ABSTRACT
The
effect of ethanol extract of Dennettia
tripetala on rats exposed to carbon tetrachloride was investigated. Ethanol extract of the plant was prepared
using standard procedure. Sets of 30 female wistar albino rats were divided
into 6 groups containing five animals each and were treated orally with increasing
doses of ethanol extract of Dennettia
tripetala for two weeks. CCl4 was
diluted with olive oil in a 1:1 ratio and administered once by oral route at
the end of the extract administration. Results from the study showed non-
significant decreases in the levels of catalase and SOD activities (P>0.05)
in the CCl4 group compared to the control. The extract treatment
however produced a higher activity for the antioxidant enzymes compared to the CCl4
treated groups. The results also showed increased levels of MDA
concentration (P<0.05) in the CCl4 group whereas extract treated
rats showed lower concentrations of MDA. The overall results suggests that the
ethanolic extract of Dennettia tripetala
may have moderate hepatoprotective effect in the CCl4 induced rats.
TABLE OF CONTENT
Title
page ……………………………………………….……………………i
Certification………………………………………………………………….ii
Dedication……………………………………………………………………iii
Acknowledgement……………………………………………………………iv
Table
of contents………………………………………..……………………v
Abstract……………………………………………………………………….vi
CHAPTER
ONE
1.0 INTRODUCTION………………………………………………………
1
1.1LITERATURE
REVIEW……………………………..…………………..3
1.1.0
THE LIVER……………………………………………………………..3
1.1.0.1
FUNCTIONS OF THE LIVER …………………..………………….4
1.1.0.2
BIOTRANSFORMATION OF HEPATOTOXICANTS…………….5
1.2
KIDNEY…………………………………………………………………..7
1.2.1
FUNCTIONS OF THE KIDNEY………………………………………7
1.2.1.0
EXCRETION OF WASTES…………………………………………..8
1.2.1.1
REABSORPTION OF VITAL NUTRIENTS……..………………….8
1.2.1.2
ACID-BASE HOMEOSTASIS……………………………………….9
1.2.1.3
OSMOLALITY REGULATION………………………………….….9
1.2.1.4
BLOOD PRESSURE REGULATION…………………….……….10
1.2.1.5
HORMONE SECRETION…………………………………………..11
1.3
HEPATOTOXICITY………………………………………..…………..12
1.3.1
SYMPTOMS OF HEPATOTOXICITY…………………..…………..13
1.3.1.0
CLINICAL MANIFESTATION…………………………………….13
1.3.1.1
PATHOLOGICAL MANIFESTATION…………………….………14
1.4
CARBONTETRACHLORIDE………………………………………….14
1.5
OXIDATIVE STRESS…………………………………………………..18
1.5.1
EFFECT OF OXIDATIVE STRESS ON DNA……………………….18
1.5.2
EFFECT OF OXIDATIVE STRESS ON LIPIDS…………………….20
1.5.3
EFFECT OF OXIDATIVE STRESS ON PROTEINS………..……….20
1.5.4
LIPID PEROXIDATION……………………………………..………..21
1.6
ANTIOXIDANTS………………………………………………………..25
1.6.0
ENZYMATIC ANTIOXIDANTS……………………………………...25
1.6.0.1
SUPEROXIDE DISMUTASE………………………………………..25
1.6.0.2
CATALASE………………………………………………………….26
1.6.0.3
GLUTATHIONE PEROXIDASE…………………………………...27
1.6.1
NON ENZYMATIC ANTIOXIDANTS……………………………….29
1.6.1.1
VITAMIN C…………………………………………………………..29
1.6.1.2
VITAMIN E………………………………………………………….29
1.6.1.3
GLUTATHIONE…………………………………………………….30
1.7
LIVER FUNCTIONTESTS………………………………………………30
1.8
LIPID PROFILE…………………………………………………………33
1.9
DENNETTIA TRIPETALA……………………………………………............34
JUSTIFICATION…………………………………………………………….44
AIM
AND OBJECTIVE……………………………………………………..44
CHAPTER
TWO……………………………………………………………..45
2.0 MATERIALS
AND METHODS…………………………………………45
2.1
MATERIALS…………………………………………………………..…45
2.2
APPARATUS……………………………………………………………46
2.3
METHODS ………………………………………………………………47
2.3.1
EXPERIMENTAL ANIMALS………………………………………....47
2.3.2
COLLECTION AND EXTRACTION OF PLANTMATERIALS…….47
2.4
ANIMAL FEEDING EXPERIMENT……………………………………48
2.5
CATALASE……………………………………………………………...49
2.6
SUPEROXIDE DISMUTASE……………………………………………50
2.7
MALONDIALDEHYDE………………………………………………….51
CHAPTER
THREE
3.0
RESULTS……………………………………………………….53
CHAPTER
FOUR
DISCUSSION……………………………………………………….58
CONCLUSION………………………………………………………66
REFERENCES………………………………………………………..67
APPENDIX
I……………………………………………………….. 75
APPENDIX
II………………………………………………………...76
APPENDIX
III………………………………………………………77
APPENDIX
IV………………………………………………………79
APPENDIX
V……………………………………………………..…81
APPENDIX
VI………………………………………………………84
APPENDIX
VII………………………………………………………89
CHAPTER ONE
1.0 INTRODUCTION
Natural plant products and their
derivatives represent more than 50% of all the drugs in clinical use in the
world (Ben-Eric, 2002). Dennettia tripetala also
known as pepper fruit tree is a well-known Nigerian spicy medicinal
plant. It is found in the tropical rainforest region of Nigeria and
sometimes in Savanna areas (Okwu et
al., 2005). It is locally called “Nkarika” by the Efiks of
Calabar. The young leaves and fruits have distinctive spicy taste.
The mature fruits constitute the main edible portions. Some communities
in parts of Southern Nigeria also utilize the leaves and roots, in addition to
the fruits for medicinal purpose. Dennettia
tripetala has been found to contain
lots of minerals, vitamins, alkaloids and trace elements which are of medicinal
importance. It was also indicated that the rich presence of essential oil
(oleoresins) determines the aromatic flavoring, coloring and pungent properties
of pepper fruits. (Nwaogu et al., 2007)
investigated phytochemical content of Dennettia tripetala and detected the
presence of saponins, flavonoids, tannins and cyanogenic glycosides. The intake of flavonoids in any
fruit and vegetable tends to decrease cancer risk (Neuhouser, 2004; Graf et al.,
2005). Flavonoid contributes to the color of plants, their fruits and flowers.
The use of medicinal plants in traditional medicine is not intended in any way
to replace modern medical science but rather an aid in conventional therapy
(Ben-Eric, 2002).
Carbon
tetrachloride (CCl4) is an industrial chemical that does not occur
naturally. Most of the carbon tetrachloride produced is used in the production
of chlorofluorocarbons (CFCs) and other chlorinated hydrocarbons. It was once
used widely as a solvent, cleaner and degreaser, both for industrial and home
use. Today, the scientific database on the effects of haloalkanes is so vast
that it is no longer employed for such purposes although it is used as a model
of experimental liver injury (Weber et
al., 2003).
CCl4 is a well-known hepato- and nephrotoxicant (Thrall et al., 2000; Ogeturk et al., 2005), and proves highly useful
as an experimental model for the study of certain hepatotoxic effects (Muriel et al.,
2003; Moreno and Muriel, 2006). CCl4-induced toxicity, depending on
dose and duration of exposure, covers a variety of effects. At low doses,
transient effects prevail, such as loss of Ca2+ homeostasis, lipid
peroxidation, release of noxious or beneficial cytokines (Kyung-Hyun et al., 2006; Muriel, 2007) and
apoptotic events followed by regeneration. Other effects, with higher doses or
longer exposure, are more serious and develop over a long period of time, such
as fatty degeneration, fibrosis, cirrhosis and even cancer (Weber et al., 2003). In addition, acute
intoxication with CCl4 at high doses, when the hepatocellular
necrosis exceeds the regenerative capacity of the liver, fatal liver failure
will ensue. Extreme doses of CCl4 result in nonspecific solvent
toxicity, including central nervous system depression and respiratory failure
and death.
This
study aims at investigating the effect of ethanol extract of Dennettia tripetala on liver and kidney
antioxidant enzyme activity and malondialdehyde concentration of rats exposed
to CCl4.
1.1 LITERATURE REVIEW
1.1. 0 THE LIVER
The liver is the largest organ of the
human body weighing approximately 1500 g, and is located in the upper right
corner of the abdomen on top of the stomach, right kidney and intestines and
beneath the diaphragm. The liver performs more than 500 vital metabolic
functions (Naruse et al., 2007). It
is involved in the synthesis of products like glucose derived from
glycogenesis, plasma proteins, clotting factors and urea that are released into
the bloodstream. It regulates blood levels of amino acids.
Liver parenchyma serves as a
storage organ for several products like glycogen, fat and fat soluble vitamins.
It is also involved in the production of a substance called bile that is
excreted to the intestinal tract. Bile aids in the removal of toxic substances
and serves as a filter that separates out harmful substances from the
bloodstream and excretes them (Saukonen et
al., 2006). An excess of chemicals
hinders the production of bile thus leading to the body’s inability to flush
out the chemicals through waste.
Smooth endoplasmic reticulum of the
liver is the principal ‘metabolic clearing house’ for both endogenous chemicals
like cholesterol, steroid hormones, fatty acids and proteins, and exogenous
substances like drugs and alcohol. The central role played by liver in the
clearance and transformation of chemicals exposes it to toxic injury (Saukonen et al., 2006).
1.1.0.1 FUNCTIONS OF THE LIVER
The
liver has three main functions: storage, metabolism, and biosynthesis. Glucose
is converted to glycogen and stored; when needed for energy, it is converted
back to glucose. Cholesterol uptake also occurs in the liver. Fat, fat-soluble
vitamins and other nutrients are also stored in the liver. Fatty acids are
metabolized and converted to lipids, which are then conjugated with proteins
synthesized in the liver and released into blood stream as lipoproteins.
Numerous functional proteins such as, enzymes and blood-coagulating factors are
also synthesized by the liver. In addition, the liver, which contains numerous
xenobiotic metabolizing enzymes, is the main site of xenobiotic metabolism
(Hogson and Levi, 2004).
1.1.0.2 BIOTRANSFORMATION OF HEPATOTOXICANTS
Liver
plays a central role in biotransformation and disposal of xenobiotics.
The
close association of liver with the small intestine and the systemic
circulation enables it to maximize the processing of absorbed nutrients and
minimize exposure of the body to toxins and foreign chemicals. The liver may be
exposed to large concentrations of exogenous substances and their metabolites.
Metabolism of exogenous compounds can modulate the properties of hepatotoxicant
by either increasing its toxicity (toxication or metabolic activation) or
decreasing its toxicity (detoxification).
Most
of the foreign substances are lipophilic thus enabling them to cross the
membranes of intestinal cells. They are rendered more hydrophilic by
biochemical processes in the hepatocyte, yielding water-soluble products that
are exported into plasma or bile by transport proteins located on the
hepatocyte membrane and subsequently excreted by the kidney or gastrointestinal
tract (Totsmann et al., 2008).
The
hepatic biotransformation involves Phase I and Phase II reactions. Phase I involves
oxidative, reductive, hydroxylation and demethylation pathways, primarily by
way of the cytochrome P-450 enzyme system located in the endoplasmic reticulum,
which is the most important family of metabolizing enzymes in the liver. The
endoplasmic reticulum also contains a NADPH-dependent mixed function oxidase
system, the flavin-containing monooxygenases, which oxidizes amines and sulphur
compounds.
Phase
I reactions often produce toxic intermediates which are rendered non-toxic by
phase II reactions. Phase II reactions involve the conjugation of chemicals
with hydrophilic moieties such as glucuronide, sulfate or amino acids and lead
to the formation of more water-soluble metabolite which can be excreted easily.
Another Phase II reaction involves glutathione which can covalently bind to
toxic intermediates by glutathione-S- transferase. As a result, these reactions are usually
considered detoxification pathways. However, this phase can also lead to the
formation of unstable precursors to reactive species that can cause
hepatotoxicity.
The
activities of enzymes are influenced by various endogenous factors and
exogenous drugs or chemicals (Lee and Boyer, 2000). Many substances can
influence the cytochrome P450 enzyme mechanism. Such substances can serve
either as inhibitors or inducers. Enzyme inhibitors act immediately by blocking
the metabolic activity of one or several cytochrome P450 enzymes. Enzyme
inducers act slowly and increase cytochrome P450 activity by increasing its
synthesis (Lynch and Price, 2007).
1.2 KIDNEY
The kidneys are
bean-shaped organs that serve several essential regulatory roles in
vertebrates. They remove excess organic molecules from the blood and their best
known function is the removal of waste products of metabolism. They serve
homeostatic functions such as the regulation of electrolytes, maintenance of
acid-base balance, and regulation of blood pressure (via maintaining the salt
and water balance). In producing urine, the kidneys excrete wastes such as urea
and ammonium. They are responsible for the reabsorption of water, glucose, and
amino acids. They also produce hormones like calcitriol and erythropoietin.
1.2.1 FUNCTIONS OF THE KIDNEY
Many of the kidney’s functions are accomplished by relatively simple
mechanisms of filtration, reabsorption, and secretion, which take place in the
nephron. Filtration, which takes place at the renal corpuscle, is the process
by which cells and large proteins are filtered from the blood to make an
ultrafiltrate that eventually becomes urine. The kidney generates 180 litres of
filtrate a day, while reabsorbing a large percentage allowing for the
generation of only approximately 2 litres of urine. Reabsorption is the
transport of molecules from this ultrafiltrate into the blood. Secretion is the
reverse process, in which molecules are transported in the opposite direction,
from blood to the urine. (Bard et al.,
2003).
1.2.1.0 Excretion of wastes
The kidneys excrete a variety of waste products produced by metabolism
into the urine. These include the nitrogenous wastes urea, from protein
catabolism, and uric acid, from nucleic acid metabolism. The ability of mammals
and some birds to concentrate wastes into a volume of urine much smaller than
the volume of blood from which the wastes were extracted is dependent on an
elaborate countercurrent multiplication mechanism. This requires several
independent nephron characteristics to operate: a tight hairpin configuration
of the tubules, water and ion permeability in the descending limb of the loop,
water impermeability in the ascending loop, and active ion transport out of
most of the ascending limb. In addition, passive countercurrent exchange by the
vessels carrying the blood supply to the nephron is essential for enabling this
function.
1.2.1.1 Reabsorption of the
vital nutrients
Glucose at normal plasma levels is completely reabsorbed in the proximal
tubule. The mechanism for this is the Na+/glucose cotransporter. A
plasma level of 350mg/dL will fully saturate the transporters and glucose will
be lost in the urine. A plasma glucose level of approximately 160 is sufficient
to allow glucosuria, which is an important clinical clue to diabetes mellitus.
Amino acids are reabsorbed by sodium dependent transporters in the
proximal tubule. Hartnup disease is a
deficiency of the tryptophan amino acid transporter which results in pellagra
(Le Tao, 2013).
1.2.1.2 Acid-base
homeostasis
Two organ systems, the kidneys and lungs, maintain acid base
homeostasis, which is the maintenance of pH around a relatively stable value.
The lungs contribute to acid-base homeostasis by regulating carbon dioxide (CO2)
concentration. The kidneys have two very important roles in maintaining the
acid-base balance: to reabsorb and regenerate bicarbonate from urine, and to excrete
hydrogen ions and fixed acids (anions of acids) into urine (Seldin et al., 1989).
1.2.1.3 Osmolality
regulation
Any significant rise in plasma
osmolality is detected by the hypothalamus, which communicates directly with
the posterior pituitary gland. An increase in osmolality causes the gland to
secrete antidiuretic hormone (ADH), resulting in water reabsorption by the
kidney and an increase in urine concentration. The two factors work together to
return the plasma osmolality to its normal levels.
ADH binds to principal cells in the collecting duct that translocate
aquaporins to the membrane, allowing water to leave the normally impermeable
membrane and be reabsorbed into the body by the vasa recta, thus increasing the
plasma volume of the body.
There are two systems that create a hyperosmotic medulla and thus
increase the body plasma volume: urea recycling and the ‘single effect’.
Urea is usually excreted as a waste product from the kidneys. However,
when plasma blood volume is low and ADH is released the aquaporins that are
opened are also permeable to urea. This allows urea to leave the collecting
duct into the medulla creating a hyperosmotic solution that attracts water.
Urea can then re-enter the nephron and be excreted or recycled again depending
on whether ADH is still present or not. The ‘single effect’ describes the fact
that the ascending thick limb of the loop of henle is not permeable to water
but is permeable to NaCl. This allows for a countercurrent exchange system
whereby the medulla becomes increasingly concentrated, but at the same time
setting up an osmotic gradient for water to follow should the aquaporins of the
collecting duct be opened by ADH (Vander, 1985).
1.2.1.4 Blood pressure
regulation
Although the kidney cannot directly sense blood, long term regulation of
blood pressure predominantly depends upon the kidney. This primarily occurs
through maintenance of the extracellular fluid compartment, the size of which
depends on the plasma sodium concentration. Renin is the first in the series of
important chemical messengers that make up the renin-angiotensin system.
Changes in rennin ultimately alter the output of this system, principally the
hormones angotensin II and aldostrone. Each hormone acts via multiple
mechanisms, but both increase the kidney’s absorption of sodium chloride,
thereby expanding the extracellular fluid compartment, and an increase in blood
pressure. Conversely, when rennin levels are low, angiotensin II and
aldosterone levels decrease, contracting the extracellular fluid compartment,
and an increase in blood pressure. Conversely, when rennin levels are low,
angiotensin II and aldosterone levels decrease, contracting the extracellular
fluid compartment, and decreasing blood pressure.
1.2.1.5 Hormone secretion
The kidneys secrete a variety of hormones, including erythropoietin, and
the enzyme rennin. Erythropoietin is released in response to hypoxia (low
levels of oxygen at tissue level) in the renal circulation. It stimulates erythropoiesis
(production of red blood cells) in the bone marrow. Calcitriol, the activated
form of vitamin D, promotes intestinal absorption of calcium and the renal
reabsorption of phosphate. Part of the renin-angiotensin-aldosterone system, renin
is an enzyme involved in the regulation of aldosterone levels (Valtin, 1983).
1.3 HEPATOTOXICITY
Hepatotoxicity refers to liver dysfunction or
liver damage that is associated with an overload of drugs or xenobiotics (Navaro
et al., 2006). The chemicals that
cause liver injury are called hepatotoxins or hepatotoxicants. Hepatotoxicants
are exogenous compounds of clinical relevance and may include overdoses of
certain medicinal drugs, industrial chemicals, natural chemicals like
microcystins, herbal remedies and dietary supplements (Willett et al., 2004).
Certain drugs may cause liver injury
when introduced even within the therapeutic ranges. Hepatotoxicity may result
not only from direct toxicity of the primary compound but also from a reactive
metabolite or from an immunologically-mediated response affecting hepatocytes,
biliary epithelial cells and/or liver vasculature (Saukkonen et al., 2006).
The hepatotoxic response elicited by
a chemical agent depends on the concentration of the toxicant which may be either parent
compound or toxic metabolite, differential expression of enzymes and
concentration gradient of cofactors in blood across the acinus. Hepatotoxic response is expressed in the form
of characteristic patterns of cytolethality in specific zones of the acinus.
1.3.1 SYMPTOMS OF HEPATOTOXICITY
Hepatotoxicity related symptoms may include a jaundice or icterus
appearance causing yellowing of the skin, eyes and mucous membranes due to high
level of bilirubin in the extracellular fluid, pruritus, severe abdominal pain,
nausea or vomiting, weakness, severe fatigue, continuous bleeding, skin rashes,
generalized itching, swelling of the feet and/or legs, abnormal and rapid
weight gain in a short period of time, dark urine and light colored stool
(Bleibel et al., 2007; Chang and
Shaino, 2007).
The symptoms of hepatotoxicity can be subdivided into clinical and
drug-induced pathological symptoms.
1.3.1.0
CLINICAL MANIFESTATION
The manifestation of drug induced hepatotoxicity is highly variable,
ranging from a symptomatic evaluation of liver enzymes to fulminant hepatic
failure. The injury may suggest a hepatocellular injury with evaluation of
aminotransferases levels as the predominant symptom or a cholestatic injury,
with elevated alkaline phosphatase levels with or without hyperbiliruminemia
being the main feature.
In addition, drugs that cause mild amino transferase elevation with
subsequent adaptation are differentiated from those that result in true
toxicity that require discontinuation.
Hepatotoxicity can be
induced in the laboratory by exposing laboratory animals to toxic chemicals
such as carbon tetrachloride.
1.3.1.1
PATHOLOGICAL MANIFESTATION
Acute hepatocellular damage
Chronic hepatocellular damage
Chronic cholestasis
Vascular lesions / venocclusive disease
Angiosarcoma (Bleibel et
al., 2007; Chang and Shaino, 2007).
1.4
CARBON TETRACHLORIDE
Carbon tetrachloride (CCl4)
is an industrial chemical that does not occur naturally. Most of the carbon
tetrachloride produced is used in the production of chlorofluorocarbons (CFCs)
and other chlorinated hydrocarbons. It was once used widely as a solvent,
cleaner and degreaser, both for industrial and home use. Today, the scientific
database on the effects of haloalkanes is so vast that it is no longer employed
for such purposes although it is used as a model of experimental liver injury
(Weber et al., 2003).
CCl4 is a well-known hepato-
and nephrotoxicant (Thrall et al.,
2000; Ogeturk et al., 2005), and
proves highly useful as an experimental model for the study of certain
hepatotoxic effects (Muriel et al.,
2003; Moreno and Muriel, 2006). CCl4-induced toxicity, depending on
dose and duration of exposure, covers a variety of effects. At low doses,
transient effects prevail, such as loss of Ca2+ homeostasis, lipid
peroxidation, release of noxious or beneficial cytokines (Kyung-Hyun et al., 2006; Muriel, 2007) and
apoptotic events followed by regeneration. Other effects, with higher doses or
longer exposure, are more serious and develop over a long period of time, such
as fatty degeneration, fibrosis, cirrhosis and even cancer (Weber et al., 2003). In addition, acute
intoxication with CCl4 at high doses, when the hepatocellular
necrosis exceeds the regenerative capacity of the liver, fatal liver failure
will ensue. Extreme doses of CCl4 result in nonspecific solvent
toxicity, including central nervous system depression and respiratory failure
and death. CCl4 can be administered orally, intravenously and
intraperitonially.
CCl4 metabolism begins
with the formation of the trichloromethyl free radical, CCl3 through
the action of the mixed function cytochrome P-450 oxygenase system of the
endoplasmic reticulum. This process involves reductive cleavage of a
carbon-chlorine bond. Free radical activation of CCl4 in
mitochondria has also been observed and may contribute significantly to its
toxicity. The major cytochrome iso-enzyme to execute biotransformation of CCl4
is cytochrome P-450 iso-enzyme 2E1 (CYP2E1). This is evidenced by the absence
of toxicity in CYP2E1 knockout mice.
In humans, CYP2E1 dominates CCl4
metabolism at environmentally relevant concentrations, but at higher
concentrations other cytochromes, particularly CYP3A, also contribute
importantly (Zanger et al.,
2000). The CCl3 radical
reacts with several important biological substances, like fatty acids,
proteins, lipids, nucleic acids and amino acids (Weber et al., 2003). CCl3
also acts by abstracting hydrogen from unsaturated fatty acids to form
chloroform. DNA adducts
is a mechanism for CCl4-induced carcinogenesis.
(FIG.
1.4) A schematic diagram explaining the
onset of steatosis involving MTP degradation of CCl4. (Pan et al., 2007) CCl4
is converted to free radicals (CCl3 and Cl) by cytochrome P450
oxygenases. MTP is covalently modified by CCl3, ubiquitinylated, and
degraded by proteasomes. This leads to increased accumulation of triglycerides
and cholesterol in the tissues. If proteasomal degradation of MTP is inhibited
by lactacystin, the CCl4 toxicity is averted in part because the
protected MTP is able to assist in lipoprotein assembly and in the secretion of
triglycerides.
1.5 OXIDATIVE STRESS
Oxidative stress occurs when the balance
between antioxidants and ROS are disrupted because of either depletion of
antioxidants or accumulation of ROS. When oxidative stress occurs, cells
attempt to counteract the oxidant effects and restore the redox balance by
activation or silencing of genes encoding defensive enzymes, transcription
factors, and structural proteins (Scandalios et al., 2004). Ratio between oxidized and reduced glutathione
(2GSH/GSSG) is one of the important determinants of oxidative stress in the
body. Higher production of ROS in body may change DNA structure, result in
modification of proteins and lipids, activation of several stress-induced
transcription factors, and production of pro-inflammatory and anti-inflammatory
cytokines.
1.5.1 EFFECTS OF OXIDATIVE STRESS ON
DNA
ROS can lead to
DNA modifications in several ways, which involves degradation of bases, single-
or double- stranded DNA breaks, purine, pyrimidine or sugar-bound modifications,
mutations, deletions or translocations, and cross-linking with proteins. Most
of these DNA modifications are highly relevant to carcinogenesis, aging, and
neurodegenerative, cardiovascular, and autoimmune diseases. Tobacco smoke,
redox metals, and non-redox metals, such as iron, cadmium, chrome, and arsenic,
are also involved in carcinogenesis and aging by generating free radicals or
binding with thiol groups. Formation of 8-OH-G is the best- known DNA damage
occurring via oxidative stress and is a potential biomarker for carcinogenesis.
Promoter regions of genes contain consensus sequences for transcription
factors. These transcription factor–binding sites contain GC-rich sequences
that are susceptible for oxidant attacks. Formation of 8-OH-G DNA in
transcription factor binding sites can modify binding of transcription
factors
and thus change the expression of related genes as has been shown for AP-1 and
Sp-1 target sequences. Besides 8-OH-G, 8,59-cyclo-29-deoxyadenosine (cyclo-dA)
has also been shown to inhibit transcription from a reporter gene in a cell
system if located in a TATA box (Marietta et
al., 2002). The TATA-binding
protein initiates transcription by changing the bending of DNA.
Oxidative
stress causes instability of microsatellite (short tandem repeats) regions.
Redox active metal ions, hydroxyl radicals increase microsatellite instability. Even
though single-stranded DNA breaks caused by oxidant injury can easily be
tolerated by cells, double-stranded DNA breaks induced by ionizing radiation
can be a significant threat for the cell survival (Caldecott et al., 2003). Methylation at CpG
islands in DNA is an important epigenetic mechanism that may result in gene
silencing. Oxidation of 5-MeCyt to 5-hydroxymethyl uracil (5-OHMeUra) can occur
via deamination/oxidation reactions of thymine or 5-hydroxymethyl cytosine
intermediates (Cooke et al.,
2003). In addition to the modulating
gene expression, DNA methylation also seems to affect chromatin organization.
Aberrant DNA methylation patterns induced by oxidative attacks also affect DNA
repair activity.
1.5.2 EFFECTS OF OXIDATIVE STRESS ON
LIPIDS
ROS
can induce lipid peroxidation and disrupt the membrane lipid bilayer
arrangement that may inactivate membrane-bound receptors and enzymes and
increase tissue permeability. Products of lipid peroxidation, such as
MDA and unsaturated aldehydes, are capable of inactivating many cellular
proteins by forming protein cross-linkages. 4-Hydroxy-2-nonenal causes
depletion of intracellular GSH and induces of peroxide production, activates epidermal growth factor
receptor, and induces fibronectin
production (Tsukagoshi et al., 2002). Lipid peroxidation products, such as
isoprostanes and thiobarbituric acid reactive substances, have been used as
indirect biomarkers of oxidative stress, and increased levels were shown in the
exhaled breath condensate or broncho-alveolar lavage fluid or lung of chronic
obstructive pulmonary disease patients or smokers (Montuschi et al., 2000).
1.5.3 EFFECTS OF OXIDATIVE STRESS ON
PROTEINS
ROS can cause fragmentation of the peptide
chain, alteration of electrical charge of proteins, cross-linking of proteins,
and oxidation of specific amino acids and therefore lead to increased
susceptibility to proteolysis by degradation by specific proteases (Kelly et al., 2003). Cysteine and methionine
residues in proteins are particularly more susceptible to oxidation. Oxidation of sulfhydryl groups or methionine
residues of proteins cause conformational changes, protein unfolding, and
degradation. Enzymes that have metals on
or close to their active sites are especially more sensitive to metal catalyzed
oxidation. Oxidative modification of enzymes has been shown to inhibit their
activities. In some cases, specific oxidation of proteins may take place. For example,
methionine can be oxidized methionine sulfoxide and phenylalanine to o-tyrosine
sulfhydryl groups can be oxidized to form disulfide bonds and carbonyl groups
may be introduced into the side chains of proteins. Gamma rays, metal-catalyzed
oxidation, HOCl, and ozone can cause formation of carbonyl groups (Shacter et al., 2000).
1.5.4 LIPID PEROXIDATION
Currently, lipid peroxidation is
considered as one the main molecular mechanisms involved in the oxidative
damage to cell structures and in the toxicity process that lead to cell death.
First, lipid peroxidation was studied for food scientists as a mechanism for
the damage to alimentary oils and fats, nevertheless other researchers
considered that lipid peroxidation was the consequence of toxic metabolites
(e.g. CCl4) that produced highly reactive species, disruption of the
intracellular membranes and cellular damage (Dianzani and Barrera, 2008).
Lipid peroxidation is a complex process
known to occur in both plants and animals. It involves the formation and
propagation of lipid radicals, the uptake of oxygen, a rearrangement of the
double bonds in unsaturated lipids and the eventual destruction of membrane
lipids, with the production of a variety of breakdown products, including
alcohols, ketones, alkanes, aldehydes and ethers (Dianzani and Barrera, 2008).
In pathological situations the reactive oxygen and nitrogen species are
generated at higher than normal rates, and as a consequence, lipid peroxidation
occurs with α -tocopherol deficiency. In addition to containing high
concentrations of polyunsaturated fatty acids and transition metals, biological
membranes of cells and organelles are constantly being subjected to various
types of damage. The mechanism of biological damage and the toxicity of these
reactive species on biological systems are currently explained by the
sequential stages of reversible oxidative stress and irreversible oxidative
damage. The lipid peroxidation reaction involves three major steps:
1.
Initiation step
2.
Propagation step
3.
Termination step
Mechanism
of lipid peroxidation (Wang, 1999)
INITIATION
Initiation is the step whereby a fatty acid radical is
produced. The initiators in living cells and most notably reactive oxygen
species (ROS) such as hydrogen peroxide and hydroxyl groups as illustrated in
the peroxidation mechanism. This reactive oxygen species combine with a
hydrogen atom in the liquid molecule to make water and a fatty acid radical
(Kanner, 1987).
PROPAGATION
The fatty acid radicals are not very
stable molecules and so, they react readily with molecular oxygen, thereby
creating peroxyl fatty acid radicals. This radical is an unstable species that
react with another free fatty acid producing a different fatty acid radical and
lipid peroxide or cyclic peroxide if it had reacted with itself. The cycle
continues as the new fatty acid radicals react in the same way (Marnett, 1999).
TERMINATION
When a radical reacts, it produces another
radical this 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 that speed up termination by
catching free radicals and therefore protect cell membrane.
1.6 ANTIOXIDANTS
The human body is equipped with a
variety of antioxidants that serve to counterbalance the effect of oxidants.
For all practical purposes, these can be divided into 2 categories: enzymatic
and non-enzymatic.
1.6.0 ENZYMATIC ANTIOXIDANTS
The major enzymatic antioxidants of
the lungs are SODs, catalase, and GSH-Px. In addition to these major enzymes,
other antioxidants, including heme oxygenase-1, and redox proteins, such as
thioredoxins (TRXs), peroxiredoxins (PRXs) and glutaredoxins, have also been
found to play crucial roles in the pulmonary antioxidant defenses.
1.6.0.1 SUPEROXIDE DISMUTASE
Since superoxide is the primary ROS
produced from a variety of sources, its dismutation by SOD is of primary
importance for each cell. All three forms of SOD, that is, CuZn- SOD, Mn-SOD,
and EC-SOD, are widely expressed in the human lung. Mn-SOD is localized in the
mitochondria matrix. EC-SOD is primarily localized in the extracellular matrix,
especially in areas containing high amounts of type I collagen fibers and around
pulmonary and systemic vessels. It has also been detected in the bronchial
epithelium, alveolar epithelium, and alveolar macrophages (Kinnula et al., 2003)
Overall, CuZn- SOD and Mn-SOD are
generally thought to act as bulk scavengers of superoxide radicals. The
relatively high EC-SOD level in the lung with its specific binding to the
extracellular matrix components may represent a fundamental component of lung
matrix protection (Zelko et al.,
2003).
1.6.0.2 CATALASE
H2
O2 that is produced by the action of SODs or the action of oxidases,
such as xanthine oxidase, is reduced to water by catalase and the GSH-Px.
Catalase exists as a tetramer composed of 4 identical monomers, each of which
contains a heme group at the active site. Degradation of H2 O2
is accomplished via the conversion between 2 conformations of
catalase-ferricatalase (iron coordinated to water) and compound I (iron
complexed with an oxygen atom). Catalase also binds NADPH as a reducing
equivalent to prevent oxidative inactivation of the enzyme (formation of
compound II) by H2 O2 as it is reduced to water.69
Enzymes in the redox cycle responsible for the reduction of H2 O2
and lipid hydroperoxides (generated as a result of membrane lipid peroxidation)
include the GSH-Pxs.
1.6.0.3 GLUTATHIONE PEROXIDASE
(GSH-Px)
The
GSH-Pxs are a family of tetrameric enzymes that contain the unique amino acid
selenocysteine within the active sites and use low-molecular-weight thiols,
such as GSH, to reduce H2 O2 and lipid peroxides to their
corresponding alcohols. Four GSH- Pxs have been described, encoded by different
genes: GSH- Px-1 (cellular GSH-Px) is ubiquitous and reduces H2 O2
and fatty acid peroxides, but not esterified peroxyl lipids (Arthur et al., 2000) Esterified lipids are
reduced by membrane-bound GSH-Px-4 (phospholipid hydroperoxide GSH-Px), which
can use several different low-molecular-weight thiols as reducing equivalents.
GSH-Px-2 (gastrointestinal GSH-Px) is localized in gastrointestinal epithelial
cells where it serves to reduce dietary peroxides. GSH-Px-3 (extracellular GSH-Px) is the only
member of the GSH-Px family that resides in the extracellular compartment and
is believed to be one of the most important extracellular antioxidant enzyme in
mammals. Of these, extracellular GSH-Px is most widely investigated in the
human lung. In addition, disposal of H2
O2 is closely associated with several thiol-containing enzymes,
namely, TRXs (TRX1 and TRX2), thioredoxin reductases (TRRs), PRXs (which are thioredoxin peroxidases),
and glutaredoxins (Gromer et al.,
2004).
Two
TRXs and TRRs have been characterized in human cells, existing in both cytosol
and mitochondria. In the lung, TRX and TRR are expressed in bronchial and
alveolar epithelium and macrophages. Six different PRXs have been found in
human cells, differing in their ultra-structural compartmentalization.
Experimental studies have revealed the importance of PRX VI in the protection
of alveolar epithelium. Human lung expresses all PRXs in bronchial epithelium,
alveolar epithelium, and macrophages (Kinnula et al., 2002). PRX V has
recently been found to function as a peroxy-nitrite reductase, which means that
it may function as a potential protective compound in the development of
ROS-mediated lung injury (Holmgren et
al., 2000). Common to these antioxidants is the requirement of NADPH as a
reducing equivalent. NADPH maintains catalase in the active form and is used as
a cofactor by TRX and GSH reductase, which converts GSSG to GSH, a co-substrate
for the GSH-Pxs. Intracellular NADPH, in turn, is generated by the reduction of
NADP1 by glucose-6-phosphate dehydrogenase, the first and rate-limiting enzyme
of the pentose phosphate pathway, during the conversion of glucose- 6-phosphate
to 6-phosphogluconolactone. By generating NADPH, glucose-6-phosphate
dehydrogenase is a critical determinant of cytosolic GSH buffering capacity (GSH/
GSSG) and therefore, can be considered an essential, regulatory antioxidant
enzyme (Dickinson et al., 2002).
GSTs, another antioxidant enzyme family, inactivate secondary metabolites, such
as unsaturated aldehydes, epoxides, and hydroperoxides. Three major families of
GSTs have been described: cytosolic GST, mitochondrial GST, and
membrane-associated microsomal GST that has a role in eicosanoid and GSH
metabolism.
1.6.1 NON-ENZYMATIC ANTIOXIDANTS
Non-enzymatic antioxidants include
low-molecular-weight compounds, such as vitamins (vitamins C and E),
b-carotene, uric acid, and GSH, a tripeptide (L-g-glutamyl-L-cysteinyl-L-
glycine) that comprise a thiol (sulfhydryl) group.
1.6.1.1 Vitamin C (Ascorbic Acid)
Water-soluble
vitamin C (ascorbic acid) provides intracellular and extracellular
aqueous-phase antioxidant capacity primarily by scavenging oxygen free
radicals. It converts vitamin E free radicals back to vitamin E. Its plasma
levels have been shown to decrease with age.
1.6.1.2 Vitamin E (α-Tocopherol)
Lipid-soluble vitamin E is
concentrated in the hydro- phobic interior site of cell membrane and is the
principal defense against oxidant-induced membrane injury. Vitamin E donates
electron to peroxyl radical, which is produced during lipid peroxidation.
α-Tocopherol is the most active form of vitamin E and the major membrane-bound
antioxidant in cell. Vitamin E triggers apoptosis of cancer cells and inhibits
free radical formations.
1.6.1.3 Glutathione
GSH is highly abundant in all cell compartments and is
the major soluble antioxidant. GSH/GSSG ratio is a major determinant of
oxidative stress. GSH shows its antioxidant effects in several ways. It
detoxifies hydrogen peroxide and lipid peroxides via action of GSH-Px. GSH
donates its electron to H2 O2 to reduce it into H2
O and O2. GSSG is again reduced into GSH by GSH reductase that uses
NAD(P)H as the electron donor. GSH-Pxs are also important for the protection of
cell membrane from lipid peroxidation. Reduced glutathione donates protons to membrane
lipids and protects them from oxidant attacks. GSH is a cofactor for several
detoxifying enzymes, such as GSH-Px and transferase. It has a role in
converting vitamin C and E back to their active forms. GSH protects cells
against apoptosis by interacting with pro-apoptotic and anti-apoptotic
signaling pathways (Masella et al.,
2005).
1.7 LIVER FUNCTION TESTS
When the liver is damaged, it cannot
carry out its functions effectively. Certain enzymes and metabolites can
provide a basis for the test of liver functionality. Alanine aminotransferase (ALT), aspartate
aminotransferase (AST), alkaline phosphatase (ALP), Ƴ-glutamyl transpeptidase
(GGT), are among a class of enzymes called liver marker enzymes. Although not
entirely specific to the liver, these enzymes along with molecules like albumin
and bilirubin are used as markers for liver function.
1.7.1 ALANINE AMINOTRANSFERASE (ALT)
Perhaps
the most commonly used indicator of liver (hepatocellular) damage are the ALT
and AST, formerly called SGPT and SGOT respectively. These enzymes are normally
found in liver cells, but a dysfunction or injury could lead to a leakage of
these enzymes into the blood. The ALT is thought to be a more specific
indicator of liver inflammation as AST is also found in other organs such as
the heart and skeletal muscle. In acute injury to the liver, as in viral
hepatitis, the level of ALT and AST may be used as a general measure of the
degree of liver inflammation or damage. The higher the ALT level, the more cell
death or inflammation of the liver enzyme occurred (Dial, 1995).
1.7.2 ASPARTATE AMINOTRANSFERASE
(AST)
AST
is the enzyme that is produced by both the liver and the muscle. The level of
AST also increases in cases of heart attack. In many cases of liver
inflammation, the ALT and AST levels are equally elevated. In some conditions
such as hepatitis, AST levels may be higher than ALT level. AST levels can be
normal yet there can be liver damage occurring. This test adds one more
perspective to the picture of liver disease (Dial, 1995).
1.7.3 ALKALINE PHOSPHATASE
It
is produced in the bile duct, kidney placement, and bones. This enzyme is
measured or determined to help the physicians to know if the disease is
concentrated in the bile duct or in the liver. When this enzyme level is high,
and the level of ALT and AST are fairly normal, problems associated with the
bile duct may be determined (Wright, 1993).
1.7.4 BILIRUBIN
Bilirubin
is the main bile pigment in humans which, when elevated causes the yellow
discoloration of the skin called jaundice. It is a reddish yellow pigment
formed by the breakdown of hemoglobin in worn out red blood cells. The liver
helps to conjugate bilirubin with glucuronic acid, forming bilirubin di-glucuronide
(its conjugated form). The level of bilirubin in the blood can be elevated due
to over production, decreased uptake by the liver, decreased conjugation,
decreased secretion from the liver or blockage of the bile duct. In cases of
increased production, decreased secretion from the liver or bile duct
obstruction, de-conjugated or indirect bilirubin will be primarily elevated.
Many different liver diseases can cause elevated bilirubin levels (Crook,
2006).
1.7.5 ALBUMIN
Albumin
is produced entirely in the liver and constitutes about 60% of total serum
protein. It is important in regulating the flow of water between the plasma and
tissue fluid by its effect on plasma colloid osmotic pressure (oncotic
pressure). When the concentration of albumin is significantly reduced, the
plasma osmotic pressure is insufficient to draw water from the tissue spaces
back into the plasma. This leads to a build-up of fluids within the tissue
spaces, referred to as oedema.
1.8 LIPID PROFILE
1.8.1 TOTAL CHOLESTEROL
Cholesterol
is a waxy fat like substance that is important for normal body functioning.
Cholesterol is used for cellular functions and for the production of hormones.
Your body, in most cases will produce enough cholesterol to maintain normal
body needs. The liver is the major production factory for cholesterol (about
70%). Diets high in saturated fats significantly increase the amount of
cholesterol in the blood stream. Research indicates that diets high in
saturated and total fat play a significant role in the process of
atherosclerosis (plaque build-up on the artery walls). A cholesterol value of
220mg/dl correlates to nearly two-fold elevation in incidence of coronary heart
disease as compared to 180mg/dl (Tymoczko et
al., 2010).
1.8.2 TRIGLYCERIDES
It
is an ester derived from glycerol and 3 fatty acids. It is a main constituent
of vegetable oil and animal fats (Jonker et
al., 2003). Triglycerides are a major component of very low density
lipoproteins and chylomicrons, and they play a role in metabolism as energy
source and transporters of dietary fats.
High level of triglycerides in the blood stream has been linked to atherosclerosis
and by extension, the risk of heart disease and stroke (Jonker et al., 2002).
1.9 DENNETIA TRIPETALA
Dennettia
tripetala also known as pepper fruit tree is a well-known Nigerian spicy
medicinal plant. It is found in the tropical rainforest region of Nigeria
and sometimes in Savanna areas (Okwu et
al., 2005). It is locally called “Nkarika” by the Efiks of
Calabar. The young leaves and fruits have dinstinctive spicy taste.
The mature fruits constitute the main edible portions. Some communities
in parts of Southern Nigeria also utilize the leaves and roots, in addition to
the fruits for medicinal purpose. Dennettia
tripetala is used as
masticators, which when chewed produces unique peppery effect. The peppery
spicy taste of mature Dennettia tripetala fruits usually serves as a mild stimulant
to the consumer.
1.9.1
METHOD OF PROPAGATION
Dennettia
tripetala is propagated by direct seedling and use of root
suckers. Dennettia tripetala is also
propagated by natural regeneration.
1.9.2 ETHNOMEDICINAL USES OF DENNETIA TRIPETALA
Dennettia
tripetala is commonly known as pepper fruit by the English,
“mmimi” by the Igbos, “Nkaika” by the Ibibio and Efik, “Imako” by the Urhobo
tribe of the Niger-Delta region, and “Igberi” by the Yorubas.). Dennettia tripetala (pepper fruit) is a
medium sized tree found commonly in the tropical rainforest region of Nigeria
and sometimes in savannah areas. Dennettia
tripetala, or pepper fruit tree, is a well-known Nigerian spicy medicinal
plant. It is an indigenous medium sized or small woody shrub. It is commonly
found in the rain-forest and occasionally in the savanna. (Timothy and Okere, 2008). The tree grows up
to 12m-15m in height and 0.6m in girth, with a dense compact crown. The wood is
soft, white coloured and prone to termite attack. It has a fibrous bark which
has a strong scent. The leaves are 3 – 6 inches long by 1.5–2.5 inches broad,
elliptic to ovate, shortly acuminate broadly connate to rounded at the base.
The flowers are light brown outside, reddish inside and usually in small
clusters on the young or older wood. The fruits are green at first but
eventually turn reddish pink when ripe with finger-like carpel constricted
between the seeds. The fruits are edible and rich in vitamin C , both the
fruits and young leaves have a distinctive spicy taste, the bark of Dennettia
tripetala fruits is mixed with food to create variation in the taste and
flavour of different foods. (Okafor, 1980). It has been reported that the peppery fruits
of Dennettia tripetala usually find
application in food meant for pregnant women. Dennettia tripetala fruit contains several nutrients and
biologically active components that prolong and enhance life. It is a good
source of ascorbic acid, riboflavin, thiamine and niacin. Therefore, this fruit
is nutritionally necessary for a well-balanced diet because it contributes
important vitamins such as vitamin C (ascorbic acid) which can be used for the
treatment of the common cold and the control of other diseases such as prostate
cancer. The bark of the tree is mixed
with food to create variation in the taste and flavour of different food .The
leaves are used in folk medicine for the treatment of fever, cough, asthma,
catarrh, toothache, diarrhoea and rheumatism the fruit reduces the risk of
blindness caused by glaucoma.
1.9.3 BOTANICAL DESCRIPTION OF DENNETIA
TRIPETALA
Fig 1 Fruits of Dennettia. tripetala
Dennettia
tripetala (pepper fruit) is an indigenous fruit tree of the
family Annonaceae (Etukudo, 2000). It is a medium- sized or
small tree which spreads throughout the rain forest and sometimes found in
forest within the Savanna areas matured fruits constitute the main edible
portion. The leaves, fruit, bark and root of the plants possess strong peppery
and pungent spicy taste with a characteristic aroma and fragrance. The young
leaves and fruits have instinctive spicy taste. The fruits are chewed in
different forms (fresh green, fresh ripened red, black dry fruit and dry seed). The bark is smooth to roughly
scaly, grey to brown, often with some distinct purple layers.
1.9.4 COMMERCIAL / ECONOMIC
IMPORTANCE OF DENNETTIA TRIPETALA
The parts used include the leaves, fruits, seeds,
roots and stem. A survey of existing literatures shows that pepper fruits
contain essential oils and phenolic acids, ethanol, alkaloid, ethyl acetate,
flavonoids, tannins and glycosides. Indications show that the rich presence of
a type of Dennettia essential oil
called oleoresins determines the aromatic flavouring, colouring and pungent
properties of Dennettia tripetala.
Medicinally, the leaves and fruits are used for cough treatment and enhancing
appetite. In Igbo land, the fruits and seed are signs of hospitality for
visitors. The wood is white and soft which yields a good fuel wood. The fruit tree is a tropical tree common in
the mangrove forests of the west coast of Africa. The fruit of Dennettia tripetala is quite popular in
Southern Nigeria where it serves for cultural entertainment of guests,
particularly during coronation, the new yam festival and marriage ceremonies.
1.9.5
NUTRITIONAL EVALUATION OF UNRIPE AND RIPE PEPPER FRUIT (DENNETTIA TRIPETALA)
The nutritional evaluation of unripe Dennettia
tripetala in percentage wet basis (Table 1) revealed protein (6.59%),
moisture content (15.26%), fat (5.52%), ash (4.13%), fiber (17.05%) and
carbohydrate (51.45). Its mineral content comprises of calcium (181.69mg/g),
magnesium (229.78mg/g), iron (0.2mg/g), phosphorus (285.8mg/g), potassium
(360.8mg/g) and Sodium (6.12m/mg). The vitamins include ascorbic acid
(85.65mg/g), niacin (0.40mg/g), thiamine (0.10mg/g), riboflavin (0.05mg/g) and
vitamin A (65.58mg/g) while ripe Dennettia tripetala showed protein
(4.67%), moisture content (18.73%), fat (5.78%), Ash (3.18%), fiber (14.32%)
and carbohydrate (53.32%). Evaluation of the ripe pepper fruit for its
mineral content indicated calcium (138.94mg/g), magnesium (173.68mg/g),
iron (0.23mg/g), phosphorus (243.8mg/g), potassium (324.27mg/g) and
sodium (5.47mg/g). The study also revealed ascorbic acid (115.57mg/g), niacin
(0.37mg/g), thiamine (0.08mg/g), riboflavin (0.05mg/g) and vitamin A
(388.10mg/g). (Okwu and Morah, 2004).
Table 1. Proximate composition
Dennettia Tripetala (Pepper fruit)
% Samples
Nutrient Unripe Ripe
Moisture content 15.26 ± 0.07 18.73 ± 0.02
Protein 6.59 ± 0.08 4.67 ± 0.08
Fat 5.52 ± 0.3 5.78 ± 0.08
Ash 4.13 ± 0.02 3.18 ± 0.03
Fiber 17.05 ± 0.7 14.32 ± 0.3
Carbohydrate 51.45 ± 0.015 53.32 ± 0.02
(Values
are means of triplicate analysis)
(Ihemeje et al., 2013)
The
unripe pepper fruit (Dennettia tripetala) showed higher value in protein
(6.59%), ash (4.13%) and fiber (17.05%) than the ripe Dennettia tripetala which
had protein (4.67%), ash (3.18%) and fiber (14.32%). Ripe Dennettia
tripetala showed high moisture content (18.73%), fat (5.78%) and
carbohydrate (53.32%) than the unripe. Increased moisture content in a food
material increases the chances of microbial attack. This justifies why ripe Dennettia
tripetala is more prone to microbial spoilage than the unripe. The high ash
content of unripe pepper fruit is a reflective of its greater mineral content
than the ripe (Table 2) which makes it important for little children. Minerals
enhance the important functions of maintaining acid-base balance and proper
osmotic pressure in the body (Onimawo and Egbekun, 1998). Minerals are required
for normal functioning of the nerves and also muscular contraction and
relaxation. Calcium and phosphorus are highly required by growing children,
pregnant women and nursing mothers (Norman and Joseph, 2006). Hence Dennettia
tripetala could be a fair and cheap source of these essential minerals.
TABLE
2. MINERAL COMPOSITION OF DENNETTIA
TRIPETALA (PEPPER FRUIT)
Mineral (mg/g) Unripe Ripe
Ca 181.69 ± 1.89 138.94 ± 1.89
Mg 229.78 ± 2 173.68 ± 6
Fe 0.27 ± 0.06 0.23 ± 0.05
P 285.8 ± 0.1 243.8 ± 0.2
K 360.8 ± 2 324.27 ± 0.4
Na 6.12 ± 0.03 5.47 ± 0.02
Mean of triplicate
experiment
(Ihemeje et al., 2013)
The
result also showed that ripe Dennettia tripetala contains higher vitamin
C and A than the unripe (Table 3). The increase in vitamin could be attributed
to the effect of ripening. This corresponds with the work of (Adebayo et
al., 2010) on changes in the total phenol content and antioxidant
properties of pepper fruit (Dennettia tripetala) with ripening.
TABLE
3. VITAMIN COMPOSITION OF DENNETTIA
TRIPETALA (PEPPER FRUIT)
Vitamin (mg/g) Unripe Ripe
Vitamin C 85.65 ± 0.82 115.5 ± 0.82
Niacin 0.40 ± 0.01 0.37 ± 0.01
Thiamine 0.10 ± 0.03 0.08 ± 0.01
Riboflavin 0.05 ± 0.02 0.05 ± 0.02
Vitamin A 65.58 ± 0.29 388.10 ± 0.38
Mean of triplicate values
(Ihemeje et al., 2013)
1.9.6
ANTI-NUTRITIONAL EVALUATION OF UNRIPE AND RIPE PEPPER FRUIT (DENNETTIA TRIPETALA)
The
anti-nutritional evaluation of pepper fruit (Table 4) revealed that unripe Dennettia
tripetala contains phenol(1.2mg/g), saponin (0.27mg/g), tannin(0.36mg/g),
flavonoid (3.10mg/g) and alkaloid(0.33mg/g) while the ripe D. tripetala
showed phenol (1.6mg/g), saponin (0.15mg/g), tannin (0.18mg/g),
flavoniod (1.2mg/g) and alkaloid (0.14mg/g).
Unripe D.tripetala had higher saponin, tannin, flavonoid and alkaloid
than the ripe while the ripe had higher phenol content (1.6mg/g) than the
unripe (1.2mg/g) pepper fruit (Table 4).
The
significant difference (p<0.05) in phenol content of ripe and unripe pepper
fruit may be due to the physiological changes that accomplish ripening which
brings about changes in pigments (Oboh et
al.,2007; Materska and Perucka, 2005). This result corresponds with the
study of Adebayo et al., (2010).
There was significant difference (P<0.005) in flavonoid content of the
unripe (3.10mg/g) and ripe (1.25mg/g) pepper fruit in the study while the study
of (Adebayo et al., 2010) revealed no significant difference between the
unripe (0.1m/g) and ripe (0.1mg/g). Flavonoids are the class of widely
distributed phytochemicals with antioxidant and biological activity. They are
constituents of plant foods that have been implicated in the reduction of
cancer risk (Wolfe and Liu, 2008). In the zutphen elderly study, flavonoid
intake from fruits and vegetables was inversely associated with all-cause
cancer risk and cancer of alimentary and respiratory tracts
(Hertog
et al., 1994). Many other epidemiological studies have shown a trend of
decreased cancer risk with high flavonoid consumption (Neuhouser, 2004; Graf et
al., 2005).
TABLE 4. ANTI-NUTRIENT COMPOSITION OF RIPE AND
UNRIPE DENNETTIA TRIPETALA
Antinutrient Unripe Ripe
Phenol 1.2 ± 0.16 1.6 ± 0.16
Saponin 0.27 ± 0.01 0.15 ± 0.01
Tannin 0.18 ± 0.005 0.36 ± 0
Flavonoid 3.10 ± 0.04 1.25 ± 0.01
Alkaloid 0.33 ± 0.01 0.14 ± 0.02
Mean of triplicate analysis
(Ihemeje
et al., 2013)
JUSTIFICATION
Dennettia tripetala is a well-known forest fruit and
spicy indigenous plant and has been found to contain several
antioxidants which may be helpful in combating the induced hepatic damage which
results from exposure to toxic chemicals such as carbon tetrachloride.
AIM AND OBJECTIVE
The study is aimed at investigating the
effect of ethanol extracts of Dennettia
tripetala on carbon tetrachloride induced rats.
The main objectives of the study
include:
1.
Determination
of antioxidant enzyme levels
2. Determination of malondialdehyde
concentrations
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