INTRODUCTION- Liver diseases are considered to be serious health disorders. The
liver has one of the highest value of importance for the systemic
detoxification and deposition of endogenous and exogenous substances. Liver
dysfunction challenges not only health care professionals but also the
pharmaceutical industry and drug regulatory agencies. Drug-induced liver injury
(DILI) possesses a major clinical problem. DILI has become the leading cause of
acute liver failure and transplantation in Western countries. Hepatotoxicity
associated with most other drugs are idiosyncratic, which implies by definition
that DILI develops in only a small proportion of subjects exposed to a drug in
therapeutic doses [1]. The risk of acute liver failure associated with
idiosyncratic hepatotoxin is usually less than 1 per 10000 exposed patients.
However, more than 1000 drugs and herbal products have been associated with
idiosyncratic hepatotoxicity.
Diclofenac is Non-steroidal
anti-inflammatory drugs (NSAIDs) which are the centerpiece of pharmacotherapy
for most rheumatological disorders, and are used in large numbers as analgesics
and antipyretics, both as prescription drugs and over the counter purchases. It is the most important cause of the
drug induced toxic injury to several organ systems, including well known injury
to gastrointestinal tract, liver, heart and kidneys [2]. Diclofenac
cardiotoxicity is an archetype of idiosyncratic DIHI (Drug induced heart
injury) [2]. About 15% of those
patients regularly taking diclofenac develop elevated levels of heart enzymes,
and a threefold rise in transaminase levels has been reported in 5%. Diclofenac
is associated with a predominantly hepatocellular and cardiovascular pattern of
liver and heart injury.
Products of higher plant origin have been known to be effective sources
of chemotherapeutic agents without any underlying effects. Plants continue to
be a major source of medicines, as they have been throughout human history. The
use of medicinal plants all over the world predates the introduction of
antibiotics and other modern drugs [3,4]. A medicinal plant is any
plant with one or more of its organs containing substances that can be used for
therapeutic purposes or which are the precursors for the synthesis of useful
drugs [5]. They are of great importance to the health of individuals
and communities; the medicinal values of certain plants lie in some chemical
substances that produce definite physiological action on the human body [6].
The most important of these bioactive constituents of plants are flavonoids,
tannins, alkaloids and foods plants sometimes added to foods. Mistletoe
is a general term for woody shoot parasites in several plant families,
especially in Loranthaceae and Viscaceae families [6]. The
common European mistletoe grows on various trees, usually apples and junipers.
It is an evergreen plant with small, greenish flowers and berries. Similar
mistletoe, American mistletoe, found in the United States, grows on deciduous
trees, particularly red marple elm, from eastern Texas to Florida and northward
to Missouri and New Jersey [6]. The leafless flowering dwarf mistletoes depend
entirely on the host tree for nourishment. These scrubs are lethal parasites of
conifers, such as pine, spruce, fir and hemlock. The plant leaves and berries
contain toxic chemicals that can be poisonous and the plant should be kept out
of reach of young children who may be tempted to eat the berries [7,8]. Carica
papaya L., (pawpaw),
belongs to the family of Caricaceae. Papaya
is not a tree but an herbaceous succulent plant that possess self-supporting
stems [9]. Papaya is a large perennial herb with a rapid growth
rate. The plants are usually short-lived, but can produce fruit for more than
20years. The papaya has a rather complicated means of reproduction. The plants
are male, hermaphrodite, or female [9]. The male trees are uncommon,
but sometimes occur when homeowners collect their own seeds. Hermaphrodite
trees (flowers with male and female parts) are the commercial standard,
producing a pear shaped fruit. These plants are self-pollinated. Carica
papaya plants produce natural compounds (annonaceous acetogenins) in leaf
bark and twig tissues that possess both highly anti-tumor and pesticidal
properties. It was suggested that a potentially lucrative industry based simply
on production of plant biomass could be developed for production of anti-cancer
drugs, pending Food and Drug Agency approval, and natural (botanical)
pesticides [10]. The high level of natural self-defence compounds in
the tree makes it highly resistant to insect and disease infestation. Carica
papaya L. leaf tea or
extract has a reputation as a tumor-destroying agent. The papaya fruit, as well
as all other parts of the plant, contain a milky juice in which an active
principle known as papain is present. Aside from its value as a remedy in
dyspepsia and kindred ailments, it has been utilized for the clarification of
beer. The juice has been in use on meat to make it tender. The seed is used for
intestinal worms when chewed. The root is chewed and the juice swallowed for
cough, bronchitis, and other respiratory diseases. The unripe fruit is used as
a remedy for ulcer and impotence [11]. Fresh, green pawpaw leaf is
an antiseptic, whilst the brown, dried pawpaw leaf is the best as a tonic and
blood purifier. Chewing the seeds of ripe pawpaw fruit also helps to clear
nasal congestion, [12]. The
green unripe pawpaw has a therapeutic value due to its antiseptic quality. It
cleans the intestines from bacteria, more so that (only a healthy intestine is
able to absorb vitamin and minerals, especially vitamin B12). The tea, prepared
with the green papaya leaf, promotes digestion and aids in the treatment of ailments such as chronic indigestion,
overweight and obesity, arteriosclerosis, high blood pressure and weakening of
the heart [13]. In view of the above, the present study was
planned to determine the antioxidant
and hepatoprotective activity of papaw leaf and cocoa mistletoes against diclofenac-induced
hepatotoxicity in Rats.
MATERIALS AND METHODS
Collection
of Samples- The cocoa mistletoes plants and dry
pawpaw leaf were obtained from Ifaki local farm Ekiti State, Nigeria. Some
parts of the plants were taken to the Department of Plant Science in Ekiti
State University, Ado-Ekiti, Ekiti State, Nigeria for identification/ Authentication.
Experimental Protocol- The
study was performed on fourty (40) wistar albino rats (all males) housed in
ventilated cages in the Animal House of College of Medicine, Ekiti State
University, Ado-Ekiti, Nigeria. They were acclimatized for two weeks before
administration of the drugs. Animals were divided into ten groups of four rats
each as follows
Group
1 Positive Control (Normal) animals
Group
2 Negative control (50mg/kg
Diclofenac treated) animals
Group
3 Negative control (100mg/kg
Diclofenac treated) animals
Group
4 Negative control (150mg/kg
Diclofenac treated) animals
Group
5 50mg/kg Diclofenac treated animals + mistletoes extract
Group
6 100mg/kg Diclofenac treated
animals + mistletoes extract
Group
7 150mg/kg Diclofenac treated
animals + mistletoes extract
Group
8 50mg/kg Diclofenac treated
animals + dry pawpaw leaf
Group
9 100mg/kg Diclofenac treated
animals + dry pawpaw leaf
Group
10 150mg/kg Diclofenac treated
animals + dry pawpaw leaf
Chemicals/ Reagent kits-
All chemicals and drugs used were obtained commercially and of analytical
grade. All the diagnostic kits are products of Fortress Chemical Ltd. United
Kingdom.
Preparation
of Organs homogenate- The animals were quickly dissected,
the organs was removed. 10% of each organs homogenate was then prepared in 6.7
mM potassium phosphate buffer, (pH 7.4) using the Teflon homogenizer. The
homogenate was centrifuged at 10,000rpm for 10 minutes at 40C to
obtain a clear supernatant which was stored at 80C and used for
measurement of biochemical contents.
Biochemical Assay- Standard Fortress kits were used to determine
Cholesterol, Total protein, Alkaline Phosphatase (ALP), Aspartate Transaminase
(AST) and Alkaline Transaminase.
Determination
of Alanine aminotransferase
GPT
∝-oxoglutarate + L-alanine
→ L-glutamate + pyruvate
Alanine
aminotransferase is measured by monitoring the concentration of pyruvate
hydrazone formed with 2, 4-dinitrophenylhydrazine.
Determination
of AST
GOT ∝-oxoglutarate
+ L-
aspartate → L-glutamate + oxaloacetate
AST is measured by the monitoring
the concentration of oxaloacetate hydrazone formed with 2,
4-dinitrophenylhydrazine.
Determination
of ALP- Plasma alkaline phosphatase (ALP) was determined by
kinetic method according to Abubakar et al. [13].
Determination
of Plasma Malondialdehyde (MDA)- Lipid
peroxidation was determined by measuring the formation of thiobarbituric acid
reactive substances (TBARS). An aliquot of 0.4 mL of the plasma or other organ
homogenates was mixed with 1.6 mL of Tris-KCl buffer to which 0.5 mL of 30%
trichloroacetic acid (TCA) was added. Then 0.5mL of 0.75% TBA was added and
placed in a water bath for 45 minutes at 80oC. This was then cooled
on ice and centrifuged at 3000g. The
clear supernatant was collected and absorbance measured against a reference
blank of distilled water at 532nm. The MDA level was calculated. Lipid
peroxidation in units/mg protein or gram tissue was computed with a molar
extinction coefficient of 1.56 x 105 M-1 cm 1.
MDA
(units/mg protein)= Absorbance x volume of mixture /E532 nm x volume
of sample x mg protein
Determination of Catalase activity of samples- This experiment was carried out using calorimetrically. This
method is based on the reduction of dichromate in acetic acid to chromic
acetate when heated in the presence of H2O2, with the
formation of perchromic acid as an unstable intermediate. The chromic acetate
so produced is measured calorimetrically at 570-610 nm. However, the dichromate
has no absorbance at the wavelength and its presence in the assay mixture does
not interfere with the determination. The catalase preparation is allowed to
split H2O2 for different periods of time. The reaction is
stopped at a particular time by the addition of dichromate/acetic acid mixture
and the remaining H2O2 is determined by measuring chromic
acetate calorimetrically after heating the reaction mixture.
Calculation- The mononuclear velocity constant, K, for the decomposition of H2O2
by catalase was determined by using the equation for a first-order reaction: K=
1/t log S0/S
Where S0 is the
initial concentration of H2O2 and S is the concentration
of the peroxide at t min. The values of the K are plotted against time in
minutes and the velocity constant of catalase K(0) at 0 min
determined by extrapolation.
The catalase contents of the enzyme preparation were expressed in
terms of Katalasefeiahigkeit or ‘Katf’.
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Katf =
K (0)
/ mg protein/ ml
STATISTICAL ANALYSIS: All values are presented in Tables
and the appropriate comparisons between groups were made using Student’s-test.
The difference between the groups is taken to be significant at P<0.05.
RESULTS-
Fig. 1 represents the effect of pawpaw leaf
and cocoa mistletoes on the catalase activity
in plasma, heart and liver of diclofenac-induced rat. A significantly lower
(P<0.05) plasma and liver levels
of Catalase was observed in
diclofenac-induced rats compare to normal group but when treated with the
pawpaw leaf and mistletoes extracts, the catalase activity was increased
towards to normal proportion.
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Fig. 1: Effects
mistletoes and pawpaw leaf treatment on the catalase activity in plasma, and liver of diclofenac-induced rat
Fig. 2 represents the effect of
pawpaw leaf and cocoa mistletoes on the malondialdehyde concentration in plasma
and liver of diclofenac-induced rat. A significantly higher (P>0.05) plasma,
heart and liver levels of
malondialdehyde was observed in diclofenac-induced rats when compare to normal group but when treated
with the pawpaw leaf and mistletoes extracts, the malondialdehyde concentration
was decreased towards to normal level.
.jpg)
Fig. 2: Effects Mistletoes and pawpaw leaf treatment
on the Malonaldehyde
concentration in plasm and liver of diclofenac-induced rat
Administration of diclofenac produced
significant adverse effects on the functionalities of liver and plasma, which
is evidenced by a significant elevation in the actions of ALT, AST, and ALP
enzymes in (negative group) diclofenac-induced rats group compared to (Normal
control) normal. Treatment of cardiotoxic and hepatotoxic rats with pawpaw
leaf and mistletoes
exhibited improvement in the actions of ALT, AST and ALP enzymes compared to
(negative control) rats (Fig. 3 to Fig. 5).
Fig.
5: Effects of mistletoes and pawpaw leaf on treatments on the alanine
transaminase activity in plasma of diclofenac-induced rat
DISCUSSION-
However, Fig. 1 above showed
the effects of aqueous extract of mistletoes and pawpaw leaf
treatment on the catalase activity
in plasma and liver of diclofenac-induced rat. Free radical scavenging
enzyme,such as CAT, are the first line of defense against oxidative injury. The
H2O2 formed by SOD and other processes is scavenged by
catalase that catalyzed the dismutation of H2O2 into
water and molecular oxygen. Thus, the antioxidant enzyme catalase is
responsible for detoxification of H2O2. The catalase
enzyme may also be released into the extracelular environment in which it has
the potential to function as a potent antioxidant and thereby regulated cell
survival [14]. In light of these considerations, it seems plausible
that extracellular catalase might function as an important autocrine
antioxidant and survival factor. A large number of the metabolites produced by NSAIDs are found
to generate superoxide anion and other free radicals in the biological systems [15].
However, at a higher dose of diclofenac (150 mg/kg) and consistent using of
diclofenac, intermediate metabolites accumulate and cause liver damage. The
reduction in catalase activity after induction with diclofenac is another
significant finding in this study. The decreased concentration of plasma
catalase is attributable in part to the reduced synthesis of this antioxidant
enzyme (which functions in the detoxification of hydrogen perioxide) whose
concentrations would have fallen with the diclofenac that was injected into the
animals. Though some studies have reported that no alterations in the activity
of red cell catalase. However, this study is in agreement with earlier reports
by Redmond and Redmond
[16], who reported a decreased in red blood cell catalase activity
cardiovascular and hepato-cellular disorder. While treatment with Pawpaw leaf and mistletoes extracts caused significant increase in catalase activity
in their respective groups due to bioactive compounds present in the plants
which aid the activities of the antioxidant enzymes which is in agreement with
the previous study and
can also explain the increased detoxification of the reactive metabolites
generated from the diclofenac metabolism in the liver of plant extracts treated animals.
In present studies, the Pawpaw
leaf and mistletoes co-administered rats showed
significantly decreased levels of these lipids peroxidation markers as compared
with diclofenac-induced rats.
This present study is accordance with the previous study of Sarker et
al. [17], which they found that pawpaw leaf and mistletoes
respectively contain substances that delay the rate of oxidation by directing
the breakdown of peroxides into stable substances that do not promote further
oxidation or by sweeping free radicals away. Damaged membranes were
recovered by the treatment with plans extracts by
enhancing antioxidants' status and decreasing lipid peroxidation [18].
The inhibition of lipid peroxidation by Pawpaw leaf and mistletoes extracts,
therefore, may be one of the mechanisms by which the plants exert their
protection against diclofenac sodium mediated tissue injury.
Estimating
the activities of serum marker enzymes, like AST, ALT, and ALP, can make
assessment of liver function. When liver cell plasma membrane is damaged, a
variety of enzymes normally located in the cytosol are released in to the blood
stream. Their estimation in the serum is a useful quantitative marker of the
extent and type of hepatocellular damage [19,20]. The enhanced
activities of these serum marker enzymes observed in diclofenac sodium treated
rats in present study correspond to the extensive liver damage induced by
diclofenac sodium. The tendency of these enzymes to return towards a near
normal level in group I (Normal) rats is a clear manifestation of
anti-hepatotoxic effect of mistletoes and dry pawpaw leaf. Consistent and high
dosage administration of diclofenac in this
study produced significant adverse effects on the organs functions of the rats
such as liver, heart and kidney, which is evidenced by a significant increase
in the actions of ALT, AST, and ALP enzymes in (negative groups)
diclofenac-induced rats group compared to (control) normal. Similar
result has also been recorded that
stated that when enzymes leak from the liver cytosol into the blood stream, it
lead to higher level of ALP in serum is an indicative of hepatotoxic of
diclofenac-induced rats [20]. High levels of ALP in serum indicate
liver damage which is similar to this present study where, the increase in ALP
activity in diclofenac-induced rats shows the liver damage, as a result of
metabolic changes such as administration of toxin, liver cirrhosis, hepatitis,
and cancer of the liver [20]. Thus, it can be used involved as
markers to estimate the extent of liver damage. Also Aminotransferases (ALT and AST)
occupy a central position in the metabolism of amino acids as they help in
retaining amino groups (to form new ones) during degradation of amino acids.
They are also involved in the biochemical regulation of amino acid pool and in
providing necessary intermediate to predict possible toxicity in some organs
such as kidney cytolysis and the heart and liver of animals [21], which
similar result occurred this present study. The measurement of the activities
of these enzymes is of clinical and toxicological significance. Since the
enzymes were only being released into cellular flow when organs are damaged,
the reduction in the levels of these enzymes in the plasma of the animals
maintained on the dry pawpaw leaf and mistletoes extracts
treated groups.
CONCLUSION-
The oxidative
stress has been
imposed due to
the imbalance of biochemical processes that involves the
generation of reactive oxygen and nitrogen species (ROS and RNS) and their
neutralization by the inherent antioxidant (enzymatic or non-enzymatic) defense
system of the cells. Mistletoes and pawpaw leaf are one of the most abundant
natural antioxidants with variety of applications. The present study shows that
the Hepatotoxicity in rats treated with diclofenac was markedly reduced by
simultaneous administration of mistletoes and pawpaw leaf extract. The results
of this study show protective effect of mistletoes and
pawpaw leaf on plasma and liver function which might be due to the presence of
some bioactive compound in the extract. Further investigation should be
conducted to identify this bioactive compounds present in mistletoes.
ACKNOWLEDGMENTS-
This study was supported by all the
authors financially, morally, and physically.
CONTRIBUTION
OF AUTHORS- Substantial contributions to the design
of the work were done by Oseni AO and Odesanmi EO, then the collection Data by
Odesanmi EO, while Data analysis and interpretation for the work were done by
Odesanmi EO, Also Drafting of the article was carried out by Oloyede OI,
Critical revision of the article for important intellectual content was done by
Adebayo OD and Ogundare MAB, and Final approval of the version to be published
was done by Oseni AO and Oloyede OI.
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