Antioxidant Hepatoprotective Activities Carica Papaya Loranthus Bengwensis Diclofenac Induced Hepatotoxicity PDF

Int. J. Life. Sci. Scienti. Res.
eISSN: 2455-1716
Oseni et al., 2018
DOI:10.21276/ijlssr.2018.4.5.3
Research Article
Antioxidant and Hepatoprotective Activities of Carica papaya

(Papaw Leaf) and Loranthus bengwensis (Cocoa Mistletoes) against
Diclofenac Induced Hepatotoxicity in Rats
2 1 1 1 1
Oseni OA , Odesanmi EO *, Oloyede OI , Adebayo OD , Ogundare MAB
1
Department of Biochemistry, Faculty of Science, Ekiti-State University, Ado-Ekiti, Nigeria
2
Department of Medical Biochemistry, College of Medicine, Ekiti-State University, Ado-Ekiti, Nigeria
*Address for Correspondence: Mr. Odesanmi Elijah Olalekan, Department of Biochemistry, Faculty of Science,
Ekiti-State University, Ado-Ekiti, Nigeria
Received: 29 Apr 2018/ Revised: 28 Jun 2018/ Accepted: 14 Aug 2018
ABSTRACT
In this study, the effect of diclofenac sodium induced liver damage in rats was investigated, biochemically. Damaging effects of
reactive oxygen species (ROS) on living systems are well documented, they include oxidative attack on vital cell constituents.
Administration of diclofenac sodium at different concentrations (50 mg/kg, 100 mg/kg, and 150 mg/kg/d) for 14 days produced
severe liver injury, as demonstrated by dramatic elevation of plasma hepato specific markers like aspartate amino transaminase
(AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP). In addition, diclofenac sodium administration caused
oxidative stress in rats, as evidence by increased ROS production and malondialdehyde (MDA) concentrations in the liver of rats,
along with a remarkable reduction in hepatic catalase (CAT) activity. However, simultaneous treatment with aqueous extract of
dry pawpaw leaf (30%) and cocoa mistletoes (30%) significantly attenuated diclofenac sodium induced hepatotoxicity. The results
showed that serum marker enzymes and hepatic MDA content as well as ROS production were reduced dramatically and CAT
activity content were restored remarkably when treated with the extracts, as compared to the diclofenac sodium treated rats. It
is, therefore suggested that dry pawpaw leaf and cocoa mistletoes employed in this study can provide a definite protective effect
against acute hepatic injury caused by diclofenac sodium in rats, which may mainly be associated with its antioxidative effect.
Key-words: Diclofenac, oxidative stress, Hepatotoxicity, Antioxidant, Drug-induced liver injury
INTRODUCTION
Liver diseases are considered to be serious health Hepatotoxicity associated with most other drugs are
disorders. The liver has one of the highest value of idiosyncratic, which implies by definition that DILI
importance for the systemic detoxification and develops in only a small proportion of subjects exposed
deposition of endogenous and exogenous substances. to a drug in therapeutic doses [1]. The risk of acute liver
Liver dysfunction challenges not only health care failure associated with idiosyncratic hepatotoxin is
professionals but also the pharmaceutical industry and usually less than 1 per 10000 exposed patients. However,
drug regulatory agencies. Drug-induced liver injury (DILI) more than 1000 drugs and herbal products have been
possesses a major clinical problem. DILI has become the associated with idiosyncratic hepatotoxicity.
leading cause of acute liver failure and transplantation in Diclofenac is Non-steroidal anti-inflammatory drugs
Western countries. (NSAIDs) which are the centerpiece of pharmacotherapy
for most rheumatological disorders, and are used in large
How to cite this article
Oseni OA, Odesanmi EO, Oloyede OI, Adebayo OD, Ogundare MAB. numbers as analgesics and antipyretics, both as
Antioxidant and Hepatoprotective Activities of Carica papaya prescription drugs and over the counter purchases. It is
(Papaw Leaf) and Loranthus bengwensis (Cocoa Mistletoes) against
Diclofenac Induced Hepatotoxicity in Rats. Int. J. Life. Sci. Scienti.
the most important cause of the drug induced toxic
Res., 2018; 4(5): 1974-1982. injury to several organ systems, including well known
injury to gastrointestinal tract, liver, heart and kidneys [2].
Access this article online Diclofenac cardiotoxicity is an archetype of idiosyncratic
www.ijlssr.com DIHI (Drug induced heart injury) [2]. About 15% of those
patients regularly taking diclofenac develop elevated
Copyright © 2015 - 2018| IJLSSR by Society for Scientific Research under a CC BY-NC 4.0 International License Volume 04 | Issue 05 | Page 1974
Int. J. Life. Sci. Scienti. Res. eISSN: 2455-1716
Oseni et al., 2018
DOI:10.21276/ijlssr.2018.4.5.3
levels of heart enzymes, and a threefold rise in shaped fruit. These plants are self-pollinated. Carica
transaminase levels has been reported in 5%. Diclofenac papaya plants produce natural compounds
is associated with a predominantly hepatocellular and (annonaceous acetogenins) in leaf bark and twig tissues
cardiovascular pattern of liver and heart injury. that possess both highly anti-tumor and pesticidal
Products of higher plant origin have been known to be properties. It was suggested that a potentially lucrative
effective sources of chemotherapeutic agents without industry based simply on production of plant biomass
any underlying effects. Plants continue to be a major could be developed for production of anti-cancer drugs,
source of medicines, as they have been throughout pending Food and Drug Agency approval, and natural
human history. The use of medicinal plants all over the (botanical) pesticides [10]. The high level of natural self-
world predates the introduction of antibiotics and other defence compounds in the tree makes it highly resistant
modern drugs [3,4]. A medicinal plant is any plant with to insect and disease infestation. Carica papaya L. leaf
one or more of its organs containing substances that can tea or extract has a reputation as a tumor-destroying
be used for therapeutic purposes or which are the agent. The papaya fruit, as well as all other parts of the
precursors for the synthesis of useful drugs [5]. They are plant, contain a milky juice in which an active principle
of great importance to the health of individuals and known as papain is present. Aside from its value as a
communities; the medicinal values of certain plants lie in remedy in dyspepsia and kindred ailments, it has been
some chemical substances that produce definite utilized for the clarification of beer. The juice has been in
physiological action on the human body [6]. The most use on meat to make it tender. The seed is used for
important of these bioactive constituents of plants are intestinal worms when chewed. The root is chewed and
flavonoids, tannins, alkaloids and foods plants the juice swallowed for cough, bronchitis, and other
sometimes added to foods. Mistletoe is a general term respiratory diseases. The unripe fruit is used as a remedy
for woody shoot parasites in several plant families, for ulcer and impotence [11]. Fresh, green pawpaw leaf is
especially in Loranthaceae and Viscaceae families [6]. The an antiseptic, whilst the brown, dried pawpaw leaf is the
common European mistletoe grows on various trees, best as a tonic and blood purifier. Chewing the seeds of
usually apples and junipers. It is an evergreen plant with ripe pawpaw fruit also helps to clear nasal congestion,
[12]
small, greenish flowers and berries. Similar mistletoe, . The green unripe pawpaw has a therapeutic value
American mistletoe, found in the United States, grows on due to its antiseptic quality. It cleans the intestines from
deciduous trees, particularly red marple elm, from bacteria, more so that (only a healthy intestine is able to
eastern Texas to Florida and northward to Missouri and absorb vitamin and minerals, especially vitamin B12).
New Jersey [6]. The leafless flowering dwarf mistletoes The tea, prepared with the green papaya leaf, promotes
depend entirely on the host tree for nourishment. These digestion and aids in the treatment of ailments such as
scrubs are lethal parasites of conifers, such as pine, chronic indigestion, overweight and obesity,
spruce, fir and hemlock. The plant leaves and berries arteriosclerosis, high blood pressure and weakening of
contain toxic chemicals that can be poisonous and the the heart [13]. In view of the above, the present study was
plant should be kept out of reach of young children who planned to determine the antioxidant and
may be tempted to eat the berries [7,8]. Carica papaya L., hepatoprotective activity of papaw leaf and cocoa
(pawpaw), belongs to the family of Caricaceae. Papaya is mistletoes against diclofenac-induced hepatotoxicity in
not a tree but an herbaceous succulent plant that Rats.
possess self-supporting stems [9]. Papaya is a large
perennial herb with a rapid growth rate. The plants are MATERIALS AND METHODS
usually short-lived, but can produce fruit for more than Collection of Samples- The cocoa mistletoes plants and
20years. The papaya has a rather complicated means of dry pawpaw leaf were obtained from Ifaki local farm Ekiti
reproduction. The plants are male, hermaphrodite, or State, Nigeria. Some parts of the plants were taken to
female [9]. The male trees are uncommon, but sometimes the Department of Plant Science in Ekiti State University,
occur when homeowners collect their own seeds. Ado-Ekiti, Ekiti State, Nigeria for identification/
Hermaphrodite trees (flowers with male and female Authentication.
parts) are the commercial standard, producing a pear
Oseni et al., 2018
DOI:10.21276/ijlssr.2018.4.5.3
Experimental Protocol- The study was performed on Biochemical Assay- Standard Fortress kits were used to
fourty (40) wistar albino rats (all males) housed in determine Cholesterol, Total protein, Alkaline
ventilated cages in the Animal House of College of Phosphatase (ALP), Aspartate Transaminase (AST) and
Medicine, Ekiti State University, Ado-Ekiti, Nigeria. They Alkaline Transaminase.
were acclimatized for two weeks before administration
Determination of Alanine aminotransferase-
of the drugs.
GPT ∝-oxoglutarate + L-alanine→ L-glutamate + pyruvate
Animals were divided into ten groups of four rats each as
follows- Alanine aminotransferase is measured by monitoring the
Group 1 Positive Control (Normal) animals concentration of pyruvate hydrazone formed with 2,
4-dinitrophenylhydrazine.
Group 2 Negative control (50 mg/kg Diclofenac
treated) animals Determination of AST-
GOT ∝- oxoglutarate + L- aspartate → L-glutamate +
oxaloacetate
treated) animals
AST is measured by the monitoring the concentration of
oxaloacetate hydrazone formed with 2, 4-
treated) animals dinitrophenylhydrazine.
Group 5 50 mg/kg Diclofenac treated animals + Determination of ALP- Plasma alkaline phosphatase
mistletoes extract (ALP) was determined by kinetic method according to
Group 6 100 mg/kg Diclofenac treated animals + Abubakar et al. [13].
mistletoes extract Determination of Plasma Malondialdehyde (MDA)-

Group 7 150 mg/kg Diclofenac treated animals + Lipid peroxidation was determined by measuring the
formation of thiobarbituric acid reactive substances
mistletoes extract
(TBARS). An aliquot of 0.4 mL of the plasma or other
Group 8 50 mg/kg Diclofenac treated animals + dry organ homogenates was mixed with 1.6 mL of Tris-KCl
pawpaw leaf buffer to which 0.5 mL of 30% trichloroacetic acid (TCA)
was added. Then 0.5mL of 0.75% TBA was added and
Group 9 100 mg/kg Diclofenac treated animals +
placed in a water bath for 45 minutes at 80oC. This was
dry pawpaw leaf then cooled on ice and centrifuged at 3000g. The clear
Group 10 150 mg/kg Diclofenac treated animals + supernatant was collected and absorbance measured
against a reference blank of distilled water at 532nm.
dry pawpaw leaf
The MDA level was calculated. Lipid peroxidation in
units/mg protein or gram tissue was computed with a
Chemicals/ Reagent kits- All chemicals and drugs used
molar extinction coefficient of 1.56 x 105 M-1 cm 1.
were obtained commercially and of analytical grade. All
the diagnostic kits are products of Fortress Chemical Ltd. MDA (units/mg protein)=
United Kingdom.
Absorbance x volume of mixture
Preparation of Organs homogenate- The animals were E532 nm x volume of sample x mg protein
quickly dissected, the organs was removed. 10% of each
organs homogenate was then prepared in 6.7 mM Determination of Catalase activity of samples- This
potassium phosphate buffer, (pH 7.4) using the Teflon experiment was carried out using calorimetrically. This
homogenizer. The homogenate was centrifuged at method is based on the reduction of dichromate in acetic
10,000rpm for 10 minutes at 40C to obtain a clear acid to chromic acetate when heated in the presence of
supernatant which was stored at 80C and used for H2O2, with the formation of perchromic acid as an
measurement of biochemical contents. unstable intermediate.
Oseni et al., 2018
DOI:10.21276/ijlssr.2018.4.5.3
The chromic acetate so produced is measured The catalase contents of the enzyme preparation were
calorimetrically at 570-610 nm. However, the dichromate expressed in terms of Katalasefeiahigkeit or ‘Katf’.
has no absorbance at the wavelength and its presence in
the assay mixture does not interfere with the Katf= K (0) / mg protein/ ml
determination. The catalase preparation is allowed to
split H2O2 for different periods of time. The reaction is Statistical Analysis- All values are presented in Tables
and the appropriate comparisons between groups were
stopped at a particular time by the addition of
made using Student’s-test. The difference between the
dichromate/acetic acid mixture and the remaining H2O2
groups is taken to be significant at P<0.05.
is determined by measuring chromic acetate
calorimetrically after heating the reaction mixture. RESULTS
Fig. 1 represents the effect of pawpaw leaf and cocoa
Calculation- The mononuclear velocity constant, K, for
mistletoes on the catalase activity in plasma, heart and
the decomposition of H2O2 by catalase was determined
liver of diclofenac-induced rat. A significantly lower
by using the equation for a first-order reaction-
(P<0.05) plasma and liver levels of Catalase was
K= 1/t log S0/S observed in diclofenac-induced rats compare to normal
group but when treated with the pawpaw leaf and
Where S0 is the initial concentration of H2O2 and S is the mistletoes extracts, the catalase activity was increased
concentration of the peroxide at t min. The values of the towards to normal proportion.
K are plotted against time in minutes and the velocity
constant of catalase K(0) at 0 min determined by
extrapolation.
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 rats when compare to normal group but when treated
mistletoes on the malondialdehyde concentration in with the pawpaw leaf and mistletoes extracts, the
plasma and liver of diclofenac-induced rat. A significantly malondialdehyde concentration was decreased towards
higher (P>0.05) plasma, heart and liver levels of to normal level.
malondialdehyde was observed in diclofenac-induced
Oseni et al., 2018
DOI:10.21276/ijlssr.2018.4.5.3
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 control) normal. Treatment of cardiotoxic and

adverse effects on the functionalities of liver and plasma, hepatotoxic rats with pawpaw leaf and mistletoes
which is evidenced by a significant elevation in the exhibited improvement in the actions of ALT, AST and
actions of ALT, AST, and ALP enzymes in (negative group) ALP enzymes compared to (negative control) rats (Fig. 3
diclofenac-induced rats group compared to (Normal to Fig. 5).
Fig. 3: Effects of mistletoes and pawpaw leaf on treatment on the alkaline phosphatase activity in plasma and liver
of diclofenac-induced rat
Oseni et al., 2018
DOI:10.21276/ijlssr.2018.4.5.3
Fig. 4: Effects of mistletoes and pawpaw leaf on treatments on the alkaline phosphatase activity in plasma and liver
of diclofenac-induced rat
Fig. 5: Effects of mistletoes and pawpaw leaf on treatments on the alanine transaminase activity in plasma of
diclofenac-induced rat
DISCUSSION enzyme catalase is responsible for detoxification of H2O2.

However, Fig. 1 above showed the effects of aqueous The catalase enzyme may also be released into the
extract of mistletoes and pawpaw leaf treatment on the extracelular environment in which it has the potential to
catalase activity in plasma and liver of diclofenac- function as a potent antioxidant and thereby regulated
induced rat. Free radical scavenging enzyme,such as CAT, cell survival [14]. In light of these considerations, it seems
are the first line of defense against oxidative injury. The plausible that extracellular catalase might function as an
H2O2 formed by SOD and other processes is scavenged by important autocrine antioxidant and survival factor. A
catalase that catalyzed the dismutation of H2O2 in large number of the metabolites produced by NSAIDs are
to water and molecular oxygen. Thus, the antioxidant found to generate superoxide anion and other free
Oseni et al., 2018
DOI:10.21276/ijlssr.2018.4.5.3
[15]
radicals in the biological systems . However, at a type of hepatocellular damage [19,20]. The enhanced
higher dose of diclofenac (150 mg/kg) and consistent activities of these serum marker enzymes observed in
using of diclofenac, intermediate metabolites diclofenac sodium treated rats in present study
accumulate and cause liver damage. The reduction in correspond to the extensive liver damage induced by
catalase activity after induction with diclofenac is diclofenac sodium. The tendency of these enzymes to
another significant finding in this study. The decreased return towards a near normal level in group I (Normal)
concentration of plasma catalase is attributable in part rats is a clear manifestation of anti-hepatotoxic effect of
to the reduced synthesis of this antioxidant enzyme mistletoes and dry pawpaw leaf. Consistent and high
(which functions in the detoxification of hydrogen dosage administration of diclofenac in this study
perioxide) whose concentrations would have fallen with produced significant adverse effects on the organs
the diclofenac that was injected into the animals. Though functions of the rats such as liver, heart and kidney,
some studies have reported that no alterations in the which is evidenced by a significant increase in the actions
activity of red cell catalase. However, this study is in of ALT, AST, and ALP enzymes in (negative groups)
agreement with earlier reports by Redmond and diclofenac-induced rats group compared to (control)
Redmond [16], who reported a decreased in red blood cell normal. Similar result has also been recorded that stated
catalase activity cardiovascular and hepato-cellular that when enzymes leak from the liver cytosol into the
disorder. While treatment with Pawpaw leaf and blood stream, it lead to higher level of ALP in serum is an
mistletoes extracts caused significant increase in catalase indicative of hepatotoxic of diclofenac-induced rats [20].
activity in their respective groups due to bioactive High levels of ALP in serum indicate liver damage which
compounds present in the plants which aid the activities is similar to this present study where, the increase in ALP
of the antioxidant enzymes which is in agreement with activity in diclofenac-induced rats shows the liver
the previous study and can also explain the increased damage, as a result of metabolic changes such as
detoxification of the reactive metabolites generated administration of toxin, liver cirrhosis, hepatitis, and
from the diclofenac metabolism in the liver of plant cancer of the liver [20]. Thus, it can be used involved as
extracts treated animals. In present studies, the Pawpaw markers to estimate the extent of liver damage. Also
leaf and mistletoes co-administered rats showed Aminotransferases (ALT and AST) occupy a central
significantly decreased levels of these lipids peroxidation position in the metabolism of amino acids as they help in
markers as compared with diclofenac-induced rats. This retaining amino groups (to form new ones) during
present study is accordance with the previous study of degradation of amino acids. They are also involved in the
Sarker et al. [17], which they found that pawpaw leaf and biochemical regulation of amino acid pool and in
mistletoes respectively contain substances that delay the providing necessary intermediate to predict possible
rate of oxidation by directing the breakdown of toxicity in some organs such as kidney cytolysis and the
peroxides into stable substances that do not promote heart and liver of animals [21], which similar result
further oxidation or by sweeping free radicals away. occurred this present study. The measurement of the
Damaged membranes were recovered by the treatment activities of these enzymes is of clinical and toxicological
with plans extracts by enhancing antioxidants' status and significance. Since the enzymes were only being released
decreasing lipid peroxidation [18]. The inhibition of lipid into cellular flow when organs are damaged, the
peroxidation by Pawpaw leaf and mistletoes extracts, reduction in the levels of these enzymes in the plasma of
therefore, may be one of the mechanisms by which the the animals maintained on the dry pawpaw leaf and
plants exert their protection against diclofenac sodium mistletoes extracts treated groups.
mediated tissue injury.
CONCLUSIONS
Estimating the activities of serum marker enzymes, like
The oxidative stress has been imposed due to the
AST, ALT, and ALP, can make assessment of liver
imbalance of biochemical processes that involves the
function. When liver cell plasma membrane is damaged,
generation of reactive oxygen and nitrogen species (ROS
a variety of enzymes normally located in the cytosol are
and RNS) and their neutralization by the inherent
released in to the blood stream. Their estimation in the
antioxidant (enzymatic or non-enzymatic) defense
serum is a useful quantitative marker of the extent and
Oseni et al., 2018
DOI:10.21276/ijlssr.2018.4.5.3
system of the cells. Mistletoes and pawpaw leaf are one [5] Fahey JW, Zhang Y, Talalay P. Proc. Natl. Acad. Sci.
of the most abundant natural antioxidants with variety USA, 1997; 94: 10367-10372
of applications. The present study shows that the [6] Gill LS, Onyibe HI. Mistletoes on rubber trees in
Hepatotoxicity in rats treated with diclofenac was Nigeria. Haustorium, 23: 1-2. Am Eurasian
markedly reduced by simultaneous administration of 2007. Journal of Sustainable Agriculture, 1990; 5:
mistletoes and pawpaw leaf extract. The results of this 360–361.
study show protective effect of mistletoes and pawpaw [7] Kearney P, Baigent C, Godwin J,Halls H, Emberson J,
leaf on plasma and liver function which might be due to Patrono C. Do selective cyclo-oxygenase-2 inhibitors
the presence of some bioactive compound in the extract. and traditional non-steroidal anti-inflammatory
Further investigation should be conducted to identify drugs increase the risk of atherothrombosis? Meta-
this bioactive compounds present in mistletoes. analysis of randomised trials. BMJ, 2006; 332(7553):
1302-1308.
ACKNOWLEDGMENTS [8] Khammash A. “Holly Parasite or Christmas
This study was supported by all the authors financially, Mistletoe” Jordan Times Weekender. Copyright ©
morally, and physically. 2005, http: www.jordanflora.com/holly.htm., 2005.
[9] Kingsbury JM, Redmond WA. “Poisonous Plants.”
CONTRIBUTION OF AUTHORS
Microsoft ® Student [DVD]. Microsoft Corporation,
Research concept- Oseni OA
2007.
Research design- Odesanmi EO
[10]Kokowaro J O. Asparagus africanus. Medicinal plants
Supervision- Oloyede OI
of East Africa, 1976; p. 239.
Materials- Odesanmi EO
[11]Mitchell JR, Jollow DJ, Potter WZ, Gillette JR, Brodie
Data collection - Oseni OA
BB. Acetaminophen inducedhepatic necrosis.
Data analysis and interpretation- Odesanmi EO
Protective role of glutathione. Pharmacology and
Literature search- Odesanmi EO
Experimental Therapeutics 1973; 187: 211–215.
Writing article- Odesanmi EO
[12]National Cancer Institute. Mistletoe Extract (PDG)
Critical review- Oloyede OI
National Cancer Institute. Website. Accessed on July
Article editing- Ogundare MAB
3, 2007.
Final approval- Oseni OA
[13]Abubakar MS, Musa AM, Ahmed A, Hussaini IM. The
Research support- Adebayo OD
Perception and Practice of Traditional Medicine in
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Int. J. Life. Sci. Scienti. Res.

Antioxidant and Hepatoprotective Activities of Carica papaya

Received: 29 Apr 2018/ Revised: 28 Jun 2018/ Accepted: 14 Aug 2018

mistletoes extract Determination of Plasma Malondialdehyde (MDA)-

Administration of diclofenac produced significant control) normal. Treatment of cardiotoxic and

DISCUSSION enzyme catalase is responsible for detoxification of H2O2.

Open Access Policy:

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