University of Nigeria Research Publications

Aut

hor

NJOKU, Ugochi Olivia

PG/M.Sc/03/34258

Title

Antioxidant Activity of Seed Extract and Fractions of Monodora Tenuifolia

(Annonaceae)

Facu

lty

Medicine

Dep

artm

ent

Pharmacology and Toxicology

Dat

e

February, 2007

Sign

atur

e

ANTIOXIEANT ACTIVITY OF SEED EXTRACT AND FRACTIONS OF Monodora tsnuifolia

(Annonaceae)

NJOKU, UGOCHI OLlVlA PG/M.Sc/03/34258

A PROJECT SUBMITTED TO THE DEPARTMENT OF PHARMACOLOGY~~OXlCOLOGY, UNIVERSITY OF NIGERIA, NSUKKA IN PARTIAL FULFILLMENT OF

THE REQUIREMENT FOR THE AWARD OF MASTERS OF SCIENCE (M.Sc) DEGREE.

TITLE PAGE

ANTIOXIDANT ACTIVITY OF SEED EXTRACT AND FRACTIONS OF Monodora tenuifolia

(Rnnonaceae)

A PROJECT SUBMITTED TO THE DEPARTMENT OF PHARMACOLOGY/TOXlCOLOGY, UNIVERSITY OF NIGERIA, NSUKKA IN PARTIAL FULFILLMENT OF

THE REQUIREMENT FOR THE AWARD OF MASTERS OF SCIENCE (M.Sc.) DEGREE

NJOKU, UGOCHI OLlVlA DEPARTMENT OF PHARMACOLOGY/TOXlCOLOGY

UNIVERSITY OF NIGERIA, NSUKKA

SUPERVISOR: Prof. P. A. AKAH DEPARTMENT OF PHARMACOLOGY/TOXlCOLOGY UNIVERSITY OF NIGERIA, NSUKKA

CERTIFICATION

NJOKU, Ugochi Olivia, a postgraduate student in the Department

of PharmacologyiToxicology and with registration number

PG/M.Sc/03/32458 has satisfactorily completed the requirement of

research work for the degree of Masters of Science (M.Sc) in

PharmacologyiToxicology.

This work embodied in this project is original and has not been

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

other University. 5 F t i ,. .

roject Supervisor s Head of Department

iii

DEDICATION

To my beloved husband, great and wonderful kids: Onyii, Obi. Jnr. &

Chioma, parents and loved ones.

ACKNOWLEDGEMENT

My most profound gratitude goes to my supervisor, Prof. P.A.

Akah for his warmly guidance and supervision. His unreserved kindness

and understanding not only inspired me but also encouraged me

throughout the trying moments of this work.

1 also wish to express my indebtedness to Dr. Charles Okoli of the

Department of Pharmacology and Toxicology who by close supervision

and assistance contributed in no small measure to the completion of this

work.

My gratitude goes to Okoye and Ajaba for their unrelentful

assistance throughout my laboratory work.

To my husband, Prof. 0. U. Njoku, my kids: Onyinye, Obi Jnr.,

and Chioma, I really appreciate your moral and financial support.

My'thanks also goes to my caring colleagues, friends who have

contributed inestimably throughout the course of this work. In this

respect, I am particularly grateful to Ada Onyirioha, Thelma Ihedioha,

Ada Ibiam, Uju Udeogaranya, Prof. Mrs. E. Okeke, and a host of others.

I thank my parents: Elder and Elder (Mrs.) Ibe, my parents' in-law:

Sir and Lady Rufus Njoku, my siblings: Paul, Uloma, Nwajiulo, Ngozi,

Jude, Chizoba, and Tochukwu who have been an everlasting source of

love, strength and encouragement.

Finally, above all, to my gracious heavenly father for the grace,

strength and ability, for perfecting all that concerneth me to him be all the

glory thanksgiving and honour.

TABLE OF CONTENTS Page

List of Figures .................................................................................... viii

................................................................................................ Abstract X

CHAPTER ONE

1 . 1 Antioxidant: An Ovef-View ................................................... 1

1 . 1 . 1 Natural Antioxidant of low and high molecular weight 2

1 . 1 . 2 Mechanism of action of antioxidants .............................. 5

1.2 Lipid Pef-oxidation ................................................................. 6

1.3 Mechanisms of lipid peroxidation induction .................. 7

........................................................................... 1 ~ 3 - 1 Autoxidation

1.3.2 Photo-oxidation .....................................................................

1 a3.3 Enzymatic peroxidation ......................................................

1.4 General antioxidant actions ...............................................

1.4.1 Free f-adicals ..........................................................................

1 a4.2 Antioxidant mokcules .........................................................

1.5 Oxidative stress .................................................................... 1.6 Diseases associated with free radicals ..........................

Flavonoids ..............................................................................

Glycosides ..............................................................................

Cardiac glycosides ...............................................................

Steroidal aglycone ................................................................

Saponins .................................................................................

Tannins ....................................................................................

Carbohydrates ....................................................................... Literature survey of Monodora tenuifolia plant ............

Characteristics/Morphology of Monodora tenuifolia ...

HabitaVEcology of Monodora tenuifolia .........................

Local names ........................................................................... Pharmacological uses of Monodora tenuifolia .............

Aim of research ..................................................................... CHAPTER TWO

Materials and Methods ....................................................................

2.1 Materials .................................................................................. 2.1 . 1 Phnt materials .......................................................................

2.1 2 Animals ....................................................................................

2- 1 a3 Chemicals ............................................... : ...............................

2.1 -4 EquipmenVApparatus .........................................................

2.1.5 Preparation of reagents for Phytochemical Analysis ..

2-2 Methodology .......................................................................... 2.2. Extraction of oil from Monodora tenuifolia seeds .am.....

2.3 Thin-layer chromatographic technique ..........................

2.3.1 Determination of lipid components .................................. 2.4 Determination of vitamin in Monodora tenuifolia .........

2.5 Phytochemical analysis of Monodora tenuifolia seed

2.6 c o h l n chromatographic technique ............................... 2.6.1 Elution of the column ...........................................................

2.7 Nitric oxidellipid peroxidation scavenging assay ........

2-7-1 Scavenging of nitric oxide test ..........................................

vii

2.7.2 Carbon tetrachloride induced lipid peroxidation test

2.8 Statistical analysis ................................................................

CHAPTER THREE

Results ....................-......................~~....--.......-....-.-~~.....-.-....-.--....*..-..... 3-1 Percentage yield of extract .--*.-.......-.----....----....-.--.**.----....-

3.2 The result of the phytochemical analysis of Monodora tenuifolia seed ..................................................

3.3 The result of acute toxicity test: Acute toxicity (LDS0) of the extract ..........................................................................

3.4 Nitric oxidellipid peroxidation scavenging ...---.-...----.-..--

3.4.1 Effect of pet-ether extract and fractions F1 and F p on Carbon tetrachloride-induced lipid peroxidation in rat Liver homogenate .................................................................

3.4.2 Effect of nitric oxide scavenging activity of pet-ether extract and the fractions ....----.....---.-..-.---. * .----...----..*.---......-

3.5 Result of the lipid compounds present in the seed extract ......................................................................................

CHAPTER FOUR

viii

LIST OF FIGURES

Figure 1 : Shows a balance between Oxidative Stress and defense system ........................................................................ 2

Figure 3: Effect of extract and fraction on carbon-tetrachloride- Induced lipid peroxidation in rat liver Homogenate ....... 75

Figure 4: Effect of Extract and fractions on nitric oxide Scavenging activity ................................................................. 78

LIST OF TABLES

Table 1: Shows the Radical Reaction Potentials ........................... 20

Table 3.1 Results of Phytochemical Test ............................................ 70

Table 3.3 Effect of Crude Extract and Fractions on CCI4-lnduced Lipid Peroxidation ........................................ 74

Table 3.4 Effect of Nitric Oxide Scavenging activity of Crude Extract and Fractions ............................................................. 77

Table 3.5 Result of Vitamin Assay of the Extract ......................... 79

Table 3.6 Result of the Lipid Components present in the seed extract 80

ABSTRACT

The antioxidant activity of the seed extract and fractions of

Monodora tenuifolia (Fam. Annonaceae) was evaluated. The Monodora

tenuifolia seed was extracted with pet ether 40-60°C to produce the

crude extract. Fractionation of the extract by column chromatography

using pet ether 60-80°C and diethyl-ether produced 2 fractions (F1) and

(F2). Phytochemical analysis of Monodora tenuifolia seed extract

showed the presence of some plant secondary metabolites, viz:

alkaloids, flavonoids, proteins, carbohydrates, saponins, glycosides,

cyanogenic glycosides, cardiac glycosides, tannins, steroidal aglycon

while, 0 and C glycosides, anthracene glycosides and reducing sugar

were absent. The 3 fractions showed the presence of vitamin A and

vitamin El The pet-ether extract and the fractions (F1 and F P ) reduced

CCle-induced lipid peroxidation in rat liver homogenate. They also

exhibited significant antioxidant activity in nitric oxide induced lipid

peroxidation. The crude extract and diethylether fraction (F2) produced

dose-dependent protective effect against lipid peroxidaiton and free

radical generation in liver homogenate. The acute toxicity study with the

crude extract showed no signs of obvious toxicity up to a dose level of

5000 mglkg. These results suggest that Monodora tenuifolia seed

extract possessed significant antioxidant properties and could be used

for the treatment of diseases associated with free radical generation.

CHAPTER ONE

1 .I Antioxidant: An overview

Antioxidants are a group of substances, which when present at

low concentrations, in relation to oxidizable substrates, significantly

inhibit or dctlay oxidative processes, while often being oxidized

themselves (Kanner et al., 1999).

The application of antioxidants are widespread, in industries they

are used in preventing polymer from oxidative degradation, rubber and

plastic from losing strength, gasoline from autooxidation, synthetic and

natural pigments from discolouration and as additives to cosmetics, food

(especially food with high fat content) beverages and baking products

(Kanner et a/, 1999).

In recent years there has been an increase in the application of

antioxidant in medicine as information is constantly gathered linking the

development of human diseases to oxidative stress (Halliwell et a/.,

1999). The generally accepted hypothesis in any biological system is

that, an important balance must be maintained between the formation of

reactive oxygen and nitrogen species (ROS and RNS, respectively). The

reactive species such as superoxide (02)) hydrogen peroxide (H202),

hydroxyl radical (OH), nitrogen oxide (NO), and hypochlorous acid

(HOCI), are all products of normal metabolic pathways of human organs,

but under certain condition, when in excess they can exert harmful

effects.

To maintain an oxido/redox balance, organs protect themselves

from the toxicity of excess ROSlRNS in different ways, including the use

of endogenous and exogenous antioxidants.

OXIDATIVE STRESS DEFENSE SYSTEM I I

- Mitochondria - Radiation - ~araoxonase ' - Vitamin C ' - Peroxisomes - Ozone - Glutathione- - Flavonoids

* -Inflammatory Cells - Xenobiotics peroxidase - Glutathione -Superoxide

dimutase

Fig. 1: Shows a balance between oxidative stress and defense system

. 11.1 Natural antioxidant of low and high molecular weight

Naturally occurring antioxidants of high or low molecular weight,

can differ in their mechanism and site of action (Sahart, 2001). They can

be divided into the following categories: -

(a) Enzymes

(b) High molecular weight proteins

(c) Low molecular weight antioxidants

a) Enzymes: The best studied cellular antioxidants are the enzymes,

superoxide dimutase (SOD), catalase and glutathione peroxidase (GPx).

These attenuate the generation of reactive oxygen species by removing

potential oxidants or by transforming ROSIRNS into relatively stable

compounds. SOD, which was discovered in the late 60s, catalyzes the

transformation of the superoxide radical into hydrogen peroxide, which

can then be further transformed by the enzyme catalase into water and

molecular oxygen (Sahart, 2001). Glutathione peroxidase (GPx)

reduces lipid peroxides (ROOH), formed by the oxidation of poly-

unsaturated fatty acid (PUFA) to a stable, non-toxic molecule hydroxyl

fatty acid (ROH) (Sahart, 2001). Less well studied (but probably just as

important) enzymatic antioxidants are the peroxiredoxins and the

recently discovered sulfiredoxin. Other enzymes that have antioxidant

properties (though this is not their primary role) include Paraxonase,

Glutathione - strainsferases, and aldehyde, dehydrogenases (Current

Medicinal Chemistry, 2005).

' b) High molecular weight proteins: These preventive antioxidants

hinder the formation of new ROS. These antioxidants are protein that

binds ROS to protect essential proteins. The group includes albumin,

metallothonine, transferring, ceruloplasmin, myoglobin, happtoglobin and

ferritin (Current Medicinal Chemistry, 2005).

These are all present in plasma and bind to redox active metals

and limit the production of metal - catalyzed free radicals (Current

Medicinal Chemistry, 2005). Metals such as iron, copper, chromium,

vanadium and cobalt are capable of redox cycling in which a single

electron may be accepted or donated by the metal (Current Medicinal

Chemistry, 2005). Albumin and ceruloplasmin can bind copper, ions, and

transferin binds free iron. Haptoglobin binds heme-containing protein

and can thus clear them from the circulations (Current Medicinal

Chemistry, 2005). Both free and heme associated protein have pro-

"oxidant properties due to their reaction with H202 to form ferry1 species,

which can easily initiate lipid peroxidation (Current Medicinal Chemistry,

2005).

c) Low molecular weight antioxidants: These are subdivided into

lipid-soluble antioxidants (Tocopherol, carotenoids, quinines, bilirubin

and some poly-phenols) and water-soluble antioxidant (ascorbic acid,

uric acid and some polyphenols) (Niki, 1987).

a-Tocopherol (vitamin E) and p-carotene have considerable

support as lipid-soluble antioxidants; tocopherol might act synergistically

with ascorbate. Vitamin C in living organisms regenerates vitamin E by

reducing the tocopherol radical that is produced when vitamin E

scavenges a peroxyl radical (Niki, 1987). Uric acid is another antioxidant

in primates as their blood has a higher concentration than that of other

mammals; uric acid might serve to scavenge reactive free radicals (Re)

and therefore account for the prolonged life span of humans (Ames et

al., 1981). Some carotenoids, including p-carotene, quench highly

reactive singlet oxygen under certain conditions and can block free

radical-mediated reactions (Bendich, 1989).

1 .I .2 Mechanism of action of antioxidants

Two principle mechanisms of action have been proposed for

antioxidant. The first is chain-breaking mechanism, by which primary

antioxidants donate an electron to the free radical present in the system.

The second mechanism involves removal of ROSIRNS initiators

(secondary antioxidants) by eliminating chain-initiating catalyst (Murray,

Electron donation

Primary antioxidants are compounds that are able to donate

hydrogen atom rapidly to a lipid radical forming a new radical, more

stable than the initial one (Murray et a/., 1990). Biological organs

contain many polyunsaturated fatty acids (PUFA), such as linoleic,

linolenic and arachidonic acid, mainly in the form of ester with

cholesterol. These PUFA can undergo lipid peroxidation that can be

interrupted by the primary antioxidant by the donation of electrons.

The whole process can be depicted as follows.

RH + O2 (Singlet oxygen) - - - - -ROOH

ROOH + ~ e ' ~ - - - - - RO. + HO- + ~ e ' ~

ROOH + ~ e ' ~ - - - - ROO. + H' + ~ e ' ~

ROO.+a-TO. - - - - Non radical products.

RH = Polyunsaturated fatty acid (PUFA)

ROOH = PUFA hydroperoxide

RO. = Alkoxyl radical

ROO. = Peroxyl radical

a - TO. = Tocopheryl radical

Metal Chelation

Secondary antioxidant can retard the effect of ROSIRNS radical

initiated action by means of initiator removal or elimination. This can be

accomplished by deactivation of high-energy species, absorption of UV

light, scavenging of oxygen and thus reducing its concentration (Omenn

et a/, 1996). Chelation of metal catalyzes free radical reaction or inhibits

peroxidase, such as NADPH oxidase, xanthine oxidase, dopamine-p-

hydroxylase or lipoxygenases (Omenn et al., 1996).

The ability of antioxidant to chelate transition metal ions can be

followed spectroscopically. High molecular weight proteins bind directly

or indirectly to redox active metals and thus inhibit the production of

metal-catalyzed free radicals. Some low molecular weight compounds,

such as polyphenols, in addition to their ability to donate hydrogen atom

and thus act as chain-breaking antioxidant, can also chelate transition

metal ions and hence inhibit free radical formation (Omenn et a/., 1996).

1.2 Lipid peroxidation

Peroxidation (autooxidation) of lipids exposed to oxygen is

responsible not only for deterioration of food (rancidity) but also for

damage to tissues in vivo, where it may be a cause of cancer,

inflammatory diseases, atherosclerosis, aging, etc (Murray et al., 1990).

The deleterious effects are initiated by free radicals (ROO., RO., OH.)

produced during peroxide formation from fatty acids containing

methylene interrupted double bonds, i.e., those found in the naturally

occurring polyunsaturated fatty acid (PUFA) (Murray et a/., 1990).

Lipid peroxidation is a chain reaction providing a continuous

supply of free radicals that initiate further peroxidation. Since the

molecular precursor for the initiation process is generally the

hydroperoxide product (ROOH), lipid peroxidation is a branching

reaction with potentially devastating effects. To control and reduce lipid

peroxidaiton both humans and nature involves the use of antioxidants.

1.3 Mechanisms of lipid peroxidation induction

Three different mechanisms are able to induce lipid peroxidation.

1 .3.l Autoxidation

This is a radical - chain process involving three sequences:

(a) Initiation: In a peroxide-free lipid system, the initiation of a

peroxidation sequence refers to the attack of a l iOS (with sufficient

reactivity) able to abstract a hydrogen atom from a methylene group

(-CH2-); these hydrogen having very high mobility (Morel, 1997). This

attack generates easily free radical from polyunsaturated fatty acids: OH

is the most efficient ROS to do that attack, whereas 02. is sufficiently

reactive.

The carbon radical tends to be stabilized by a molecular

rearrangement to form a conjugated diene. Under aerobic conditions

conjugated dienes are able to combine with O2 to give a peroxyl (or

peroxyl) radical, ROO'.

(b) Propagation: A peroxyl radical is able to abstract hydrogen from

another lipid molecule (adjacent fatty acid), especially in the presence of

metals such as copper or iron, thus causing an autocalalytic chain

reaction. The peroxyl radical combines with hydrogen to give a lipid

hydroperoxide. This reaction characterizes the propagation stage

(Morel, 1997).

(c) Termination: Termination (formation of a hydroperoxide is most

often achieved by reaction of a peroxyl radical with a-tocopherol, which

is the main lipophilic "chain-breaking molecule" in the cell membranes.

Furthermore, any kind of alkyl radical (lipid free radical) can react with a

lipid peroxide to give non-initiating and non-propagating species such as

the relatively stable dimers (Morel, 1997).

1.3.2 Photo-Oxidation

As singlet oxygen ('02.) is highly electrophilic, it can react rapidly

with unsaturated lipid but by a different mechanism than free radical

autoxidation. In the presence of sensitizers (chlorophyll, porphyrins,

myoglobin, riboflavin, methylene blue, etc.) a double bond interacts with

singlet oxygen produced from O2 by light. Oxygen is added at either

ends of carbon of a double bond, which takes the trans configuration.

Thus, one possible reaction of singlet 0 2 with a double bond between

C12 and Cl3 of one fatty acid is to produce 12- and 13-hydroperoxide.

The inhibition of photosensitized oxidation is efficiently inhibited by

an antioxidant present and rich in green leafy carotenoids, vegetables

and many coloured fruits (Khachik et a/., 1986). The inhibitory

mechanism is thought to be through an interference with the formation of

' singlet oxygen from the oxygen molecule. In contrast tocopherols

inhibits its oxidation by quenching the previously formed singlet oxygen,

and this forms stable additional products (Morel, 1997).

1.3.3 Enzymatic Peroxidation

Lipoxygenase enzymes (from plants and animals) catalyze

reaction between O2 and polyunsaturated fatty acid, such as arachidonic

acid (20 : 4, - 6), containing methylene interrupted double bonds. When

20: 4,-6 is the substrate, these hydroperoxides are known as HpETES,

which can be transformed into hydroxyl products (HETEs). These

HETEs are also formed directly via cytochrome P450 induced reactions.

Cycloxygenase enzymes (in plants and animals) catalyzed the

addition of molecular oxygen to various polyunsaturated fatty acid, they

are thus converted into biologically active molecule called endoperoxide

(PGG, PGH), intermediates in the transformation of fatty acid to

prostaglandin (Morel, 1997).

1.4 General antioxidant actions

1.4.1 Free radicals

Every cell has chemical reactions involving the oxidation and

reduction of molecules. These reaction or redox pathways can lead to

the production of free radicals.

A free radical is any chemical species capable of independent

existence possessing one or more unpaired electrons. Biological free

radicals are thus highly unstable molecules that have electrons available

to react with various organic substrates (Sahart, 2001).

Many free radicals are generated from naturally occurring

processes such as oxygen metabolism and inflammatory processes.

For example, when cells use oxygen to generate energy, free radicals

are created as a consequence of ATP production by the mitochondria

(Sahart, 2001). Exercise can increase the levels of free radicals as can

environmental stimuli such as ionizing radiation (from industry, sun

exposure, cosmic rays, and medical x-rays), environmental toxins,

altered atmospheric conditions (e.g. hypoxia and hyperoxia), ozone and

nitrous oxide (primarily from automobile exhaust). Lifestyle stressors

such as cigarette smoking and excessive alcohol consumption are also

known to affect levels of free radicals (Omenn et a/., 1996).

It has been noted that certain organ systems are predisposed to

greater levels of oxidative or nitrosative stress. Those organ systems

most susceptible to damage are the pulmonary system (exposed to high

levels of oxygen), the brain (exhibits intense metabolic activity yet has

lower levels of endogenous antioxidants), the eye (constantly exposed to

damaging UV light), circulatory system (victim to fluctuating oxygen and

nitric oxide levels) and reproductive systems (at risk from the intense

metabolic activity of sperm cells). Nearly every organ system can be

found to have an Oxidative or Nitrosative "Achilles heel" (Omenn, et a/.,

1996).

Reactive Oxygen Species (ROS)

ROS is a term collectively describing radicals and other non-

radical reactive oxygen derivatives. These intermediates may participate

in reactions giving rise to free radicals or that are damaging to organic

substrates. ROS in living organisms include the following:

Radicals Non-Radicals

Hydroxyl OH* Peroxynitrite ONOO-

Superoxide 02*- Hypochloric acid HOCl

Nitric oxide NO* Hydrogen peroxide H202

Thyl RS* Singlet oxygen A&-' 02

Peroxyl ROzO Ozxone 0 3

Lipid peroxyl LOO* Lipid peroxide LOOH

Reactive Nitrogen Species (RNS)

RNS are nitrogen-based molecules that can act to facilitate

nitrosylation reactions. Reactive nitrogen species (RNS) include:

Nitrous oxide NO*

Peroxynitrite OONO-

Peroxynitrous acid ONOOH

Nitroxyl anion NO'

Nitryl chloride N02CI

Nitrosyl cation NO'

Nitrogen dioxide NO2*

Dinitrogen trioxide N203

Nitrous acid HN02

The most reactive and damaging free radicals are the OH* and OONO-

(Sahart, 2001). Many other radical species can be formed by biological

reactions, for example: phenolic and other aromatic species are often

formed during xenobiotic metabolisms as part of natural detoxification

mechanisms (Sahart, 2001). Most of the free radicals are produced by

mitochondria and most of the free radical damage is to mitochondria

membranes and mitochondria1 DNA (Wei and Lee, 2002). Between one

and five percent of the oxygen used by mitochondria to generate energy

results in the formation of superoxide radicals (Wei and Lee, 2002).

Although mitochondria are the major source of free radicals, there

are numerous other sources. A green peroxidase of phagocytic cells (as

neutrophils and monocytes) are another source of free radicals. They

(neutrophils and monocytes) assist in bacteriocidal activity by catalyzing

the oxidation of ionic halogen to free halogen (Buetner and Jurkiewicz,

1996).

Myeloperoxidase enzyme, which is a peroxidase found in the

lysosomal granules of myeloid cells, particularly macrophages and

neutrophils, responsible for generating potent bacteriocidal activity by the

hydrolysis of hydrogen peroxide (produced in the metabolic burst) in the

presence of halide ions (Buettner and Jurkiewicz, 1996). Free radicals

are generated by eicosanoids from arachidonic acid during Ischemia-

reperfusion injuries. During reperfusion the endothelial enzyme xanthine

oxidase converts oxygen to superoxide, which can react with nitric oxide

to produce peroxynitrite (Buettner and Jurkiewicz, 1996). Free radicals

from tobacco smoke and air pollution can cause oxidative damage to

* lungs, blood vessels and other body tissue (Bendich and Olson, 1989).

Reactive free radicals ( R - ) ~ appear to have a role in the general

process of aging and in tissue damage that results from radiation,

reactive oxygen metabolite and carcinogen metabolism (Rose and Bode,

1993):

Details of underlying chemistry of ascorbate (Levine and Morita,

1985) and free radical generation (Grisham and McCord, 1986) are

available. Although many or most R' that are generated in the body are

metabolized to non-reactive species (Fig. 2), cellular damage is initiated

under some conditions.

Animals have evolved intricate and interrelated processes for

protecting against the effects of R'. The enzymatic reactions of

superoxide dimutase (SOD), catalase, glutathione peroxidase are not

100% effective in eliminating the formation of all free radicals. For

example, the very reactive hydroxyl free radical, HO', is not eliminated

by these mechanisms (Rose and Bode, 1993).

At the body's non-enzymatic protective mechanisms is a

scavenging reaction in which some endogenous compounds with the

inherent trait of entering into redox reactions contributes an electron to

fill the outer shell of R' and thereby neutralize it to a non-reactive

species. In principle, many chemicals could serve this purpose because

the high reactivity of R* results in it extracting an electron from almost

any available molecule. A few of the compounds shown to have this

property are: Mannitol (Caughey and Watkins (1 985) heamoglobin

(Giulivi and Davies, 1990), estrogens (Niki and Nakon, 1990), bile acids

and derivatives (Stocker et a/., 1990) and serotonin (Jovanovic et a/.,

1990).

For a substance to function biologically, it must do more than

simply react with R*. The present emphasis is on water-soluble

compounds that might have been useful throughout the long

evolutionary development from microbes to mammals. Particular

emphasis is on primates, as they are subjected to threat from R' over

long lifespan.

It must be considered that the source of R* changed over the last

10 years, with the threat from reactive oxygen species increasing (due to

plant generation of 02) and the threat from solar radiation diminishing

(due to emergence of the earth's stratospheric ozone layer) (Rose and

Bode, 1993). Carcinogen metabolites tend to be electron-deficient or

electrophilic (Cavalieri and Rogan, 1984).

Properties of an ideal free radical scavenger

Protection is thought to be available in the form of endogenous

compounds that react with and thereby "scavenge" the R*. Because

many R* are reactive forms of oxygen, an effective scavenger is often

referred to as an antioxidant.

To be an effective antioxidant physiologically, a substance must

have certain chemical and biological properties.

(a) It must be present in adequate amounts in the body

In that most R* have a brief half-life in the body and diffuse only

over short distances, the probability that they react with any given

antioxidant is proportional to the antioxidant's concentration in the

immediate environment where the R* is generated. Most potential

scavengers are present in the mammalian body at a low concentration.

Some have appeared only recently in evolution.

For instance, the introduction of hemoglobin coincided with the

appearance of animals having a closed circulatory system; most earlier

forms of aerobic life depended on cutaneous respiration and did not

have the possibility of respiratory pigments protecting them from free

radical threats (Rose and Bode, 1993).

(b) It must be versatile

The ideal scavenger should combine with a wide variety of free

radicals, i.e., it must be readily oxidized. One limitation of superoxide

dismutase (SOD) in eliminating free radicals is its lack of versatility; it

has but one substrate (Rose and Bode, 1993).

(c) It must be suitable to be compartmentalized

The antioxidant must be suitable for the body to translocate it

between tissues and must accumulate within compartments where a

need for protection exists at the time. A frequent cellular mechanism for

directing substrates to specific sites of the body is membrane transport

, e.g. through polarized cells of the gastrointestinal tract, renal tubule,

liver, placenta etc. (Rose and Bode, 1993). Molecualr size of the

antioxidant is important. Small molecules may be so permanent that

even if they were recognized by a transport mechanism, they would

. readily diffuse out of any membrane-bound compartment. Very large

compounds may not be transported across cellular membranes at rates

great enough to be useful (Rose and Bode, 1993).

(d) It must have tolerable toxicity

Ideally, the antioxidant would be non-toxic, both before and after it

performs the scavenging reaction. If toxicity is a possibility, careful

management of the toxic form must be accomplished under normal

conditions (Rose and Bode, 1993).

(e) It must be available

If the compound is to be accessible to all animal species, it should

either be synthesized de novo or acquired in the diet. A particular

antioxidant might be produced by some animal species or acquired in

the diet by others. If some organisms became devoid of synthetic

capability (e.g. primates, in the case of ascorbic acid) the compound

must be suitable to be ingested as food. Therefore, it must exist in plant

products and be stable for periods of days or weeks after harvest. It

must also be suitable for the normal processes of ingestion, digestion,

and intestinal absorption.

- (f) It might be suitable for regeneration

The process of neutralizing a R' results in the scavenger

becoming oxidized to a form that has less capacity to react with

additional Re. Thus a scavenger would be particularly useful if it is

' recycled so that dietary acquisition does not become prohibitively

expensive. The compound must have a biologically convenient reducing

mechanism, which could be either a specific enzyme or a direct chemical

reaction.

(g) It must be conserved by the kidneys

If the compound is filtered in the glomerular of the kidney, it must

be suitable for reabsorption. Because renal clearance of small

compounds that are filtered but not reabsorbed is high in most animals

(with the half-life of plasma disappearance < 1 hr), large urinary losses

would occur in the absence of active reabsorption (Rose and Bode,

1993).

1.4.2 Antioxidant molecules

We can accept that many substances interact with free radicals or

at least with the most reactive of them. This is not surprising, as some

R' (such as HOD) are so electrophylic that they strip an electron or

hydrogen atom from almost any compound with which they come in

contact. Some of these reactions immediately result in products that are

stable, thus terminating the free radical activity. Many of those

compounds, however, have few of the properties listed above. For

instance, mannitol is present in plants but it is not synthesized in

animals. It is not recognized by mammalian membrane receptors or

transporters; thus it is not absorbed in the gastrointestinal tract or

directed to specific sites of the body. Mannitol does not enter most

animal cells, as evidenced by its use in research as an extracellular

space marker (Rose and Bode, 1993).

a-Tocopherol (Vitamin E) and p-carotene have considerable

support as lipid-soluble antioxidants; tocopherol might act synergistically

with ascorbate. Vitamin C in living organisms regenerates vitamin E by

reducing the tocopherol radical that is produced when vitamin E

scavenges a peroxyl radical (Niki, 1987).

ASCH- + 'TOC TOC + 'ASC-

ASCH- -ASC-

Fig. 2: Ascorbate free Radical Formation

R H

This interaction is consistent with the results of a 1 3 ~ - ~ ~ ~ study,

which showed that the phenolic head group of a-tocopherol in

unilamellar vesicles is located very close to the lipid-water interface

(Perly et a/. , 1985).

Urate is another likely candidate for an antioxidant role in

primates, as their blood has a higher concentration than that of other

mammals. Urate might serve to scavenge R' and thereby account for

the prolonged life span of humans (Ames eta/., 1981).

Free radicals can be listed by one-electron reduction potentials in

millivolts (mV) at pH 7.0. The reduced form of each radical is capable of

neutralizing (reducing) free radicals having a higher potential. As can be

seen from the Table 1, the hydroxyl radical (,OH) has the highest

potential and is the most destructive (reactive) of biological free radicals.

20

Table I: Radical Reaction Potentials

Radical

'OH (hydroxyl)

'LO (alkoxyl)

LOO'

'GS (glutathione)

'HU (Urate)

'TOC (Tocopherol)

'ASC (Ascorbate)

I ~ e ~ ' - EDTA

Vitamin C (Ascorbate ASCH-)

Vitamin C can donate a hydrogen atom to a free radical molecule

(R') thereby neutralizing the free radical while becoming an ascrobate

radical itself. It accumulates in many tissues, both in animal species that

produce it and in those that absorb it as a vitamin. Considering the levels

of ascorbate in humans compared with the plasma, it is highly

concentrated in leukocytes, adrenal, pituitary and compartments of the

eye (Evans et a/, 1982).

Table 1 shows that the ascorbate radicallascorbate

thermodynamic couple is low compared with the reduction potential of

the a-tocopherol free radical, the glutathione radical, the aliphatic alkoxyl

and alkyl peroxyl radicals, and the hydroxyl free radical. Because of this,

ascorbate will act as an antioxidant in each system, and also with

superoxide (Nishikimi, 1975), the urate free radical and other radicals not

prevalent in the body such as nitroxides (Melhorn, 1991). The biological

damaging reactive oxidative species come from a variety of sources,

including ionizing radiation, oxygen metabolism and carcinogen

metabolism. The ease with which ascorbate is oxidized has resulted in

significant commercial utility; ascorbate, or its stereoisomers form, D-

Isoascorbate, is effective in preventing (or reversing) oxidation in a wide

variety of food products. The evidence is also strong that ascorbate has

high reactivity with R' in body fluids (Buettner and Jurkiewicz, 1993).

Ascorbate's efficacy as a scavenger depends on the reactivity of

the ascorbyl free radical (AFR). If AFR were highly reactive with other

substances at the biological pH, temperature, electrolyte composition,

, etc. the chain of free radical reactions would be propagated to

completion as with other intermediate forms of R* in the cell.

In addition to being well suited for an antioxidant role in biology,

ascorbate has also been shown to have a pro-oxidant role in vitro (Borg

and Schaich, 1989).

Chelation of ~ e ~ ' with EDTA actually enhances the reactivity of

iron toward superoxide, thus favouring the Haber-Weiss Reaction

(Buettner and Jurkiewicz, 1996). F~~'-EDTA chelate can catalyze the

Fenton Reaction to generate hydroxyl ion without reduction of Fe3' to

~ e * ' on the other hand, ~ e ~ ' and F~~'-EDTA can be reduced to Fe2' by

ascorbate (ASCH-) to generate the ascorbate radical ('ASC-). The

reduced Iron can then generate a hydroxyl radical by the Fenton

Reaction. Copper ion (cu*') is 80 times more efficient at reacting with

ascorbate than ~ e ~ ' (Buettner and Jurkiewicz, 1996). Thus vitamin C

can be a powerful antioxidant as long as metal ions are not present, but

small amounts of vitamin C in the presence of metal ions can make

vitamin C a dangerous pro-oxidant. Large amounts of vitamin C can

restore the antioxidant function. (Vitamin C has been called an

'. "oxymoron antioxidant") (Buettner and Jurkiewicz, 1996).

Ascorbate is present in many plants, microorganisms, and

animals; it therefore appears to have been present throughout animal

evolution. Evidence that early forms of life use ascorbate comes from

the finding of ascorbate oxidase isoenzymes in tea leaves (Chen and

Asada, 1989).

There are three (3) principal reasons for suggesting that

ascorbate serves an important role as scavenger of free radicals in the

human body:

(a) It is chemically suited to react with oxidizing free radicals;

(b) It is present in the body at sufficiently high concentrations to

be effective;

(c) It fits into the physiology of cellular transport and metabolism.

This combination of properties is well suited for this antioxidant

molecule to contribute to the extended life span potential of

humans (Cutler, 1984).

The most effective singlet oxygen quenchers are carotenoids,

phytochemicals, which plants produce to protect themselves from singlet

oxygen produced by ultraviolet light (Cutler, 1984). rruur" rc-*-

Carotenoids F L * . ~ .b -. ' -

Of 600 carotenoids from natural sources that have been

characterized, fewer than 10% serve as precursors of vitamin A. Many

dietary carotenoids, both with and without provitamin A activity, are

formed in the blood and tissues of humans (Bendich and Olson, 1989).

p-carotene, the most nutritionally active carotenoid, comprises

15-30% of total serum carotenoids (Bendich and Olson, 1989).

Green leafy vegetables and many coloured fruits are rich in

carotenoids and polyenes, (Khachik et a/., 1986). In animal models,

carotenoids have been implicated as chemo-protective or chemo-

preventive agents in several kinds of cancer (Peto et a/., 1981),

particularly skin cancer. Epstein (1977) first showed that injected P-

carotene slowed the growth of skin tumors in hairless mice exposed to

ultraviolet light (UV-A, UV-B). Similarly, feeding either p-carotene,

canthaxanthin, or phytoene to hairless mice exposed to UV-B irradiation

delayed the appearance of skin tumors and reduced their number

(Mathews-Roth, 1982).

Carotenoids may protect cells from oxidative stress by quenching

free radicals capable of causing cellular damage. Unsaturated lipids in

cell membranes are prime targets for free radical reactions. A free

radical-mediated attack on lipid membranes can initiate a chain reaction

that results first in lipid peroxidation and ultimately in functionally

significant damage to membranes, enzyrnes and nucleic acids

(Benedich and Olson, 1989).

Both in vivo and in vitro, p-carotene has been shown to protect

isolated lipid membranes from peroxidation, LDL-containing lipids from

oxidation, and liver lipids from oxidation induced by carbon tetrachloride-

induced free radicals.

In chemical studies, the possible basis for the protective actions of

carotenoids have been examined. Although p-carotene primarily has

been studied, theoretically all carotenoids with a similar conjugated

double bond system should act similarly (Krinsky and Deneke, 1982). In

purely chemical studies, p-carotene interacts with peroxyl radicals

irreversibly to form a carbon-centered carotenoid radical (Burton, 1989).

It is difficult to extrapolate directly from chemical and biological

systems. For example, although antioxidant effects in a chemical

system were noted at a carotenoid concentration of 50 pM, maximal

inhibition of peroxidation was observed at 0.5 mM (Burton and Ingold,

1984). On the other hand, as singlet oxygen quenchers, low

concentrations of carotenoids with nine or more conjugated double

bonds can inhibit the peroxidation of linolenate (Burton, 1989).

At high oxygen tensions, a-tocopherol is the most effective

antioxidant (Burton, 1989).

More than 20 epidemiological studies, both prospective and

retrospective types, have shown that the risk of developing or dying from

certain types of cancer (usually in both men and women) is inversely

associated with the intake of carotenoid-containing fruits and vegetables

and with higher levels of serum p-carotene concentration (Ziegler, 1989).

In the case of lung cancer, which has been most closely associated with

intake of fruits and vegetables, individuals with the lowest carotenoid

intake or serum p-carotene concentrations were at a two- to sevenfold

higher risk of developing neoplasms than those in the highest intake and

highest serum level groups (Ziegler, 1989).

1.5 Oxidative stress

Oxidative stress is a medical term for damage to animal or plant

cells (and thereby the organs and tissues composed of those cells) that

occurs in normal metabolic processes through the production of "free

radicals" caused by reactive oxygen species (Current Medicinal

Chemistry, 2005). In the quest to find a "mate" and become stable, free

radicals interact with the nearest molecule, targeting proteins, fats, or

even DNA. These actions can be so violent that they create bursts of

light within the body. If not neutralized rapidly, they may crate more free

radicals or cause damage to vessel and cell walls, lipids, proteins and

even the nucleus (DNA) of the cell, processes which can lead to cell

death (apoptosis) by induction of mitochondria1 membrane permeability

transition and release of apotogenic factors such as cytochrome C (Wei

and Lee, 2002).

Oxidative stress could be defined as an imbalance between pro-

oxidants prevailing (Current Medicinal Chemistry, 2005). Superoxide is

produced deleteriously by I-electron transfers in the mitochondria1

electron transfer chain. Other enzymes capable of producing superoxide

are xanthine oxidase, NADPH oxidases and cytochrome P450(s).

Hydrogen peroxide is produced by a wide variety of enzymes including

monoxygenases and oxidases (Current Medicinal Chemistry, 2005).

Meals such as Iron, Copper, Chromium, Vanadium and Cobalt are

capable of redox cycling in which a single electron may be accepted or

donated by the metal. This action catalyzes reactions that produce

reactive radicals and can produce reactive oxygen species such as

hydroxyl radical in reactions like Fenton's reaction. The hydroxyl radical

then can lead to modifications of amino acids (e.g. meta-tyrosine and

ortho-tyrosine formation from phenyl-alanine) carbohydrates, initiate lipid

peroxidation (Current Medicinal Chemistry, 2002).

Most enzymes that produce reactive oxygen species contain one

of these metals. The presence of such metals in biological systems in

an unsequestered form (not in an enzyme or other protein) can

significantly increase the level of oxidative stress (Current Medicinal

Chemistry, 2002). Under normal circumstances, there is a delicate

balance between the production of oxidants and antioxidants. However,

during periods of increase pro-oxidant production, antioxidants can

become depleted in the process of detoxification, leaving the body with

little or no defense against the next attack of free radicals (Ray, 2006).

Oxidative stress is a combination of hereditary, environment and

lifestyle factors. While it is possible to change lifestyle and some

environmental factors through diet, physical activity and antioxidant

supplementation, we cannot change our genes (Ray, 2006).

Intense exercise can actually increase oxidative stress in humans,

as the body struggles to detoxify free-radicals while muscles use oxygen

at 100 - 200 times their normal rate (Stauth ef a/., 2002). In this type of

intense exercise for example exercise by ultramarathon runners, who

have competed in races of 50 kilometers, or more than 30 miles, it is

found that here is increased oxidative stress and depleted levels of

vitamin E. When running or doing other intense exercise, there is a 10-

20 fold increase in whole body oxygen consumption; this can produce

reactive oxygen species, which can be harmful, at rates that exceed the

body's natural capacity to detoxify them (Stauth et a/, 2002).

a-ketoglutarate dehydrogenase (a-KGDH), a key enzyme in the

Kreb's cycle is a crucial early target of oxidative stress (Tretter and

Adam-Vizi, 2000). The study on the generation of ROS in the reaction

catalyzed by a-ketoglutarate dehydrogluase demonstrates that a-KGDH

is able to generate H202 and thus, could also be a source of ROS in

mitochondria. Isolated a-KGDH with coenzyme A (HS-CoA) and

thiamine pyrophosphate started to produce H202 after addition of a-

ketoglutarate in the absence of nicotinamide adenine dinucleotide-

oxidized (NAD'). NAD', which proved to be a powerful inhibitor of a-

KGDH-mediated H202 formation, switched the H202 form mode of the

enzyme to the catalytic [nicotinamide adenine dinucleotide-reduced

(NADH) forming] mode (Treter and Adam-Vizi, 2000). In contrast, NADH

stimulated H202 formation by a-KGDH, and for this, neither a-

ketoglutarate nor HS-CoA were required. When all of the substrates and

cofactors of the enzyme were present, the NADHINAD' ratio determined

the rate of H202 production. The higher the NADHINAD' ratio the higher

the rate of H202 production. H202 production as well as the catalytic

function of the enzyme was activated by ca2'. In synaptosomes, using

a-keto-glutarate as respiratory substrate, the rate of H202 production

increased by 2.5-fold, and a conitase actikity decreased, indicating that

a-KGDH can generate H202 in in siu mitochondria. Thus a-KGDH is not

only a target of ROS but could significantly contribute to generation of

oxidative stress in the mitochondria (Tretter and Adam-Vizi, 2000).

1.6 Diseases associated with free radicals

Certain organ systems are predisposed to greater levels of

oxidative or nitrosative stress. Those organ systems most susceptible to

damage are the pulmonary system (exposed to high levels of oxygen),

the brain (exhibits intense metabolic activity yet has lower levels of

endogenous antioxidants), the eye (constantly exposed to damaging UV

light), circulatory system (victim to fluctuating oxygen and nitric oxide

levels) and reproductive systems (at risk from the intense metabolic

activity of sperm cells) (Wee and Lee, 2002).

Oxidative stress definitely contributes to tissue injury following

irradiation and hyperoxia and is suspected (though not proven) to be a

cause of neuro degenerative diseases including Lou Gehrigls disease

(aka MND or ALS), Parkinson's disease, Alzheimer's disease and

Huntington's disease. Oxidative stress is thought to be linked to certain

cardiovascular disease, since oxidation of LDL in the endothelium is a

precursor to plague formation. Oxidative stress also contributes to many

diseases including inflammation, cancer, autoimmune diseases (Current

* Medicinal Chemistry, 2005).

These diseases associated with free radicals are: -

1.6.1 Neurodegenerative diseases

a) Parkinson's disease

Parkinson's disease is a degenerative disorder of the central

nervous system that affects the control of muscles, and so may affect

movement, speech and posture. Parkinson's disease belongs to a

group of conditions called movement disorders, characterized by muscle

rigidity, tremor, a slowing of physical movement (bradykinesia), and in

extreme cases, a loss of physical movement (akinesia) (Parkinson,

2002).

Current concepts of the pathogensis of Parkinson's disease (PD)

center on the formation of reactive oxygen species and the onset of

oxidative stress leading to oxidative damage to substantia nigra pars

compacta (Jenner and Olanow, 1996). Extensive postmortem studies

have provided evidence to support the involvement of oxidative stress in

the pathogenesis of Parkinson's disease; in particular, these include

alterations in brain iron content, impaired mitochondria1 function,

alteration in the antioxidant protective systems (most notably superoxide

dismutase [SODJ and reduced glutathione [GSH]), and evidence of

oxidative damage to lipids, proteins and DNA (Jenner and Olanow,

1996).

Iron can induce oxidative stress, and intranigral injections have

been shown to induce a model of progressive Parkinsonism (Klein and

Ackerman, 2003). A loss of GSH is associated with incidental lewy body

disease and may represent the earliest biochemical marker of nigral cell

loss. GSH depletion alone may not result in damage to nigral neurons

but may increase susceptibility to subsequent toxic or free radical

exposure (Jenner and Olanow, 1996). The nature of the free radical

species responsible for cell death in Parkinson's disease remains

unknown, but there is evidence of involvement of hydroxyl radical (OH.),

peroxynitrite, and nitric oxide. Indeed, OH. and peroxynitrite formation

may be critically dependent on nitric oxide formation (Jenner and

Olanow, 1996).

Central to many of the processes involved in oxidative stress and

oxidative damage in Parkinson's disease are the actions of monoamine

oxidase-B (MAO-B). MAO-B is essential for the activation of l-methyl-4-

phenyl-I ,2,3,6-tetrahydropyridine to 1-methyl-4-phenylpyridinium ion, for

a component of the enzymatic conversion of dopamine to hydrogen

peroxide (H202), and for activation of other potential toxins such as

isoquinolines and beta-carbolines (Nakamura et a/., 2000).

"b) Huntington's Disease (HD)

Huntington's disease (HD), also known as Huntington disease and

previously as Huntington's chorea and chorea major, is a rare inherited

neurological disorder affecting up to 8 people per 100,000 (Vessie,

1932).

Huntington's disease is caused by a rinucleotide repeat expansion

in the Huntingtin (tt) gene and is one of several polyglutamine (or polyQ)

diseases (Vessie, 1932). This produces and extended form of protein,

mutant Huntingtin (mHtt), which causes cell death in selective areas of

the brain. HD's most obvious symptoms are abnormal body movements

called chorea, but also affects a number of mental abilities (Graham et

a/. , 2006).

The gene's coding involved in HD, called the HD gene, is used to

produce a 348 KDa cytoplasmic protein called Huntingtin (Htt). Htt has a

characteristic sequence of fewer than 40 glutamine amino acid residues

in the normal form, more than this and a mutated form of Htt that causes

the disease, mHtt, is produced (Ridley et a/., 1988).

The severity of the disease is generally proportional to the number

of extra residues. The continuous aggregation of the mHt molecules in

neuronal cells causes them to die off in selected regions of the brain.

Although the full function of Htt is unknown, it acts as a transcription

factor in upregulating the expression of Brain-derived neurotrophic factor

(BDNF). With mHtt, there is suppression of this transcription regulatory

function of Htt and hence underexpression of BDNF (Ridley et a/., 1998).

It is suspected that the cross-linking of Htt results in aggregates, which

are toxic, and can lead to dysfunction of the proteasome system. This

mitochondria1 dysfunction can lead to excitotoxicity and oxidative stress

(Graham et a/., 2006). The neurodegeneration caused by mHtt 8is

related to the caspase-6 enzyme clearing the Htt protein (Graham et a/.,

2006).

c) Autism

Autism is a complex neurological disorder and oxidative

imbalance is one feature of the autistic syndrome (Pratico, 2006).

Autism, an early onset neurological disorder, is characterized by

impaired social interactions, limited verbal and non-verbal

communication and repetitive and restricted behavioural patterns

(Pratico, 2006). Children with autism shows signs of abnormal blood-

vessels function and damaging levels of oxidative stress compared to

healthy children. Previous studies on Autism have shown that autistic

patients have reduced cerebral blood flow, presumably due to

constricted blood vessels in the brain, versus healthy controls. Urinary

samples of autistic children who were similar in age and healthy controls

were provided by the Pfeiffer Treatment Center where patients were

diagnosed with autism disorder and evaluated. Patients were excluded

from analysis if they had ever received anti-oxidant treatments or

medicine with any known anti-oxidant effect; if they suffered from chronic

illness, such as depression, psychosis, or inflammatory disorders; andlor

if they were sick at the time of the sample collection.

Isoprostane, a biomarker for oxidative stress; thromboxane, an

index of platelets activation; and prostacyclin, a measure of blood vessel

activation in the samples were measured. It was observed that the rates

thromboxane and prosacyclin synthesis re both not significantly

increased in autism, but are closely related with the rate of oxidative

stress. Compared with controls, children with autism had significantly

higher urinary levels of isoprostane, thromboxane and prostacyclin

(Pratico, 2006).

In patients with autism, the levels of isoprostane (the chemical

byproduct of free radicals attacking fat cells) are nearly double the level

of oxidative stress than the levels in healthy controls (Pratico, 2006).

In autistic patients, there is a biochemical imbalance in the

patients' blood vessels, resulting in high levels of thromboxane (an

indicator of platelet activity) and prostacyclin (an indicator of constricting

endothelial cells). During normal function, thromboxane and prostacyclin

work together to maintain the integrity of vessels. In response to

different kinds of stress, platelets release thomboxane, which causes

vessels to contract. The endothelium responds to elevated levels of

thromboxane by releasing prostacyclin. This event counterbalances the

effect on vessels, inducing dilation of the vessels and, in turn, more

'blood flow (Pratico, 2006). Oxidative imbalance may also play a role in

this disease: autism is characterized by an impaired antioxidant defense

system, higher free-radical production, and improvement of behavioural

symptoms after taking anti-oxidants (Pratico, 2006).

1.6.2 Cardiovascular disease

Cardiovascular disease refers to the class of diseases that involve

the heart and/or blood vessels (arteries and veins). While the term

technically refers to any disease that affects the cardiovascular system,

it is usually used to refer to those related to artherosclerosis (arterial

disease) (Andraws et a/., 2005).

Cardiovascular disease usually occurs as a result of arterial

damage. In coronary heart disease, atherosclerotic plaques (inflamed

fatty deposits in the blood vessel wall) obstruct the coronary arteries

(blood vessels supplying the heart). Narrowing of arteries is called

arterial stenosis. When the blockages become severe enough, the

blood flow to the heart is restricted (cardiac ischemia), especially during

increased demand (i.e. during exertion or emotion). This disease occurs

when one of the plaques ruptures, forming thrombus (blood clot) that

acutely occludes the whole artery. The portion of the heart muscle

supplied by that artery dies; this is known as a myocardial infarction (or a

heart attack in lay parlance). This may result in the death of the patient if

the affected area is large enough. If the patient survives, congestive

heart failure may result. (Andraws et a/., 2005).

Similarly, inflammation and blood clots may obstruct the cerebral

arteries (those supplying the brain). As the disease progresses, an

artery may be transiently blocked, causing cerebral ischemia. These

results in a transient ischemic attack (TIA) (Leaf et a/., 2003) called a

mini-stroke in lay parlance. If the obstruction is severe, a

cerebrovascular accident or stroke may result, due to the death of brain

tissue supplied by the artery (Leaf et a/., 2003).

In peripheral artery disease, obstruction occurs in the arteries of

the arms or legs. This results initially in pain, during temporary

obstruction, and finally in tissue death and gangrene if not treated

(Andraws et a/., 2005). There are many specific illnesses that may occur

in association with these and other cardiovascular disease. In addition

to the ones mentioned above, these include hypertension (high blood

pressure), arterial aneurysms (arterial enlargement and weakening),

cardiomegaly (abnormal enlargement of the heart), tachycardia1

braqdycardialarrhythmia (fast/slowlirregular heart rates), cardiac arrest

(heart stoppage), cardiomyopathy (heart muscle weakness), heart valve

regurgitation (leakage), and heart valve stenosis (narrowing) (Andraws

et a/., 2005).

There are many risk factors, which predispose to various forms of

cardiovascular disease. These include the following: -

- Age

- Absence of key nutritional elements, such as polyphenol

antioxidants

- Diabetes mellitus

- Hypercholesterolemia (elevated cholesterol levels) and less than

excellent lipoprotein particle profile (cholesterol subtypes)

- Tobacco smoking

- Obesity, especially central or male-type obesity; apart from being

linked to diabetes, this form of obesity independently increases

cardiovascular risk, presumably by inducing an inflammatory and

procoagulant state.

- Genetic factors/family history of cardiovascular disease

- High blood pressure (Leaf et a/., 2003).

1.6.3 Aging

Human cells rely on ATP for growth, differentiation, and response

to physiological stimuli and environmental changes. It has been

established that mitochondria make ATP by the coupling of respiration-

generated proton gradient with the proton-driven phosphorylation of ADP

by F,, F1 ATPase (Wei and Lee, 2002). Mitochondria do not only

produce less ATP, but they also increase the production of reactive

oxygen species (ROS) as byproducts of aerobic metabolism in the aging

tissues of the human and animals (Wei and Leo, 2002). It is now

generally accepted that aging-associated respiratory function decline

can result in enhanced production of ROS in mitochondria. Moreover,

the activities of free radical-scavenging enzymes are altered in the aging

.. process. The concurrent age-related changes of these two systems

result in the elevati.on of oxidative stress in aging tissues. Within a

certain concentration range, ROS may induce stress response of the

cells by altering expression of respiratory genes to uphold the energy

metabolism to rescue the cell. However, beyond the threshold, ROS

. may cause a wide spectrum of oxidative damage to various cellular

components to result in cell death or elicit apoptosis by induction of

mitochondrial membrane permeability transition and release of

apoptogenic factors such as cytochrome C. (Wei and lee, 2002).

Moreover, oxidative damage and large-scale deletion and

duplication of mitochondrial DNA (MTDNA) have been found to increase

with age in various tissues of the human. Mitochondria act like a

biosensor of oxidative stress and they enable cell to undergo changes in

aging and age-related diseases. On the other hand, it has recently been

demonstrated that impairment in mitochondrial respiration and oxidative

phosphorylation elicits an increase in oxidative stress and causes a host

of mtDNA rearrangements and deletions (Wei and Lee, 2002). This

aging of the skin is an outward manifestation of "Oxidative stress" which

is occurring within every cell in our body (Ray, 2006).

1.6.4 Cancer

Cancer is caused in all or almost all instances by mutation of

cellular genes that control cell growth and cell mitosis. The mutated

genes are called oncogenes (Antoniades and Owen, 1982). Usually two

or more different oncogenes must occur in a cell before the cell will

become cancerous. Mutation can occur as a result of exposure to

certain chemical, physical, or biological factors (Hall, 1984). Some of

these are the following:

(1) It is well known that ionizing radiation such as x-rays, gamma

rays, and particle radiations from radioactive substances and

even ultraviolet light, can predispose to cancer (Antoniades and

Owen, 1982). Ions formed in tissue cells under the influence of

such radiation are highly reactive and can rupture DNA strands,

thus causing many mutations (Hall, 1984).

(2) Chemical substances of certain types also have a high propensity

for causing mutations (Hall, 1984). Historically, it was long ago

discovered that various aniline dye derivatives are very likely to

cause cancer so that workers in chemical plants producing such

substances, if unprotected, have a special predisposition to

cancer (Hall, 1984). Chemical substances that can cause

mutation are called carcinogens. The carcinogens that cause by

far the greatest number of deaths in our present day society are

those in cigarette smoke. These can cause about one quarter of

all cancer deaths (Ziegler, 1989).

(3) Physical irritants can also lead to cancer, such as continued

abrasion of the linings of the intestinal tract by some types of food

(Gey et a/., ,1987). The damage to the tissue, leads to rapid

mitotic replacement of the cells. The more rapid the mitosis, the

greater the chance for mutation (Lerman, 1984).

1.7 Pharmacological options

Parkinson's disease is a chronic disorder that requires broad-

based management including patient and family education, support

group services, general wellness maintenance, exercise and nutrition

(de Lau et a/., 2004). The most widely used form of treatment is L-dopa

in various forms. L-dopa is transformed into dopamine in the

dopaminergic neurons by L-aromatic amino acid decarboxylase (often

known by its former name dopa-decarboxylase) (Jenner and Olanow,

1996). However, only 1-5% of L-DOPA enters the dopaminergic

neurons (de Lau et a/., 2004).

Carbidopa and benserazide are dopa decarboxylase inhibitors.

They help to prevent the metabolism of L-dopa before it reaches the

dopaminergic neurons and are generally given as combination

preparations of carbidopa/levodopa (co-careldopa) (e.g. sinemet

(http;//www.sinemet.com/parcopa) and benserazide/levodopa (co-

beneldopa) (e.g. Madopar) (de Lau et al., 2004).

There are also controlled release versions of Sinemet and

Madopar that spread out the effect of the L-dopa. Duodopa is a

combination of Levodopa and Carbidopa, dispersed as a viscous gel.

Using a patient-operated portable pump, the drug is continuously

delivered via a tube directly into the upper small intestine, where it is

rapidly absorbed (Marras et al., 2005). Talcopone inhibits the COMT

enzyme, thereby prolonging the effects of L-dopa, and so has been used

to complement L-dopa. Stalevo another drug containing Levodopa,

Carbidopa and Entacopone Stalevo (http://www.stalevo.com/info./sp).

Mucuna prurious, is a natural source of therapeut6ic quantities of L-dopa

, (de Lau et al., 2004).

The Dopamine-agonists bromocriptine (parlodel), pergolide

(permax), pramipexole (Mirapex), ropinirole (Requip), cabergoline

(Cabazer), apomorphine (Apokyn), and Lisuride (Revanil), are

. moderately effective (Mona, 2000).

Dopamine agonists can be useful for patients experiencing on-off

fluctuations and dyskinesian as a result of high doses of L-dopa.

Apomorphine can be administered via subcutaneous injection using a

small pump, which is carried by the patient. A low dose is automatically

administered throughout the day, reducing the fluctuations of motor

symptoms by providing a steady dose of dopaminergic stimulation

(Michael, 2005). Apomorphine is also available in a more acute dose as

an autoinjector pen for emergency doses such as after a fall or first thing

in the morning (Marras eta/., 2005).

Selegiline (Eldepryl) and rasogiline (Azilect) reduce the symptoms

of Parkinson's disease by inhibiting monoamino oxidaze-B (MAO-B),

which inhibits the breakdown od dopamine secreted by the dopaminergic

neurons (Jenner and Olanow, 1996).

MAO-B is essential for the activation of l-methyl-4-phenyl-I ,2,3,6-

tetrahydropyridine to 1-methyl-4-phenylpyridinium ion, for a component

of the enzymatic conversion of dopamine to hydrogen peroxide (H202),

and for the activation of other potential toxins such as isoquinolines and

beta-carbolines (Jeriner and Olanow, 1996).

Thus the inhibition of MAO-6 by drugs such as selegiline may

protect against activation of some toxins and fee radicals formed from

the MAO-B oxidation of dopamine. In addition, selegiline may act

through a mechanism unrelated to MAO-6 to increase neurotrophic

factor activity and upregulate molecules such as glutathione, superoxide

dimutase (SOD), catalase, and BCL-2 protein, which protect against

oxidant stress and apostosis. Consequently, selegiline may be

advantageous in the long-term treatment of Parkinson's disease (Jenner

and Olanow, 1996).

There are standard treatments to alleviate emotional symptoms of

Huntington's disease. These include the use of antidepressants and

sedatives with antipsychotics (in low doses) for psychotic symptoms

(Graham et a/., 2006). Nutrition is an important part of treatment, most

Huntington's disease sufferers need two-three times the calories than

the average person to maintain body weight. Note, an average calorie

intake is between 2000 (women) to 2500 (children and men) (Graham et

a/., 2006).

To aid swallowing, thickener can be added to drinks when

* swallowing becomes hazardous the option of using a stomach PEG for

intake of nutrients is often chosen, this reduces the chances of

pneumonia due to aspiration of food and increases the amount of

nutrients that can be given (Sirna therapeutics, 2006). Other agents and

measures that have shown promise in initial experiments include

. dopamine receptor blockers, creatine, CoQ10, the antibiotic Minocycline,

Trehalose, exercise, antioxidant-containing foods and nutrients (Sirna

Therapeutics, 2006).

Treatment of cardiovascular disease depends on the specific form

of the disease in each patient, but effective treatment always includes

preventive lifestyle changes, which take the form of modifying risk

factors (Leaf, et a/., 2003). Some, such as gender (male or female), age,

and family history cannot be modified. Smoking cessation (or

abstinence) is one of the most effective and easily modifiable changes

(Andraws et a/., 2005). Regular cardiovascular exercise (aerobic

exercise) complements the healthful eating habits. Medications, such as

blood pressure reducing medications, aspirin and the statin cholesterol-

lowering drugs may be helpful (Leaf et a/., 2003).

Sometimes, the combination of diet and exercise will improve

lipoprotein (cholesterol) levels. Aspirin has been shown to decrease the

clot formation that may lead to myocardial infarctions and strokes; it is

routinely prescribed for patients with one or more cardiovascular risk

factors (Andraws et a/., 2005). Eating oily fish at least twice a week may

help reduce the risk of sudden death and arrhythmias (Leaf et a/., 2003).

Studies of individual heart cells showed that the fatty acids blocked

excessive sodium and calcium currents in the heart, which could

otherwise cause dangerous, unpredictable changes in its rhythm (Leaf et

a/., 2003).

Epidemiological evidence suggests that vitamin E

supplementation decreases the incidence of ALS (bou Gehrig's disease)

and Alzheimer's (Andraws, et a/., 2005).

In epidemiological studies, the intake of caroteroid-rich, fruits and

vegetables has been correlated with protection from some forms of

cancer, particularly lung cancer (Bendich and Olson, 1989). In the

treatment of cancer, some classes of chemotherapeutic agents are used

such agents like Alkylating agents (e.g. Nitrogen Mustards,

Ethylenimines and Methylmelamines, Folic acid analogs),

Antimetabolites, (e.g. pyrimidine analogs, purine analogs and related

inhibitors) Natural products, (e.g. Antibiotics, Enzymes, biological

response modifiers, Anthracenedione etc.) and hormones and

antagonists (e.g. Estrogens, Antiestrogen) androgens, antiandrogen,

Gonadotropin-Releasing hormone analog) (Chang et a/., 1993).

1.8 General review of phytochemistry

1.8.1 Alkaloids

They are a group of basic secondary plant substance, which

usually possesses an n-containing heterocyte. Alkaloids exist in plants

as salts, amine or n-oxides. Dicotyledonous plants are the real

producers of alkaloids (Evans, 1989).

They appear in large members and in many variation in these

plants. They are bitter to taste, so when present in plants, insects and

predators tend to move away from such plants. They also protect the

plant from the effect of singlet oxygen (Bonner and Varner, 1965).

Alkaloids at high concentration, produces a variety of toxic effects on

animals.

Their pharmaceutical and medicinal importance can be seen to

act on the cardiovascular system and some have been resorted to be

antihypertensive. Alkaloids also contribute to liver disease and

hepatocellular tumor (Antoniodes and Owen, 1982). Alkaloids of

Catharanthus roseus are used in cancer chemotherapy.

1.8.2 Flavonoids

The origin of the names is from a Latin word "FLAVUS" meaning

yellow. They are a series of related water soluble phenolic glycosides

having in common a basic structural unit. The CI5 skeleton of flavones.

The flavones are sap-soluble (Bonner and Varner, 1965). The phonetic

compound contributes to the colour of soft fruits, which are scarlet,

crimson and purple anthocyamins e.g. cyamidin-3-rutinoside. They are

widely distributed in nature but are more common in the higher plants

and in young tissues, where they occur in the cell sap. Flavonoids

contribute to the taste and flavour of foodstuffs (Bonner and Varner,

.. 1965). Flavonoids when consumed in certain quantity could lead to

serious disorder in the system.

1.8.3 Glycosides

These are the products obtained after condensation of sugar with

different types of organic hydroxyl compounds. These are referred to as

the cardiac-active or cardio-tonic glycosides examples include amygdalin

(Stryer, 1975). In small doses, glycosides promote mild gastric irritation

causing a reflux from the bronchioles. This can be attributed to its wide

usage but in larger dose, they lead to vomiting (Evans, 1989). A larger

number of glycosides and their aglycone have antimicrobial activities.

1.8.4 Cardiac glycosides

This is a derivative of glycosides. It is normally seen in urine,

fruits. It is made up of a non-sugar moiety called aglyone and this non-

sugar moiety, determines the pharmacological effectiveness. The sugar

causes the compounds to be soluble and also power glycosides fixation

to the cardiovascular muscle (Evans, 1989).

I .8.5 Steroidal aglycone

These are non-sugar moiety compounds. They are important

because of their relationship with compounds like the sex hormones,

cortisone, vitamin D and cardiac glycosides (Evans, 1989).

These compounds possess both toxic and play active roles when

they are in the forms of steroids; they have drastic effect on animals.

I .8.6 Saponins

Saponins are useful in the production of soft drinks, beers,

confectioneries, shampoos, soaps, fibre extinguishers and beverages

and this is attributed to its foaming ability (Liener, 1972). They are quite

toxic when injected into the bloodstream and are harmless when taken

by mouth since the sarsaparilla is rich in saponins but is used in the

preparation of non-alcoholic beverages (Evans, 1989).

The highest sapogenin concentration occurs in the reproductive

parts of the plants, the seeds containing 18% trigonenin (Bonner and

Varner, 1965).

Saponin have some medicinal properties, since it has been

reported to have anti-inflammatory, anti-fungal, antimycolic,

bacteriostatic and other biological activities. When ground in a

powdering form, causes violent sneezing.

I .8.7 Tannins

The word "tannin" signifies substances present in plant extracts,

which are able to combine with protein of animal hides, prevent their

putrefaction and the conversion to leather (Evans, 1989). Those tannins

are responsible for the taste qualities of wines, tea and coffee. They are

astrigent and styptic (i.e. the dry sensation felt in the mouth). Tannins

due to their antiseptic properties prevent fungal attacks (Bonner and

Varner, 1965; Evans, 1989). They also have tumorigenic and

carcinogenic effects.

1.8.8 Carbohydrates

Carbohydrates being the most abundant biomolecule with starch

as its precursor is considered of very high pharmaceutical importance. It

is found most abundantly in roots, rhizomes, fruits and seeds. It is also

located in the chlorophyll containing tissues of plants (Evans, 1989).

Monosaccharide, one of the three major size classes of starch are

simple sugars and consist of a single polyhydroxy aldelyde or ketone

unit. It can be oxidized by relatively mild oxidizing agents such as ferric

(I=e3+) or cupric (cu2*) ion. Glucose and other sugars capable of reducing

ferric (Fe3') or Cupric (cu2') ion are termed "Reducing Sugars". This

property is the basis of Fehling's reaction, a qualitative test for the

presence of reducing sugar.

1.9 Literature Survey of Monodora tenuifolia Plant

Taxonomical Classification - Monodora tenuifolia belongs to the:

Kingdom - Plantae

Phylum/Division - Spermatophytae

Class - Dicotyledonae

Subclass - Angrospermal

Order - Archichlamydae

Family - Annonaceae

Gems - Monodora

Specie - Tenuifolia

1.9.1 Characteristics/Morphology of Monodora tenuifolia

Monodora tenuifolia is a widespread and attractive small tree with

very decorative flowers appearing just before the leaves. The fruit is

suspended on a long green stalk with numerous seeds embedded in

whitish sweet smelling pulp. The seed is oblong and pale brown when

fresh with a thin seed coat and hard kernel (Dalziel, 1948). The seed

production is seasonal occurring between April to June. The fruits are

globular and ovoid; 3-4 inch long and about 3-5 inch diameter. The

wood is hard. The seeds are contained in a hard shell and have a very

strong aroma (Neilson, 1965). This plant is commonly called Orchid

flower tree in English, Ehuru Ofia in lgbo (Okafor, 1989) (www.Terv-uren

Xylariumwood Database.com). Monodora tenuifolia is a specie of

calabash nutmeg, the edible seeds yield a nutmeg-flavoured oil which is

used in West Africa for cooking (Eggeling, 1952).

1.9.2 HabitatlEcology of Monodora tenuifolia

Monodora tenuifolia is tropically distributed. It is cultivated in East

India, Malaysia, Sri Lanka, West lndies and Africa. It could be

propagated by stem culturing and budding (Okafor, 1989). The

Monodora species are also found in West Africa and are cultivated in the

southern parts of Nigeria. The trees are very common in Anambra, Abia

and Enugu States.

I .9.3 Local Names

The plant is usually called Orchid flower and is also referred and

called:

Ehuru Ofia - lgbo name

Ehinawosin - lkale name

Lakosin - Yoruba name

Uyenghen - Edo name

Keay, 1989 (www.TervurenXylariumwoodDatabse.com)

I .9.4 Pharmacological uses of Monodora tenuifolia

The plant is widely used in ethnomedicine, especially to relieve

toothache as well as in the treatment of dysentery (Nielson, 1979). When

roasted and ground, the seeds are rubbed on the skin for (unspecified)

skin diseases (Irvine, 1961). This suggests that the seeds of M.

tenuifolia plant could be germicidal or antiseptic.

The roasted ground seeds are chewed, then spat into the hand

and then rubbed across the forehead to relieve headache. The seeds

are also crushed and used as insecticide, while the root relieves

toothache when crushed (Oguntinein et a/, 1989).

Monodora tenuifolia seeds are also used for the treatment of

constipation and as a stimulant (Irvine, 1961). The essential oil from

Monodora tenuifolia seed is used in pharmaceutical and dental

preparation (Talalaji, 1965).

1 . I0 Aim of research

The aim and objective of this research is to:

(1) Investigate the antioxidant activity profile of Monodora tenuifolia

seed extract; and possibly determine the antioxidant principles.

CHAPTER TWO

MATERIALS AND METHODS

2.1 MATERIALS

2.1 .I Plant material

The fresh seeds of the Monodora tenuifolia were collected within

the surroundings of the University of Nigeria, Nsukka Campus, Enugu

State in the month of April to June and the seeds were identified and

authenticated by Mr. A. 0 . Ozioko of Bioresources Development and

Conservation Programme (BDCP), Nsukka.

2.1.2 Animals

Twenty-seven mice (100-120 g) of either sex and 8 Swiss albino

rats (170 - 200 g) of either sex were used for the LD5(-), nitric oxide

scavenging activity and CC14 lipid peroxidation scavenging activity.

These mice and rats were bought from the Department of

Pharmacology & Toxicology Animal House, University of Nigeria,

Nsukka. Animals were kept in cages at 25OC + Z°C. All the animals were

acclimatized to the laboratory conditions for a week before use. They

were fed with standard animal feed and water ad libitum.

2.1.3 Chemicals

All the chemical used in this work were all of analytical grade

commercially available.

The chemicals include:

Absolute ethanol

Acetic acid

BDH

Vicker

Acetone

Ammonia

Ammonium Moybdate

Ammonium solution

Ascorbic acid

Carbon-tetrachloride

Chloroform

Diethyl-ether (40-60°C)

Ethyl acetate

Ferric Chloride

Ferrous Sulphate

Hydrochloric acid (Conc.)

Lead acetate

Methanol

Naphthylethylenediamine dihydrochloride

n-Hexane

Nitric Oxide

Petroleum ether (40-60°C, 60-80°C)

Phosphoric acid

Potassium chloride

Potassium hydroxide

Potassium Iodide

Silica gel

Sodium hydroxide

Sodium nitroprusside

Sulphanilamide

Sulphuric acid (Conc.)

Thiobarbituric acid

Trichloroacetic acid

Tween 80

Vicker

BDH

Merck

Sigma

BDH

Numex

May and Baker

Merck

BDH

Vicker Lab. Ltd.

BDH

Merck

Sigma

Fluka

BDH

Riedel-de Haen

Merck

Frutaran

BDH

BDH

BDH

Merck

Merck

MAY AND BAKER

BDH

Sigma

BDH

Sigma

Sigma

Sigma

2.1.4 EquipmentlApparatus

The equipment used for this includes:

Column

Glass wares (test tubes, beakers etc.)

Mortar and Pestle

Refrigerator

Rotary evaporator

Spatula

Separating funnel

Soxhlet apparatuss

Spectrophotometer

Spreader & TLC applicator

TLC plates

Weighing balance

Water bath

2.1.5 Reagents for phytochemical analysis

Aluminium chloride solution (0.5%)

Ammonium solution

Dilute sulphuric acid solution

Dragendorff's reagent

Ethanol (45%)

Ferric chloride solution (5%)

Lead sub-acetate solution (1 5%)

Mayer's reagent

Pyrex

Pyrex

Life Scan Company

Thermocool

Mecitherm (PFV)

Pyrex

Giant Instrument

Mecitherm (PFV)

Novospec (LKB)

Merck

Merck

Meter HAS 6

Mecitherm (PFV)

Wagner's reagent

2.1.6 Reagents for nitric oxidellipid peroxidation scavenging assay

Ferrous sulphate (1 5 mM)

Naphthylene diamine dihydrochloride (0.1 %)

Phosphoric acid (2%)

Potassium chloride (0.15 m)

Potassium sulphate buffer (pH 7.4)

Sodium nitroprusside (5 pm)

Standard phosphate buffer (0.025 m, pH 7.4)

Thiobarbituric acid (0.67%)

Thiobarbituric acid (0.67%) in acetic acid (50%)

Trichloroacetic acid (1 0%)

. 2.1.7 Preparation of reagents for thin-layer chromatographic separation of lipid components

Preparation of chromatoplates

The clean thin-layer chromatographic plates were cleaned with

acetone and placed on an applicator. The silica gel was spread on the

plates using a spreader.

The plates were allowed to dry in air and stored in a dust free

desiccator. Before usage, the plates were activated in an oven at I 10°C

for an hour and left to cool before usage.

55

Solvents for chromatographic studies

Petroleum ether, diethylether and acetic acid solvent mixture

(1 80:20:2 vlvlv)

n-hexane, diethylether and acetic acid solvent mixture (80:20:2

vlvlv).

n-hexane and benzene (85:l5 vlv) solvent mixture

Chloroform, methanol and water (95:20:2.5 vlvlv) solvent mixture

Chloroform, .acetone, methanol and acetic acid (50:32O: 10: 10.5

v/v/v/v) solvent mixture.

n-hexane, diethylether and acetic acid (69:29:2 vlvlv) solvent

mixture.

Methodology

' 2.2.1 Extraction of oil from Monodora tenuifolia Seeds

The freshly collected seeds of the Monodora tenuifolia were sun

dried and powdered using a pistle and mortar. The powder was defatted

with petroleum ether (40 - 60°C) using soxhlet apparatus.

The whole filtrate was allowed to evaporate at room temperature

leaving the oil.

% yield of oil = Weight of Oil 100 -X-

Weight of Sample I

2.3 Thin-Layer chromatographic technique

The extracted oil of Monodora tenuifolia was prepared with

chloroform (1:5) (0.1 ml of oil was dissolved in 0.5 ml chloroform). This

56

solution was spotted 2 cm from the base of the chromatographic plates.

Before developing in the appropriate solvent system, they were allowed

to dry. Micropipetes were used for spotting of the sample.

2.3.1 Determination of lipid components

A chromatographic tank is used for the thin layer chromtography

and the solvent sy'stem (pet-ether, diethylether and acetic acid (180:20:2

V/V/V) was poured inside with vigorous shaking so that it will systemize.

This solvent was allowed to equilibrate for about 1 hr. The spotted

plates were placed inside the tank containing the solvent system and

allowed to develop for 45 minutes.

The spotted plates were left for a while to allow for separation

shown by relative distance moved by different components of the oil.

Development of the plate was done in an iodine tank with iodine vapour.

On observation, it was discovered that there were seven distinct

components. Their relative distance was measured and the solvent front

was determined. From the measurements, specific Rf values were

calculated using the formular:

Rf = Distance moved by each component Distance moved by solvent front

On using different chemical agents, series of thin-layer

chromatography were conducted to determine the presence of

aminolipids, glycolipids, free fatty acids and cholesterol.

Determination of aminolipids

The spotted plates were placed in the chromatotank containing

the solvent system to separate. The plates were then sprayed with

ninhydrin reagent (0.2 g of ninhydrin dissolved in 100 mi of ethanol).

The sprayed plates were heated at 120°C for 5 minutes in a humid

atmosphere. A red violet colour indicates the presence of aminolipids.

Determination of free fatty acids

After spotting the chromatographic plates with the lipid sample, it

was placed in the chomatotank containing the solvent system to

separate. The plate was then sprayed with ferric chloride solution (1.0 g

of ferric chloride in 100 ml of H20). A Rose-Violet spot on the plate

shows the presence of free fatty acid.

Determination of glycolipids

The plate having been spotted with the oil dissolved in chloroform

was developed in the solvent system. After air-drying the plates, was

sprayed with a-naphthol reagent (0.5 g of a-naphthol dissolved in a

solution containing equal volumes of 25 ml methanol and H20) and

subsequently heated in an oven at 120°C for 5 minutes. A purple blue

spot indicates the presence of glycolipids.

Determination of c.holesterol

The plate from the chromatotank was air-dried and sprayed with

acidic ferric chloride solution. The sprayed plate was heated for 2 - 3

minutes, in an oven at 100°C. Red violet colour/spots indicate the

presence of cholesterol.

2.4 Determination of vitamin in Monodora tenuifolia

Preparation of plant extract

In preparation of plant extract, a quantity of 0.1 g of the sample

were each dissolved in 10 ml of chloroform and 10 ml of ethanol in 2

different test tubes. The mixture was shaken for 30 minutes, filtered with

filter paper and the filtrate was used for determination of the presence of

Vitamins A and E.

Vitamin A assay

Two drops of the extracts were added to 5 drops of chloroform

using a pipette with the addition of 2 drops of concentrated H2SO4. A

' brown colour indicates the presence of Vitamin A.

Vitamin E assay

In assaying for vitamin E, a quantity of 0.1 g of the extracts from

the sample was added to 1 ml of ethanol and 10 drops of concentrated

HN03. The presence of red colour indicates the presence of Vitamin E.

2.5 Phytochemical analysis of Monodora tenuifolia Seed

Test for alkaloids

Some quantity of the Monodora tenuifolia sample was ground in a

mortar and some quantity of about 0.2 g of the macerated sample was

boiled with 5 ml of 2% HCI on a steam bath. This was filtered and 1 ml

portion of the filtrate was treated with 2 drops of the following reagent:

Dragendorff's Reagent (Bismuth potassium-Iodide Solution: A

red precipitate was observed indicating the presence of

alkaloids.

Mayer's Reagent (Potassium Mercuric Iodide Solution): A

creamy white coloured precipitate indicates the presence of

alkaloids.

Wagner's Reagent (Iodine in Potassium Iodide Solution): A

reddish brown precipitate indicates the presence of alkaloids.

Picric Acid (1%): A yellow precipitate indicates the presence

of alkaloids. The following colours were observed using the

reagents: Dragendorffs reagent (red precipitate), Mayer's

reagent (white precipitate), 1 % picric acid (yellow precipitate

and Wagner's reagent (brown precipitates).

' Test for the presence of flavonoids

To test for the presence of flavonoids, 0.2 g of the macerated

sample was heated with 10 ml of ethylacetate in a boiling water for 3

minutes. The mixture was filtered and the filtrate used for the following

tests:

(i) 4 ml of the filtrate was shaken with 1 ml of 1% aluminium

chloride solution and observed for light yellow coloured

ethylacetate layer. A yellow coloration observed in the

ethylacetate layer indicates the presence of Flavonoids.

(ii) 4 ml of the filtrate was shaken with 1 ml of dilute ammonia.

The layers were allowed to separate. A yellow coloration

Dragendorffs Reagent (Bismuth potassium-Iodide Solution: A

red precipitate was observed indicating the presence of

alkaloids.

Mayer's Reagent (Potassium Mercuric Iodide Solution): A

creamy white coloured precipitate indicates the presence of

alkaloids.

Wagner's Reagent (Iodine in Potassium Iodide Solution): A

reddish brown precipitate indicates the presence of alkaloids.

Picric Acid (1%): A yellow precipitate indicates the presence

of alkaloids. The following colours were observed using the

reagents: Dragendorff's reagent (red precipitate), Mayer's

reagent (white precipitate), 1% picric acid (yellow precipitate

and Wagner's reagent (brown precipitates).

* Test for the presence of flavonoids

To test for the presence of flavonoids, 0.2 g of the macerated

sample was heated with 10 ml of ethylacetate in a boiling water for 3

minutes. The mixture was filtered and the filtrate used for the following

tests:

(i) 4 ml of the filtrate was shaken with 1 ml of 1% aluminium

chloride solution and observed for light yellow coloured

ethytacetate layer. A yellow coloration observed in the

ethylacetate layer indicates the presence of Flavonoids.

(ii) 4 ml of the filtrate was shaken with 1 ml of dilute ammonia.

The layers were allowed to separate. A yellow coloration

observed at the ammonia layer indicates the presence of

Flavonoids.

Test for the presence of glycosides

To test for the presence of glycosides, 2.0 g of the ground sample

was added to 10 ml of water, the solution was heated on a water bath for

5 minutes, filtered and used for the following tests:

(i) To 5 ml of the filtrate was added 0.2 ml of Fehlings solution A

and Fehlings solution B until it turned alkaline (tested with

litmus paper) and heated on a water bath for 2 minutes. A

brick red colour was observed.

(ii) 15 ml of dilute H2SO4 instead of water was used, the above

process was repeated and the quantity of the precipitate

formed was compared with that of the former experiment.

Test for the presence of cardiac glycosides

To test for the presence of cardiac glycosides, 0.1 g of the sample

was warmed with 5 ml of chloroform in a water bath and decanted. The

decanted solution was evaporated to dryness on a water bath and the

residue dissolved in 3 ml glacial acetic acid containing a drop of ferric

chloride. The concentrated sulphuric acid was poured carefully by the

side of the tube. A brown ring was observed at the interface.

Test for presence of cyanogenic glycosides

In the test for the presence of cyanogenic glycosides, 0.1 g of the

ground sample was placed in a conical flask and enough water was

added to cover the sample. A piece of sodium picrate paper was

suspended in the flask placed on a water bath for one hour. A colour

change from yellow to white was observed.

Test for presence of anthracene glycosides

The test for the presence of anthracene glycosides, 5 ml dilute

sulphuric acid and 5 ml ferric chloride solution was added to 0.1 g of the

ground sample of Monodora tenuifolia. The resultant mixture was boiled

for 5 minutes, cooled and filtered into a 50 ml separating funnel. The

filtrate was shaken with equal volume of carbon tetrachloride. The lower

organic layer was carefully separated into a test tube and 5 ml of dilute

ammonia solution was added to it and was gently shaken. A brown

coloration in the ammonium layer was observed.

Test for presence of 0- and C-glycosides

To test for the presence of 0- and C-glycosides, 0.1 g of the

ground sample was heated with 5 ml of water in a boiling water bath for

15 minutes, cooled and filtered. The filtrate was treated with 5 ml of 25%

HCI and the solution heated once more for 15 minutes, cooled with 10 ml

of diethylether in separating funnel. Both phases were checked for

purple red colour in the aqueous layer.

The combined ether extract was shaken with 5 ml of 3.5% dilute

ammonia solution and observed. The acid phase was titrated with 0.5 g

of ferric chloride and then heated for about 30 minutes in a water bath

and cooled. The solution was extracted with 10 ml of chloroform. The

layer was washed with 5 ml of water and then 3 ml of 3.5% dilute

ammonia solution was added. If a dark red precipitate is observed, then

it shows a positive result.

Test for presence of tannins

In the test of tannins, 20 g of the ground sample was boiled with 5

ml of 45% ethanol for 5 minutes, cooled and filtered, the filtrate was used

for the following tests:

(i) Lead Sub-Acetate:

In the lead sub-acetate test, 0.1 ml of the filtrate was

added a few drops of lead sub-acetate solution and observed

for a gelatinous white precipitate.

(ii) Bromine Water:

For the bromine water test, 1.0 ml of the filtrate was

added 0.5 ml bromine water and a pale yellow precipitate was

observed.

(iii) Ferric Chloride:

For the ferric chloride test, 0.1 ml of filtrate was diluted

with distilled water and a few drops of Fed3 solution was

added. A thick yellow colour was observed, which later

turned black.

Test for presence of saponins

The test for the presence of saponins, 0.1 g of the ground sample

was boiled with 5 ml of distilled water for 5 minutes and decanted while

still hot. The filtrate was used for the following tests:

(i) Frothing Test:

In the frothing test, 1 ml of the filtrate was diluted with

4 ml of distilled water, shaken vigorously. A stable froth was

observed on standing the test tube.

(ii) Emulsion Test:

For the emulsion test, 1 ml of the filtrate was added to

2 drops of olive oil. The mixture was shaken and there was

formation of an emulsion.

Test for presence of reducing Sugar

To test for reducing sugars, 1.0 g of the ground sample was

shaken vigorously with 5 ml of distilled water and filtered. About 1 ml

portion of the filtrate was added to equal volume of Fehlings solutions A

and B and shaken vigorously. A brick red precipitate was observed.

Test for carbohydrate (Molish Test)

The test for carbohydrates, 0.1 g of the ground sample was

shaken vigorously with water and filtered. To the aqueous filtrate was

added few drops of Molish reagent followed by vigorous shaking.

Then 1 ml of concentrated sulphuric acid was carefully added to

form a layer below the aqueous solution. A brown ring was observed at

the interface.

Test for steroidal aglycon

To test for the presence of steroidal aglycon, 10 ml of lead acetate

solution (9% wlv) and 20 ml aqueous ethanol were added to about 1 gm

of the ground material in a 100 ml conical flask. This was placed in a

boiling water bath for about 2 minutes, then cooled and filtered. The

filtrate was extracted twice with 15 ml chloroform. The lower chloroform

layer was retained while 5 ml of the chloroform extract was evaporated

to dryness on a water bath. To the residue, 2 ml of 3,5-diintrobenzoic

acid solution (2% on ethanol) and 1 ml NaOH solution were added and

then observed for pinkish coloration.

2.6 Column chromatographic technique

The column was set up by clamping in a vertical position, a small

quantity of glass wool was placed in the bottom of the column. These

retain solid materials in the column while allowing liquid to percolate

freely through them. Air trapped in the glass wool was removed by

pushing it with a glass rod.

The medium i.e. dry silica gel was poured in, such that it allowed

uniform packing of the column and a free flow of solvent through it in

order to attain optimum results. The silica gel was poured into the tube a

little at a time and allowed to separate under gravity until a column of the

correct height was attained. A little of the solvent was poured into the

column to a depth of a few inches.

Finally, the tap at the bottom of the column was opened to allow

the liquid to run out until it just covers the top of the silica gel.

2.6.1 Elution of the column

The extract to be separated is mixed with some quantity of the

silica gel until a fine mixture is gotten. Thus mixture is introduced into

the packed column to a depth of a few inches.

Some of the silica gel is further introduced to a depth of few

inches covering the extract - silica gel mixture. The first eluting solvent

petroleum ether is introduced and allowed to flow steadily through the

medium adjusting the clips to give the required rate. The flow is

continued until the mixture is completely separated into its components.

After the lSt elution, using petroleum-ether (60-80°C), which

readily must have eluted some proportion of the component in the

'mixture further elution is carried out with a second solvent diethyl-ether.

Here, the separated compounds emerge in the eluate in different

fractions. This results in different fractions of eluant viz: petroleum ether

(60-80°C) fraction (F1) and Diethyl ether Fraction (F2).

2.7 Nitric oxidellipid peroxidation scavenging assay

The crude extract as well as fractions F1 and F2 were evaporated

to dryness to remove the solvents and suspended in 20% (vlv) of Tween

80 and then used for the nitric oxidellipid peroxidation scavenging tests.

2.7.1 Scavenging o f nitric oxide

Sodium nitroprusside 1 ml of 15 pglml was incubated with

different concentrations (100, 200, 400, 800 pglml respectively) of F1

dissolved in standard phosphate buffer (0.025 m, pH 7.4) and the tubes

were incubated at 25OC for 5 hours. After 5 hrs, 0.5 ml of incubation

solution was removed and diluted with 0.5 ml of Griess reagent

(prepared by mixing equal volume of 1% suphanilamide in 2%

phosphoric acid and 0.1% naphthylene diamine dihydrochloride in

water). The chromophore formed was read at 546 nm in a

spectrophotometer. The control experiment was also carried out in

similar manner, using distilled water in the place of F1. The experiment

was repeated in same manner using F p and crude pet-ether extract. The

tests with each of the extracts - F1, Fp and crude extract were carried out

in triplicates and % scavenging activity was calculated using the

following formular:

Abs T = Mean Absorbance of the Extracts (Fl, F2 and crude

extract)

Abs C = Mean Absorbance of the control (Distilled water).

The activity of each fraction and extract was compared with

ascorbic acid of different concentrations (100, 200, 400 and 800 pglmi),

which was used as a standard antioxidant.

2.7.2 Carbon tetrachloride induced lipid peroxidation test

The method of (Comporti, 1989) was used in the CCL4 induced

lipid peroxidation assay. Eight adult Swiss albino rats weighing 170 -

200 g were anaesthetized in chloroform and dissected to remove the

liver and their livers were carefully removed.

The total liver from the rats weighing 8 g was homogenized with

20 ml of ice-cold potassium chloride. Aliquots of 0.5 ml of homogenates

were taken in different test tubes. These test tubes with different

concentrations of Fi (100, 200, 400 and 800 pg/ml) were incubated at

37OC in a constant shaker water bath for 45 minutes with 1.5 ml of

potassium sulphate (pH 7.4), 2 ml of potassium chloride and finally 10 ml

of carbon tetrachloride (CC14) was added.

The reaction was stopped by the addition of 2 ml of 10% (wlv)

trichloroacetic acid and after incubation, the contents of each test tubes

were centrifuged at 4000 rpm for 10 minutes and 2 ml of the clear

supernatant was transferred to another test tubes and finally 2 ml of

0.67% (wlv) of thiobarbituric acid was added to the test tubes and

heated in a boiling water bath for 15 minutes. Each of the test tubes was

cooled and the mixture adjusted to pH 12.5 with potassium hydroxide.

The chromophore was measured at 543 nm in a Novaspec

Spectrophotometer. The control test was carried out in same manner

but distilled water was used in place of F1. The test was also carried

with different concentrations (100, 200, 400, 800 pglml) of the other

extracts (F2 ands crude extract) all in triplicate and the %

scavenginglinhibition was calculated using the following:

where:

Abs T = Mean Absorbance of the Extracts (F1, F2 and crude

extract)

Abs C = Mean Absorbance of the control (Distilled water).

Again activities of each extracts (F1, F2 and crude extract) were

compared with that of ascorbic acid of different concentration (100, 200,

400, 800 pglml), which was used as a standard antioxidant drug.

2.8 Statistical analysis

The results were analyzed with one-way ANOVA expressed as

Mean + SEM. The FISCHER LSD Posthoc test was used to test the

differences between mean of treated and control groups and were

regarded significant at pc 0.05.

CHAPTER THREE

RESULTS

3.1 Percentage yield of extract

In the present study, the % yield of the crude extract was found to

be 18.9%.

3.2 The result of the phytochemical analysis of Monodora tenuifolia seed

The result shows that the Monodora tenuifolia seed contains

alkaloids, carbohydrates, cardiac glycosides, cyanogenic glycosides,

flavonoids, glycosides, proteins, saponins, steroidal aglycon and tannins

while anthracene glycosides, 0 and C glycosides and reducing sugar

were absent (Table 3.1).

Table 3.1: Results of phytochemical tests

COMPONENTS

Alkaloids

Anthracene glycosides

Carbohydrate

Cardiac glycosides

Cyanogenic glycosides

Flavonoids

Glycosides

0 and C glycosides

Protein

Reducing sugar

Saponins

Steroidal aglycones

Tannins

RESULTS -

Present

Absent

Present

Present

Present

Present

Present

Absent

Present

Absent

Present

Present

Present

3.3 The result of acute toxicity test: (LDS0) of the extract

In the acute toxicity test, administration of 10, 100 and 1000

mglkg body weight of the extract caused no death in the first phase of

the test. In the second phase of the test, no death was recorded even

up to 5000 mglkg body weight.

The LD50 was therefore greater than 5000 mglkg (Table 3.2).

Table 3.2: Result of acute toxicity test

1 ST PHASE

2ND PHASE

CRUDE EXTRACT

Crude extract

Crude extract

DOSE (mglkg)

10

MORTALITY GROUP

013

3.4 Nitric oxidellipid peroxidation scavenging

3.4.1 Effect of pet-ether extract and fractions fl and f2 on carbon tetrachloride-induced lipid peroxidation in rat liver homogenate

Lipid peroxide formation by CC14 was inhibited by the pet-

ether(40-60°C) extract, the pet-ether fraction (60-80°C) (F1) and

diethylether fraction (F2) at all tested doses (100 - 800 pglml). The

percentage inhibition of peroxide formation was increased in a,dose

dependent manner for the pet-ether (40-60°C) extract and diethylether

fraction (F2). While for the pet-ether fraction (F1) the percentage

inhibition showed a result independent of the dose (Table 3.3)..

Table 3.3: Effect of extract and fractions on CCL4 induced lipid peroxidation

EXTRACT

Pet-ether fraction (F,)

Diethlether fraction (F2)

Pet-ether Extract (crude)

Vitamin C (standard)

BLANK

CONC. pglml

100

200

400

800

100

200

400

800

100

200

400

800

100

200

400

800

ABSORBANCE (Mean f SE)

0.49 + 0.03*

0.54 + 0.06*

0.48 + 0.04*

0.45 + 0.01*

0.64 + 0.01*

0.59 + O.OO*

0.45 + O.OO*

0.43 + 0.01*

0.64 + 0-OO*

0.49 + 0.01*

0.45 + O.OO*

0.40 f 0.00*

0.41 + 0.04"

0.34 + 0.02*

0.24 + O.OO*

0.12 _+ o.oo*

1.30 + 0-00* $ BLANK

INHIBITION ( O W

62.07

57.93

63.33

65.40

51.03

54.33

64.40

66.67

50.80

62.30

65.67

69.50

68.47

73.87

81.50

90.53

0 100 200 300 4 00 500 GO0 700 000 900

Concentration (uglnil)

Fig. 3: Effect of,extract and fractions on carbon tetrachloride-induced lipid peroxidation in the rat liver homogenate

--+a- Pel Elher Fraction (FI ) -0- Oielhylelher fraclion (F2)

-A- Oil (Pel elhcr exlracl) - -n- - Vilamin C (Slandard)

3.4.2 Effect of nitric oxide scavenging activity of pet-ether extract and the fractions

The Monodora tenuifolia pet ether (40-60°C) seed extract and the

fractions; pet-ether (60-80°C) fraction (Fl), and diethylether fraction (F2)

showed significant free radical, scavenging action against nitric oxide

(NO) induced release of free radicals. Diethylether fraction and the pet-

ether (40-60°C) extract showed an increased percentage inhibition,

which was dose dependent while the pet-ether (60-80°C) fraction (F1)

showed a percentage inhibition, which was independent of the dose.

Table 3.4: Effect of nitric oxide scavenging activity of pet-ether extract and fractions

EXTRACT

Pet-ether fraction (F1)

Diethlether fraction (F2)

Pet-ether Extract (crude)

Vitamin C (standard)

BLANK

CONC. ( pglml)

100 200 400 800 100 200 400 800 100 200

400

800 100 200 400 800

P < 0.05 \ S BLANK

ABSORBANCE (Mean f SE)

0.30 + 0.02" 0.40 + O.OO* 0.43 + 0.01* 0.38 + 0.02* 0.64 + 0.02* 0.57 + 0.01* 0.55 + O.OO* 0.44 + 0.01* 0.33 + 0.03* 0.31 + 0.03*

0.22 + O.OO* 0.17 + 0.00" 0.38 f 0.00* 0.32 + O.OO* 0.25 + 0.01* 0.22 + O.OO*

INHIBITION (%)

79.43 72.60 64.77 73.73 55.90 60.73 62.07 70.10 77.17

78.53 84.64

88.17 74.00 78.10 82.87 84.67

0 100 200 300 100 ' 500 600 700 000 900

Concenlralibn (uglm)

Fig. 4: Effect of extract and fractions on nitric oxide scavenging activity

--a- Pel Elher Fraction ( F I ) --I+-- Dielhylether fraction (F2)

-a- Oil (Pel elhcr cxlract) . W . . Vilarn~n C (Standard)

3.5 Results of qualitative determination of vitamins

The result showed the presence of vitamin A and Vitamin E in the

pet-ether extract (crude), pet-ether fraction (F,) and the diethlether

fraction (F2) (Table 3.5).

Table 3.5: Qualitative result of vitamin assay

Vitamin A I + I + I + I

VITAMINS

Key: + = Positive

RESULT

Vitamin E

Pet-ether Extract (crude)

+

Pet-ether Fraction (F,)

Diethylether Fraction (F2)

+ +

3.6 Lipid Compounds Present inthe Seed Extract

The result shows the different classes of lipids present in

Monodora tenuifolia seed. The result shows the presence of

aminolipids, free fatty acids, glycolipids, phospholipids and sterols. This

is shown in Table 3.6.

Table 3.6: Result of lipid components present in the seed extract

"

Key: +ve = Positive

Free fatty acids Glycolipids Phospholipids Sterols

LIPID COMPONENT Aminolipids

+ve +ve +ve +ve

RESULTS +ve

CHAPTER FOUR

DISCUSSION

In traditional system of medicine, certain herbs are claimed to

have medicinal properties ranging from the relief of pains to curing of

diabetes etc. The claimed therapeutic effects have to be verified in a

scientific manner. In the present study, one such drug Monodora

tenuifolia seed extract was studied for its possible antioxidant

properties..

Monodora tenuifolia is a tropical plant found in forest regions and

they appear in colonies (Dalziel, 1948) and is mostly used as spices or

condiments in soup as well as in pharmaceutical preparations as a

dental cure (Talalaji, 1965). From the study, it is evident that the extract

of seeds of Monodora tenuifolia has promising antioxidant activity.

In the present study, the percentage (%) yield of the extract was

found to be 18.9%, which is relatively low when compared to a previous

study on the plant. In a previous study (Esuoso et al, 2000) reported

that Monodora tenuifolia seed extract had a yield of 34.7-68.8%.

The possible difference in the O h yield could be as a result of

geographical and climatic factors, which has been found to affect plant

constituents, or time of collection of the seed, method of storage, the

variety of the parent plant and the nature of the soil on which it is planted

(Alam et all 1982).

In the acute toxicity test, administration of up to 5000 mg/kg

caused no death, suggesting that the extract could be safe even at very

high doses. Monodora tenuifolia seed was found to contain a lot of

secondary plant metabolites namely: alkaloids, carbohydrates, cardiac

glycosides, cynogenic glycosides, flavonoids, glycosides, proteins,

saponins, steroidal aglycones and tannins. Alkaloids at high

concentration, has been found to produce a variety of toxic effects on

" animals. They also protect the plant from the effect of singlet oxygen

(Bonner and Varner, 1965). These plant constituents are known to be

biological active, eliciting a variety of pharmacological actions such as

antioxidant effects (Bauer et a/., 1996). It can also be concluded that the

antioxidant activity of the extract could be attributed to flavonoids, which

. are fourid in antioxidant plant such as Aspidium cacutarium (Ghoghari et

all 2006), Phyllanthus debilis klein ex Willd (Kumaran and Karunakaran,

2006) as well in Tephrosia purpurea (Jain ef al., 2006).

In the Nitric oxide scavenging test, systemic administration of the

pet-ether (40-60°C) extract, pet-ether (60-80°C) fraction and

diethylether fraction showed significant free radical scavenging action

against nitric oxide (NO) induced release of free radicals.

Sodium nitroprusside serves as a chief source of the free radicals.

The absorbance of the chromophore formed during diazotization of the

nitrite with sulphanilamide and subsequent coupling with napthyethylene

diamine is used as the marker of NO scavenging activity (Mukherjee,

1989). The chromophore formation was not complete in the presence of

Monodora tenuifolia seed extract (pet-ether (40-60°C) extract),

diethyether fraction (F2) and pet-ether (60-80°C) fraction, which

scavenged the NO formed from the sodium nitroprusside. The

absorbance for the pet-ether extract and the diethylether fraction (F2)

decreases as their concentration increases in a dose-dependent

manner.

In terms of the vitamin content, Monodora tenuifolia seed extract

contained vitamin A and vitamin E (Table 3.6). These vitamins offer

protective measures from oxidative stress by quenching free radicals

capable of causing cellular damage. Vitamin A in the form of

carotenoids can exhibit maximum inhibition of peroxidation of linolenate

. at low concentration (Krinsky and Deneke, 1982). At high O2 tensions,

a-tocopherol is the most effective antioxidant (Burton, 1989).

Many antioxidant agents modify oxidative degradation in the body

by accelerating the destruction or antagonizing the action of mediators of

oxidative processes e.g. the reactive species superoxide (02-), hydrogen

peroxide (H202), hydroxyl radical (HO.), nitrogen oxide (NO.) and

hypochlorous acids (HOCI) (Akah et al., 1997). These free radicals play

an important role in the genesis of various diseases such as

inflammation, arthrosclerosis, arthritis, rheumatism, cirrhosis of liver,

lung damage, cataract, aging and ischemia (Halliwal, 1994). There are

various pharmacological options in the treatment of these diseases,

which are geared towards protecting the human body from damage due

to free radicals (Osawa et a/., 1990).

Lipid peroxidation has been implicated in the apthogenesis of

various diseases including arthritis. It is well established that

bioenzymes are very much susceptible to lipid peroxidation, which is

considered to be the starting point of many toxic as well as degradative

processes. Thus the presence of flavonoids and alkaloids, as well as

vitamins A and E may be responsible for the antioxidant effects

observed with Monodora tenuifolia seed extract in the presence study.

The lipids present in this study are important viz., aminolipids, free

fatty acids, glycolipids, phospholipids and sterols. They equally play

important roles in both cellular functions and in the free radical

- reactions.The results of present study with Monodora tenuifolia seed

extract and fractions have good correlation with the therapeutic use of

Monodora tenuifolia in the possible treatment of oxidative stress

conditions. Plants that belong to Annonaceae family are rich in

flavonoids and bioflavonoids and are known to have antioxidant activity.

Monodora tenuifolia seed extract contains important pharmacological

compounds, alkaloids, flavonoids, and vitamins A and E as well as many

important lipids; arhinolipids, free fatty acids, glycolipids, phospholipids

and sterols.

REFERENCES

Akah, P. A,, Gamaniel, K. S., Wambebe, C. O., (1997). Evaluation of Nigerian Traditional Medicine: Effects of Gkani, a herbal anti- asthmatic drugs. Journal of Ethnopharmacology 55: 87-92.

Alam, M. N., Farug, M. O., Hossain, M. E. and Alam, M. J. (1982). Investigation of Rubber seed oil Bangladesh. J. Sci. Ind. Res. xvii, (3-4), 200-204.

Ames, B. N., ~a thcar t , R., Schwiers, E., and Hochstein, P. (1981). Uric acid provides an antioxidant defense in humans against oxidant and radical caused aging and cancer; a hypothesis. Proc. Natl. Acad. Sci. USA. 78: 6858-6862.

Andraws, R., Berger, J. S. and Brown, D. L. (2005). Effects of antibiotic therapy on outcomes of patients with coronary artery disease. JAMA. 293: 2641-7.

Antoniades, H. N. and Owen, A. J. (1982). Growth factors and regulation of cell growth. Ann. Rev. Med., 33: 445.

Bauer, R., Probosthe, A., Latter, H. Wasgherredecker, W., Matheissen, U. (1996). Phytomedicine, J. Pharm. Col. 4: 305-308.

Bendich A. and Olson, J. A. (1989). Biological actions of Carotenoids. FASEB J. 3: 1927-1932.

Bendich, A. (1989). Carotenoids and Immune Response. J. Nutr. 119: 112-115.

Bonner, J. and Varner, J. E. (1965). Plant Biochemistry Academic Press London, pp. 252-703.

Boogerd, W., Vander Sande, J. J. and Van Zandwijk, N. (1999). Teniposide sometimes effective in brain metastases from non- small cell lung cancer. J. Neurooncol. 41 : 285-289.

Borg, D. C. and Schaich, K. (1989). Pro-oxidant action of antioxidants. CRC Handbook of Free Radicals and ~ntioxidants in Biomedicine. (Miquel, J., Quintanilha, A. T., and Weber, H., eds) CRC Press, Boca Raton, Florida. Vol. I. pp. 12-45.

Borg, D. M., (1993). Oxygen free radicals and tissue injury; a reference in oxygen free Radical in Tissue Damage (Tarr, M. and Sampson, eds.) C. V. Press, Mosby, by St. Louis, pp. 10-32.

Buettner, G. R., and Jurkiewicz, B. A. (1993). Ascorbate free radical as a marker of oxidative stress: an EPR study. Free Rad. Biol. Med. 14; 49-55.

Buettner, G. R., and Jurkiewicz, B. A. (1996). Catalytic Metals, Ascorbate and Free Radicals: Combination to Avoid. Radiation Res. 145: 532-541.

Burton, G. W. (1989). Antioxidant action of carotenoids. J. Nutr. 119: 109-111. .

Burton, G. W., and Ingold, K. U. (1984). Beta-Carotene: an unusual type of lipid antioxidant . Science 224; 569-573.

Caughey, W. S., and Watkins, J. A. (1985). Oxy-radical and Peroxide formation by haemoglobin and myoglobin. In CRC Handbook of Methods for oxygen Radical Research (Greenwald R. A., ed): pp. 95-1 04.

Cavalieri, E. L. and Rogan, E. G. (1984). One-electron and two-electron oxidation in aromatic hydrocarbon carcinogenesis. In Free Radicals in Biology (Pryor, W. A., ed.) I : 323-370.

Chang, T. K., Weber, G. F., Crespi, C. L., and Waxman, D. J. (1993). Differential activation of cyclophosphamide and ifophamide by cytochromes p-450 23 and 3A in human liver microsomes. Cancer Res. 53: 5629-5637.

Chen, G. X., and Asada, K. (1989). Ascorbate peroxidase in tea leaves: Occurrence of two lsozymes and the differences in their enzymatic and molecular properties. Plant Cell Physiol. 30: 987- 998.

Comporti, M. (1989). Three models of free radical induced cell injury, Chem. Biol. Interact, 72: 1-56.

Current Medicinal Chemistry (2005). Metals, toxicity and oxidative stress. Free Radical heory of aging. 12(10): 1 I 61 -1 208.

Cutler, R. G. (1984). Antioxidants, aging, and longevity. In Free Radicals in Biology, Academic, Orlando, Florida. Vol. I. pp. 371-428.

Dalziel, F. M. (1948). The useful plant of West Tropical Africa, being an appendix to the flora of West Tropical Africa by Hutchinson. J. and Dalziel, J. M. The Crown Agent for the Colonies, London. S.W. T. pp. 5-9.

De Lau, L., Giesbergen, P., Rijk, M., Hofman, A., Koudstaal, P. and Breteler, M. (2004). Incidence of Parkinsonism and Parkinson disease in a general population: the Rockenstein study. Neurology 63(7): 1240-4.

Eggeling, W. J. (1952). The Indigenous Trees of the Uganda protectorate (Revised and enlarged by Ivan R. Dale). Government printer, Entebbe Uganda, Crown Agents for the Colonies London. pp. xiii and 491.

Epstein, J. H. (1977). Effects of Beta-Carotene on Ultraviolet induced cancer formation in the hairless mouse skin. Photochem. Photobiol. 25: 21 1-21 3.

',Esuoso, K. O., Lutz, H., Bayer, E., Kutubuddin, M. (2000). Unsaponifiable lipid constituents of some underutilized tropical seed oil. J. Agric. Fd.Chem. 48(2): 231-234.

Evans, R. M., Currie, L., and Campbell, A. (1982). The distribution of ascorbic acid between various cellular components of blood, in normal individuals, and its relation to the plasma concentration. Br. J. Nature 47: 473-482.

Evans, R. W. (1989). Pharmacognosy (13'~ ed); Baillere Tindall, London. pp. 527-665.

Gey, K. F., Braubacher, G. B. and Stahelin, H. B. (1987). Plasma levels of antioxidant vitamins in relation to ischemic heart disease and cancer. Am. J. Clin. Nutri., 45: 1368-1 377.

Ghoghari, A. M., Bagul, M.S., Anandjiwala, S., Chauhan, M. G. and Rajani, M. (2006). Free radical scavenging activity of Aspidium cicutarium rhizome. J. Natural Remedies, 6(2): 1 31 -1 34.

Graham, R. K., Deng, Y., Slow, E. J., Haigh, B., Bissada, N., Lu, G., Pearson J., Shehadh, J., Bertram, L. and Murphy, Z. (2006). Cleavage at the caspase-6 site is required for neuronal dysfunction and degeneration due to mutant Huntingtin. Cell., 125: 1179-1191.

Grisham, M. B. and McCord, J. M. (1986). Chemistry and cytotoxicity of reactive oxygen metabolites. In: Physiology of Oxygen Radicals (Taylor, A. E., Matalon, S. and Ward, P., eds.). Am. Physiological Society. Bethesda, Md. Pp. 248-261.

Guilivi, C. and Davies, K. J. A. (1990). A novel antioxidant role for haemoglobin. The com-proportionation of ferrylhaemoglobin with oxyhaemoglobin. J. Biol. Chem. 265: 19453-1 9460.

Hall, A. (1 984). Oncogenes-implications for human cancer. A review. J. Res. Soc. Med., 77: 41 0.

Halliwal, H. (1 994). Lancet. 344: 721-724.

Halliwell, B., Gutteridge, J. M. C. and Holbrook, N. J. (1999). Free radical in Biology and Medicine, Oxford University Press. London, p. 625.

Irvine, F. R. (1961). Woody Plants of Ghana with special reference to their uses. Oxford University Press, London pp. 13-23.

Jain, A., Singhai, A. K. and Dixit, V. K. (2006). In vitro evaluation of Tephrosia purpurea pers for antioxidant activity. J. Natural remedies. 6(1): 162-1 64.

~enner, P. and Olanow, C. W. (1996). Oxidative stress and the pathogenesis of Parkinson's disease. Neurology. 47: 161-1 70.

Jovanovic, S. V., Steenken, S. and Simic, M. G. (1990). One electron reduction potentials of 5-lndoxyl radicals. A pulse radiolysis and laser photolysis study. J. Phys. Chem. 94: 3583-4588.

Kanner, P. H., Guyton, K. E., and Gorospe, M. (1999). Oxidative stress and the molecular biology of antioxidant defense (Scandolios, J. G. ed) Cold Spring Harbor Laboratory Press, New York, pp. 247- 272.

Khachik, F., Beecher, G. R., and Whittaker, N. F. (1986). Separation, Identification and quantification of the major Carotenoid and Chlorophyll Constituents in extracts of several green vegetables by liquid chromatography. J. Agr. Food Chem. 34: 603-616.

Klein, J. a. and Ackerman, S. L. (2003). Oxidative stress, cell cycle and neurodegeneration. J. Clin. Invest; 11 l(6): 785-793.

Krinsky, N. I. and Deneke, S. M. (1982). Interaction of Oxygen and Oxyradicals with Carotenoids. J. Natl. Cancer lnst. 69: 206-210.

Kumaran, A. and Karunakaran, J. (2006). Antioxidant activity of polyphenols from phyllantus debilis klein ex Willd. J. Natural Remedies. 6(2): 141 -146.

Leaf, a., Kang, J. X., Xiao, Y. F. and Billman, G. E. (2003). Clinical prevention of sudden cardiac death by n-3 polyunsaturated acids and mechanism of prevention of arrhythmias by n-3 fish oils. Circulation: 107: 2646-2652.

Lerman, L.S. (1 984). Sequence determined DNA separations. Ann. Rev. Biophys. Bioeng. 13: 399.

Levine, M.. and Morita, K. (1985). Ascorbic acid in endocrine systems. Vitamin. Horm:42

Liener, I. E. (1972). Toxic Constituents of Plant Food Stuff, Academic Press, London, pp. 143-1 55.

Marras, C., McDermott, M., Rochon, P., Tanner, C., Naglie, G., Rudolph, A., and Lang, A. (2005). Survival in Parkinson disease: thirteen- year follow-up of the DATATOP Cohort. Neurology, 64(1): 87-93.

Mathews-Roth, M. M. (1982). Antitumor activity of beta-carotene, Canthaxanthin and Phytoene. Oncology, 39: 33-37.

Melhorn, R. J. (1991). Ascorbate and dehydroascorbic acid mediated reduction of free radicals in the human erythrocylc. J. Biol. Chem. 266: 2724-273 1.

Michael, J. F. (2005). Dynamic dopamine modulation in the basal ganglia: A neurocomputational account of cognitive deficits in medicated and non-medicated Parkinsonism. J. Cognitive Neuroscience; 1 7: 5 1 -73.

. Mona, T. (2000). The Nigrostriatal Dopaminergic system as a preferential target of repeated exposures to combined paraqual and manels: implications for Parkinson's disease. J. Neuroscience 20(24): 9207-921 4.

Morel, C. 0 . (1997). Mechanisms of Fatty Acid Oxidation. http:llwww.atozofhealth.com. Downloaded on the 1 lth ~ u n e , 2005.

Mukherjee, K. 1. (1 989). Medical laboratory technology. Tata McGraw Hill Publishing Company Limited, New Delhi, 1" edition, pp. 1123- 1124.

Murray, P. E., Vaya, J. and Aviran, M. (1990). Nutritional Antioxidant: Mechanisms . of Action, Analyses of Activities and Modern Application. Bentham Science Publishers Ltd. New York, pp. 99- 110.

Nakamura, K., Bindokas, V. P., Marks, J. D., Wright, D. A., Frim, D. M., Miller, R. J. and Kang, U. J. (2000). The selective toxicity of 1- methyl-4-phenylpyridinium to dopaminergic neurons: The role of mitochondria1 complex I and reactive oxygen species revisted. Mol. Pharmacol. 58(2): 271 -278.

Neilson, M. (1965). Introduction to flowering plants of West Africa, University of London Press Ltd, London. pp. 90-105.

Niki, E. (1987). Interaction of ascorbate and a-toco-pherol. In: Third Conference on Vitamin C. (Burres, J. J., Rivers, J. M. and Machlin, L. J., eds). Ann. N. Y. Acad. Sci. 498: 186-199.

Niki, E. and Nakon, M. (1990). Estrogens as antioxidants. Methods Enzymol. 186: 330-333.

Nishikimi, M. (1975). Oxidation of Ascorbic acid with superoxide amion generated by the Xanthine-Xanthine Oxidase System. Biochem. Biophys. Res. Commun. 63: 463-468.

Oduyemi, 0 . (1993). Insecticidal properties of certain indigenous plant oils against Sitophlius zeamais mots. Appl. Entomol. and Phytopathol., 1 : 1 9-27.

Oguntimein, B., Ekundayo, O., Laasko, I. and Hitunen, R. (1989). Constituents of the essential oil of Monodora tenuifolia. Flav. and Fragr. J., 4: 193-1 95.

Okafor, J. C. (1989). Some useful Tropical Plans in Health Care Delivery, Forestry Commission HQ, Enugu. pp. 3-9.

Dmenn, G. S., Goodman, G. E., Thornquist, M. D., and Cullen, M. R. (1996). Free radicals and Antioxidants. Bio. Med., 334: 11 50- 11 55.

Osawa, T., Kavakishi, S. and Namiki, M. (1990). In: Kuroda, Shankal D. M., waters MD. (Eds). Antimutagenesis and anticarcinogenesis mechanisms II. New York, pp. 139.

Parkinson, J. (2002). As essay on the shaking palsy. J. Neuropsychiatry Clin. Neuroscience; l4(2): 223-236.

Perly, B., Smith, I.C.P. and Hughes, L. (1 985). Estimation of the Location of natural alpha-tocopherol in lipid bilayers by ' 3 ~ - ~ ~ ~ spectroscopy. Biochem. Biophys. Acta, 81 9: 131 -1 35.

Peto, R., Doll, R., Buckley, J. D. and Sporn, M. B. (1981). Can dietary beta-carotene materially reduce cancer rates? Nature, London. 290: 201-208.

Pratico, D. (2006). Link between autism and abnormal blood-vessel function, oxidative stress. http://www.med.upenn.edu/ Downloaded on the 2dth sept., 2006.

Ray, D. S. (2006). Oxidative stress. www.bionutrition.org. downloaded on the 24" Sept., 2006.

Ridley, R. M., Frith, C. D., Crow, T. J. and Conneally, P. M. (1988). Anticipation in Huntington's disease is inherited through the male line but may originate in the female. J. Medical Genetics. 25: 589- 595.

Robbelen, G., Downey, R. K. and Ashri, A. (1989). Oil Crops of the World, McGraw-Hill Publishing Company, New York, pp. 107-1 08.

Rose, R. C. and Bode, A. M. (1993). Biology of free radical scavengers: An evaluation of Ascorbate. FASEB J. 7: 11 35-1 141.

"

Sahart, M. H. (2001). Free radicals and Antioxidant. Biol. Med., 28: 1685-1 696.

Sawa, T., Kavakishi, S. and Namiki, M. (19900. In: Kuroda, Shankal D.M., Waters, M. D. (Eds.) Antimutagenesis and anticarcinogenesis mechanisms I!. New York; p. 139.

Sirna Therapeutics (2006). Huntington's disease overview. http://www.Sirna.com/wt/page/neurology. downloaded on the 24th Sept., 2006.

Stauth, D., Traber, M. and Mastaloudis, A. (2002). Marathon runners deplete vitamins, raise oxidative stress. Free Radical Biol. & Medicine. 54: 737-797.

Stocker, R., McDonagh, A F., Glazer, A. N. and Ames, B. N. (1990). Antioxidant activities of bile pigments: bilverdin and bilirubin. Methods Enzymol. 186: 301-309.

Stryer, L. (1988). Biochemistry 3rd ed. W. A. Freeman and Company, New York pp. 331 and 470.

Talalaji, S. J. (1 965). Essential oil from Monodora myristica grown in Ghana. West African Pharmacist, 4: 64-65.

Tretter, L. and Adam-Vizi, V. (2000). Generation of reactive oxygen species in the raction catalyzed by a-ketoglutarate dehydrogenase. http:www.neurosci.orcr/cai/contentlfu11/24/36/777l

Vessie, P. R. (1932). On the transmission of Huntington's chorea for 300 years - the Bures Family Group. J. Nen/ous and Mental Disease. 76: 553-573.

Wei, Y. N., and Lee, H. C. (2002). Oxidative Stress and Mitochondria1 DNA mutations in Human Aging. Proc. Soc. Exp. Biol. Med. 227: 671 -682.

Ziegler, R. G. (1989). A review of epidemiologic evidence that carotenoids reduce the risk of cancer. J. Nufr. 119: 116-122.