitis

and various cholestatic disorders and chronic liver disease;

37

2. Assess severity – they are helpful to asses the severity and predict the

outcome of certain diseases like primary biliary circhosis and

3. Follow up – they are helpful in the follow-up of certain liver diseases and also

helpful in evaluating response to therapy like autoimmune hepatitis (Thapa

and Anuj, 2007).

Classification of Liver Function Tests

A. Test of the capacity of the liver to transport organic anions and to metabolize

drugs (serum bilirubin, urine bilirubin and urobilirubin).

B. Test that detect injury to hepatocytes (serum enzyme tests) – aminotransferases,

alkaline phosphatase, glutamyl transpeptidase, 5–nucleotidase, leucine

aminopeptidase.

C. Test of the biosynthetic capacity of the liver – serum proteins, albumin,

prealbumin, serum ceruloplasmin, procollagen III peptide, a lantitrypsin, a feto

protein, prothrombin time (Thapa and Anuj, 2007).

An initial step in detecting liver damage is a simple blood test to determine the

presence of certain liver enzymes in the blood. Under normal circumstances, these

enzymes reside in the cells of the liver. But when the liver is injured these enzymes are

spilled into the blood stream (Sultana et al., 2004). Among the most sensitive and widely

used of these liver enzymes are the aminotransferases – aspartate aminotransferases

(AST) and alanine aminotransferases (ALT). These enzymes are normally contained

within liver cells. If the liver is injured, the cells spill the enzymes into the blood stream,

raising the enzymes level in the blood and signaling their damage (Medicinenet, 2007b).

A. Test of the capacity of the liver to transport organic anions and to metabolize

drugs.

Serum bilirubin

Bilirubin is an endogenous anion derived from haemoglobin degradation from the

red blood cell (RBC). The cellular components of the blood- the white blood cells, red

blood cells and platelets are suspended in the plasma. The normal total circulating blood

38

volume is about 8% of the body weight (5600 in a 70kg man). About 55% of this volume

is plasma. RBCs biconcave discs having a mean diameter of about 7.8micrometer and

thickness of 2.5 mm at the thickest point and 1 micrometer or less in the centre. WBC

consist of polymorph nuclear neutrophils, polymorph nuclear eosinophils polymorph

nuclear basophils, monocytes, lymphocytes and occasionally plasma cells (Guyton and

Hall, 2006; Gonong, 2003). The classification of bilirubin into direct and indirect

bilirubin is based on the original Van der Beigh method of measuring bilirubin. Bilirubin

is a yellow fluid produced in the liver when worn-out red blood cells are broken down at

the end of their 120 day lifespan (Mason, 2004). Bilirubin is a major product of

haemoglobin. During splenic degradation of red blood cells, hemoglobin is separated out

from iron and cell membrane components. Haemoglobin is transferred to the liver where

it undergoes further metabolism in a process called conjugation. Conjugation allows

haemoglobin to become more water-soluble. The water solubility of bilirubin allows the

bilirubin to be excreted into bile (Jensen, 2007). In the blood, unconjugated (or indirect)

bilirubin is carried by albumin to the liver. It is conjugated to make it more water-soluble,

before it is excreted in bile. Conjugated bilirubin is also called direct bilirubin. The

concentration of bilirubin in the serum therefore reflects the balance between the amount

produced by erythrocyte destruction and that removed by the liver. As the liver becomes

irritated, the total bilirubin may rise (Medicines Information Bulletin, 2002).

1.4.1 Aminotransferases

The aminotransferases (formerly called transaminases) are the most frequently

utilized indicators of hepatocellular necrosis. These enzymes–aspartate aminotransferase

(AST formerly serum glutamate oxaloacetic transaminase–SGOT) and alanine amino

transferase (ALT formerly serum glutamic pyruvate transaminase–SGPT) catalyse the

transfer of the amino acids of aspartate and alanine respectively to the keto group of

ketoglutaric acid (Wikipedia, 2007b). ALT is primarily localized in the liver but the AST

is present in a wide variety of tissues like heart, skeletal muscle, kidney, brain and liver.

AST: aspartate + α-ketoglutarate Oxaloacetate + glutamate

ALT: alanine + α-ketoglutarate Pyruvate + glutamate

39

Whereas the AST is present in both the mitochondria and cytosol of hepatocytes.

ALT is localized in the cytosol. The cytosolic and mitochondrial forms of AST are true

isoenzymes and immmunologically distinct. About 80% of the AST activity in human

liver is contributed by the mitochondrial isoenzyme, whereas most of the circulating AST

activity in normal people is derived from the cytosolic isoenzyme (Thapa and Anuj,

2007).

1.4.2 Alanine aminotransferase (ALT)

The enzyme ALT is present in high concentrations in the liver. It is also found in

cardiac and skeletal muscle. However, ALT is considered as a specific marker of

hepatocellular damage because levels are generally only significantly raised in liver

damage. ALT is present in the heart and muscles in much lower concentrations – only

marginal elevations occur in acute myocardial infarction. People with acute liver damage

have particularly high ALT levels and those with chronic liver disease and obstructive

jaundice have more modestly raised levels. Low ALT (and AST) levels suggest vitamin

B6 deficiency. The level of ALT abnormality is increased in conditions where cells of the

liver have been inflamed or undergone cell death. As the cells are damaged, the ALT

leaks into the blood stream leading to a rise in the serum level (Mason, 2004). Any form

of hepatic cell damage can result in an elevation in the ALT. ALT is the most sensitive

marker for liver cell damage (Mason, 2004). Elevations are often measured in multiples

of the upper limit of normal (ULN). Reference range 5 to 40 IU/L (Wikipedia, 2008).

1.4.3 Aspartate aminotransferase

Aspartate aminotransferase (AST) is more widely distributed than ALT. It is

present in the liver, heart, kidneys, skeletal muscle and red blood cells. AST levels are

raised in shock (Wikipedia, 2007b). It is less specific for liver disease and is not included

in liver function profiles by all laboratories (Wikipedia, 2007b). AST levels are also

raised in pregnancy and after exercise. Ratios between ALT and AST are useful to

physicians in assessing the etiology of liver enzyme abnormalities and also useful in

differentiating between causes of liver damage (Mason, 2004).

40

Mild, Moderate and Severe Elevations of Aminotransferases

1. Severe (>20 times, 1000 U/L): The AST and ALT levels are increased to some

extent in almost all liver diseases. The highest elevations occur in severe viral

hepatitis, drug or toxin induced hepatic necrosis and circulatory shock.

2. Moderate (3–20 times): The AST and ALT activities are moderately elevated in

acute hepatitis, neonatal hepatitis, chronic hepatitis, autoimmune hepatitis, drug-

induced hepatitis, alcoholic hepatitis and acute biliary tract obstructions. The ALT

is usually more frequently increased as compared to AST except in chronic liver

disease.

3. Mild (1–3 times): These elevations are usually seen in sepsis induced neonatal

hepatitis, extra hepatic biliary atresia (EHBA), fatty liver, cirhosis and even after

vigorous exercise.

4. ALT exceeds AST in toxic hepatitis, viral hepatitis, chronic active hepatitis and

cholestatic hepatitis.

5. Mitochondrial AST: Total AST ratio: This ratio is characteristically elevated in

alcoholic liver disease (Thapa and Anuj, 2007).

1.4.4 Alkaline Phosphatase (ALP)

Alkaline phosphatases are a family of zinc metaloenzymes, with a serine residue

at the active centre. They release inorganic phosphate from various organic

orthophosphates and are present in nearly all tissues. ALP is produced in the lower bile

duct, bone and gut and are widely distributed in the body. In liver, alkaline phosphatase is

found histochemically in the microvilli of bile canaliculi and on the sinusoidal surface of

hepatocytes. Alkaline phosphatase from the liver, bone and kidney are thought to be from

the same gene but that from intestine and placenta are derived from different genes

(Thapa and Anuj, 2007). In the liver, two distinct forms of alkaline phosphatase are also

found but their precise roles are unknown. ALP is a hydrolase responsible for removing

phosphate groups from many types of molecules, including nucleotides, proteins and

alkaloids (Wikipedia, 2008).

The process of removing the phosphate group is called dephosphorylation. As the

name suggests, alkaline phosphatases are most effective in an alkaline environment.

41

Alkaline phosphatase lines the cells in the biliary ducts of the liver. ALP levels in plasma

will rise with large bile duct obstruction, intrahepatic cholestasis or infiltrative diseases of

the liver. It is present in the bone and placenta, so it is higher in growing children (as their

bones are being remodeled) and elderly patients with Paget’s disease (Mason, 2004).

Highest levels of alkaline phosphatase occur in cholestatic disorders. Elevations occur as

a result of both intrahepatic and extrahepatic obstruction to bile flow. ALP is also raised

in cirrhosis and liver cancers, but levels can be within the reference range or only slightly

raised in acute hepatitis.

The normal range is 39 – 120 IU/L. High ALP levels can show that the bile ducts

are blocked. Lowered levels of ALP are less common than elevated levels. The

mechanism by which alkaline phosphatase reaches the circulation is uncertain. Leakage

from the bile canaliculi into hepatic sinusoids may result from leaky tight junctions and

the other hypothesis is that the damaged liver fails to excrete alkaline phosphatase made

in bone, intestine and liver. Isoenzymes studies using electrophoresis can confirm the

sources of ALP. Heat stability also distinguishes bone and liver isoenzymes.

1.5 Reactive Oxygen Species

Mitochondrial respiration is considered to produce reactive oxygen species (ROS)

as product of regular electron transfer (Nohl et al., 2004). Reactive oxygen species such

as superoxide anion radical (O2–), hydroxyl radical (OH

•) and hydrogen peroxide (H2O2)

(Table 3) are considered important factors in the etiology of several pathological

conditions such as cardiovascular diseases, diabetes, inflammation and cancer (Nohl et

al., 2004)

The reactive oxygen species produced in cells include hydrogen peroxide (H2O2),

and free radicals such as the hydroxyl radical (OH•) and the superoxide anion (O2

–).

42

Table 3: Reactive Oxygen Species

Reactive Oxygen Species

O2– Superoxide Radical

OH• Hydroxyl Radical

ROO• Peroxyl Radical

H2O2 Hydrogen Peroxide

•O2 Singlet Oxygen

NO• Nitric Oxide

ONOO– Peroxynitrite

The hydroxyl radical is particularly unstable and will react rapidly and non-

specifically with most biological molecules (Liu et al., 1997). These oxidants can damage

cells by starting chemical chain reactions such as lipid peroxidation or oxidizing DNA or

proteins. Damage to DNA can cause mutations while damage to proteins causes

degradation, inhibition, denaturation and protein degradation (Karam and Simic, 1989).

Free radicals induce the cell damage by altering the biological activities of lipids,

proteins, DNA and carbohydrates (Fig. 10). Damaged cells function incorrectly (Ghauri

et al., 1993). This results to a further escalation of the oxidative stress and to enhanced

tissue damage. Simultaneous ionic disbalance, mitochondrial dysfunction and activation

of the caspase/calpine cascades result in cell death (Traykova and Kostova, 2005).

The generation of ROS is associated with life under aerobic conditions and

reactive intermediates are produced under physiological and patho-physiological

conditions (Stahl and Sies, 2002).

43

Iron

Fe2+

an electron reduction

(e.g respiratory chain)

Superoxide

dismutase

Fig. 10: Implications of reactive oxygen species (ROS), reactive nitrogen species (RNS)

and oxidative stress (OS) in the cell damage.

O2 Arginine

O2– NO

•

ONOO–

H2O H2O2 H+

OH •OH + NO2

•

Fig. 11: Overview of formation of reactive oxygen species

Catalase

x-ray

Amadori products

ATP, NAD(P)H

depletion

ROS RNS

Lipids Proteins DNA

Lipid

peroxidation

Thiols

oxidation

Carbonyls

formation

Polli ADP

riboxylation

Membrane

damage

Damaged ion

transport/

activation

Altered gene

expression

Deactivation

of enzyme

Cell damage

Age

Schift base Carbohydrates

44

Reactive oxygen species are either radicals e.g. hydroxyl radical, peroxyl radical

or reactive non-radical compounds such as singlet oxygen, peroxynitrite or H2O2. Their

half-lives vary from a few nanoseconds for the most reactive compounds to seconds and

hours for rather stable radicals (Stahl and Sies, 2002).

The superoxide radical anion appears to play a central role as other reactive

intermediates are formed from it (Fig. 11). ROS are also produced in the organism as part

of the primary immune defense. Phagocytic cells such as neutrophils or macrophages

defend against foreign organisms by generating O2- and nitric oxide as a part of the

killing mechanism. Both compounds can combine to form peroxynitrite (ONOO-), a

reactive species capable of inducing lipid peroxidation in lipoproteins (Goldstein et al.,

1989).

The hydroxyl radical is the most reactive oxygen species. It is formed in vivo

upon high energy radiation e.g. X-rays, by haemolytic cleavage of water or from

hydrogen peroxide. Due to its high reactivity, this radical immediately reacts with

surrounding target molecules at the site where it is generated (Karam and Simic, 1989).

•O2 is electronically excited oxygen which is found in biological system via

photosensitization reactions. It should be noted that the organism is also exposed to

reactive oxygen species from external sources e.g quinone, cigarette smoke (Stahl and

Sies, 2002). Lipid peroxidation, a general mechanism of tissue damage by free radicals is

known to be responsible for cell damage and may induce pathological events. During

pulmonary inflammation, increased amount of ROS and RN intermediates are produced

as a consequence of phagocytic respiratory burst (Reddy et al., 2004).

Chemical compounds and reactions capable of generating potential toxic oxygen

species can be referred to as pro-oxidants (Murray et al., 2003). On the other hand,

compounds and reactions disposing of these species, scavenging them, suppressing their

formation or opposing their actions are antioxidants and include compounds such as

NADPH, GSH, ascorbic acid and vitamin E.

In a normal cell, there is an appropriate pro-oxidant– antioxidant balance. This

can however be shifted toward pro-oxidants when production of oxygen species is

increased greatly e.g following ingestion of certain chemicals or drugs or when levels of

45

antioxidants are diminished. This state is called oxidative stress and can result in serious

cell damage if stress is massive or prolonged (Murray et al., 2003).

Free radicals play a major role in the causation of the health problems of the

industrialized world, including cardiovascular diseases, cancer, neurological diseases,

atherosclerosis and ageing. Under certain stress conditions, there is an increase in the

cellular levels of ROS. Under normal physiological conditions, there is a balance between

the formation of ROS and the protective antioxidant mechanisms of cells. But when the

generation of ROS overcomes the cellular antioxidant capacity, cells undergo oxidative

stress situations which can eventually produce cell death (Euroconference, 1998).

As oxidative stress is implicated in development of many pathologies, one

possible aspect of the therapeutic effect of coumarins might be related to their antioxidant

action (Raj et al., 1998).

The balance between formation and elimination of free radical determines the

overall stability of a living body. Free radicals originate from a large variety of normal

and pathological metabolic transformations from host-defense against undesirable

invasion (chemical or biological) and from host response to a disturbance of the integrity

of the tissues (Copeland, 1986).

Free-radical chain reactions in the body are initiated by reactive species (RS –

molecules, ions, free radicals) possessing oxygen (ROS) or nitrogen (RNS) atom with

unpaired electron. If more RS are formed than needed for the normal redox-signaling and

self defense of the host, oxidative stress (OS) occurs leading to an oxidative cellular

damage, even to cellular death. The oxidative damage of the hosting tissues is restrained

by endogenous antioxidant defense (Raj et al., 1998).

1.6 Antioxidants

An antioxidant is a molecule capable of slowing or preventing the oxidation of

other molecules (Potter and Hotchkiss, 1996). Oxidation is a chemical reaction that

transfers electrons from a substance to an oxidizing agent. Antioxidants are often

reducing agents such as thiols or polyphenols. Although oxidation reactions are crucial

for life, they can also be damaging (Wikipedia, 2007c). Natural antioxidants present in

foods include lecithin, vitamin C and E and certain sulphur-containing amino acids.

46

Antioxidants are intimately involved in the prevention of cellular damage – the common

pathway for cancer, aging and a variety of diseases. They act as a major defence against

radical-mediated toxicity by protecting the damages caused by free radicals. Inhibition of

free radical generation can serve as a facile system for identifying cancer preventive

agents (Nayana and Janardhanan, 2000).

Diets rich in fruits and vegetables decrease the risk of chronic degenerative

diseases. The pathogenesis of these diseases are hypothesized to be by harmful effects of

free radicals on DNA, protein and small intercellular molecules. Recently, the search for

natural antioxidants and other preparation of plants concerning the beneficial effect for

good health has been intensified (Wikipedia, 2007c).

Both oxidants and antioxidants are needed in the biochemical economy of human

cells. We need to inhale oxygen because it is a fundamental oxidant we use and without

it, we die. Cells walk a balance between essential oxidant and essential antioxidant

process (Wikipedia, 2007c).

Antioxidants are powerful allies in combating inflammation and lowering heart

disease and cancer risk. They promote strong immune systems and help tip the odds

toward graceful aging. Some antioxidants are manufactured in our bodies, but our innate

capacity to synthesize antioxidants becomes less efficient as we age (Organic

Chemistry.org 2008).

The cellular antioxidant defence system operates through enzymatic and non-enzymatic

components (Altinyazar et al., 2006). Antioxidants from dietary sources include lipid-

soluble vitamins such as vitamin E and carotenoids as well as the water-soluble vitamin C

(Chaudiere and Ferrari-llious, 1999). Vitamin E appears to be one of the most important

free radical-scavenging antioxidants within membranes and lipoproteins. It is an effective

chain breaking antioxidant that protects polyunsaturated lipids from peroxidation by

scavenging peroxyl radicals, gets oxidized itself and is converted to a radical. The

resulting vitamin E radical is suggested to be regenerated to vitamin E by other

antioxidants, primarily vitamin C. The regeneration of vitamin E by other antioxidants is

one part of the intricate cooperation that exists between different antioxidants in the

antioxidant defense system (Joshua and Nwodo, 2010).

47

1.6.1 Glutathione

Glutathione (GSH) present in plants, animals and some bacteria, often at high

levels can be thought of as a redox buffer, it is derived from glycine, glutamate and

cysteine (Sies, 1999). The -carboxyl group of glutamate is activated by ATP to form an

acyl phosphate intermediate, which is then attacked by the α-amino group of cysteine. A

second condensation reaction follows, with the -carboxyl group of cysteine activated to

an acyl phosphate to permit reaction with glycine. The oxidized form of glutathione

(GSSG), produced in the course of its redox activities contains two glutathione molecules

linked by a disulphide bond. Glutathione probably helps maintain the sulfhydryl groups

of proteins in the reduced state and the iron of heme in the ferrous (Fe2+

) state (Murray et

al., 2003) (Fig. 13).

2GSH + R-O-O-H GSSG + H2O + R-OH

This reaction is catalyzed by glutathione peroxidase (Nelson and Cox, 2005). In

the erythrocytes, the pentose phosphate pathway provides NADPH for the reduction of

oxidized glutathione catalyzed by glutathione reductase. Reduced glutathione removes

H2O2 in a reaction catalyzed by glutathione peroxidase, an enzyme that contains selenium

as selenocysteine at the active site.

Fig. 12: Reduction of glutathione

Glutathione (Fig. 13) is found in most forms of aerobic life. It has antioxidant

properties since the thiol group in its cysteine merely is a reducing agent and can be

reversely oxidized and reduced. In cells, glutathione is maintained in the reduced form by

the enzyme glutathione reductase and in turn reduces other metabolites and enzyme

systems as well as reacting directly with oxidants (Wikipedia, 2007c).

G – S – S – G

Pentose

Phosphate

Pathway

FAD Glutathione

Reductase

Se Glutathione

Peroxidase

2H

NADPH + H+

2G – SH NADP+ H2O2

2H2O

48

Fig. 13: Structure of glutathione

Glutathione cannot enter most cells directly, and therefore must be made inside

the cell (Janaky et al., 1999). Its three constituent amino acids are glycine, glutamate and

cysteine. These three amino acids are obtained from food or through supplementation.

The rate at which glutathione can be made depends on the availability of cysteine.

Cysteine molecule has a sulphur – containing portion which gives the whole glutathione

molecule its biochemical activity (Lomaestro and Malone, 1995).

Glutathione also plays roles in catalysis, metabolism, signal transduction, gene

expression and apoptosis (Aw et al., 1991). It is a cofactor for glutathione S-transferase,

which is involved in the detoxification of xenobiotics, including carcinogenic

genotoxicants, and for the glutathione peroxidases, crucial selenium – containing

antioxidant enzymes (Samiec et al., 1998).

Glutathione values decline with age and higher values in older people are seen to

correlate with better health (Lands et al., 1999). Glutathione (reduced) is known

chemically as N-(N-L-gamma-glutamyl-L-cysteinyl) glycine and abbreviated as GSH. Its

molecular formula is C10H17N3O6S and the molecular formula for GSSG is

C20H32N6O12S2 (Betram, 2001).

1.6.1.1 Pharmacology and Mechanism of Action of Glutathione

Glutathione has antioxidant activity. It may have detoxyfying and

immunomodulatory activities and may have beneficial effects on sperm motility and in

the protection against noise-induced hearing loss (Janaky et al., 1999). Glutathione is a

nucleophilic scavenger and an electron donor via the sulfhydryl group of its active

residue, cysteine. Its reducing ability maintains molecules such as ascorbate and proteins

NH2

N

H HO

O O SH

O

O

OH

H

N

49

in their reduced state. Glutathione is the cofactor for the selenium – containing

glutathione peroxidase, which is the major antioxidant enzymes (Nayana and

Janardhanan, 2000).

Absorption of orally administered glutathione has been observed in some animals

e.g. rats, mice and guinea pigs. Oral glutathione has been demonstrated to reverse age-

associated decline in immune responsiveness in mice. Parenterally administered

glutathione was found to improve sperm motility in a small human trial (Sies, 1999). This

effect was thought to be due to the antioxidant activity of this substance (Sies, 1999).

1.6.1.2 Pharmacokinetics of Glutathione

The pharmacokinetics of oral glutathione in humans is not well understood. It

appears that in some animals, serum glutathione levels do increase following its oral

administration. The case is not the same with human studies. Gamma glutamyl

transferase hydrolyzes orally administered glutathione in the intestine. Studies have

shown that there is an occasional increase in circulating glutathione after oral

administration, but apparently this is rapidly metabolised by hepatic gamma-glutamyl

transferase (Sies, 1999).

1.7 Oxidative Stress and Radical Reactions

Oxidative stress has been defined as a disturbance in the balance between

antioxidants and prooxidants (free radicals and other reactive species), with increased

levels of prooxidants leading to potential damage. This imbalance can be an effect of

depletion of endogenous antioxidants, low dietary intake of antioxidants and/or increased

formation of free radicals and other reactive species (Stahl and Sies, 2004).

Radicals can react with other molecules in several ways. When two free radicals

meet, their unpaired electrons can form a shared electron pair in a covalent bond and both

radicals are lost. When a radical gives one electron to, takes one electron from or simply

adds on to a non-radical, that non-radical becomes a radical. Since most molecules

present in living organisms are non-radicals, any free radicals produced in the body will

most likely react with a non-radical and generate a new radical. Hence, free radical

reactions in vivo tend to proceed as chain reactions (Betterige, 2001).

50

1.7.1 Lipid Peroxidation and Free Radicals

Lipid peroxidation is a source of free radicals. Peroxidation (auto-oxidation) of

lipids exposed to oxygen is responsible not only for deterioration of foods (rancidity) but

also for damage to tissues in vivo, where it may be a cause of cancer, inflammatory

diseases, atherosclerosis, aging etc (Halliwell, 1994).

A free radical may in simple terms be defined as an atom or molecule that

contains one or more unpaired electrons and is capable of independent existence. An

unpaired electron is an electron that occupies an orbital alone (indicated by •). Electrons

usually associate in pairs in orbitals of atoms and molecules. Free radicals are generally

more reactive than non-radicals due to their unpaired electron, but different types of free

radicals vary widely in their reactivity. The oxygen molecule qualifies as a free radical

because it contains two unpaired electrons, but is not particularly reactive due to a special

electron arrangement that makes the reactions with oxygen spin restricted (Sies, 1997).

However, when oxygen is partly reduced, several different reactive oxygen species, both

radicals and non-radicals, may be produced. Examples of reactive oxygen species are

hydroxyl radicals (OH•), superoxide anion (O2

•) and hydrogen peroxide (H2O2). The

hydroxyl radical is an extremely reactive free radical. It is very unstable and attacks a

large array of molecules in the nearby environment. Example of other radicals are the two

gaseous radicals nitric oxide (NO•) and nitrogen dioxide (NO2

•), the carbon-centred (R

•),

alkoxyl (RO•) and peroxyl radicals (ROO

•) formed during peroxidation of lipids (Fig. 14)

and the trichloromethyl radical (CCl3•) formed by the metabolism of carbon tetrachloride

(CCl4•) in the liver (Halliwell et al., 1995).

51

PUFA

– H• Loss of H• to a free radical

R

• Carbon

•

centred radical

Molecular rearrangement

R•

Conjugated

diene •

+O2 Uptake of oxygen

ROO•

Peroxyl

radical O

O• +H• Abstraction of a H• from

An adjacent fatty acid

ROOH

Hydro-

Peroxide O

O

H

Fig. 14: Mechanism of Non-enzymatic Lipid Peroxidation.

52

Since the molecular precursor for the initiation process is generally the

hydroperoxide product ROOH, lipid peroxidation is a chain reaction with potentially

devastating effects. To control and reduce lipid peroxidation, both humans in their

activities and nature invoke the use of antioxidants. Propyl gallate, butylated

hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) are antioxidants used as

food additives. Naturally occurring antioxidants include vitamin E (tocopherol), which is

lipid-soluble as well as urate and vitamin C, which are water-soluble. Beta-carotene is an

antioxidant at low pO2. Antioxidants fall into two classes: (1) preventive antioxidants,

which reduce the rate of chain initiation; and (2) chain-breaking antioxidants, which

interfere with chain propagation. Preventive antioxidants include catalase and other

peroxidases that react with ROOH and chelators of metal ions such as DTPA

(diethylenetriaminepentaacetate) and EDTA (ethylenediamine-tetraacetate). Chain-

breaking antioxidants are superoxide dismutase, which acts in the aqueous phase to trap

superoxide free radicals (O2•); perhaps urate; and vitamin E, which acts in the lipid phase

to trap ROO• radicals.

Peroxidation is also catalyzed in vivo by heme compounds and by lipoxygenases

found in platelets and leukocytes, etc.

1.7.2 Lipid Peroxidation and Tissue Damage

Lipid peroxidation is a well established mechanism of cellular injury in human

and is used as an indicator of oxidative stress in cells and tissues. Lipid peroxides derived

from PUFA are unstable and decompose to form a complex series of compounds

(Halliwell, 1995). These include reactive carbonyl compounds and the most abundant is

malondialdehyde (MDA).

Antioxidants inhibit lipid peroxidation (LPO) by preventing peroxidation chain

reaction or by accumulating the reactive oxygen. Antioxidants are divided into four

subgroups, according to their effect: scavenging, quenchers, repairing and chain breaking.

Lipid peroxidation is one of the molecular mechanisms for cell injury and is associated

with a decrease of cellular antioxidants such as glutathione, superoxide dismutase (SOD)

and catalase (CAT) (Hijora et al., 2005).

53

Free radicals are released by activated leucocytes which cause peroxidation of

membrane lipids. There is a rupture of the lysosomal membranes, the release of

lysosomal enzymes, necrosis of the cell and destruction of parenchymal tissue. All these

processes culminate in an increase in serum MDA levels. Hence, increased serum MDA

could be used as a marker for the free radical mediated destruction of liver parenchymal

cells. Liver disease is accompanied by an increased production of free radicals.

Hepatocyte membranes are rich in PUFA (polyunsaturated fatty acids) which are

susceptible to attack by free radicals. The PUFA of the membranes undergo peroxidation

(Pratibha et al., 2004).

The occurrence of impaired oxidative status is highlighted by several biomarkers,

among them is MDA, a terminal compound of lipid peroxidation which is commonly

used as an index of oxidative stress (Leuratti et al, 1998). Exposure of lipids in cell

membrane to free radicals stimulates the process of lipid peroxidation. The products of

lipid peroxidation are themselves reactive species and lead to extensive membrane

organellar and cellular damage. The free radical activities and the extent of tissue damage

are related quantitatively to the amount of lipid peroxide level in the blood. MDA is one

of the end products of lipid peroxidation and the extent of lipid peroxidation is measured

by estimating MDA levels most frequently. Increased serum level of MDA has been

reported in cardiovascular, neurological and other diseases (Lata et al., 2004).

Biological membranes are particularly prone to reactive oxygen species (ROS)

effect. The peroxidation of unsaturated fatty acids in biological membranes leads to a

decrease of membrane fluidity and to a disruption of membrane integrity and function

which is implicated in serious pathological changes. Several endogenous protective

mechanisms have been evolved to limit ROS and the damage caused by them. However,

since this protection may not be complete, or when the formation of ROS is excessive, an

additional protective mechanism by dietary antioxidants may be of great importance

(Ulicna et al., 2003).

54

1.7.3 Malondialdehyde

Since free radicals are potentially toxic, they are usually inactivated or scavenged

by antioxidants before they can inflict damage to lipids, proteins or nucleic acids

(Rukmini et al., 2004).

The human body has a complex antioxidant defense system that includes the

antioxidant enzymes, superoxide dismutase, glutathione peroxidase and catalase. These

block the initiation of free radical chain reaction. The non-ezymatic antioxidant

components consist of molecules such as glutathione, alpha-tocopherol, ascorbic acid and

beta-carotene that react with activated oxygen species and thereby prevent the

propagation of free radical chain reaction (Rukmini et al., 2004). However, when free

radicals are generated in excess or when the cellular antioxidant defense system is

defective, they can stimulate chain reaction by interacting with proteins, lipids and

nucleic acids causing cellular dysfunction and even death. Malondialdehyde is an end

product of lipid peroxidation. The determination of lipid peroxide products like MDA has

been used as measure of oxygen radical activation (Pratibha et al., 2004). Lipid

peroxidation is followed by a loss of structural integrity of plasma membranes. MDA is

also a product of prostaglandin biosynthesis (Leuratti et al., 1998). In biological matrices,

MDA exist in both free (fMDA) and bound (bMDA) to SH and/or NH2 groups of

proteins, nucleic acids and lipoproteins. Chemically reactive fMDA is an index of recent

and potential damage, while bMDA excreted by the kidney is a marker of an older injury

(NDT, 2009).

MDA is a carbonyl compound generated by lipid peroxidation and during

arachidonic acid metabolism for the synthesis of prostaglandins. MDA is mutagenic and

carcinogenic to rats (Leuratti et al., 1998).

Increased levels of lipid peroxidation products have been associated with a variety

of chronic diseases in both humans and model systems (Sarayme et al., 2004).

MDA is formed during oxidative degeneration as a product of free oxygen

radicals (Yazar et al., 2004). It has the ability to interact with lipoproteins and so has

received particular attention in pharmacological studies (Sarka & Rautary, 2009).

55

1.7.4 Superoxide Dismutase

Superoxide dismutase is an enzyme whose function is to protect against the

potentially damaging reactivities of the superoxide radical (O2–) generated by aerobic

metabolic reactions. Two types of SOD have been found in all mammalian cells except

erythrocytes. Cu, Zn-SOD was present in both the cytosol and the intermediate

membrane space of the mitochondria, and Mn-SOD was present in the mitochondrial

matrix (Takada et al., 1982).

Non-sulfur Fe enzyme known as superoxide dismutase (SOD) catalyze

disproportion of

O2 : 2O2– + 2H

+ H2O2 + O2

FeSOD is found in bacteria, especially the more primitive ones, the chloroplast of

plants, a few protists and possibly eukaryotes. The homologous MnSODs are found in

bacteria and mitochondria, and are believed to protect DNA from endogenous oxidative

stress, whereas FeSOD may serve as a housekeeping enzyme and provide resistance to

environmental oxidative stress by virtue of its periplasmic location. With catalases and

peroxides, SOD caused by the chemical progeny of O2– : H2O2 and OH

•. Atomic

absorption spectroscopy reveals that SOD monomer contains one metal ion and no other

cofactors (Miller, 2001).

1.7.5 Albumin

Albumin is a key export protein of the liver. In plasma, albumin has numerous

functions including maintaining colloid and osmotic pressure and buffering. Additional

functions of albumin include the transport of fatty acids, cortisol, thyroxine and bilirubin

and the binding of endotoxins (Preedy et al., 2002).

The liver produces most of the plasma proteins in the body, and albumin is a

protein made specifically by the liver. Albumin is quantitatively the most important

protein in plasma synthesized by the liver and is a useful indicator of hepatic function.

Since the half life of albumin in serum is as long as 20 days, the serum albumin level is

not a reliable indicator of hepatic protein synthesis in acute liver disease (Orhue et al.,

2005). Albumin synthesis is affected not only in liver diseases but also by nutritional

56

status, hormonal balance and osmotic pressure. Liver is the only site of synthesis of

albumin (Thapa and Anuj, 2007).

The serum levels are typically depressed in patients with cirrhosis and ascites.

Normal serum values range from 3-5g/dl to 4.5g/dl. The average adult has approximately

300 to 500g of albumin. The serum levels at anytime reflect its rate of synthesis,

degradation and volume of distribution. The serum albumin levels tend to be normal in

diseases like acute viral hepatitis, drug related hepatotoxicity and obstructive jaundice.

Decreased levels of serum proteins are found in renal disease, malnutrition albuminuria

and terminal liver failure. Although, the concentration of the serum albumin is reduced in

severe liver diseases, that of the globulins is usually increased so that the total protein

concentration is rarely low (Wurochekke et al., 2008).

Consequently, decreased albumin levels also may be associated with liver disease.

Albumin levels also may indicate general health and nutritional status (Carepath, 2010).

The normal half life of albumin is an average of 21 days and therefore a decrease in

serum albumin is usually not apparent early in the course (Orhue et al., 2005).

1.7.6 Serum Proteins

Measurement of serum proteins and fractions reflects nutritional and health status

(Pervaiz et al., 2001). Globulin is one of the main groups found in blood. The alpha and

beta globulins are produced by the liver, whereas the gamma-globulins (antibodies that

play an important role in the body’s ability to defend itself against disease) are produced

by some white blood cells and plasma cells. The level of serum globulin is often elevated

in liver disease, collagen connective tissue diseases and myeloma (Carepath, 2010).

Reduced serum albumin (SA) levels are used as prognostic indicators in several

diseases, including cancer, kidney disease, human immunodeficiency virus infections,

chronic liver diseases, autoimmune diseases and even in healthy geriatric individuals (Al-

Jaudi, 2005).

57

1.9 Aim and Objectives of Study

This study was primarily designed to compare the effects of esculetin and

scopoletin on paracetamol- induced liver damage in male Wistar rats.

Several parameters would be assayed using the serum of the experimental animals

from the various groups. These parameters are indicators of liver function status of

animals. These parameters are:

Aspartate aminotransferase (AST)

Alanine aminotransferase (ALT)

Alkaline phosphatase (ALP)

Total Bilirubin (TB)

Conjugate Bilirubin (CB)

Superoxide dismutase

Albumin

Lipid Peroxidation

58

CHAPTER TWO

MATERIALS AND METHODS

2.1 Materials

2.1.1 Animals

The experimental animals used in this study were male Wistar albino rats of

between 4 and 8 weeks old with average range of 150 –230g. The rats were obtained

from the Faculty of Biological Sciences and Faculty of Pharmaceutical Sciences, Animal

house, University of Nigeria, Nsukka (UNN).

2.1.2 Chemicals/Reagents/Samples

Dimethyl sulphoxide (DMSO) and scopoletin were bought from Serva,

Heidelberg/New York, while esculetin was bought from Aldrich, Germany. Paracetamol

(500mg) was a product of Emzor Pharmaceutical Industries Ltd., Lagos, Nigeria. All

other chemicals and reagents except where stated were bought from reputable companies

or their representatives. Reagents used for all the assays were commercial kits and

products of Randox Laboratory, Grumlin, CO, Antrin, UK and QCA, Spain.

2.1.3 Equipment/Instruments

Equipment Manufacturer

Digital colorimeter E1 (312 Model) Japan

Micro Haematocrit tubes Vasekaer, Harlev Denmark

Centrifuge Chikpas, England

Water bath Gallenkamp, England

2.2 Methods

The albino rats were administered 6mg/kg body weight of esculetin and 6mg/kg

body weight of scopoletin respectively dissolved in 10% dimethyl sulphoxide (DMSO)

intragastrically for 28 days. They also received 500mg/kg body weight of paracetamol

(single dose).

59

2.2.1 Experimental Design

Thirty-five male Wistar rats were housed in separate cages, acclimatized for seven

days and then divided into five groups of seven rats each. The animals were maintained

on a 12h light and dark cycle under tropical conditions. They had free access to water and

were fed with normal rat diet ad libitum. The normal rat diet was purchased from Bendel

Feed and Flour Mills Limited., Ewu, Edo State, Nigeria.

GROUP I animals were administered 6mg/kg(as single dose) body weight of

esculetin via gastric intubation daily for 28 days. This group also received 500mg/kg

body weight of paracetamol on the 28th day.(as a single dose).

GROUP II animals were administered 6mg/kg body weight of scopoletin via

gastric intubation in 10% DMSO also daily for 28 days. This group also received

500mg/kg body weight of paracetamol on the 28th day.

GROUP III animals represented positive control group which were administered

single dose (500mg/kg body weight) of paracetamol via gastric intubation on the 28th

day.

GROUP IV animals administered 10% DMSO for 28 days represented negative

control group

GROUP V animals administered distilled water only for 28 days represented

negative control group

The rats were weighed at the beginning of the experiment in order to

predetermine dosage. The animals in Groups I and II were pretreated with esculetin and

scopoletin respectively, daily, for 28 days.

The rats were exposed to the toxicant (paracetamol) on the 28th day and allowed

to survive for 48 h before they were sacrificed. Bleeding of the animals was done by

ocular puncture.

2.2.2 Preparation of Scopoletin Solution

Scopoletin solution was prepared by dissolving 36mg of scopoletin in 6ml of 10%

DMSO.

60

2.2.3 Preparation of Esculetin Solution

This was prepared by dissolving 36mg esculetin in 6ml of 10% DMSO.

2.2.4 Preparation of Paracetamol Solution

Paracetamol (3125.0mg) was dissolved in 50ml of distilled water.

2.2.5 Preparation of Liver Samples

The animals were made unconscious using chloroform. While still unconscious,

they were dissected and the liver immediately excised and preserved in 10% formal

saline.

2.2.6 Preparation of Serum Samples

Whole blood was collected from the animals in different groups through ocular

puncture and introduced into a clean non-anticoagulated blood sample containers. The

blood sample was centrifuged at a speed of 3000 rpm in order to get the supernatant

(serum). The different sera obtained were used immediately for biochemical analyses.

2.2.7 Assay Methods

2.2.7.1 Assay of Aspartate aminotransferase Activity

A Randox Commercial Enzyme kit according to the method of Reitman and

Frankel (1957) and Schmidit and Schmidit (1963) was used.

This method is based on the principle that oxaloacetate is formed from the

reaction below:

–Oxoglutarate + L – aspartate L-glutamate + oxaloacetate

Glutamic-oxaloacetic acid transaminase (aspartate aminotransferase) activity was

measured by monitoring the concentration of oxaloacetate hydrazone formed with 2,4-

dinitrophenyl hydrazine.

61

Reagents

Content Initial concentration of solution

1.

Phosphate buffer 100mmol/l, pH 7.4

L–Aspartate 100mmol/l

–Oxoglutarate 2mmol/l

2. 2, 4–dinitrophenyl hydrazine 2mmol/l

Sodium hydroxide 0.4mol/l

1. Measurement against Reagent Blank

The AST substrate phosphate buffer (0.5ml each) was pipetted into both the

reagent blank (B) and sample (T) test tubes respectively. The serum sample (0.6ml) was

added to the sample (T) test tubes only and mixed thoroughly. Then 0.1ml of distilled

water was added to the reagent blank (B). The entire reaction medium was well mixed

and incubated for 30min in a water bath at 37oC.

Immediately after incubation, 2,4-dinitrophenyl-hydrazine (0.5ml) was added to

the reagent blank (B) and the sample (T) test tubes, mixed thoroughly and allowed to

stand for exactly 20min at 25oC. Finally, 5.0ml of sodium hydroxide (0.4 mol/l) solution

was added to both the blank and the reagent test tubes respectively and mixed thoroughly.

The absorbance of sample was read at a wavelength of 550nm against the reagent

blank after 5min.

2. Measurement against Sample Blank

The AST substrate phosphate buffer (0.5ml each) was pipetted into the sample

blank (B) and sample (T) test tubes respectively. The serum sample (0.1ml) was added to

the sample test (T) only and mixed immediately then incubated in a water bath for

exactly 30min at 37oC.

2,4 – Dinitrophenylhyrazine was added to both sample blank (B) and sample (T)

test tubes immediately after incubation. Also, 0.1ml of the sample was added to the

sample blank (B) only. Each medium was mixed and allowed to stand for exactly 20min

at 25oC. Finally, 5.0ml of sodium hydroxide (NaOH) solution 0.4 mol/l was added to

62

both the sample blank (B) and sample (T) test tubes and mixed thoroughly. Absorbance

of the sample was read at a wavelength of 550nm against the sample blank after 5min.

Table 4: Serum Normal Values of AST in Humans

The activity of AST in the serum was obtained from the Table below.

Absorbance U/L Absorbance U/L

0.020 7 0.120 47

0.030 10 0.130 52

0.040 13 0.140 59

0.050 16 0.150 67

0.060 19 0.160 76

0.070 23 0.170 89

0.080 27

0.090 31

0.100 36

0.110 41

Source: (Reitman and Frankel, 1957) and Schmidit and Schmidit (1963)

Normal values in human, serum up to 12U/L

2.2.7.2 Assay of Alanine aminotransferase Activity

A Randox Commercial Enzyme Kit based on the methods of Reitman and Frankel

(1957) and Schmidt and Schmidt (1963) was used.

Alanine aminotransferase assay is based on the principle that pyruvate is formed

from:

–Oxoglutarate + L-alanine L-glutamate + pyruvate

Alanine aminotransferase is measured by monitoring the concentration of

pyruvate hydrazine formed with 2, 4–dinitrophenyl hydrazine.

63

Reagents


Phosphate buffer 100mmol/l pH 7.4

L–alanine 200mmol/l

–oxoglutarate 2.0mmol/l

2, 4–dinitrophenyl hydrazine 2.0mmol/l

Sodium hydroxide 0.4mol/l

Procedure

Measurement against Sample Blank

The ALT substrate phosphate buffer (0.5ml each) was pipetted into two sets of

test tubes labeled B (sample blank) and T (sample test) respectively. The serum (0.1ml)

sample was added to the sample test (T) only and mixed properly, then incubated for

exactly 30min in a water bath at a temperature of 37oC.

2, 4 – dinitrophenyl hydrazine (0.5ml) was added to both test tubes labeled T

(sample test) and B (sample blank) immediately after the incubation. Also, 0.1ml of

serum sample was added to the sample blank (B) only. The entire medium was mixed

thoroughly and allowed to stand for exactly 20min at 25oC. Then, 5.0ml of sodium

hydroxide (NaOH) solution (0.4 mol/l) was added to both test tubes and also mixed

thoroughly. Absorbance of the sample read at a wavelength of 550nm after 5min.

Measurement against Reagent Blank

The ALT substrate phosphate buffer (0.5ml each) was pipetted into both the

reagent blank (B) and sample (T) test tubes respectively. The serum sample 0.1ml was

added to the sample (T) test tube only and mixed thoroughly. Then 0.1ml of distilled

water was added to the reagent blank (B). The entire medium was mixed and incubated

for exactly 30min in a water bath at 37oC. Immediately after incubation, 2,4 –

dinitrophenyl – hydrazine (0.5ml) was added to both reagent blank and sample (T) test

tubes. The contents of the tubes were mixed thoroughly and allowed to stand for exactly

20min at 25oC. Finally, 5.0ml of sodium hydroxide solution (0.4 mol/l) was added to both

64

the blank and reagent test tubes respectively. Each was mixed thoroughly and absorbance

of sample read at a wavelength of 550nm against the reagent blank after 5min.

Table 5: Serum Normal Values of ALT in Humans

Activity of ALT in serum was obtained from the Table below.

Absorbance U/L Absorbance U/L

0.025 4 0.275 48

0.050 8 0.300 52

0.075 12 0.325 57

0.100 17 0.350 62

0.125 21 0.370 67

0.150 25 0.400 72

0.175 29 0.425 77

0.200 34 0.450 83

0.225 39 0.475 88

0.250 43 0.500 94

Source: (Reitman and Frankel, 1957; Schmidt and Schmidt, 1963).

Normal Values in humans serum up to 12 U/L

2.2.7.3 Assay of Alkaline Phosphatase Activity

This was done using the QCA Commercial Enzyme Kit which is based on the

phenolphthalein monophosphate method of Klein et al. (1960); Babson (1965) and

Babson et al. (1966).

Principle

Serum alkaline phosphatase hydrolyzes a colourless substrate of phenolphthalein

monophosphate giving rise to phosphoric acid and phenolphthalein which at alkaline pH

values, turns to pink colour that can be photometrically determined.

65

The concentrations in the reagent solution are;

2-Amino-2-methly-1-propanol pH 11 7.9mM

Phenolphthalein monophosphate 63mM

Na2PO4 80mM

Stabilizers and preservatives

Procedure

Distilled water (1ml) was pipetted into 2 sets of test tubes labeled SA (sample)

and ST (standard) respectively. Then one drop of each of the chromogenic substrates was

added to the distilled water in the two sets of test tubes. Their contents were mixed and

incubated at 37oC for 5min.

A standard solution of 0.1M (alkaline phosphate) was added to the standard test

tube (ST) only, followed by the addition of 0.1ml of the serum sample to the sample test

tube (SA). The contents of the test tubes were mixed and incubated at 37oC for 20 min in

a water bath. A colour developer (phenolphthalein monosulphate) (5.0ml each) was

added to both sets of test tubes.

Absorbance of the sample against the blank (water) was read at a wave length of

550nm.

The activity of alkaline phosphatase in the serum was obtained from the formula

below:

SA O.D x 30 = U/L of Alkaline phosphatase

ST O.D

SA O.D = Sample Optical Density

ST O.D = Standard Optical Density

Normal values

Adults = 9 – 35 U/L

Children = 35 – 100 U/L

2.2.7.4 Assay of Bilirubin

A colorimetric method with a kit supplied by Randox® was used. (Jendrassik and

Grof, 1938; Sherlock, 1951).

66

Principle

Direct (conjugated) bilirubin reacts with diazotized sulphanilic acid in alkaline

medium to form a blue coloured complex. Total bilirubin is determined in the presence of

caffeine, which releases albumin bound bilirubin, by the reaction with diazotized

sulphanilic acid.

Reagent


Sulphanilic acid 29mmol/L

Hydrochloric acid 0.17N

Sodium Nitrate 25mmol/L

Caffeine 0.26mol/L

Sodium benzoate 0.52mol/L

Tartrate 0.93mol/L

Sodium hydroxide 1.9N

Procedure

Total Bilirubin (TB)

Reagent 1 (sulphanilic acid, hydrochloric acid), 0.20ml, was pipetted into two

different cuvettes labeled sample blank (B) and sample (A) respectively and then a drop

(0.05ml) of reagent was introduced. Then a drop of 0.05ml of reagent was pipetted into

the cuvette containing sample (A) only.

Afterwards, 1.0ml of reagent 3 (caffeine, sodium benzoate) was pipetted into the

cuvettes containing samples B and A respectively. Serum sample (0.2ml) was then

pipetted into both cuvettes, sample blank (B) and sample (A). Their contents were

separately mixed and allowed to stand for 10min at 25oC.

This was followed by addition of 1ml of reagent 4 (Tartrate, sodium hydroxide)

into both cuvettes containing sample blank and sample. They were mixed and allowed to

stand for 30min at 25oC. Finally, absorbance of the sample against the sample blank

(ATB) was read at 560nm. Total bilirubin values were obtained using the calculation

below:

67

Total bilirubin (µmol/L) = 185 x ATB (560nm)

Total bilirubin (mg/dl) = 10.8 x ATB (560nm)

Direct Bilirubin (DB)

Reagent 1 (0.2ml) was pipetted into two cuvettes labeled sample Blank (B) and

sample (A). A drop (0.05ml) of reagent 2 was pipetted into the cuvette containing sample

(A) only. This was followed by the addition of 2.0ml sodium chloride (µg/L) into both

cuvettes containing sample blank (B) and sample (A) respectively. Serum (0.2ml) was

finally pipetted into both cuvettes, sample blank (B) and sample (A). The contents of the

two cuvettes were mixed thoroughly and allowed to stand for exactly 5min at 25oC. The

absorbance of the sample against the sample blank (ADB) was read at 550nm.

Direct bilirubin values were obtained from the following calculation.

Direct bilirubin (µmol/L) = 246 x ADB (550nm)

Direct bilirubin (mg/dl) = 14.4 x ADB (550nm)

Normal value in serum

Total bilirubin up to 17 µmol/L

up to 1 mg/dL

Direct bilirubin up to 4.3 µmol/L

up to 0.25 mg/dL

2.2.7.5 Superoxide Dismutase (SOD) Activity

The activity of SOD was evaluated by the method of Misra and Fridovich (1972).

It is based on the inhibition of epinephrine auto-oxidation to adenochrome in alkaline

environment. Auto-oxidation of epinephrine was initiated by adding 1ml of Fenton

reagent to a 4ml mixture of 0.3mm epinephrine, 1mm solution of Na2CO3, 0.3mm EDTA

and 1ml of distilled water at a final volume of 6ml. The autooxidation was monitored

spectrophotometrically at 480nm every 30secs for 5min. The experiment was repeated

with 1.0ml of the serum. A graph of absorbance against time was plotted for each sample

and initial rate of autooxidation was calculated. One unit of SOD activity was defined as

the concentration of the enzyme in the sample that cause 50% reduction in the auto

68

oxidation of epinephrine. SOD activity was then calculated for each sample and

expressed in U/l.

2.2.7.6 Serum Protein (Band et al., 1949)

Principle: The colour produced by serum protein with Biuret reagent is estimated and

compared with a protein solution of known strength. The globulin is separated by Na

sulphate and other precipitate and Albumin is determined with the aqueous layer and

globulin is estimated by difference.

Stock Biuret Reagent

Sodium potassium tartarate (45g) in 400ml of 0.3N NaOH. A quantity of 15g of

copper sulphate (CuSO4.5H2O) pentahydrate was added and stirred until solution was

complete. A quantity of 5g of KI (potassium iodinate) was added and made up to a litre

with 0.2N NaOH.

Working Biuret reagent

Stock (200ml) was diluted to a litre with 0.2N NaOH which contain 5g KI per

litre.

22.6% Na2SO4

A quantity of 226g of NaSO4 anhydrous was dissolved in a litre of H2O. It was

kept in a warm place.

Method

Serum (0.4ml) was placed in a centrifuge tube, 3.6ml Na2SO4 solution was added.

The tube was stoppered and mixed thoroughly by inversion not shaking. A volume of

1.0ml of the solution was transferred at once into a tube marked ‘total’. A volume of

1.0ml of ether was added into the remaining solution and mixed by inversion 10 times, it

was then spun for 5min at 200 rpm.

A volume of 1.0ml of aqueous solution into another tube labeled albumin.

Distilled water (1.0ml) was added into another tube marked blank. A volume of 4ml of

69

working Biuret reagent was added to all the tubes, mixed and allowed to stand for 30min

and absorbance was read at 540nm.

Total protein – Albumin = Globulin

2.2.7.7 Lipid Peroxidation Assay

Lipid peroxidation was determined spectrophotometrically by measuring the level

of the lipid peroxidation product, malondialdehyde (MDA) as described by Wallin et al.

(1993).

Principle

Malondialdehyde (MDA) reacts with thiobarbituric acid to form a red or pink

coloured complex which, in acid solution, absorbs maximally at 532nm.

MDA + 2TBA MDA:TBA adduct + H2O

Reagent Preparation

i. 1.0% Thiobarbituric acid (TBA): A quantity, 1.0 g, thiobarbituric acid was

dissolved in 83 ml of distilled water on warming and after complete dissolution the

volume was made up to 100 ml with distilled water.

ii. 25% Trichloroacetic acid (TCA): A quantity, 12.5 g, of trichloroacetic acid was

dissolved in distilled water and made up to 50 ml in a volumetric flask with distilled

water.

iii. Normal saline solution (NaCl): A quantity, 0.9 g, of NaCl was dissolved in 10

ml of distilled water and made up to 100 ml with distilled water.

Procedure

To 0.1 ml of plasma in a test tube was added 0.45 ml of normal saline and mixed

thoroughly before adding 0.5 ml of 25% trichloroacetic acid (TCA) and 0.5 ml of 1%

thiobarbituric acid. The same volume of tricholoracetic acid, and saline was added to the

blank. Distilled water (0.1ml) was also added to the blank instead of plasma. Then, the

mixture was heated in a water bath at 95 0C for 40 min. Turbidity was removed by

70

centrifugation. The mixture was allowed to cool before reading the absorbance of the

clear supernatant against reagent blank at 532 nm. Thiobarbituric acid reacting substances

were quantified as lipid peroxidation product by referring to a standard curve (Appendix

1) of MDA concentration (i.e. equivalent generated by acid hydrolysis of 1,1,3,3-

tetraethoxypropane (TEP) prepared by serial dilution of a stock solution).

Table 6: Lipid peroxidation assay procedure

Blank Test

Plasma --- 0.10 ml

Distilled water 0.10 ml ---

Normal saline 0.45 ml 0.45 ml

25% TCA 0.50 ml 0.50 ml

1% TBA 0.50 ml 0.50 ml___

2.3 Statistical Analysis

Test of statistical significance was carried out using one-way analysis of variance

together with postHoc test (Multiple comparison). Statistical difference was considered

when P≥0.05. The statistical package used was Statistical Package for Social Sciences

(SPSS).

71

CHAPTER THREE

RESULTS

3.1 Comparative Effects of Esculetin and Scopoletin on Alkaline Phosphatase

(ALP) Activity

As shown in Fig. 15 and Appendix 11, there was no significant difference

(P>0.05) between the test groups administered single dose (G.I) 6mg/kg body weight of

esculetin and scopoletin + 500 mg/kg body weight of paracetamol.

A significant difference (P<0.05) was observed between the level of ALP for

esculetin + paracetamol treated groups and the positive control. A significant difference

(P<0.05) was also observed between the level of the ALP for the scopoletin +

paracetamol treated groups and the positive control. There was a significant difference

(P<0.005) between the ALP level of the positive control and negative control (Group V).

Significant difference was observed between the positive control and the group

administered 10% DMSO only. No significant difference (P>0.05) was observed between

the ALP level of the negative control and the test groups administered (G.I) 6mg/kg body

weight esculetin/scopoletin + 500 mg/kg body weight of paracetamol.

3.2 Comparative Effects of Esculetin and Scopoletin on Alanine

Aminotransferase (ALT) Activity

It was observed (Fig.16 and Appendix II) that there was no significant difference

(P>0.05) between the ALT levels of the test groups administered 6mg/kg body weight

esculetin + 500 mg/kg body weight of paracetamol and scopoletin + paracetamol. A

significant difference (P<0.05) in the level of AST was observed between the positive

control and the test groups administered esculetin + paracetamol and scopoletin +

paracetamol. No significant difference (P>0.05) was observed between the negative

control (Group IV and V) and the test groups. A significant difference (P<0.05) was

observed between the AST level of the positive control and of the negative control

(Group V). Significant difference was observed between the AST level of the positive

control and group IV.

72

3.3 Comparative Effects of Esculetin and Scopoletin on Aspartate

Aminotransferase Activity (AST) Activity

There was neither significant increase nor decrease (P>0.05) in the AST activity

of the test group administered 6mg/kg body weight esculetin + 500mg body weight of

paracetamol compared with the test groups administered scopoletin 6mg/kg body

weight+ 500mg/kg body weight of paracetamol (Fig. 17 and Appendix 11). A

significance difference (P>0.05) in the activity of AST was observed between the

positive control and the test groups administered esculetin + paracetamol and scopoletin

+ paracetamol. However no significant difference (P>0.05) was observed between the

negative control (Group V) and the test groups. AST activity increased significantly

(P<0.05) in Group IV and II. There was significant increase (P<0.05)in the activity of

AST as observed in the animals in the positive control group when compared with the

AST activity of the animals in the negative control group given distilled water. No

significant difference (P>0.05) was observed between the AST activities of the positive

control and test groups.

3.4 Comparative Effects of Esculetin and Scopoletin on Conjugated Bilirubin

Level

The results shown in Fig.18 and Appendix II indicate that no significant

difference (P>0.05) was observed between the test groups administered 6mg/kg body

weight esculetin + 500mg/kg body weight of paracetamol and the group administered

6mg/kg body weight scopoletin + 500mg/kg body weight of paracetamol. A significant

difference (P<0.05) was observed between the positive control and the negative control

given distilled water. No significant difference (P>0.05) was also observed between the

test groups administered esculetin + paracetamol and scopoletin + paracetamol and the

negative control group (Group IV). There was a significant difference (P<0.05) observed

between the test groups and Group V. The results also show that there was no significant

difference between the positive control and the test groups administered 6mg/kg body

weight esculetin + 500mg/kg body weight of paracetamol. Significant difference

(P<0.05) was observe between the positive group administered scopoletin 6mg/kg body

weight + 500mg/kg body weight of paracetamol. There was also no significant difference

(P>0.05) observed between the positive control and the group the group administered

10% DMSO. No significant difference (P>0.05) was observed between groups IV and V.

73

3.5 Comparative Effects of Esculetin and Scopoletin on Total Bilirubin Level

From Fig. 19 and Appendix II, significant difference (P<0.05) was observed

between the total bilirubin level of the test groups administered 6mg/kg body weight

esculetin + 500mg/kg body weight of paracetamol and scopoletin 6mg/kg body weight +

500mg/kg body weight of paracetamol. No significant difference (P>0.05) was observed

between the total bilirubin level of the esculetin + paracetamol group and the positive

control. Also a significant difference (P<0.05) was observed between the total bilirubin

level of scopoletin + paracetamol group and the positive control. A significant difference

was observed between the test group, esculetin + paracetamol (Group V).

Significant difference (P<0.05) was also observed between the negative control

groups (IV and V) and the positive control. A significant difference (P>0.05) was

observed between groups IV and V.

No significant difference (P>0.05) was observed between group I and group IV.

No significant different (P>0.05) was observed between groups II and V and groups II

and IV.

3.6 Comparative Effect of Esculetin and Scopoletin on Superoxide Dismutase

Activity

No significant difference (P>0.05) was observed between the test groups

administered esculetin + paracetamol and scopoletin + paracetamol. A significant

difference (P<0.05) was observed between the test groups and the positive group

administered only paracetamol (Fig. 20 and Appendix II).

A significant difference was observed between the test groups I and II and the

group administered 10% DMSO. No significant difference was observed between the test

groups and the negative control administered only water.

A significant difference (P<0.05) was observed between group administered

paracetamol only and the groups administered 10%DMSO and water. A significant

difference (P<0.05) was observed between group IV and V.

74

3.7 Comparative Effect of Esculetin and Scopoletin on Malondialdehyde Level

As shown in Fig. 21, no significant difference (P>0.05) was observed between the

test groups administered esculetin + paracetamol and scopoletin + paracetamol. A

significant difference (P<0.05) was observed between the test groups I and II and the

group administered paracetamol only. A significant difference (P<0.05) was also

observed between the test groups and the group administered 10% DMSO.

No significant difference (P>0.05) was observed between the test groups and the

negative control. A significant difference (P<0.05) was observed between groups IV and

V. No significant difference (P>0.05) was observed between groups IV and V.

3.8 Comparative Effect of Esculetin and Scopoletin on Albumin Level

There was no significant change (P>0.05) observed in the level of serum albumin

between the test groups administered esculetin + paracetamol and scopoletin +

paracetamol as shown in Fig. 22 and Appendix II. No significant difference (P>0.05) was

also observed between the test groups and the group administered paracetamol alone. No

significant difference was observed between the group I and group IV and V.

A non significant difference (P<0.05) was between the observed groups

administered 10% DMSO and water. A significant difference was observed between the

group administered paracetamol and group V. No significant difference between group

IV and group III.

No significant difference was observed between group II and IV. There was

significant difference between group II and V. Significant difference was observed

between group III and V. Also, there was significant difference between group IV and V.

75

3.9 Comparative Effect of Esculetin and Scopoletin on Globulin Level

No significant difference was observed between the test groups I and II. No

significant difference was observed between test groups and the paracetamol group (Fig.

23 and Appendix II).

A significant difference (P<0.05) was observed between group I and IV. No

significant difference between group I and V. No significant difference between group II

and V. No significant difference between group III and group V.

No significant difference between groups (P>0.05) between groups IV and V.

76

Fig. 15: Effect of esculetin and scopoletin on the serum Alkaline

phosphatase activity in rats

0

10

20

30

40

50

60

70

80

90

Group 1 Group 2 Group 3 Group 4 Group 5

Group

Me

an

AL

P A

cti

vit

y (

IU/L

)

Fig. 15: Comparative effects of esculetin and scopoletin on the serum alkaline

phosphatase activity (ALP)

Group 1=Esculetin Group 2=Scopoletin

Group 3=Paracetamol Group 4=10% DMSO

Group 5=Water

77

Fig. 16: Effect of esculetin and scopoletin on the serum Alanine

aminotransferase activity in rats

0

5

10

15

20

25

30

35

40

45


Group

Me

an

AL

T A

cti

vit

y (

IU/L

)

Fig. 16: Comparative effects of esculetin and scopoletin on the serum alanine

aminotransferase activity (ALT)



Group 5=Water



Group 5=Water

78

Fig. 17: Effect of esculetin and scopoletin on the serum Aspartate

aminotransferase activity in rats

0

10

20

30

40

50

60

70

80

90

100


Group

Me

an

AS

T A

cti

vit

y (

IU/L

)

Fig. 17: Comparative effects of esculetin and scopoletin on the serum aspartate

aminotransferase activity (AST)



Group 5=Water

79

Fig. 18: Effect of esculetin and scopoletin on the conjugated

bilirubin concentration in rats

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7


Group

Me

an

Bil

iru

bin

Co

nc

(m

g/m

l)

Fig. 18: Comparative effects of esculetin and scopoletin on the conjugated bilirubin level



Group 5=Water

80

Fig. 19: Effect of esculetin and scopoletin on the serum Total

bilirubin concentration in rats

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7


Group

Me

an

To

tal

Bilir

ub

in C

on

c (

mg

/ml)

Fig. 19: Comparative effects of esculetin and scopoletin on the total bilirubin level



Group 5=Water

81

Fig. 20: Effect of esculetin and scopoletin on the superoxide

dismutase activity in rats

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5


Group

Me

an

SO

D A

cti

vit

y (

IU/L

)

Fig. 20: Comparative effects of esculetin and scopoletin on the superoxide dismutase

activity



Group 5=Water

82

Fig. 21: Effect of esculetin and scopoletin on the Malondialdehyde

concentration in rats

0

5

10

15

20

25

30

35

40


Group

Me

an

MD

A C

on

c (

mg

/ml)

Fig. 21: Comparative effects of esculetin and scopoletin on the malondialdehyde level



Group 5=Water

83

Fig. 22: Effect of esculetin and scopoletin on the serum Albumin


0

1

2

3

4

5

6


Group

Me

an

Alb

um

in C

on

c (

mg

/ml)

Fig. 22: Comparative effects of esculetin and scopoletin on the serum albumin level



Group 5=Water

84

Fig. 23: Effect of esculetin and scopoletin on the serum Globulin


0

0.5

1

1.5

2

2.5

3

3.5

4


Group

Me

an

Glo

bu

lin

Co

nc

(m

g/m

l)

Fig. 23: Comparative effects of esculetin and scopoletin on the serum globulin level



Group 5=Water

85

CHAPTER FOUR

4.1 DISCUSSION

The liver is an organ of paramount importance which plays an essential role in the

metabolism of foreign compounds entering the body. Human beings are exposed to these

compounds through environmental exposure, consumption of contaminated food or

during exposure to chemical substances in the occupational environment. In addition,

human beings take considerable amount of synthetic drugs during disease conditions

which are alien to the body. All these compounds produce a variety of toxic

manifestations (Atha et al., 1997). Conventional drugs used in the treatment of liver

diseases are often inadequate. It is therefore necessary to search for alternative remedies

for the treatment of liver disease. This has necessitated a radical shift to the use of plant

species for treatment (Rajesh et al., 2001).

Paracetamol, a widely used over the counter analgesic-antipyretic drug is safe

when used within therapeutic doses. When taken in large doses, it may cause

hepatotoxicity, leading to fulminate hepatic and renal tubular necrosis which are lethal to

human and many species of animals.

Paracetamol induced hepatic injuries are commonly used models for screening

hepatoprotective activity of substances and the extent of hepatic damage is assessed by

the level of increase in cytoplasmic ezymes (ALP, AST and ALT) in circulation (Janbaz

and Gilani, 2002). Esculetin and scopoletin when administered prophylactically exhibited

protection against paracetamol-induced lethality in rats, suggesting hepatoprotective

action.

Paracetamol is converted to a toxic reactive intermediate called N-acetyl-p-

benzoquinone imine (NAPQI) following metabolism by a number of isozymes of

cytochrome P-450 (cyps) i.e CYP 2E1 (Tanaka and Misawa, 2000), CYP 1A2 (Lehman,

2000), CYP 2A6 (Chen et al., 1988), CYP 3A4 and CYP 3A4 and CYP 2D6 (Dong et al.,

2000).

The massive production of reactive species may lead to depletion of protective

physiological antioxidants moieties such as glutathione and -tocopherol thereby

ensuring widespread propagation of alkylation as well as peroxidation, causing damage to

86

the macro-molecules in vital biomembranes (Aldridge, 1981). The inhibitors of CYPs are

known to curtail the toxicity of paracetamol (Tanaka and Misawa, 2000). The choice of

the coumarins (esculetin and scopoletin) for this research work was based on their

numerous pharmacological properties. The results of this research primarily reveal the

role of esculetin and scopoletin in protecting the liver from hepatic damage in

paracetamol-induced toxicity in rats. Esculetin and scopoletin were able to ameliorate the

paracetamol induced hepatocellular damage as evidenced by reduction in the levels of

serum enzyme studied (ALP, AST and ALT). The reported antioxidant activity of

esculetin might be the contributing factor for the observed hepatoprotection (Gilani et al.,

1998).

Bum-Chun et al. (2007) studied the coumarins-esculetin, umbelliferone and

scopoletin for their anti-oxidative and photo-protective effects and reported that esculetin

was the most effective. Coumarins comprise a class of natural antioxidants which are

widely distributed in plants. Among natural antioxidants, coumarins are lipophillic. The

core structure of these compounds is 2H-1-benzopyran-2-one, which are comprised of an

-pyrone-linked benzene ring and the majority have a hydroxyl group at the 6-7 position

and show blue, violet or yellow-green fluorescence under UV radiation (365nm) (Borges

et al., 2005).

In particular, the antioxidant effect of these compounds is the result of their

unique structure. They contain conjugated double bonds and an ester bond which can

serve as a powerful and stable proton donor (Bun-Chun et al., 2007).

Several hydroxyl coumarins have potent free radical scavenging activities and

strongly inhibit lipid peroxidation. In addition, Kaneko et al. (2003) reported that

catecholic coumarins have a protective effect against the cell damage caused by linoleic

acid hydroperoxide. It is also speculated that the number of hydroxyl groups in the

structure was an important factor in radical scavenging activities of coumarin (Traykova

and Kostova, 2005).

Bum-Chum et al. (2007) reported that coumarin compounds with fewer than two

hydroxyl groups did not show much antioxidant activity. These results suggest that

esculetin, which possesses two hydroxyl groups without modification may be the critical

87

antioxidant. They also proposed esculetin as being an antioxidant agent in UV-irradiated

human skin cells.

Sagrario et al. (1998) examined the effect of esculetin on the glutathione system

and lipid peroxidation in mice. They reported that reduced glutathione in liver

supernatants was increased significantly in esculetin-treated animals. The GSSG/GSH

ratio was significantly lower in esculetin groups and also reported an increase in

glutathione reductase (GR) activity. The extent of decline in the GSSG/GSH ratio was

correlated with the increase in the GR activity. This is supportive of this work, as it was

discovered that esculetin showed a significant protective effect on the hepatic cells

damaged by paracetamol when compared with the positive control.

A liver function test is a blood test that gives an indication of whether the liver

has been damaged (membrane damage). Damage to the membrane lipids leads to the

leakage of these enzymes into the blood. The liver function tests used in this study are

adopted to compare the the antioxidants properties of esculetin and scopoletin on

paracetamol- induced liver damage. Results of the liver function tests (AST, ALT and

alkaline phosphatase) of the group administered the toxicant (positive control) showed

significant increase (P<0.05) compared with the negative control – the groups

administered distilled water and DMSO only respectively. These results are consistent

with the findings of Gilani et al. (1998) who observed a significant increase in AST, ALT

and ALP after 48 hours of the administration of 600mg/kg of paracetamol in 10% DMSO

to male Wistar rats. Results in Fig. 15 and Appendix II showed that the concentration of

alkaline phosphatase (ALP) is significantly higher (P<0.05) in the group administered the

toxicant (Paracetamol) as compared with the negative control group administered only

distilled water. ALP levels in plasma will rise with increased bile duct obstruction,

intrahepatic cholestasis or infiltrative diseases of the liver. ALP is also raised in cirrhosis

and liver cancers. As seen in this result, the rise in the activity of ALP in the group

administered the toxicant (paracetamol) as compared with the group administered

distilled water only and the test groups is an indication of damage to the liver. The

reduction in the activity of ALP in the test groups pretreated with esculetin and scopoletin

as compared with group administered paracetamol indicates protection from liver damage

by these coumarins. The most commonly used markers of hepatic injury are aspartate

88

aminotransferase (SGOT). While ALT is cytosolic, AST has both cytosolic and

mitochondrial forms (Johnston, 1999). AST (SGOT) is present in large amounts in the

liver, heart and the skeletal muscle. ALT is therefore more specific for liver disease than

AST (Song et al., 2001). In normal tissue, there is a balance between the production and

scavenging of reactive oxygen metabolites (ROMS). The activities of the enzyme

markers of hepatocellular injury (AST, ALT and alkaline phosphatase) reduced

significantly (P<0.05) when pretreated with (6mg/kg body weight) of esculetin and

scopoletin for 28 days before exposure to the toxicant 500mg/kg body weight of

paracetamol. Esculetin is a phenolic antioxidant compound as reported by Gilani et al.

(1998). It has also been shown by Gilani et al. (1998) to have a protective effect on

paracetamol- induced liver damage. Oral administration of paracetamol produced liver

damage in rats. Pretreatment of rats with esculetin prevented the paracetamol-induced

rise in serum enzymes indicating that esculetin possesses anti-hepatic activity due to its

antioxidant nature. Scopoletin is also an antioxidant coumarin since it possesses an

hydroxyl group in its structure and thereby has the tendency of reversing damage to liver.

Antioxidants are compounds capable of slowing or preventing the oxidation of other

molecules. Antioxidants are often reducing agents such as thiols or poly phenols. They

act as a major defence against radical-mediated toxicity by protecting the damages caused

by free radicals.

The results from Appendix II showed that there was no significant difference

(P>0.05) in the levels of conjugated bilirubin of the groups pretreated with esculetin and

scopoletin and the group administered the toxicant (paracetamol). and also non-

significant difference (P>0.05) as compared with the groups administered DMSO only. A

significant difference (P<0.05) was however observed in the level of conjugated bilirubin

between the group administered the toxicant (paracetamol) and the group administered

distilled water only.

A significant difference (P<0.05) was also observed in the level of total bilirubin

in the groups administered esculetin and scopoletin compared with the group

administered the toxicant (paracetamol) i.e the positive control. There was also a

significant difference (P<0.05) between the positive control administered the toxicant

(paracetamol) and the negative controls administered DMSO and distilled water

89

respectively. No significant difference (P>0.05) was observed between the groups

administered DMSO only and distilled water only.

The antioxidant properties of monohydroxycoumarin have been related with

radicals–scavenging activity, inhibition of tyrosine kinases and possibly with effect on

the prostaglandin proteination. The antioxidant activity is enhanced if the OH group is

situated on the flavonoid ring while being depressed after substitution of H with methoxy

(-OCH3) or methyl (CH3) in the molecule. For example, while 7-methyl coumarin is an

antioxidant 4-methyl coumarin is a prooxidant. If the OH group is linked to a C3 atom

such as 3-hydroxyscopoletin and 3-hydroxyumbelliferone, the corresponding coumarin

would be an inhibitor of 5 lipoxygenase and -D-glucosidase (Traykova and Kostova,

2005).

Dihydroxycoumarins are better antioxidants than monohydroxy-coumarins (Holt

and Paya, 1996). If the OH group is in the ortho position, the corresponding

dihydroxycoumarin can decrease the lipid peroxidation by scavenging O2 and ROO

.. The

oxidative behaviour of these compounds is strongly determined by all aspects of their

molecular structure.

Depending on their structure and on the source of the oxidative stress, coumarins

might act as antioxidants or prooxidants. The hydroxycoumarins are typical phenolic

compounds and therefore act as potent metal chelators and/or free radical scavengers

which may result in powerful antioxidant effect.

No significant difference (P>0.05) was observed between the groups administered

6mg/kg body weight esculetin and 6mg/kg body weight scopoletin for all parameters

assayed. As shown by this result, comparing the effect of esculetin and scopoletin on

paracetamol- induced liver damage in rats, no significant difference was observed in their

effect and none among the two can be said to be more effective. This is possibly due to

the fact that both esculetin and scopoletin have their OH groups on the ortho positions

add are equally phenolic. Also as reported by Kaneko et al. (2003), catechilic coumarins

have a protective effect against cell damage. They reported that esculetin protected cells

from injury by linoleic acid hydroperoxide in cells incubated with acid hydroperoxide.

This is further confirmed in this work, since esculetin and scopoletin are catechilic

coumarins.

90

A significant difference (P<0.05) was observed in the results of the antioxidant

enzyme (superoxide dismutase SOD) and Malondialdehyde MDA of the group

administered (G.I) the toxicant (positive control) compared with the groups administered

distilled water and 10% DMSO respectively. There was also a significant difference

between the test groups and the group administered the toxicant. No significant

difference was observed between the best groups and the groups administered water and

10% DMSO. Reduced activity of SOD in paracetamol treated rats confirmed the hepatic

damage to the rat. Pre-treatment with the coumarins, esculetin and scopoletin to rats in

which hepatic damage was induced by paracetamol treatment caused significant increase

in SOD activity. Hence antilipid peroxidation and/or adaptive nature of the system as

brought about these coumarins against the damaging effects of free radical produced by

paracetamol. This result is supported by the work reported by Durairaj et al. (2008).

Increase in the serum activity of SOD is a sensitive index in hepatocellular

damage as is the most sensitive enzymatic index in liver injury. SOD has been reported as

one of the most important enzymes in the enzymatic antioxidant defense system. It

scavenges the superoxide anion to form hydrogen peroxide, hence diminishing the toxic

effect caused by this radical. There was a significant difference in the activity of SOD of

the groups pretreated with esculetin and scopoletin as compared with the group

administered the toxicant paracetamol only without the coumarins. This indicates that

esculetin and scopoletin can reduce reactive free radical and that might lessen oxidative

damage to the tissues and improve the activity of the hepatic antioxidant enzymes.

The increased MDA level of the liver reported in this work is due to lipid

peroxidation and this is in agreement with the report of Omotuyi et al. (2008). Increase in

MDA in liver suggests provoked lipid peroxidation leading to tissue damage and

depleting antioxidants protein such as glutathione, thus resulting to poor defense

mechanism. Pre-treatment with esculetin and scopoletin reversed these changes as earlier

suggested.. Hence, it may be possible that the mechanism of hepato protection of

esculetin and scopoletin is due to their antioxidant effect. This result is supported by that

reported by Durairaj et al. (2008) where they investigated the protective activity and

antioxidant potential of Lippia nodiflora extract in paracetamol induced hepatotoxicity in

rats.

91

Lipid peroxidation is one of the molecular mechanisms for cell injury and is

associated with a decrease of cellular antioxidant such as glutathione, superoxide

dismutase (SOD) and catalase (CAT) (Hijora et al., 2005). MDA is a decomposition

product of peroxidised polyunsaturated fatty acids that is widely detected during oxygen

radicals-induced pathological conditions (Olayaki et al., 2008).

Results of the serum proteins albumin and globulin showed that no significant

difference was observed between the test groups and the group administered the toxicant.

Also no significant difference was observed between the test groups and control groups.

There was also no significant difference between the test groups. This is supported by the

report of Wurochekke et al. (2008), who reported that serum albumin levels tend to be

normal in diseases like acute viral hepatitis, drug related hepatotoxicity and obstructive

jaundice. Although the concentration of the serum albumin is reduced in severe liver

diseases, that of the globulins is usually increased so that the total protein concentration is

rarely low. Orhue et al. (2005) reported that the normal half life of albumin is an average

of 21 days and therefore a decrease in serum albumin is usually not apparent early(four

weeks) enough in the course of liver disease. This report is supported by the result

obtained in this study since there was no statistically significant difference between the

test groups and the paracetamol treated group of rats.

4.2 Conclusion

In conclusion, work further confirmed that paracetamol is a potential toxicant

which induced liver intoxication. This research work also suggests that the coumarins

esculetin and scopoletin have potential efficacy in protecting against paracetamol induced

liver damage in rats. The pretreatment of rats with 6mg/kg body weight of esculetin and

scopoletin for 28 days before exposure to the toxicant (paracetamol) significantly reduced

the increased levels of liver enzyme markers (AST, ALT and alkaline phosphatase) in the

blood and total bilirubin level.

These findings support the reports that esculetin protects against paracetamol-

induced liver damage. Also, it supports the reports that dihydroxycoumarins, and

phenolic coumarins act as antioxidants. It also suggests that conscious and unconscious

exposure to different potential toxicants should be avoided.

92

Liver disease is accompanied by an increased production of free radicals.

Hepatocyte membranes are rich in PUFA which are susceptible to attacks by free

radicals. The PUFA of the membranes undergo peroxidation. Free radical reactions are

implicated in the progression of cancer, inflammation, hepato cellular damage and the

biological process of aging. The hepatoprotective action combined with antioxidant

activity has a synergistic effect to prevent the process of initiation and progress of

hepatocellular diseases. The parameters studied therefore serve to indicate that liver

diseases are accompanied by lipid peroxidation.

It can be concluded that the coumarins, esculetin and scopoletin possess good

hepatoprotective and definite antioxidant activity either through stabilization of cellular

membrane or anti-peroxidase activity. The study suggests that esculetin and scopoletin

possess hepatoprotective and antioxidanta activity.

4.3 Indications for Further Studies

Studies on hepatoprotective property of scopoletin should be carried out in which

scopoletin is not administered at once but in a progressive manner and in small doses

over a period of time.

Experiments on how scopoletin protects the liver against damage should be

conducted. Studies also on hepatoprotective property of esculetin should be carried out to

ascertain the most appropriate dose that protects the liver best from damage.

Furthermore, plant materials that contain esculetin and scopoletin should be

investigated and exploited since antioxidants have a very important role to play in

metabolism.

Finally, studies on how best to extract and preserve these coumarin compounds

from plants should conducted. In addition, studies should be conducted to know if plants

containing coumarins have antioxidant effects when consumed directly.

93

REFERENCES

Abbasi, A.M., Khan, M.A., Ahmad, M., Zafar, M., Khan, H., Muhammad, N. and

Sultana, S. (2009). Medicinal plants used for the treatment of Jaundice and

Hepatitis based on socio-economic documentation. African Journal of

Biotechnology, 8 (8): 1643-1650.

Abdulrahman, M., Omar, A. I. and Al-Arifi, M.N. (2005). Comparative effects of

Scopoletin and Menadione on aldehyde oxidase activity of guinea pig liver. Journal

of Biological Science, 40:1-6.

Aldridge, W. N. (1981). Mechanism of toxicity: New concepts are required in toxicity.

Trends in Pharmacological Science, 2:228-231.

Al-Jaudi, F.S. (2005). Prognostic value of an index of serum globulin compensation in

colon and breast cancers. Singapore Medical Journal, 46(12):710.

Altinyazar, H.C., Gurel, A., Kora, R., Armutru, F. and Unalacal, M. (2006). The status of

oxidants and antioxidants in the neutroplils of patients with recurrent aphthous

stomatitis. Turkish Journal of Medical Science, 36:87 – 91.

Arnab, K., Chatterjee, F., Dean, T., Steven, D. G. and Robert, H.G. (2003). Synthesis of

coumarins by ring-closure metathesis. Pure & Applied Chemistry, 75(4):421-425.

Arogba, S.S. (2008). Phenolics: A Class of Natures Chemical Weapons of Self

Preservation. 1st Inaugural Lecture, Kogi State University, Anyigba: Kogi State.

Athar, M., Zakir, H. S. and Hassan, N. (1997). Drug metabolizing enzymes in the liver.

In: Liver and Environmental Xenobiotics. New Delhi: Narosa Publishing House.

Available on line at http:// www.ijp-online.com/article.asp?issn=0253-

7613,year=2004; volume=36;issue=5.

Atreyee, B., Willen, G.L., Hideyuki, C., Uma, K.M., Chaidong, C., Jyofirmory, N. and

Chan, S.H.P. (1998): Induction of ATPase inhibitor protein by propylthiouracil and

protection against paracetamol hepatotoxicity in rats. British Journal of

Pharmacology, 124:1041-1047.

Australian Rheumatology Association (ARA) (2007). Patient Information on

Paracetamol. http://www.atdn.org/simple/liverfun.html. Retrieved on 12/07/2009.

Aw,T. Y., Wierzbicka, G. and Jones, D. P. (1991). Oral glutathione increases tissue

glutathione in vivo. Chem-biol interactions, 80: 89-97.

Babson, L. A. (1965): Alkaline phosphate. Clinica Chemica, 11:789.

Babson, L.A.; Greeley, S.J.; Coleman, C.M. and Philips, G.D. (1966): Alkaline

phosphates determination. Clinica Chemica, 12:482-490.

http://www.ijp-online.com/article.asp?issn=0253-7613,year=2004;

http://www.ijp-online.com/article.asp?issn=0253-7613,year=2004;

http://www.atdn.org/simple/liverfun.html

94

Band, A. G., Well, C. J. and David, M. M. (1949). Recent advances in clinical pathology.

Journal of. Biochemistry, 177:25–26.

Basaran, I., Sinan, S., Cakit, U., Buluf, M., Arslan, O. and Ozensory, O. (2007): In vitro

inhibitor of cytosolic carbonic anhydrase I and II by some New dihydroxycoumarin

compounds. Journal of Enzyme Inhibition and Medicinal Chemistry, 23(1): 32-36.

Betterige, D. J. (2002). What is oxidative stress? Metabolism, 49: 3-8.

Betram, G. K. (2001). Basic and Clinical Pharmacology. 8th Edition. McGraw Hill:

USA. p. 615.

Borges, F., Roleira, F., Mihazes, N., Santana, L. and Uriatte, E. (2005). Simple

coumarins and analogues in medicinal chemistry. Occurrence, Synthesis and

Biological Activity. Current Medicinal Chemistry, 12:887-916.

Bum-Chun, L., Soyong, L., Hwa-Jeong, L., Gwan, S.S., Jin-Hui, K., Jin Hwa, K., Young-

Ho, C., Dong-Hwan, L., Hyeong-Bae, P., Tae-Boo, C.; Dong-Cheul, M, Yeo-Pyo,

Y. and Jin-Tae, H. (2007). Anti-oxidative and photo-protective effects of coumarins

Isolated from Fraxinus chinesis. Archives of Pharmacal Research, 30(10): 1293-

1301.

Carepath (2010). A guide to general laboratory testing. testing-trifold. L 517-0907-7.

http://www.carepath.com Assessed 12/04/2010.

Chan, T.; Chan, A. and Critchely, J.A.J.H. (1996): Factors Responsible for Continuing

Morbidity after Paracetamol Poisoning in Chinese Patients in Hong Kong.

Singapore Medical Journal, 37: 275-277.

Chau, T. (2008). Drug Induced Liver Injury. An Update. Hong Kong Medical Diary.

13(3):23-26.

Chaudiere, J. and Ferrari-llious, R. (1999). Intracellular Antioxidants from Chemical to

Biochemical Mechanisms. Food Chemical Toxicology, 37: 949-962.

Chen, W.L.L., Koeing, S.J., Thompson, R.M., Peter, A.E., Retti, N.F. and Nelson, S.D.

(1988). Oxidation of acetaminophen to its toxic quinine imine and non-toxic

catechol metabolites by baculovirus – expressed and purified human Cytochrome

P450 2E1 and 2A6. Chemical Research in Toxicology, 11:295-301.

Copeland, E. S. (1986). Free radicals in carcinogenesis. Cancer Research, 46:5451.

Corbett, J. T. (1989). The Scopoletin assay for hydrogen peroxide. A review and a better

method. Journal of Biochemical and Biophysical Methods, 18(4):297-307.

http://www.carepath.com/

95

Daly, F.F.S., Fountain, J.S., Murray, L., Graduns, A. and Buckley, N.A. (2008).

Guidelines for the management of paracetamol poisoning in Australia and New

Zealand – Explanation and elaboration. Medical Journal of Australia, 18(5):296-

301.

Daud, M.D.H., Jing Li, S.G. and Masayuki, F. (2008). Suppressive effects of coumarins

on pumpkin seedling growth and glutathione S-transferase activity. Journal of Crop

Science Biotechnology, 11(3):187-192.

De-Vecchi, A.F., Bamonti, F., Novembrino, C., Ippolito, S., Guerra, L., Lonati, S., Salini,

S., Aman, C.S., Scurati-Manzoni, E. and Cighetti, G. (2010). Free and total plasma

malondialdehyde in chronic renal insufficiency and in dialysis patients.

Downloaded from http://indt.oxfordjournal.org. March 23, 2010.

Dong, H., Hainong, R.L., Thummel, K.E., Rettie, A.E. and Nelson, S.D. (2000).

Involvement of human cytochrome P450 2D6 in the bioactivation of

acetaminophen. Drug Metabolism and Disposition, 28:1397-1400.

Durairaj, A., Vaiyapur, S.T., Kanti, M.U. and Malay, G. (2008). Protective activity and

antioxidant potential of Lippia nodiflora extract in paracetamol induced

hepatotoxicity in rats. Iranian Journal of Pharmacology and Therapeutics, 7(1) :

83–89.

Elliot, S., Rowan, A.D., Carrere, S., Koshy, P., Catteral, J.B. and Cawston, T.E. (2001):

Esculetin inhibits cartilage restoration induced by interleukin in combination with

oncostalin. Annals of Rheumatic Diseases, 60:158 – 165.

Euro-Conference (1998): Oxygen, free radicals and oxidative stress in plants. Free

Radical Research Methods, 31:1–4.

Ezeanyika, L.U.S., Obidoa, O., Oluwashemire, N., Tekeudo, D.L. and Umar, I.A. (2002):

Comparative effects of scopoletin and cyanide on serum electrolytes, urea,

creatinine and some haematological parameters of rats. Global Journal of Pure and

Applied Sciences. 8(3): 311–314.

Ezeanyika, L.U.S., Obidoa, O. and Shoyinka, V.O. (1999): Comparative effects of

scopoletin and cyanide on rat brain, 1: Histopathology. Plant Foods for Human

Nutrition 53(4):351-358.

Farouk, A. and Al-Charchatchi, F.M.R. (2006). G6PDH and 6PGDH activities of

artemisia Herbal alba seeds and seedlings during germination in the presence of

esculetin. Pakistan Journal Biological Science, 9(1):210-213.

http://indt.oxfordjournal.org/

96

Ghauri, F.Y., McLean, A.E., Bealis, D.W. and Nicholson, J.K. (1993): Induction of 5-

oxoprolinuria in the Rat Following Chronic Feeding. Biochemical Pharmacology,

46(5):953–957.

Gilani, A. H., Janbaz, K. H. and Shah, B. H. (1998): Esculetin prevents liver damage

induced by paracetamol and CCl4. Pharmaceutical Research, 37(1):31–35.

Goldstein, B. D., Czernieki, B. and Witz, G. (1989). The role of free radicals in tumuor

protein. Environmental Health Perspective, 81:55–57.

Gonong, W. F. (2003). Review of Medical Physiology. 20th

edn McGraw-Hill

Companies, Inc. united States of America.

Guyton, C. and Hall, J. E. (2006). Textbook of Medical Physiology.11th

edn Elsevier,

New Delhi India.

Guzy, J., Chovanova, Z., Marekova, M., Chavkova, Z., Tomeckova, V., Mojzisova, G.

and Kusnir, J. (2004). Effect of Quercetin on Paracetamol–Induced Rat Liver

Mitochondria Dysfunction. Biologia, Brastilava. 59(3):399-403.

Halliwell, B. (1994). Free radicals and antioxidants: A Personal Review. Nutritional

Review, 52: 253–265.

Halliwell, B, Murica, M. A., Chirico, S. and Aruoma, O. I. (1995). Free radicals and

antioxidants in food and in vivo: What they do and how they work. Critical

Reviews of Food Science and Nutrition, 35: 7-20.

Hijora, E., Nistiar, F. and Sipulova, A. (2005). Changes in ascorbic acid and

malondialdehyde in rats after exposure to mercury. Bratisl lek listy. 106 (8-9) : 248-

251.

Ho, T. W. and Lam, K. C. (1996). Therapeutic misadventure with paracetamol. Hong

Kong Medical Journal, 2:434–436.

Holt, J. and Paya, M. (1996). Pharmacological and biochemical actions of simple

coumarins. Natural products with the therapeutic potential. General Pharmacology,

27:713–722.

Iscan, M. Rostami, H., Guray, T., Pelcomes, O. and Rautio, A. (1994). Interindividual

variability of coumarin, 7-hydroxylation. European Journal of Clinical

Pharmacology, 47(4): 315-318.

Janaky, R., Ogita, K. and Pasqualotta, B.A. (1999). Glutathione and signal transduction

in the mammalian CNS. Journal of Neurochemistry, 73: 889–902.

Janbaz, K. H. and Gilani, A.H. (2002). Menthol prevents liver damage induced by

Paracetamol and CCl4. Pakistan Journal Biological Science, 5(10): 1001-1103.

97

Jendrasskik, J. and Grof. P. (1938). In vitro determination of total and direct bilirubin in

serum or plasma. Biochemistry, 297: 81–89.

Jensen, G. (2007). Liver Function Test. Globetro. Hers Pocket Doc.

http://www.gastromo.com/lft.html.

Joel, A., Ricardo, L.A.D., Marcelo, S., Castilltiol, G.O. and Correa, A.G. (2005).

Preparation and evaluation of a coumarin library towards the inhibition activity of

the enzyme gGAPDH (Glyceraldehyde-3-phosphate dehydrogenate) from

Trypanosoma Cruzi Journal of Brazilian Chemical Society, 16(4): 1–2.

Johnston, D. E. (1999). Special considerations in interpreting liver function test:

American Academy of Family Physicians.

Joshi, R. and Chudasama, U. (2008). Synthesis of coumarins via pechmann condensation

using inorganic ion exchanges as solid acid catalysts. Journal of Scientific and

Industrial Research. 67:1092-1097.

Joshua, P. E. and Nwodo, O. F. C. (2010). Hepatoprotective effect of ethanolic leaf

extract of Senna hirsuta (Cassia hirsuta) against carbon tetrachloride (CCl4)

intoxication in rats. Journal of Pharmacy Research, 3(2): 310 – 316.

Kaneko, T., Baba, N. and Matsuo, M. (2003). Protection of coumarins against linoleic

acid hydrogen peroxide-Induced cytotoxity. Chemico-Biological Interactions,

142(3): 239-254.

Karam, Z.R. and Simic, M.G. (1989). Mechanism of free radical chemistry and

biochemistry of benzene. Environmental Health Perspective, 82:37-41.

Ken, B. (2004). Reversing Liver Damage: John Wiley and Sons, New York. p. 515.

Available online at http://www.publmedcentral. nihgov /article-render.

Klein, B., Read, P.A. and Babson, L.A. (1960). In vitro determination of alkaline

phosphate in serum or plasma. Clinical Chemistry, 6: 269-275.

Kostova, I. (2005). Synthetic and natural coumarin as cytotoxic agents. Current

Medicinal Chemistry–Anti-cancer Agents, 5(1) 28-45.

Kumar, G., Banu, C. S., Kannan, V. and Pandian, M. R. (2005). Antihepatotoxic effect of

beta-carotene on paracetamol-induced hepatic damage in rats. Indian Journal

Experimental Biology, 43(4): 351–355.

Kupeli, E., Deliorman, D.O. and Tesilada, E. (2006). Effects of Cistus laurifolius L. leaf

extract and flavonoids on acetaminophen-induced hepatotoxicity in mice. Journal

of Ethnopharmacology, 103(3): 455-460.

http://www.gastromo.com/lft.html

98

Kwok-nam, L., Pui-yin, L., Lai-ping, K. and Po-ki, L. (2005). Immunomodulatory effects

of esculetin (6,70dihydroxy coumarin on murine lymphocytes and peritoneal

macrophages. Chinese Society of Immunology, 2(3): 181-186.

Lands, L.C., Grey, V. L. and Smountas, A.A. (1999). Effect of supplementation with

cysteine donor on muscular performance. Journal of Applied Physiology, 87:1381-

1385. Available online at

http://www.colostate.edu/depts/entomology/courses/enso70/papers1998/karam.html

. Retrieved September 19, 2007.

Lata, H., Ahuja, G.K., Narang, A.P.S. and Walia, L. (2004). Effect of immoblisation

stress on lipid peroxidation and lipid profile in rabbits. Indian Journal of Clinical

Biochemistry, 19(2):1–4.

Lehman, D. E. (2000). Enzymatic Shunting: Resolving the Acetaminophen – Warfarin

Controversy. Pharmacotherapy, 20:1464-1468.

Leshna, M., Watson, A.J., Douglas, A.P., Hamlyn, A.N. and James, O.F. (1976):

Evaluation of Paracetamol-Induced Damage in Liver Biopsies Changes and Follow-

up Findings. Virchows Arch A. Pathol. Anat. 370(4):333-334.

Leuratti, C., Singh, R. Lagneau, C., Farmer, P.B., Plastaras, J. P., Marnett, L. J. and

Shukker, E.G. (1998). Determination of malondialdehyde induced DNA damage in

human tissues using an immunoslot blot assay. Carcinogenesis, 19(1):1919-1924.

Leung, K., Leung, P., Kong, L. and Leung, P. (2005). Immunomodu-latory Effects of

6,7-Dihydroxycoumarin on Marine Lymphocytes and Peritoneal Macrophages.

Chinese Society of Immunology, 2(3):181-186.

Liu, D., Wen, J. Liu, J. and Li, K. (1997). The role of free radicals in amyotrophic lateral

sclerosis. ROS and elevated oxidation of protein, DNA and membrane

phospholipids. Journal of the Federation of American Societies for Experimental

Biology, 13:2318-2328.

Liu, X., Zhang, L., Fu, X., Chen, K. and Qian, B. (2001). Effect of scopoletin on PC3 cell

proliferation and apoptosis. Acta Pharmacologica, 22(10):929-933.

Lomaestro, B. and Malone, M. (1995). Glutathione in health and disease.

Pharmacotherapeutic Issues. Annals of Pharmacotherapy, 29:1263–1273. Available

online at http://www.colostate.edu/depts/entomology/courses/en570/

papers1998/karam.htm. Retrieved September 19, 2007.

Macias, M.L., Rojas, I.S., Mata, R. and Hennsen, B.L. (1999): Effects of selected

coumarins in Spinach chloroplast photosynthesis. Journal of Agricultural and Food

Chemistry, 47(5): 2137-2140.




http://www.colostate.edu/depts/entomology/courses/en570/

99

Mason, P. (2004). Blood test used to investigate liver, thyroid or kidney function and

disease. Pharmaceutical Journal, 272:446-448.

Medicinenet.com (2007a). Drug-induced liver disease causes, symptoms, diagnosis and

treatment. http://www.medicinet.com/drug inducedliverdisease. Retrieved 12 July,

2007.

Medicinenet.com (2007b). Information about liver blood test (enzymes).

http://www.medicinenet.com/liverbloodtests. Retrieved 12 July, 2007

Medicines Information Bulletin (MIB) (2002). A Current Awareness Bulletin from the

Medicine Information Service.

Miller, A. (2001). Iron Superoxide Dismutase. Handbook of Metallopriteins. John Willey

and Sons Ltd. Chichester. Pp 668-682.

Misra, H. P. and Fridovich, I. (1972). Superoxide dismustase assay. Journal of Biological

Chemistry, 247: 3170–3175.

Moini, H., Parker, L. and Saris, N. (2002). Antioxidant and prooxidant activities of -

lipoic acid and dihydrolipoic acid. Toxicology and Applied Pharmacology, 182:84-

90.

Muriel, P., Garciapina, T., Perez-Alvarez, V. and Mouretter, M. (1992). Silymarin

protects against paracetamol-induced lipid peroxidation and liver damage. Journal

of Applied Toxicology, 12: 439 – 449..

Murray, R. K., Granner, D. K., Mayes, P. A. and Rodwell, V. W. (2003). Harpers

Illustrated Biochemistry. 26th Edition. McGraw-Hill, New York. Pp 214.

Nahid, T., Chattervedi, S., Aggrawal, S.S. and Nassar, A. (2005). Hepatoprotective

studies on Phyllanthus niruri on paracetamol-induced. JK Practitioners

(International Journal of Current Medical Science & Practice), 12(4):211-212.

Nayana, J. and Janardhanan, K.K. (2000). Antioxidant and anti-tumuor activity of

Pleurotus florida. Current Science, 79(7):941-943.

Nelson, D.L. and Cox, M. M. (2005). Lehninger Principle of Biochemistry. 4th Edition.

Wilt Freeman and Company, New York. pp 857-858.

Nohl, H., Gille, L. and Staniel, K. (2004). The mystery of reactive oxygen species

derived from cell respiration. Acta Biochimica Polonica, 51(1): 223-229.

100

Nwachukwu, O. (2006). Hepatoprotective activities of coconut (Cocus nucifera) water in

paracetamol-intoxicated rats. A Research Project Presented to the Department of

Biochemistry, UNN.

Obidoa, O. and Obasi, S. C. (1991). Comparative effects of scopoletin and cyanide on rat

brain. Plant Foods for Human Nutrition, 41(3) 283-289.

Obidoa, O., Ezeanyika, L.U.S. and Okoli, A. H. (1999): Effect of Scopoletin on Male

Guinea Pig Reproductive Organs: 1. Levels of Citric Acid and Fructose Nutrition

Research, 19(3):443-448.

Ojala, T. (2001). Biological Screening of Plant Coumarins. Academic Dissertation.

Helsinki.

Ojewole, J.A.O. and Adesina, S. K. (1983). Evaluation of the mechanism of the

hypotensive effect of scopoletin isolated from the fruit of Tetrapleura tetraptera

Plant. Planta Medica, 49: 46-50.

Olayaki, L. A., Ajao, S. M., Jimoh, G. A.A., Aremu I. I. and Soladoye, A.O. (2008).

Effect of vitamin C on MDA in pregnant Nigerian women. Journal of Basic and

Applied Sciences, 4(2)105-108.

Omotuiyi, I.O., Nwangwu, S.C., Okugbo, O.T., Okoye, O.T., Ojieh, G.C. and Wogu,

D.M. (2008). Hepatotoxic and hemolytic effect of acute exposure of rats and

antesunate overdose. African Journal of Biochemical Research, 2(5):107–110.

Organic.chemistry.org (2008). Coumarin Synthesis. http://www.organic.

chemistry.org/synthesis/heterocycles/coumarins. Downloaded 12 May 20008.

Orhue, N. E. J., Nwanze, E. A. C. and Okafor, A. (2005). Serum total protein and

globulin levels in Trypanosoma brucei-infected rabbits: Effects of orally

administered Scoparia dulcis. African Journal of Biotechnology, 4(10):1152-1155.

Panda, S. and Kar, A. (2006). Evaluation of the antithyroid antioxidative and

antihyperglycemic activity of scopoletin from Aegle Marmelos leaves in

hyperthyroid rats. Phytotherapy Research, 20(12):1103–1105.

Pervaiz, S., Rukh, G. and Qayyum, M. (2001). Serum proteins status in women in

different age and socio-economic background and neonates. International Journal

of Agriculture & Biology, 3(4):394–396.

Potter, N. N. and Hotchkiss, J. H. (1996): Food Science. 5th Edition. S.K. Edition. S.K.

Jain. New Delhi. p. 37.

101

Pratibha, K., Anand, U. and Agarwal, R. (2004). Serum adenosine deamina 8,5’.

nuclotidase and malondialdehyde in acute infective hepatitis. Indian Journal of

Clinical Biochemistry, 19(2):128-131.

Preedy, V.R., Koll, M. and Emery, P.W. (2002). Albumin synthesis measured in vivo.

Clinical Science, 102:115–117.

Prescott, L. F. and Matthew, H. (1974). Cysteamine for paracetamol overdose. Lancet,

1:998.

Prescott, L., Spoerke, D. G., Rumack, B. H. and Meredith, T. Y. (2006): IPCS/CEC.

Evaluation of Antidotes Series.

Prescott, L.F., Illingworth, B.N. and Critchley, J.A. (1999). Intravenous N-acetylcystene:

The treatment of choice for paracetamol poisoning. Lancet, 310(8035): 432–434.

Raj, H.G., Sharm, S.K., Gary, B.S., Parma, V. S., Jain, S.C., Goel, S., Tyagi, Y.K., Singh,

A., Olsen, C.E. and Wergel, J. (1998). Mechanisms of biochemical action of

substituted 4-methylbenzopyran2-ones Parts 3: A novel mechanism for the

inhibition of biological membrane lipid peroxidation by deoxygenated 4-

methylcoumarins medicated mixed ligand complex. Journal of Biological &

Organo-Medicinal Chemistry, 6:2205-2212.

Rajesh, M.G. and Latha, M.S. (2001). Hepatoprotection of elephantopus scabe linn in

CCl4-induced liver injury. Indian Journal of Pharmacology, 45:481-486.

Reddy, Y.N., Murthy, S. U., Krishna, D.R. and Prabhakar, M. C. (2004). Role of free

radicals and antioxidants in tuberculosis patients. Indian Journal of Tuberculosis,

51:213-218.

Reitman, S. and Frankel, S. (1957). In vitro determination of glutamic-pyruvic

transaminase in serum. American Journal of Clinical Pathology, 28:56.

Rukmini, M.S., Benedicts, D. and D’souza, V. (2004). Superoxide dismutase and catalase

activities and their correlation with malondialdehyde in schizophrenic patients.

Indian Journal of Clinical Biochemistry, 19(2):114-118.

Sagrario, O. M., Juana, M.B. and Villar, A.M. (1998): Effects of the antioxidant (6,7-

Dihydroxycoumarin) esculetin on the glutathione system and lipid peroxidation in

mice. International Journal of Clinical & Experimental Medicine, 44(1) 21-25.

Samiec, P.S., Dreirs-Botsch, C. and Flagg, E.W. (1998): Glutathione in human plasma:

Decline in association with aging, age-related macular degeneration and diabetes.

Free Radical Biology & Medicine, 24:699-704.

102

Sarayme, R., Yazar, S., Kilic, E. and Ozbilge, A. (2004). Serum malondialdehyde levels

in patients infected with trichuris trichiura. Saudi Medical Journal, 25(12):2046 –

2052.

Sarka, P.D. and Rautary, S.S. (2009). A study of serum malondialdehyde levels and

paraoxanase activity in ischemic stroke patients. Biomedical Research. 20(1):64-66.

Schmidt, E. and Schmidt, F.W. (1963). Glutamic-pyruvic transaminase enzyme. Biology

Clinicals, 3:1.

Scientific Committee on Consumer Products (SCCP) (2006). Opinion on Coumarin.

European Commission Health and Consumer Protection. pp.57.

Shaabani, A., Ghadavi, R., Rahmati, A. and Rezayar, A. H. (2009). Coumarin synthesis

via knoevengagel condensation reaction in 1,1,3,3,-N,N,N`,N`-tetramethyl

vanadium trifluoroacetate ionic liquid. Journal of the Iranian Chemical Society,

6(4):710-714.

Sherlock, S. (1951). Liver Disease. Churchill: London. p. 204.

Sies, H. (1997). Oxidative stress: Oxidant and antioxidants. Experimental Physiology,

82:291-295.

Sies, H. (1999). Glutathione and Its Role in Cellular Functions. Free Radical Boilogy &

Medicine, 27:916-921. Available online at http://www.pdf

health.com/druginfo/nmdrugprofiles/nutsupdrugs/glu0126.shtml

Song, Z., Joshi-Baive, S. and McLain, C.J. (2001). Advances in alcoholic liver disease.

Current Gastroenterology Reports, 6:71-76.

Stahl, W. and Siers, H. (2002): Reactive Oxygen Species. Research Monograhs.

Available online at http://www.uni.doesseldof.de/ medfak/phiol/chem/index.html.

Sultana, F.; Anitha, D. and Venkatesh, T. (2004): Estimation of reference valves in liver

function test in health plan individuals of an urban South Indian population. Indian

Journal of Clinical Biochemistry, 19(2):72-79.

Takada, Y., Noguchi, T., Okabe, T. and Kajiyama, M.(1982).Superoxide dismutase in

various tissues from rat carcinoma in the maxillary sinus. Cancer Research. 42:

4233-4235.

Tanaka, E.M. and Misawa, S. (2000). Cytochrome P-450 2E9. Its clinical and

toxicological role. Clinical Therapeutics, 25:165-175.

Thapa, B.R. and Anuj, W. (2007). Liver function test and their interpretation. Indian

Journal of Peadiatrics, 74(7):663-671.

http://www.pdf/

103

Tkach, H. and Lukyanets, E.A. (1992). Some new reactions of coumarins. Res. Int. Org.

Intern and Dyestuffs, 8:1053-1055.

Traykova, M. and Kostova, I. (2005). Coumarin derivatives and oxidative stress.

International Journal of Pharmaceutics, 1(1):29-32.

Ulicna, O., Greksal, M., Vancova, O., Zlatos, L., Galbavy, S., Bozek, P. and Nakano, M.

(2003). Hepatoprotective effect of Rooibos Tea (Aspalathus linearis) on CCl4 –

induced liver damage in rats. Physiological Research, 52:461-466.

Venkatesha, U. and Kala, S.K. (2000). Effects of HD-O3 on levels of various enzymes in

paracetamol-induced liver damage in rats. Indian Journal of Pharmaceutical

Sciences, 32:361-364.

Wallin, B., Rosegren, B., Shertzer, H. G. and Caeejo, G. (1993). Lipoprotein oxidation

and measurement of thiobarbituric acid reacting substances formation in a single

microtiter plate: its use for evaluation of antioxidants. Analytical Biochemistry, 208:

10–15.

Wikipedia (2007a). Hepatotoxity. From http://en.wikipedia.org/wiki/ hepatotoxity.

Accesed on 13th

May, 2008.

Wikipedia (2007b). Liver Function Test. http://en.wikipedia.org/wiki/liver function.

Accesed on 13th

May, 2008

Wikipedia (2007c). Liver. http://en.wikipedia.org/wiki/liver. Accesed on 13th

May, 2008

Wikipedia (2008). Coumarin. http://wikipedia.org/wiki/coumarin. Accesed on 13th

May,

2008

Wurochekke, A.U., Anthony, A.E. and Obidah, W. (2008). Biochemical effects on the

liver and kidney of rats administered aqueous stem bark extract of Xemenia

americana. African Journal of Biotechnology, 7(16). 2777-2780.

Yang, J., Della-fera, M. A.., Hartzell, D.L., Dooley, C.N., Hausman, B. and Baile,C.

A.(2006). Esculetin induces apoptosis and inhibits adipogenesis in 3T3-L1 cells.

Obesity, 14: 1695-1699.

Yazar, E., Oztekin, E., Sivrikaya, A., Col, R., Elmas, M. and Bas, A. (2004). Effects of

different doses of Tilmicosin on malondialdehyde and glutathione concentrations in

mice. Acta Veterinaria Brno, 73:69-72.

http://en.wikipedia.org/wiki/liver

104

APPENDICES

Appendix I: Descriptive Table of the Different Parameters

N Mean Std. Deviation Std. Error

ALB Group 1 (Esculeijn) 4 4.7000 .28284 .14142

Group 2 (Scopoletin) 4 5.0250 .35940 .17970

Group 3 (Paracetamol) 4 5.3000 .29439 .14720

Group 4 (10% DMSO) 4 4.8500 .79373 .39686

Group 5 (Water) 4 4.1500 .28868 .14434

Total 20 4.8DaO .56052 _12534

GLO Group 1 (Esculetin) 4 2.9500. .41231 .20616

Group 2 (Scopoletin) 4 3.2000 .a.a165 .04082


Group 4 (10% DMSO) 4 3.5750 .23629 .11815

Group 5 (Water) 4 3.4750 .46458 .23229

Total 20 3.2950 .38997 .08720

ALT Group 1 (Esculetin) 4 15.2475 1.03019 .51510

Group 2 (Scopoletin) 4 12.0825 1.54262 .77131

Group 3 (Paracetamol) 4 35.9090 9.54673 4.77336

Group 4 (10% DMSO) 4 26.6275 2.50500 1.25250.

Group 5 (Water) 4 13.2500 2.62996 1.31498

Total 20 20.6215 10.32593 2.30895

AST Group 1 (Esculetin) 4 24.'5725 18.29589 9.14795

Group 2 (Scopoletin) 4 10.2600 5.97641 2.98821

Group 3 (Paracetamol) 4 86.6750 10.86964 !J.43482

Group 4 (10% DMSO) 4 41.5475 1.64878 .82439

Group 5 (Water) 4 2(15000 4.79583 2.39792

Total 20 32.7110 22.17999 4.95960

ALP Group 1 (Esculetin) 4 17.9050 1.13729 .56865

Group 2 ($copoletin) 4 14.7750 2.33633 1.16816


Group 4 (10% DMSO) 4 41.6600 17.00117 8.5D058

Group 5 (Water) 4 19.3250 3.63994 1.81997

Total 20 33.2.3M .71014 5.30175

TB Group 1 (Esculetin) 4 .4675 .07500 .03750

Group 2 (Scopoletin) 4 .2975 .12038 .06019

Group 3 (Paracetamol) 4 .5550 .09183 .04392

Group 4 (10% DMSO) 4 .3225 .13841 .06921

Group 5 (Water) 4 .2800 .10985 .0S492

Total 20 .3845 .14734 .03295

CB Group 1 (Esculetin) 4 .4675 .07500 .03750


Group 3 (Paracetamol) 4 .5500 .10100 .05050

105

Group 4 (10% DMSO) 4 .3225 .13841 .06921 .

Group 5 (Water) 4 .2950 .09674 .04787

Total 20 .3880 .14292 .03196

N Mean Std. Deviation Std. Error

SOD Group 1 (Esculetin) 4 .1475 .03202 .01601



Group 4 (10% DMSO) 4 3.1875 .27035 .13518

Group 5 (Water) 4 .3700 .01155 .00577

Total 20 1.6005 1.77977 .39797

MDA Group 1 (Esculetin) 4 9.6925 6.78502 3.39251

Group 2 (Scopoletin) 4 5.9225 2.95900 1.47950


Group 4 (100Al DMSO) 4 23.2175 5.01052 2.50526

Group 5 (Water) 4 10.9150 2.34342 1.17171

Total 20 16.2130 10.69137 2.39066

95% Confidence Interval for

Mean

Lower Bound Upper Bound Minimum Maximum

ALB Group 1 (Esculetin) 4.2499 5.1501 4.30 4.90

Group 2 (Scopoletin) 4.4531 5.5969 4.50 5.30

Group 3 (Paracetamol) 4.8316 5.7684 4.90 5.60

Group 4 (10% DMSO) 3.5870 6.1130 3.90 5.60

Group 5 (Water) 3.6907 4.6093 3.80 4.50

Total 4.5427 5.0673 3.80 5.60

GLO Group 1 (Esculetin) 2.2939 3.6061 2.60 3.40



Group 4 (10% DMSO) 3.1990 3.9510 3.40 3.90

Group 5 (Water) 2.7358 4.2142 3.00 4.10

Total 3.1125 3.4775 2.60 4.10

ALT Group 1 (Esculetin) 13.6082 16.8868 13.78 16.07



Group 4 (10% DMSO) 22.6415 30.6135 23.30 29.30

Group 5 (Water) 9.0652 17.4348 11.00 17.00

Total 15.7888 25.4542 10.58 46.40

AST Group 1 (Esculetin) -4.5403 53.6853 14.55 52.00

Group 2 (Scopoletin) .7502 19.7698 3.50 15.83


Group 4 (10% DMSO) 38.9239 44.1711 40.23 43.96

Group 5 (Water) 12.8688 28.1312 16.00 27.00

Total 22.3304 43.0916 3.50 76.00

ALP Group 1 (Esculetin) 16.0953 19.7147 16.20 18.50

106



Group 4 (10% DMSO) 14.6073 68.7127 20.80 57.70

Group 5 (Water) 13.5330 25.1170 15.00 23.90

Total 22.1363 44.3297 11.50 83.10

TB Group 1 (Esculetin) .3482 .5868 .43 .58

Group 2 (Scopoletin) .1059 .4891 .14 .43

Group 3 (Paracetamol) .4089 .7011 .43 .65

Group 4 (10% DMSO) .1023 .5427 .14 .43

Group 5 (Water) .1052 .4548 .18 .43

Total .3155 .4535 .14 .65

CB Group 1 (Esculetin) .3482 .5868 .43 .58


Group 3 (Paracetamol) .3893 .7107 .43 .67

Group 4 (10% DMSO) .1023 .5427 .14 .43

Group 5 (Water) .1427 .4473 .21 .43

Total .3211 .4549 .14 .67

95% Confidence Interval for

Mean

lower Bound Upper Bound Minimum Maximum

SOD Group 1 (Esculetin) .0966 .1984 .10 .17



Group 4 (10% DMSO) 2.7573 3.6177 2.99 3.58

Group 5 (Water) .3516 .3884 .36 .38

Total .7675 2.4335 .09 4.58

MDA Group 1 (Esculetin) -1.1040 20.4890 2.55 16.05



Group 4 (100Al DMSO) 15.2446 31.1904 20.44 30.72

Group 5 (Water) 7.1861 14.6439 9.30 14.31

Total 11.2093 21.2167 2.55 38.93

107

Appendix II: Multiple Comparison of the Different Parameters and Groups

Mean

Difference

Dependent Variable (I) GROUP (J) GROUP (I-J) Std. Error

ALB LSD Group 1 (Esculetin) Group 2 (Scopoletin) -.3250 .31767

Group 3 (Paracetamol) -.6000 .31767

(3roup 4 (10% DMSO) -.1500 .31767

Group 5 (Water) .5500 .31767

Group 2 (Scopoletin) Group 1 (Esculetin) .3250 .31767


Group 4 (10% DMSO) .1750 .31767

Group 5 (Water) .8750* .31767

Group 3 (Paracetamol) Group 1 (Esculetin) .6000 .31767

Group 2 (Scopoletin) .2750 .31767

Group 4 (10% DMSO) .4500 .31767

Group 5 (Water) 1.1500* .31767

Group 4 (10% DMSO) Group 1 (Esculetin) .1500 .31767

Group 2 (Scopoletin) -.1750 .31767


Group 5 (Water) .7000* .31767

Group 5 (Water) Group 1 (Esculetin) -.5500 .31767

Group 2 (Scopoletin) -.8750* .31767

Group 3 (Paracetamol) -1.1500* .31767

Group 4 (10% DMSO) -.7000* .31767

GLO LSD Group 1 (Esculetin) Group 2 (Scopoletin) -.2500 .25380


Group 4 (10% DMSO) -.6250* .25380

Group 5 (Water) -.5250 .25380

Group 2 (Scopoletin) Group 1 (Esculetin) .2500 .25380


Group 4 (10% DMSO) -.3750 .25380

Group 5 (Water) -.2750 .25380



Group 4 (10% DMSO) -.3000 .25380

Group 5 (Water) -.2000 .25380

Group 4 (10% DMSO) Group 1 (Esculetin) .6250* .25380


Group 3 (Paracetamol) .3000 .25380

Group 5 (Water) .1000 .25380

Group 5 (Water) Group 1 (Esculetin) .5250 .25380


Group 3 (Paracetamol) .2000 .25380

108

Group 4 (10% DMSO) -.1000 .25380

ALT LSD Group 1 (Esculetin) Group 2 (Scopoletin) 3.1650 3.28287

Group 3 (Paracetamol) -20.6525* 3.28287

Group 4 (10% DMSO) -11.3800* 3.28287

Group 5 (Water) 1.9975 3.28287

Mean

Difference


ALT LSD Group 2 (Scopoletin) Group 1 (Esculetin) -3.1650 3.28287


roup 4 (10% DMSO) -14.5450* 3.28287

Group 5 (Water) -1.1675 3.28287

Group 3 (Paracetamol) Group 1 (Esculetin) 20.6525* 3.28287

Group 2 (Scopoletin) 23.8175* 3.28287

Group 4 (10% DMSO) 9.2725* 3.28287

Group 5 (Water) 22.6500* 3.28287

Group 4 (100k DMSO) Group 1 (Esculetin) 11.3800* 3.28287



Group 5 (Water) 13.3775* 3.28287

Group 5 (Water) Group 1 (Esculetin) -1.9975 3.28287

Group 2 (Scopoletin) 1.1675 3.28287


Group 4 (10% DMSO) -13.3775* 3.28287

AST LSD Group 1 (Esculetin) Group 2 (Scopoletin) 14.3125 7.17164


Group 4 (10% DMSO) -16.9750* 7.17164

Group 5 (Water) 4.0725 7.17164

Group 2 (Scopoletin) Group 1 (Esculetin) -14.3125 7.17164


Group 4 (10% DMSO) -31.2875* 7.17164

Group 5 (Water) -10.2400 7.17164



Group 4 (10% DMSO) 25.1275* 7.17164

Group 5 (Water) 46.1750* 7.17164

Group 4 (10% DMSO) Group 1 (Esculetin) 16.9750* 7.17164



Group 5 (Water) 21.0475* 7.17164

Group 5 (Water) Group 1 (Esculetin) -4.0725 7.17164



109

Group 4 (10% DMSO) -21.0475* 7.17164

ALP LSD Group 1 (Esculetin) Group 2 (Scopoletin) 3.1300 6.20022


Group 4 (10% DMSO) -23.7550* 6.20022

Group 5 (Water) -1.4200 6.20022



Group 4 (10% DMSO) -26.8850* 6.20022

Group 5 (Water) -4.5500 6.20022

Mean

Difference


ALP LSD Group 3 (Paracetamol) Group 1 (Esculetin) 54.5950* 6.20022


Group 4 (10% DMSO) 30.8400* 6.20022

Group 5 (Water) 53.1750* 6.20022

Group 4 (10% DMSO) Group 1 (Esculetin) 237550* 6.20022



Group 5 (Water) 22.3350* 6.20022

Group 5 (Water) Group 1 (Esculetin) 1.4200 6.20022



Group 4 (10% DMSO) -22.3350* 6.20022

TB LSD Group 1 (Esculetin) Group 2 (Scopoletin) .1700* .07731

Group 3 (Paracetamol) -.08i5 .07731

Group 4 (10% DMSO) .1450 .07731

Group 5 (Water) .1875* .07731

Group 2 (Scopoletin) Group 1 (Esculetin) -.1700* .07731

Group 3 (Paracetamol) -.2575* .07731

Group 4 (10% DMSO) -.0250 .07731

Group 5 (Water) .0175 .07731


Group 2 (Scopoletin) .2575* .07731

Group 4 (10% DMSO) .2325* .07731

Group 5 (Water) .2750* .07731

Group 4 (10% DMSO) Group 1 (Esculetin) -.1450 .07731



Group 5 (Water) .0425 .07731

Group 5 (Water) Group 1 (Esculetin) -.1875* .07731



110

Group 4 (10% DMSO) -.0425 .07731

CB LSD Group 1 (Esculetin) Group 2 (Scopoletin) .1625 .07715


Group 4 (10% DMSO) .1450 .07715

Group 5 (Water) .1725* .07715

Group 2 (Scopoletin) Group 1 (Esculetin) -.1625 .07715


Group 4 (10% DMSO) -.0175 .07715

Group 5 (Water) .0100 .07715


Group 2 (Scopoletin) .2450* .07715

Group 4 (100A» DMSO) .2275* .07715

Group 5 (Water) .2550* .07715

Mean

Difference

Dependent Variable (I) OUP (J) GROUP (I-J) Std. Error

CB !.SD Group 4 (10% DMSO) Group 1 (Esculetin) -.1450 .07715



Group 5 (Water) .0275 .07715

Group 5 (Water) Group 1 (Esculetin) -.1725* .07715



Group 4 (10% DMSO) -.0275 .07715

SOD LSD Group 1 (Esculetin) Group 2 (Scopoletin) .0100 .16763


Group 4 (10% DM&<:» -3.0400* .16763

Group 5 (Water) -.2225 .16763

Group 2 (Scopolettn) Group 1 (Esculetin) -.0100 .16763


Group 4 (10% DMSO) -3.0500* .16763

Group 5 (Water) -.2325 .16763

Group 3 (Paracetamol) Group 1 (Esculetin) 4.0125* .16763

Group 2 (Scopoletin) 4.0225* .16763

Group 4 (10% DMSO) .9725* .16763

Group 5 (Water) 3.7900* .16763

Group 4 (10% DMSO) Group 1 (Esculetin) 3.0400* .16763

Group 2 (Scopoletin) 3.0500* .16763


Group 5 (Water) 2.8175* .16763

Group 5 (Water) Group 1 (Esculetin) .2225 .16763



111

Group 4 (10% DMSO) -2.8175* .16763

MDA LSD Group 1 (Esculetin) Group 2 (Scopoletin) 3.7700 3.44986


Group 4 (10% DMSO) -13.5250* 3.44986

Group 5 (Water) -1.2225 3.44986



Group 4 (10% DMSO) -17.2950* 3.44986

Group 5 (Water) -4.9925 3.44986



Group 4 (10% DMSO) 8.1000* 3.44986

Group 5 (Water) 20.4025* 3.44986

Mean

Difference


MDA LSD Group 4 (10% DMSO) Group 1 (Esculetin) 13.5250* 3.44986



Group 5 (Water) 12.3025* 3.44986

Group 5 (Water) Group 1 (Esculetin) 1.2225 3.44986



Group 4 (10% DMSO) -12.3025* 3.44986

95%

Confidence

Interval

Dependent Variable (I) GROUP (J) GROUP SiQ. Lower Bound

ALB LSD Group 1 (Esculetin) Group 2 (Scopoletin) .322 -1.0021

Group 3 (Paracetamol) .078 -1.2771

Group 4 (10% DMSO) .644 -.8271

Group 5 (Water) .104 -.1271

Group 2 (Scopoletin) Group 1 (Esculetin) .322 -.3521

Group 3 (Paracetamol) .400 -.9521

Group 4 (10% DMSO) .590 -.5021

Group 5 (Water) .015 .1979

Group 3 (Paracetamol) Group 1 (Esculetin) .078 -.0771

Group 2 (Scopoletin) .400 -.4021

Group 4 (10% DMSO) .177 -.2271

Group 5 (Water) .003 .4729

Group 4 (10% DMSO) Group 1 (Esculetin) .644 -.5271

112



Group 5 (Water) .044 .0229

Group 5 (Water) Group 1 (Esculetin) .104 -1.2271

Group 2 (Scopoletin) .015 -1.5521


Group 4 (10% DMSO) .044 -1.3771

GLO LSD Group 1 (Esculetin) Group 2 (Scopoletin) .340 -.7910


Group 4 (10% DMSO) .026 -1.1660

Group 5 (Water) .056 -1.0660



Group 4 (10% DMSO) .160 -.9160

Group 5 (Water) .296 -.8160



Group 4 (10% DMSO) .256 -.8410

Group 5 (Water) .443 -.7410

Group 4 (10% DMSO) Group 1 (Esculetin) .026 .0840



Group 5 (Water) .699 -.4410

Group 5 (Water) Group 1 (Esculetin) .056 -.0160



Group 4 (10% DMSO) .699 -.6410

ALT LSD Group 1 (Esculetin) Group 2 (Scopoletin) .350 -3.8323


Group 4 (10% DMSO) .003 -18.3773

Group 5 (Water) .552 -4.9998

95%

Confidence

Interval

Dependent Variable (I) GROUP (J) GROUP Sig. Lower Bound

ALT LSD Group 2 (Scopoletin) Group 1 (Esculetin) .350 -10.1623


Group 4 (10% DMSO) .000 -21.5423

Group 5 (Water) .727 -8.1648

Group 3 (Paracetamol) Group 1 (Esculetin) .000 13.6552

Group 2 (Scopoletin) .000 16.8202

Group 4 (10% DMSO) .013 2.2752

Group 5 (Water) .000 15.6527

Group 4 (10% DMSO) Group 1 (Esculetin) .003 4.3827

113



Group 5 (Water) .001 6.3802




Group 4 (10% DMSO) .001 -20.3748

AST LSD Group 1 (Esculetin) Group 2 (Scopoletin) .064 -.9735


Group 4 (10% DMSO) .032 -32.2610

Group 5 (Water) .579 -11.2135

Group 2 (Scopoletin) Group 1 (Esculetin) .064 -29.5985


Group 4 (10% DMSO) .001 -46.5735

Group 5 (Water) .174 -25.5260



Group 4 (1()o DMSO) .003 9.8415

Group 5 (Water) .000 30.8890




Group 5 (Water) .010 5.7615



Group 3 (Paracetamol) .000 ..01.4610

Group 4 (10% DMSO) .010 -36.3335

ALP LSD Group 1 (Esculetin) Group 2 (Scopoletin) .621 -10.0855

Group 3 (Paracetamol) .000 ..07.8105

Group 4 (10% DMSO) .002 -36.9705

Group 5 (Water) .822 -14.6355



Group 4 (10% DMSO) .001 -40.1005

Group 5 (Water) .474 -17.7655

95%

Confidence

Interval

DeDendent Variable (I) GROUP (J) GROUP Sig. Lower Bound

ALP LSD Group 3 (paracetamol) Group 1 (Esculetin) .000 41.3795


Group 4 (10% DMSO) .000 17.6245

Group 5 (Water) .000 39.9595



114


Group 5 (Water) .003 9.1195



Group 3 (Paracetamol) .000 6.3905

Group 4 (10% DMSO) .003 -35.5505

TB LSD Group 1 (Esculetin) Group 2 (Scopoletin) .044 .0052


Group 4 (10% DMSO) .080 -.0198

Group 5 (Water) .028 .0227



Group 4 (10011I DMSO) .751 -.1898

Group 5 (Water) .824 -.1473



Group 4 (10% DMSO) .009 .0677

Group 5 (Water) .003 .1102

Group 4 (10% DMSO) Group 1 (Esculetin) .080 -.3098



Group 5 (Water) .591 -.1223




Group 4 (10% DMSO) .591 -.2073

CB LSD Group 1 (Esculetin) Group 2 (Scopoletin) .052 -.0019


Group 4 (10% DMSO) .080 -.0194

Group 5 (Water) .041 .0081



Group 4 (10% DMSO) .824 -.1819

Group 5 (Water) .899 -.1544



Group 4 (10% DMSO) . .010 .0631

Group 5 (Water) .005 .0906

115

95%

Confidence

Interval

Dependent Variable (I) GROUP (J) GROUP Sic. Lower Bound

CB LSD Group 4 (10% DMSO) Group 1 (Esculetin) .080 -.3094



Group 5 (Water) .726 -.1369




Group 4 (10% DMSO) .726 -.1919

SOD LSD Group 1 (Esculetin) Group 2 (Scopoletin) .953 -.3473


Group 4 (10% DMSO) .000 -3.3973

Group 5 (Water) .204 -.5798



Group 4 (10% DMSO) .000 -3.4073

Group 5 (Water) .186 -.5898



Group 4 (10% DMSO) .000 .6152

Group 5 (Water) .000 3.432'r




Group 5 (Water) .000 2.4602




Group 4 (100AJ DMSO) .000 -3.1748

MDA LSD Group 1 (Esculetin) Group 2 (Scopoletin) .292 -3.5832


Group 4 (10% DMSO) .001 -20.8782

Group 5 (Water) .728 -8.5757



Group 4 (10% DMSO) .000 -24.6482

Group 5 (Water) .168 -12.3457



Group 4 (10% DMSO) .033 .7468

116

Group 5 (Water) .000 13.0493

95%

Confidence

Interval

Dependent Variable (I) GROUP (J) GROUP Sig. Lower Bound

MDA LSD Group 4 (10% DMSO) Group 1 (Esculetin) .001 6.1718



Group 5 (Water) .003 4.9493




Group 4 (10% DMSO) .003 -19.6557

95%

Confidence

Interval

Dependent Variable (I) GROUP (J) GROUP Upper Bound

ALB LSD Group 1 (Esculetin) Group 2 (Scopoletin) .3521

Group 3 (Paracetamol) .0771

Group 4 (10% DMSO) .5271

Group 5 (Water) 1.2271

Group 2 (Scopoletin) Group 1 (Esculetin) 1.0021


Group 4 (10% DMSO) .8521


Group 3 (Paracetamol) Group 1 (Esculetin) 1.2771

Group 2 (Scopoletin) .9521

Group 4 (10% DMSO) 1.1271


Group 4 (10% DMSO) Group 1 (Esculetin) .8271




Group 5 (Water) Group 1 (Esculetin) .1271

Group 2 (Scopoletin) -.1979

Group 3 (Paracetamol) -.4729

Group 4 (10% DMSO) -.0229

GLO LSD Group 1 (Esculetin) Group 2 (Scopoletin) .2910


Group 4 (10% DMSO) -.0840

Group 5 (Water) .0160

Group 2 (Scopoletin) Group 1 (Esculetin) .7910


117

Group 4 (10% DMSO) .1660


Group 3 (Paracetamol) Group 1 (Esculetin) .8660


Group 4 (10% DMSO) .2410


Group 4 (10% DMSO) Group 1 (Esculetin) 1.1660




Group 5 (Water) Group 1 (Esculetin) 1.0660



Group 4 (10% DMSO) .4410

ALT LSD Group 1 (Esculetin) Group 2 (Scopoletin) 10.1623

Group 3 (paracetamol) -13.6552

Group 4 (10% DMSO) . -4.3827


95%

Confidence

Interval


ALT LSD Group 2 (Scopoletin) Group 1 (Esculetin) 3.8323

Group 3 (Paracetamol) -16.8202

Group 4 (10% DMSO) -7.5477



Group 2 (Scopoletin) 30.8148

Group 4 (10% DMSO) 16.2698









Group 4 (10% DMSO) -6.3802

AST LSD Group 1 (Esculetin) Group 2 (Scopoletin) 29.5985


Group 4 (10% DMSO) -1.6890




118

Group 4 (10% DMSO) -16.0015




Group 4 (10% DMSO) 40.4135




Group 3 (paracetamol) -9.8415





Group 4 (10% DMSO) -5.7615

ALP LSD Group 1 (Esculetin) Group 2 (Scopoletin) 16.3455


Group 4 (10% DMSO) -10.5395




Group 4 (10% DMSO) . -13.6695


95%

Confidence

Interval


ALP LSD Group 3 (Paracetamol) Group 1 (Esculetin) 67.8105


Group 4 (10% DMSO) 44.0555









Group 4 (10% DMSO) -9.1195

TB LSD Group 1 (Esculetin) Group 2 (Scopoletin) .3348


Group 4 (10% DMSO) .3098


Group 2 (Scopoletin) Group 1 (Esculetin) -.0052


119

Group 4 (10% DMSO) .1398




Group 4 (10% DMSO) .3973


Group 4 (10% DMSO) Group 1 (Esculetin) .0198




Group 5 (Water) Group 1 (Esculetin) -.0227



Group 4 (10% DMSO) .1223

CB LSD Group 1 (Esculetin) Group 2 (Scopoletin) .3269


Group 4 (10% DMSO) .3094




Group 4 (10% DMSO) .1469




Group 4 (10% DMSO) .3919


95%

Confidence

Interval


CB LSD Group 4 (10% DMSO) Group 1 (Esculetin) .0194




Group 5 (Water) Group 1 (Esculetin) -.0081



Group 4 (10% DMSO) .1369

SOD LSD Group 1 (Esculetin) Group 2 (Scopoletin) .3673


Group 4 (10% DMSO) -2.6827




120

Group 4 (10% DMSO) -2.6927




Group 4 (100Ai DMSO) 1.3298






Group 5 (Water) Group 1 (Esculetin) .5798



Group 4 (10% DMSO) -2.4602

MDA LSD Group 1 (Esculetin) Group 2 (Scopoletin) 11.1232


Group 4 (10% DMSO) -6.1718




Group 4 (10% DMSO) -9.9418




Group 4 (10% DMSO) 15.4532


95%

Confidence

Interval


MDA LSD Group 4 (10% DMSO) Group 1 (Esculetin) 20.8782



G'roup 5 (Water) 19.6557




Group 4 (10% DMSO) -4.9493

MUSA, UNEKWU-OJO.pdf - University Of Nigeria Nsukka

Documents