ABSTRACT
The study was carried out to determine pharmacognostic standards, phytochemical constituents,
evaluate the antioxidant and hepatoprotective activities of the extract and fractions of Annona
senegalensis Pers. (Annonaceae) by employing both the in vitro and in vivo experimental
models. The acute toxicity tests result showed the drug is safe at 5000mg/kg doses.
The effect of DPPH free radical scavenging, ABTS radical scavenging, Hydroxyl radical
scavenging, Hydrogen peroxide scavenging, lipid peroxidation assay, assay of catalase,
superoxide Dismutase assay, total protein, β-carotene bleaching, FRAP scavenging, liver and
superoxide anion radical scavenging activities were evaluated. Hepatoprotective effects of the
extract was evaluated against CCl4 induce liver damage. Carbon tetrachloride (CCl4) induce
hepatotoxicity was evaluated by significant increase ( p < 0.05 ) in serum AST, ALT, ALP
activity and bilirubin level accompanied by significant decrease ( p > 0.05 ) lipid peroxidation,
and catalase activity in liver tissue. All these parameters were also evaluated using the n–hexane,
ethyl acetate, methanol fractions.
The results showed that the extract/fractions of stem bark of A. senegalensis had better
antioxidant activities at high concentrations when compared to the standard. Co-administration
of the extract/fractions (400mg/Kg) protects the CCl4 – induced lipid peroxidation, restored
altered serum elevated enzymes. It showed that it is dose dependent.
The results obtained in the present study indicate that the stem bark is a potential source of
natural antioxidants.
TABLE OF CONTENTS
TITLE PAGE ————————————————————————————- i
Approval page ———————————————————————————-ii
Dedication ————————————————————————————— iii
Acknowledgement ——————————————————————————iv
Table of contents —————————————————————————- — v
List of tables ——————————————————————————— —viii
List figures ————————————————————————- ————– ix
Abstract —————————————————————————— ———– x
CHAPTER ONE
1.0.0 Introduction ————————————————————————- —1
1.1.0 Plant Pharmacognostic profile —————————————- —————- 4
1.2.0 Botanical description ——————————————————————— 6
1.2.1 Reported active constituents ————————————————————- 7
1.2.2 Ethnomedicinal uses of A. senegalensis ——————————————– 7
1.3.0 General review of antioxidant and hepatoprotective studies ———————– 8
1.3.1 Oxidative stress (OS) ——————————————————————- 8
1.3.2 Reactive oxygen species (ROS) —————————————————-10
1.3.3 Lipid peroxidation and free radicals ————————————————- 12
1.3.4 Biological uses of Reactive Oxygen species ————————————— 16
1.3.5 Consequences of Oxidative Stress. —————————————————- 17
1.3.6 Antioxidants —————————————————————————–18
1.3.7 Biotransformation of carbon tetrachloride ——————————————- 27
1.3.8 Serum enzyme determinations as a diagnostic tool ——————————– 28
1.3.9 Enzymes in the diagnostic pathology: AST, AST AND ALP ——————– 28
1.4.0 Rationale of study ———————————————————————- 29
1.4.1 Aim of study —————————————————————————– 30
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CHAPTER TWO
2.0.0 Materials and methods ————————————————————— 31
2.1.0 Plant Collection ————————————————————————- 31
2.2.0 Preparation of Extract —————————————————————– 31
2.3.0 Experimental animals ——————————————————————- 31
2.4.0 Fractionation Procedures —————————————————————– 32
2.5.0 Phytochemical screening —————————————————————– 32
2.6.0 Microscopic Examinations ————————————————————— 36
2.7.0 Determination of Total Flavonoids —————————————————– 37
2.7.1 Determination of Total Phenolics ——————————————————- 37
2.8.0 Determination of some Pharmacognostic Standards ——————————- – 37
2.9.0 In vitro anti-oxidant analysis ———————————————————– 40
2.9.1 In vivo anti-oxidant analysis ———————————————————— 44
2.9.2 Acute Toxicity Study ——————————————————————– 47
2.9.3 Treatment of animals ——————————————————————- 47
2.9.4 Biochemical Tests ——————————————————————– 48
2.9.5 Statistical analysis ———————————————————————— 48
CHAPTER THREE
3.0.0 RESULTS ———————————————————————————- 49
3.1.0 Extract and fractions ——————————————————————— 49
3.2.0 Acute Toxicity Test and mean lethal dose ——————————————– 49
3.3.0 Phytochemical analysis ——————————————————————- 49
3.4.0 Microscopic examinations. ————————————————————– 51
3.5.0 Total Flavonoid and Total Phenolic Contents —————————————- 54
3.6.0 Pharmacognostic Standards ————————————————————- 54
3.7.0 Spectrophotometric reading for anti-oxidant —————————————- 55
vii
3.8.0 Anti-oxidant in vitro study (IC50Values )——————————————- 56
3.9.0 Anti-oxidant in vivo study————————————————————–57
3.9.1 Biochemical tests (Liver function tests) ———————————————-58
CHAPTER FOUR
4.0 DISCUSSION AND CONCLUSION —————————————————– 59
4.1 Discussion ————————————————————————————- 59
4.2 Conclusion ———————————————————————————— 65
References ——————————————————————————–
CHAPTER ONE
1.0 INTRODUCTION
There is amazing abundance of plant life in rain forest and nature has blessed the mankind with a
treasure of herbal remedy secrets that offer new approaches to health and healing. It is quite
interesting to discover that different herbs can be indicated for a vast number of health problems.
Therefore, a lot of scientific screening and research have been going on into investigating the
various constituents of plants that are responsible for a particular activity or more, despite all
odds. Many drugs have been discovered by the exploitation of traditional medicine since the
early dates of human existence (Nwaogu, 1997). Plants have invariably been a rich source for
new drugs and some antioxidant drugs in use today were either obtained from plants or
developed using their chemical structures as templates(Nwaogu, 1997).
Currently, there is an increasing awareness of the value of traditional medicine and the necessity
for improving its standard. Indeed, the Organization of African Unity (O.AU) has in the last few
years, held lots of international symposia and these were on a particular aspect of a subject,
notably medicinal plants. It has been observed that many plants contain a variety of
phytochemical substances, which have appreciable physiological and pharmacological actions on
man and animals. Researches on natural products over the years have revealed enormous
potentials of plants as source of medicinal agents. Plants are no longer being cultivated for food
alone, but also as sources of drugs.
Herbal medicine, which is the oldest form of healthcare known to mankind, involves the use of
herbs (medicinal plants) for therapeutic or medicinal purposes. Herbal medicine can be broadly
classified into various systems: traditional Chinese herbalism, which is part of the traditional
2
oriental medicine; Ayurveda herbalism, which is derived from Ayurveda; and Western
herbalism, which originally came from Greece and Rome to Europe and then spread to North and
South America.
The medicinal plants which may be leaves, stems, roots, flowers, seeds, fruits or whole plant or
any combination of these parts are prepared in various forms for therapeutic purposes. From a
scientific approach, most of the preparations are considered unscientific since they are not
pharmacologically authenticated or standardized and are seen as unrefined.
Many plants have varied pharmacological effects which have been confirmed. Extracts of
Digitalis spp, Colchicumautomnale, Catharanthusroseus and Peyotecactus had cardio – active,
anti – inflammatory, anti – neoplastic and central nervous system actions respectively. It is
already estimated that 122 drugs from 94 plants species have been discovered throughethno
botanical leads. Plants commonly used in traditional medicines assumed to be safe due to their
long usage in the treatment of disease according to knowledge accumulated over centuries.
However, recent scientific findings had shown that many plants used as food or in traditional
medicine are potentially toxic, mutagenic and carcinogenic (Schimmer et al., 1994).
Cancer chemoprevention by using antioxidant approaches has been suggested to offer a good
potential in providing important fundamental benefits to public health, and is now considered by
many clinicians and researchers as a key strategy for inhibiting, delaying, or even reversal of the
process of carcinogenesis. The cancer chemopreventive activities of naturally occurring
phytocompounds are of great interest.
Liver diseases such as jaundice, cirrhosis and fatty liver diseases are very common and large
public health problem in the world. Jaundice and hepatitis are two major hepatic disorders that
3
account for a high death rate. There is no rational therapy available for treating liver disorders
and management of liver diseases is still a challenge to the modern medicine. The modern
medicines have little to offer for alleviation of hepatic ailments whereas most important
representatives are of phytoconstituents .The traditional system of medicine like Ayurveda and
Siddha system of medicine, Unani system, Chinese system of medicine, Kampoo (Japanese)
system of medicine have a major role in the treatment of liver ailments.
Some medicinal plants are used in treatment of hepatobiliary pathologies. Many Nigerian ethno
botanic traditions propose a rich repertory of medicinal plants used by the population for
treatment of liver diseases. However, there were not enough scientific investigations on the
hepatoprotective activities conferred to these plants. One of such plant from Nigerian flora is
Annona senegalensisPers.It is believed in folkore that the fruit obtained from this multipurpose
plant is widely used locally in the treatment of two commonly energy deficiency syndrome
known as kwashiorkor and marasmus. Dalziel, (1995) made report about the plant to be of great
medicinal value and its used in native medicine to treat headache and body ache, eyelid swelling.
The stem bark of A. senegalensis is used by local populations all over Africa in treating guinea
worms, diarrhea and especially in northern Nigeria, gastroenteritis, snake bites, toothache,
respiratory infections and malaria. Awa and colleagues (2012) reported the use of leaves in the
treatment of pneumonia, and as a stimulant to improve health. A decoction from the roots is used
to stop chest colds, venereal diseases, stomach ache and dizziness (Jiofack. et al., 2010).
Many indigenous herbal plants of regional interest have been used popularly as folk medicines
in Nigeria or other African countries; however, their bioactivities or pharmacological effects are
to be investigated.
4
1.1 PLANT PHARMACOGNOSTIC PROFILE
1.1.1 TAXONOMY
Kingdom: Plantae
Division: Magnoliophyta
Class: Magnoliopsida
Order: Magnoliales
Family: Annonaceae
Genus: Annona
Species: senegalensis
Authority: Pers
5
Fig 1: A. senegalensis
6
1.2.0 BOTANICAL DESCRIPTION
Annona senegalensis is a shrub or small tree 2 – 6m tall but may reach 11m under favorable
conditions, It has a bark smooth to roughish, silvery-grey or grey-brown, with leaf scars and
roughly circular flakes exposing paler patches of under bark. Young branches with dense, brown,
yellow or grey hairs that are lost later. The leaves are alternate, simple, oblong, ovate or elliptic,
6 – 18.5cm x 2.5 – 11.5cm, green to bluish green, almost without hairs on top, but after with
brownish hairs or underside. They have net veination which may be green or reddish on both
surfaces. The apex is round or slightly notched with base square to slightly lobed base. The
margined is entire; petiole short, 0.5 – 2.5cm thick set (Ketende et al. 1995). Flowers up to 5cm
in diameter, on stalk, 2cm long, solitary or in groups of 2 – 4, arising above the leaf axils; 6
fleshly cream to yellow petals in 2 whorls, greenish outside, creamy or crimson, 0.8 – 1.5cm x
0.9 – 1.1cm, glabrous or minutely papillose within; 3 in number, free, smaller than the petals, 3-
4×4 – 5cm; stamens 1.7 – 2.5mm long. Fruits formed from many fused compels, fleshy, lumpy,
egg shaped, 2.5 – 5×2.5 – 4cm, ovoid or globose; unripe fruit green turning yellow to orange or
ripening stalk 1.5 – 5cm long; seeds numerous, cylindrical, oblong, orange brown. The genus
name, “Annona”, is from the Latin word “anon”, meaning “yearly produce”, referring to the
production habits of fruits of the various species in the genus. The specific name means “of
Senegal”, which is where the type specimen was collected (Beentje, 1994).
7
1.2.1 REPORTED ACTIVE CONSTITUENTS
A. senegalensis has been shown to contain a lot of constituents which are responsible for its
various pharmacological properties. These secondary metabolites which include; alkaloids (-);
roemerine, an aporphine), tannins, flavonoids, resins, glycosides, carbohydrates and
saponins.Others constituents reported include aliphatic ketones, alkanes, fatty acids, and sterols
from the leaves, monoterpenoids and sesquiterpenoids from the essential oil of the leaves and
fruits, amino acids from the stem bark; and ent-kaurenoids from the root back (Silva,et al, 1995).
1.2.2 ETHNOMEDICINAL USES OF A. SENEGALENSIS
Several plant parts of A. senegalensis are used in traditional medicine in various countries of
tropical Africa for the treatment of many diseases and symptoms including: cancer, convulsions,
diarrhea, dysentery, Malaria fever and filariasis, male impotency, pain of the chest and intestines,
inflammations, trypanosomiasis, venereal diseases and snake bite. Root extracts of A.
senegalensis have been found to exhibit antineoplastic activity in mice bearing sarcoma 180
ascites tumor cell, and antiprotozoal activity in mice infected with Trypanosomabrucei
(Silva,1995).
. The leaves are sometimes used as vegetables, while the edible white pulp of the ripe fruit has a
pleasant, pineapple like taste (FAO, 1983). An effective insecticide is obtained from the bark.
The bark is used for treating guinea worms and other worms, gastroenteritis, toothache and
respiratory infections. Gum from the bark is used in sealing cuts and wounds. The leaves are
used for treating pneumonia and as a tonic to promote general wellbeing. Roots are used for
stomach-ache, chest colds and dizziness. Various plant parts are combined for treating
dermatological diseases and ophthalmic disorders. In South Africa, roots are said to cure
8
madness, and in Mozambique, they are fed to small children to induce them to forget the breast
and thus hasten weaning. It has also been claimed that leaves picked on a Thursday morning and
thrown over the right shoulder brings good luck (Anon 1986).
1.3.0 GENERAL REVIEW OF ANTIOXIDANT AND HEPATOPROTECTIVE STUDIES
1.3.1 OXIDATIVE STRESS (OS)
Oxidative stress (OS) is a general term used to describe the steady state level of oxidative
damage in a cell, tissue, or organ, caused by the reactive oxygen species (ROS). This damage can
affect a specific organ or the entire organism. ROS such as free radicals and peroxides, represent
a class of molecules that are derived from the metabolism of oxygen and exist inherently in all
aerobic organisms.
OS is caused by an imbalance between the production of reactive oxygen species and detoxifier
(antioxidants). All forms of life in normal state maintain an equilibrium redox reaction.
Distortion of this normal redox state can cause toxic effects through the production of peroxides
and free radicals that can damage components of the cell, including proteins, lipids, and DNA
(Aroma 1993).
The level of oxidative stress is determined by the imbalance between the rate at which oxidative
damage is induced and the rate at which it is efficiently repaired and removed. The rate at which
damage is caused is determined by how fast the reactive oxygen species are generated and then
inactivated by endogenous defense agents called antioxidants. The rate at which damage is
removed is dependent on the level of repair enzymes. The determinants of oxidative stress are
regulated by an individual’s unique heredity factors, as well as his/her environment and
characteristic lifestyle. Unfortunately, under the present day life-style conditions many people
9
run an abnormally high level of oxidative stress that could increase their probability of early
incidence of decline in optimum body functions (Aroma 1993).
In humans, oxidative stress is involved in many diseases, such as atherosclerosis, Parkinson’s
disease and Alzheimer’s disease and it may also be important in ageing. However, reactive
oxygen species can be beneficial, as they are used by the immune system as a way to attack and
kill pathogens and as a form of cell signaling (Rice-Evans, et al., 1995).
In chemical terms, oxidative stress is a large increase in the cellular reduction potential, or a
large decrease in the reducing capacity of the cellular redox couples, such as glutathione. The
effects of oxidative stress depend upon the size of these changes, with a cell being able to
overcome small perturbations and regain its original state (Seis, 1997).
A particularly destructive aspect of oxidative stress is the production of reactive oxygen species,
which include free radicals and peroxides. Some of the less reactive of these species (such as
superoxide) can be converted by redox reactions with transition metals or other redox cycling
compounds including Quinone into more aggressive radical species that can cause extensive
cellular damage (Valko, et al., 2005). Most of these oxygen- derived species are produced at a
low level by normal aerobic metabolism and the damage they cause to cells is constantly
repaired. However, under the severe levels of oxidative stress that cause necrosis, the damage
causes ATP depletion, preventing controlled apoptotic death and causing the cell to simply fall
apart (Lelli, et al., 1998).
10
1.3.2 REACTIVE OXYGEN SPECIES (ROS)
Reactive oxygen species are chemical species which are responsible for toxic effects in the body
through various tissue damages. They are formed either by the loss of a single electron from a
non-radical or by the gain of a single electron by a non-radical.
Examples of ROS are listed in table 1
Table 1: Description of oxidants
Oxidant Description
.O2 superoxide anion One-electron reduction state of O2 , formed in many
autoxidation reactions and by the electron transport chain.
Rather unreactive but can release Fe2+ from iron-sulphur
proteins and ferritin. Undergoes dismutation to form H2O2
spontaneously or by enzymatic catalysis and is a precursor
for metal-catalyzed .OH formation.
H2O2, hydrogen peroxide Two-electron reduction state, formed by dismutation of .O2
–
or by direct reduction of O2. Lipid soluble and thus able to
diffuse across membranes.
.OH, Hydroxyl radical Three-electron reduction state formed by Fenton reaction
and decomposition of peroxynitrite. Extremely reactive, will
attack most cellular components.
ROOH, organic hydro peroxide Formed by radical reactions with cellular components such as
lipids and nucleobases.
RO., alkoxy and ROO., Peroxy
radicals.
Oxygen centered organic radicals. Lipid forms precipitates in
lipid peroxidation reactions. Produced in the presence of
oxygen by radical addition to double bonds or hydrogen
abstraction.
HOCL, hypochlorous acid. Formed from H2O2 by myeloperoxidase. Lipid soluble and
highly reactive. Will readily oxidize protein constituents,
including thiol groups, amino groups and methionine.
OONO-, peroxynitrite Formed in a rapid reaction between .O2
– and NO. Lipid soluble
and similar in reactivity to hypochlorous acid. Protonation
forms peroxynitrous acid, which can undergo hemolytic
cleavage to form hydroxyl radical and nitrogen dioxide.
The most important source of reactive oxygen species under normal conditions in aerobic
organisms is probably the leakage of activated oxygen from mitochondria during normal
oxidative respiration. Other enzymes capable of producing superoxide are xanthine oxidase,
reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and cytochromes
11
P450. Hydrogen peroxide is produced by a wide variety of enzymes including monoxygenases
and oxidases. Reactive oxygen species play important roles in cell signaling, a process termed
redox signaling. Thus, to maintain proper cellular homeostasis, a balance must be struck between
reactive oxygen production and consumption (Aroma 1993).
Cell damage is induced by reactive oxygen species (ROS). ROS are either free radicals, reactive
anions containing oxygen atoms, or molecules containing oxygen atoms that can either produce
free radicals or are chemically activated by them. Under normal conditions, ROS are cleared
from the cell by the action of superoxide dismutase (SOD), catalase, or glutathione (GSH)
peroxide. The main damage to cells results from the ROS-induced alteration of macromolecules
such as polyunsaturated fatty acids in membrane lipids, essential proteins, and DNA (Nishikimi,
et al 1972).
Exogenous sources of ROS include exposure to cigarette smoke, environmental pollutants such
as emission from automobiles and industries, consumption of alcohol in excess, asbestos,
exposure to ionizing radiation, and bacteria, fungi or viral infections.
Poor nutrition in general contributes to OS. When the body is fed poorly, it slowly starves and all
of its systems suffer. Weak organ systems are prime targets for free radical attack.
Even psychological and emotional stress can contribute to OS. When the body is under stress, it
produces certain hormones that generate free radicals. Moreover, the liver must eventually
detoxify them and that process also generates free radicals.
Heightened OS has also been observed in athletes after intensive workouts due to the physical
stress placed on the body. Both physical and emotional stress also prompts the release of
12
endogenous cortisol, an adrenal hormone that reduces inflammation, but also suppresses the
immune system (Seis, 1997).
Endurance exercise can increase oxygen utilization from 10 to 20 times over the resting state.
This greatly increases the generation of free radicals, prompting concern about enhanced damage
to muscles and other tissues (Rice – Evans et al 1995).
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 metal ion or metal. The most important
reactions are probably Fenton’s reaction and the Haber-Weiss reaction, in which hydroxyl
radical is produced from reduced iron and hydrogen peroxide (www.en.wikipedia.org/oxidativestress).
The hydroxyl radical then can lead to modifications of amino acids (e.g. meta-tyrosine
and ortho-tyrosine formation from phenylalanine, carbohydrates, initiate lipid peroxidation, and
oxidize nucleobases. Most enzymes that produce reactive oxygen species contain one of these
metals. The presence of such metals in biological systems in an uncomplexed form can
significantly increase the level of oxidative stress (Valko, et al2005).
Certain organic compounds in addition to metal redox catalyst can also produce reactive oxygen
species. One of the most important classes of these is the quinones. Quinones can redox cycle
with their conjugate semiquinones and hydroquinones, in some cases catalyzing the production
of superoxide from dioxide or hydrogen peroxide from superoxide (Valko et al2005).
1.3.3 LIPID PEROXIDATION AND FREE RADICALS
Lipid peroxidation refers to the oxidation degradation of lipids. It is the process whereby free
radicals “steal” electrons from the lipids in cell membranes, resulting in cell damage. Lipid
13
hydroperoxides are non-radical intermediates derived from unsaturated fatty acids,
phospholipids, glycolipids, cholesterol esters and cholesterol itself. Their formation occurs in
enzymatic or non –enzymatic reactions involving activated chemical species known as “reactive
oxygen species” (ROS) which are responsible for toxic effects in the body via various tissue
damages. They are formed either by the loss of a single electron from a non-radical or by the
gain of a single electron by a non-radical. They can easily be formed when a covalent bond is
broken if one electron from each of the pair shared remains with each atom, this mechanism is
known as hemolytic fission. In water, this process generates the most reactive species, hydroxyl
radicals OH. Chemists know well that combustion which is able at high temperature to rupture
C – C, C – H or C – O bonds is a free radical process. The opposite of this mechanism is the
heterolytic fission in which, after a covalent break, one atom receives both electrons (this gives a
negative charge) while the other remains with a positive charge. This process proceeds by a free
radical chain reaction mechanism. It most often affects polyunsaturated fatty acids, because they
contain multiple double bonds in between which lies methylene –CH2- groups that possess
especially reactive hydrogen (McCay et al., 1984).
As with any radical reaction, the reaction consists of three major steps: initiation, propagation
and termination. Initiation is the step whereby a radical is produced. The initiators in living cells
are most notably reactive oxygen species (or ROS), such as OH, which combines with a
hydrogen atom to make water and a fatty acid radical(www.wikipedia.org/wiki/lipidperoxidation).
The fatty acid radical is not a very stable molecule, so it reacts readily with molecular oxygen,
thereby creating a peroxyl-fatty acid radical. This too is an unstable species that’s reacts with
another free fatty acid producing a different fatty acid radical and a hydrogen peroxide or cyclic
14
peroxide if it had reacted with itself. This cycle continues as the new fatty acid radical reacts in
the same way. This is the propagation stage(www.wikipedia.org/wiki/lipid-peroxidation).
In termination stage a radical reacts with another radical, which is why the process is called a
“chain reaction mechanism”. The radical reaction stops when two radicals react and produce a
non-radical species. This happens only when the concentration of radical species is high enough
for there to be a high probability of two radicals actually colliding. Living organisms have
evolved different molecules to catch free radicals and protect the cell membrane. One important
such antioxidant is alpha-tocopherol, also known as vitamin E (www.wikipedia.org/wiki/lipidperoxidation).
Free radicals are highly unstable molecules that interact quickly and aggressively with other
molecules in our bodies to create abnormal cells. Free radicals are atoms or groups of atoms with
an odd (unpaired) number of electrons and can be formed when oxygen interacts with certain
molecules. Their instability causes them to react almost instantly with any substance in their
vicinity. Oxygen, or oxyl, free radicals are especially dangerous. Once formed, these highly
reactive radicals can start a chain reaction, like dominoes. Their chief danger comes from the
damage they can do when they react with important cellular components such as DNA, or the
cell membrane; enzymes. Cells may function poorly or die if this occurs. They are capable of
penetrating into the DNA of a cell and damaging its “blueprint” so that the cell will produce
mutated cells that can the replicate without normal controls. They accelerate aging and contribute
to the development of many diseases, including cancer and heart disease (Zhang, et al, 1993).
Surprisingly, however, free radicals are involved in many cellular functions and are a normal part
of living. When, for example, mitochondrion within cell burns glucose for fuel, the mitochondria
15
oxidize the glucose and in so doing generates free radicals. White blood cells also use free
radicals to attack and destroy bacteria, viruses and virus-infected cells. The detoxifying actions
of the liver also require free radicals (Lennon, et al., 1991).
It is important to note that free radicals are also released in the body from the breaking down or
detoxification of various chemical compounds; drugs, artificial food colorings and flavorings,
smog, preservatives in processed foods, alcohol, cigarette smoke, chlorinated drinking water,
pesticides, radiation, cleaning fluids, heavy metals such as cadmium and lead, and assorted
chemicals such as solvent traces found in processed foods and aromatic hydrocarbons such as
benzene and naphthalene (found in moth balls). Additionally, certain foods contain free radicals
which when eaten, enter the body and damage it. The major sources of dietary free radicals are
chemically altered fats from commercial vegetable oils, vegetable shortening and all oils heated
to very high temperatures (Buege, and Aust, 1978).
Some free radicals arise normally during metabolism. Sometimes the body’s immune system’s
cells purposefully create them to neutralize viruses and bacteria. However, environmental factors
such as pollution, radiation, cigarette smoke and herbicides can also spawn free radicals (Nathan
et al., 2000).
Because it is not possible to directly measure free radicals in the body, scientists have
approached the questions of how effectively can athletes defend against the increased free
radicals from exercise by measuring the by-products that result from free radical reactions. If the
generation of free radicals exceeds the antioxidant defenses then one would expect to see more
of these by-products. These measurements have been performed in athletes under a variety of
conditions (Ellman, 1959).
16
Several interesting concepts have emerged from these types of experimental studies. Regular
physical exercise enhances the antioxidant defense system and protects against exercise induced
free radical damage. This is an important finding because it shows how smart the body is about
adapting to the demands of exercise. These changes occur slowly over time and appear to
parallel other adaptations to exercise.
On the other hand, intense exercise in untrained individuals overwhelms defenses resulting in
increased free radical damage. Thus, the “weekend warrior” who is predominantly sedentary
during the week but engages in vigorous bouts of exercise during the weekend may be doing
more harm than good. To this end there are many factors that may determine whether exercise
induced free radical damage occurs, including degree of conditioning of the athlete, intensity of
exercise and diet (Sies, 1997).
Normally, the body can handle free radicals, but if antioxidants are unavailable, or if the freeradical
production becomes excessive, damage can occur. Of particular importance is that free
radical damage accumulates with age.
1.3.4 Biological uses of Reactive Oxygen species
The immune system uses the lethal effects of oxidants as a central part of its mechanism of
killing pathogens; with activated phagocytes producing both ROS and reactive nitrogen species
(Nathan, et al., 2000). Although the use of these highly reactive compounds in the cytotoxic
response of phagocytes causes damage to host tissue, the non-specificity of these oxidants is an
advantage since they will damage almost every part of their target cell (Rice-Evans, et al.,
1995).this prevents a pathogen from escaping this part of immune response by mutation of a
single molecular target.
17
More recently, it has become apparent that ROS also have important roles as signaling
molecules. A complex network of enzymatic and small molecule antioxidants controls the
concentration of ROS and repairs oxidative damage, and research is revealing the complex and
subtle interplay between ROS and antioxidants in controlling plant growth, development and
response to the environment.
1.3.5 Consequences of Oxidative Stress.
Oxidative stress contributes to tissue injury following irradiation and hyperoxia. It has been
implicated in disease states, such as neurodegenerative diseases including Lou Gehrig’s disease
(aka MND or ALS), Parkinson’s disease, Alzheimer’s disease, Huntington’s disease and aging.
Oxidative stress is thought to be linked to certain cardiovascular disease, since oxidation of low
density lipoprotein (LDL) in the vascular endothelium is a precursor to plaque formation.
Oxidative stress also plays a role in the ischemic Cascade due to oxygen reperfusion injury
following hypoxia. This cascade includes both strokes and heart attacks.
Other disease conditions associated with oxidative stress include: Diabetes, pancreatitis, liver
damage, and leaky gut syndrome, hypertension and multiple sclerosis, atherosclerosis (Steinberg,
et al., 1989), coronary thrombosis, asthma, emphysema, chronic pulmonary disease, cataracts,
retinopathy, macular degeneration, rheumatoid arthritis (Aroma, 1993), glomerulonephritis,
vitiligo, wrinkles (Pryor W.A., 1991), cancer, autoimmune diseases, inflammatory states
(Symons and Dowling, 1987), AIDS and Lupus (Montagnier, Oliveier, and Pasquier, 1998).
However, more severe oxidative stress can cause cell death and even moderate oxidation can
trigger apoptosis, while more intense stresses may cause necrosis (Lennon, et al., 1991).
18
1.3.6 ANTIOXIDANTS
To prevent free radical damage the body has a defense system of antioxidants. Antioxidants are
intimately involved in the prevention of cellular damage the common pathway for cancer, aging,
and a variety of diseases. Fortunately, the body maintains a sophisticated system of chemical and
biochemical antioxidants scavenge free radicals, that is, they stabilize the unstable free radicals
by giving them the electron they need to “calm down”. The antioxidants are usually consumed or
used up in this process, i.e., they sacrifice themselves.
Antioxidants are molecules that can safely interact with free radicals and terminate the chain
reaction before vital molecules are damaged. Although there are several enzyme systems within
the body that scavenge free radicals, the principle micronutrient (vitamins) antioxidants are
vitamin E, beta-carotene, and vitamin C. Additionally, Selenium, a trace metal that is required
for proper function of one of the body’s antioxidant enzyme systems, is sometimes included in
this category. The body cannot manufacture these micronutrients so they must be supplied in the
diet. Therefore the main antioxidants are vitamins A, E, and C, beta-carotene, glutathione,
bioflavonoids, selenium, Zinc, CoQ10 (ubiquinone), and various phyto-chemicals from herbs
and foods. Green tea, for example, is rich in polyphenols-powerful antioxidants that help fight
cancer.
The best studied cellular antioxidants are the enzymes superoxide dismutase (SOD), catalase,
and glutathione peroxidase. 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 paraoxonase,
gluthione-S transferases, and aldehyde dehydrogenases.
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Biochemical antioxidants not only scavenge free radicals, but also inhibit their formation inside
the body. These include lipoic acid, and repair enzymes such as catalase, superoxide dismutase
(SOD), glutathione peroxidase. Melatonin, a hormone produced by the pineal gland, is also a
potent antioxidant. Cholesterol, produced by the liver, is another major antioxidant, which the
body uses to repair damaged blood vessels. It is probably for this reason that serum cholesterol
levels rise as people age. With age comes more free radical activity and in response the body
produces more cholesterol to help contain and control the damage (Seis, 1997).
Of all the antioxidants, glutathione appears to be pivotal. Made up of three amino acids (cysteine,
glycine, and glutamic acid), glutathione is part of the antioxidant enzyme glutathione peroxidase
and is the major liver antioxidant. It is a basic tenet of natural medicine that health cannot exist if
the liver is intoxicated. Not surprisingly, extremely low levels of glutathione are found in people
suffering from severe OS. People with AIDS, cancer and Parkinson’s disease, for example,
typically have low glutathione levels.
As noted earlier, oxidative stress occurs when the amount of free radicals in the body exceeds its
pool of available antioxidants. Obviously, knowing the varied sources of free radicals and
avoiding them in an important part of minimizing their harmful effects.
Diet can be a major source of free radical stressors with processed or highly heated oils being the
main offenders. Replace these harmful fats with natural, cold pressed oils such as olive oil
(which can be used for cooking) and small amounts of flax oil or walnut oil (which should never
be heated). Food grade, unrefined coconut oil and organic butter are also excellent choices,
especially for cooking. Both of these naturally saturated fats are rich in certain fatty acids that
have proven activity against bacteria, harmful yeasts, fungi and tumor cells.
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Additionally, since saturated fats (from animal foods and the tropical oils) and monounsaturated
oils (from olive oil and cold-pressed nut oils) are more chemically stable, they are much less
susceptible to oxidation and rancidity than their polyunsaturated analogues, which are mostly
found in vegetable oils. As a general rule, then, although the body does require a small amount of
naturally occurring polyunsaturated oils in the diet each day, it is best not to consume too much
of them as they are more prone to free radical attack in the body. As Linus Pauling, noted: “A
diet high in unsaturated fatty acids, especially the polyunsaturated ones, can destroy the body’s
supply of vitamin E and cause muscular lesions, brain lesions, and degeneration of blood vessels.
Care must be taken not to include a large amount of polyunsaturated oil in the diet” (Linus
Pauling, 1998).
The best food sources for polyunsaturated are fish, flax oil, sesame oil, walnut oil and dark
green, leafy vegetables. One caveat: canola oil is not recommended due to its chemical instability
and its content of trans-fatty acids (TFAs), formed during processing. TFAs are increasingly
being linked with cancer, immune system dysfunction and heart disease.
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A. VITAMIN C
fig.2
L – Ascorbic acid
Ascorbic acid is a water- soluble vitamin present in citrus fruits and juices, green peppers,
cabbage, spinach, broccoli, kale, cantaloupe, kiwi, and strawberries. The RDA is 60mg per day.
Intake above 2000 mg may be associated with adverse side effects in some individuals. Vitamin
C is the most abundant water-soluble antioxidant in the body and acts primarily in cellular fluid.
It is of particular note in combating free-radical formation caused by pollution and cigarette
smoke. Also helps return Vitamin E to its active form (Hickey, and Roberts, 2004).
The vitamins C and E are thought to protect the body against the destructive effects of free
radicals. Antioxidants neutralize free radicals by donating one of their own electrons, ending the
electron- “stealing” reaction. The antioxidant nutrients themselves don’t become free radicals by
donating an electron because they are stable in either form. They act as scavengers, helping to
prevent cell and tissue damage that could lead to cellular damage and disease (Padayatty,et al.,
2003).
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B. BETA-CAROTENE
fig.3
Carotene is a terpene, synthesized biochemically from eight isoprene units. It comes in two
primary forms designated by characters from the Greek alphabet: alpha- carotene (-carotene) and
beta-carotene (-carotene). Gamma, delta and epsilon (- carotene) also exist. Beta-carotene is
composed of two retinyl groups, and is broken down in the mucosa of the small intestine by
Beta-carotene dioxygenase to retinol, a form of vitamin A. carotene can be stored in the liver and
converted to vitamin A as needed, thus making it a provitamin.
Beta-carotene is a precursor to vitamin A (retinol) and is present in liver, egg yolk, milk, butter,
spinach, carrots, squash, broccoli, yams, tomato, cantaloupe, peaches, and grains. Because betacarotene
is converted to vitamin A by the body, there is no set requirement. Instead the RDA is
expressed as retinol equivalents (RE), to clarify the relationship. (NOTE: Vitamin A has no
antioxidant properties and can be quite toxic when taken in excess). In people who smoke, betacarotene
may increase cardiovascular mortality (Todd, et al., 1999, Omenn, et al., 1998).in men
who smoke and have had a prior myocardial infarction (MI), the risk of fatal coronary heart
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disease increases by as much as 43% with low doses of beta-carotene. There are some evidence
that beta-carotene in combination with selenium, vitamin C and vitamin E might lower highdensity
lipoprotein 2 (HDL2) cholesterol levels. HDL levels are protective so this considered
being a negative effect. Dizziness, reversible yellowing of palms, hands, or soles of feet and to a
lesser extent the face (called carotenoderma) can occur with high doses of beta-carotene. Loose
stools, diarrhea, unusual bleeding or bruising and joint pain have been reported.
C. GLUTATHIONES
fig. 4
Glutathione (gamma-glutamyl-cysteinyl-glycine; GSH) is the most abundant low-molecularweight
thiol within cells. Two cytosolic enzymes, gamma-glutamylcysteine synthetase and
glutathione synthetase catalyze the synthesis of glutathione from glutamate, cysteine, and
glycine. Compelling evidence shows that glutathione synthesis is regulated primarily by gammaglutamylcysteine
synthetase activity, cysteine availability, and glutathione feedback inhibition.
Animal and human studies demonstrate that adequate protein nutrition is crucial for the
maintenance of glutathione homeostasis.
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In aerobic cells, free radicals are constantly produced mostly as reactive oxygen species. Once
produced, free radicals are removed by antioxidant defenses including the enzymes catalase,
glutathione peroxidase, and superoxide dismutase. Reactive oxygen species, including nitric
oxide and related species, commonly exert a series of useful physiological effects. Imbalance
between prooxidant and antioxidant defenses in favor of prooxidants results in oxidative stress,
this results in damage to lipids, proteins, and nucleic acids. Alone or in combination with
primary factors, free radicals are involved in the cause of hundreds of diseases.
Glutathione – or L Glutathione – is a powerful antioxidant found within every cell. Glutathione
plays a role in nutrient metabolism, and regulation of cellular events including gene expression,
DNA and protein synthesis, cell growth, and immune response. Glutathione taken as a
supplement may not be able to cross the cell membrane and thus may not be effective. Consider
acetylcysteine instead because it is the N-acetyl derivative of the amino acid, L-cysteine, and is a
precursor in the formation of the antioxidant glutathione in the body. The thiol (sulfhydryl)
group confers antioxidant effects and is able to reduce free radicals and also acetylcysteine is a
good alternative since it can help produce more glutathione.
This antioxidant, made from the combination of three amino acids cysteine, glutamate, and
glycine, forms part of the powerful natural antioxidant glutathione peroxidase that is found in our
cells. Glutathione peroxidase plays a variety of roles in cells, including DNA synthesis and
repair, metabolism of toxins and carcinogens, enhancement of the immune system, and
prevention of fat oxidation. However, glutathione is predominantly known as an antioxidant
protecting our cells from damage caused by the free radical hydrogen peroxide. Glutathione also
helps the other antioxidants in cells stay in their active form. Brain glutathione levels have been
found to be lower in patients with Parkinson’s disease (Zhang, 1993).
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Glutathione is found in foods, particularly fruits, vegetables and meats. Cyanohydroxybutene, a
chemical found in broccoli, cauliflower, Brussels sprouts and cabbage, is also thought to increase
glutathione levels. Various herbs for instance cinnamon and cardamom have compounds that can
restore healthy levels of glutathione. Although glutathione is available in pill form over the
counter, its utilization by the body is questionable since we don’t know if it can easily enter cells,
even after it is absorbed in the bloodstream. Certain nutrients help raise tissue levels of
glutathione including acetylcysteine, methyl donors, alpha lipoic acid, polyphenols such as
pycnogenol, and vitamin B12 (Silva, et al., 1995).
An excellent review article in the April 1998 issue of Alternative Medicine Review summarizes
the known effects of acetylcysteine. The author writes, “N- acetylcysteine is an excellent source
of sulfhydryl groups, and is converted in the body into metabolites capable of stimulating
glutathione synthesis, promoting detoxification, and acting directly as a free radical scavenger.
Acetylcysteine has historically been as a mucolytic [mucus dissolving] agent in a variety of
respiratory illness; however, it appears to also have beneficial effects in conditions characterized
by decreased glutathione or oxidative stress, such as HIV infection, cancer, heart disease, and
cigarette smoking”. The frequent use of acetaminophen (paracetamol) depletes glutathione
peroxidase levels. There appear to be feedback inhibition in glutathione synthesis. This means
that if glutathione levels are excessively increased with the help of the nutrients, the body may
decrease its natural production (Kelly, 1998).
Glutathione is solid in pills with dosages ranging from 50 to 250mg. Glutathione is a promising
antioxidant. However, due to the inconsistence in the medical literature on the ability of
glutathione to enter tissues and cells when ingested orally, its beneficial effect to oral dosing may
be questionable. Oral administration is poorly tolerated, owing to high doses required (due to low
26
oral bioavailability), very unpleasant taste and odor, and adverse effects (particularly nausea and
vomiting). In a research conducted by Baker, it was concluded that oral N-acetylcysteine was
identical in bioavailability to cysteine precursors. Glutathione deficiency contributes to oxidative
stress, which plays a key role in aging and the worsening of many diseases including
Alzheimer’s disease, Parkinson’s disease, liver disease, cystic fibrosis, sickle cell anemia, HIV,
AIDS, cancer, heart attack, and diabetes. The concentration of glutathione declines with age and
in some age-related diseases (Liu, et al., 2004).
Staying on top of oxidative stress is a necessity in our increasingly toxic world. Taking care to
avoid those toxins as much as possible and to enrich our diets with life-giving antioxidants is a
wise step to take in our endless quest for wellness.
FOOD SOURSES OF ANTIOXIDANTS
CoQ10 (ubiquinone): Beef heart, beef liver, sardines, spinach, peanuts.
Beta carotene: All orange and yellow fruits and vegetables; dark green vegetables.
Zinc: Oysters, herring, lamb, whole grains.
Selenium: Butter, meats, seafood, whole grain.
Vitamin A: Cold liver oil, butter, liver, all oily fish.
Vitamin E: Cold-pressed, unrefined nut and seed oils; wheat germ oil.
Vitamin C: Berries, greens, broccoli, kale, kiwi, parsley, guava.
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Glutathione (GSH): Fresh fruits and vegetables, fresh meats, low-heat dried whey.
Bioflavonoids: Most fruits and vegetables, buckwheat.
Polyphenols: Greentea,berries.
Herbal sources: Milk thistle, Ginkgo biloba, turmeric, curry (Padma 28, a packaged Ayurvedic
herbal formula, is a special blend of herbal antioxidants).
1.3.7 BIOTRANSFORMATION OF CARBON TETRACHLORIDE
Metabolism of carbon tetrachloride is initiated by cytochrome P-450 mediated transfer of an
electron to the C – Cl bond forming an anion radical that eliminates chloride, trichloromethyl
radical (Pohl et al., 1981). This radical may undergo both oxidative and reductive
biotransformation. The isoenzymes implicated in this process are the cytochrome P2E1,
cytochrome P2B1 and cytochrome P2B2 ( Gruebeleet al., 1996). Some isoforms may
preferentially be susceptible to degradation of carbon tetrachloride. Evidence that carbon
tetrachloride inactivates CYP2E, and reduces total CYP2E protein has been obtained by Dai and
Cederbaum (1995). When protein synthesis is blocked, inactivation and degradation of CYP2E1
by carbon tetrachloride are more pronounced.
The formation of carbon tetrachloride – cytochrome P-450 complexes has been demonstrated.
The most important pathway in the elimination of trichloromethyl radicals is the reaction with
molecular oxygen, resulting in the formation of trichloromethyl peroxyl radicals as proposed by
McCayet al., (1984).
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Carbon tetrachloride has been reported to be metabolized to CO2 in the liver homogenates. The
biotransformation of carbon tetrachloride to carbon IV oxide in vivo has been reported by
Reynolds et al., (1984).
1.3.8 SERUM ENZYME DETERMINATIONS AS A DIAGNOSTIC TOOL
Normally most enzymes reside within cells, where they function in various phases of
intermediary metabolism and only small quantities are present in the serum. During certain acute
physiologic insults such as myocardial infarction or acute hepatitis, cellular content escapes with
extra cellular fluid and eventually reaches the serum in high concentration.
1.3.9 ENZYMES IN THE DIAGNOSTIC PATHOLOGY: ALT, AST AND ALP
I. Alanine Transaminase (ALT)
Alanine Transaminase (ALT) formerly called Glutamate-Pyruvate Transaminase (GPT) is an
enzyme present in hepatocytes (liver cells), and in less amount in kidney, heart and skeletal
muscle. When a cell is damaged, it leaks this enzyme into the blood, where it is measured. ALT
rises dramatically in acute liver damage, such as viral hepatitis than AST (Song, et al., 2010).
II. Aspartate Transaminase (AST)
Aspartate Transaminase (AST) formerly called Glutamate-Oxaloacetate Transaminase (GOT) is
similar to ALT in that it is another enzyme associated with liver parenchymal cells. It is raised in
acute liver damage, myocardial infarction, myopathies muscular disease (muscular dystrophy,
rhabdomyolisis) or trauma but is also present in red cells, brain, cardiac and skeletal muscles. It
is therefore less specific to liver disease.
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III. Alkaline phosphatase (ALP)
Alkaline phosphatase (ALP) is an enzyme in the cells lining the biliary ducts of the liver. ALP
levels in plasma will rise with bile duct obstruction, intra-hepatic cholestasis or infiltration
disease of the liver. ALP is also present in bone and placental tissue, so it is higher in growing
children.
IV. Bilirubin
Increased total bilirubin causes jaundice and its increased production causes hemolytic anemia
and internal hemorrhage. Deficiencies in bilirubin metabolism can cause cirrhosis and viral
hepatitis, while its deficiencies in excretion can bring about obstruction of the bile duct (Schmidt
and Schmidt, 1963).
1.4.0 Rationale of study
Most of the health benefits observed in people that use the extracts of AnnonasenegalensisPers.
(Annonaceae) stem bark for the management of many ailments are attributed to its
pharmacological and medicinal properties. This study is aimed at understanding the baseline
pharmacological and toxicological effects of the extracts of A. Senegalensis stem bark in
hepatotoxic and normal rats. The rationale of this work is linked to the hepatoprotective effect of
A.senegalensisstem bark extract on the liver (Dalziel, 1995).
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1.4.1 Aim of study
u To determine pharmacognostic standards of Annona Senegalensis Pers.
u To determine phytochemical constituents of pulverized bark of Annonasenegalensis.
u To evaluate the antioxidant activities of the extract and fractions of Annona senegalensis.
u To evaluate the hepatoprotective activities of the extract and fractions of Annona
senegalensis.
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