International Research Journal of Plant Science (ISSN: 2141-5447) Vol. 4(2) pp. 55-63, February, 2013
Available online http://www.interesjournals.org/IRJPS
Copyright © 2013 International Research Journals
Full Length Research Paper
Assessment of hepatoprotective and antioxidant
activity of nauclea latifolia leaf extract against
acetaminophen induced hepatotoxicity in rats
*1Effiong, G.S, 1Udoh, I.E, 2Udo, N.M, 3Asuquo, E. N, 4Wilson, L. A, 4Ntukidem, I.U. and
4
Nwoke, I.B.
1
Department of Clinical Pharmacy and Biopharmacy, Faculty of Pharmacy, University of Uyo, Nigeria
2
Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Uyo, Nigeria
3
Department of Biochemistry, Faculty of Basic Medical Sciences, University of Calabar, Nigeria
4
Department of Chemistry, Akwa Ibom State College of Arts and Science, Nung Ukim Ikono, Nigeria
Abstract
The present study was carried out to evaluate the hepatoprotective and antioxidant effect of the ethanol
extract of Nauclea latifolia (NL) leaf in Wistar albino rats. Protective action of (NL) leaf extracts was
evaluated using animal model of hepatotoxicity induced by Acetaminophen (Paracetamol) (2 g/kg, bw
p.o). Liver marker enzymes and antioxidant status were assayed in serum. Histopathology was also
studied while silymarin was used as the standard drug for this study. Levels of aspartate
aminotransferase (AST) and alanine aminotransferase (ALT) were increased and the levels of total
protein and albumin were decreased in Acetaminophen treated rats. NL leaf at (400 mg/kg, bw) doses
decreased the elevated levels of the transaminases and restored the normalcy of total protein (TP) and
albumin significantly. The activities of catalase (CAT), glutathione Peroxidase (GPx) and superoxide
dismutase (SOD) were decreased in hepatotoxic rats but administration with NL leaf extract increased
the GPx, CAT and SOD. Histopathology shows restoration of the Acetaminophen damaged liver with NL
administration. From this study it can be concluded that the NL leaf showed significant
hepatoprotective and antioxidant action.
Keywords: Nauclea latifolia, Acetaminophen, Liver, transaminase, Antioxidant, Hepatoprotective.
INTRODUCTION
Medicinal plants have formed the basis of health care
throughout the world since the earliest days of humanity
and are still widely used, and have considerable
importance in international trade (Ahmed et al., 2006). In
developed countries such as United States, it is
estimated that plant drugs constitute as much as 25% of
the total drugs, whereas, in developing countries
including China and India, the contribution is as much as
80% (Joy et al., 1998). This underscores the increased
research interest in medicinal plants and traditional
medicine all over the world. Plant derived natural
products such as flavonoids, terpenoids, carbohydrates,
tannins, saponins, steroids, proteins, amino acids
(Begum et al., 2004) and Vitamin C (Sunttornusk and
*Corresponding author: E-mail: graceffiong2003@yahoo.com
Quantitation, 2002) etc have received considerable
attention in recent years due to their diverse
pharmacological properties including antioxidant and
hepatoprotective activity (Roy et al., 2006).
Human beings are exposed on a daily basis to
certain toxic chemicals and pathogens, which cause
certain serious health problems. Certain chemicals and
reagents that were thought to be health friendly have
been proved to have serious adverse effects on health.
Amongst these toxic substances is acetaminophen.
Acetaminophen has thus been taken as test model to
screen the anti-hepatotoxic activity of indigenous drugs
(Reid et al., 2005 and Knight et al., 2002).
Acetaminophen, a most commonly used analgesics,
effectively reduces fever and mild-to moderate pain, and
is considered to be safe at therapeutic doses. However,
acetaminophen overdose causes severe hepatotoxicity
that leads to liver failure in both humans and
experimental animals (Masubuchi et al, 2005; Kaplovitz,
56 Int. Res. J. Plant Sci.
2005). The concern here is the adverse effect of
acetaminophen to the liver, having visualized the
prominent functions of the liver for survival.
Hepatic diseases or injuries are modelly induced
experimentally by administration of acetaminophen since
it is known that acetaminophen overdose causes severe
hepatotoxicity (Masubuchi et al, 2005; Kaplovitz, 2005).
These hepatotoxic effects can be minimized or prevented
or eliminated by certain active compounds serving as
valuable antioxidants obtainable from natural plant
resources (Akhtar and Ali, 1984).
A small amount of acetaminophen is metabolized
together with cytochrome P450. As a result, N-acetyl-pbenzoquinone
imine
(NAPQI)
or
Nacetyl-pbenzosemiquinone imine (NAPSQI) appears in the
body‟s system (Bessems andVermeulen, 2001). Both
these compounds are very active chemically and their
chemical structures indicate that they are capable of
taking part in free radical reactions. Consequently,
acetaminophen overdose can lead to a number of
unfavorable consequences, especially those affecting the
liver (James et al., 2003, Kurtovic, and Riordan, 2003). A
large dose of this drug causes depletion of the cellular
glutathione (GSH) level in liver because NAPQI reacts
rapidly with glutathione (Masubuchi et al 2005; Oz et al.,
2004), which consequently exacerbates oxidative stress
in conjunction with mitochondrial dysfunction. Thus, the
GSH depletion, especially occurring in acute
hepatotoxicity, affects liver functions and leads to
massive hepatocyte necrosis, liver failure or death. Since
oxidative stress and GSH depletion contributed
acetaminophen induced liver injury; the agent(s) with
antioxidant property and/or GSH reserving ability may
provide preventive effect against the progression of
hepatocellular injury (Baudrimont et al., 1997).
Modern medical science does not have, at present, a
therapeutic agent which could cure the different liver
disorders. In fact; the available remedies are from the
traditional system of medicine. In view of severe
undesirable side effects of synthetic agents, and the
absence of a reliable liver protective drug in modern
medicine, there is growing focus to follow systematic
research methodology and to evaluate scientific basis for
the traditional herbal medicines that are claimed to
possess hepatoprotective activity (Chatterjee, 2000).
Nauclea latifolia (family: Rubiaceae) commonly
known as pin cushion tree is a straggling shrub or small
tree native to tropical Africa and Asia. The plant could
have some augmentary or protective effect against
certain hepatocellular injury. Lagnika et al., (2011)
reported that the ethanolic extract of Nauclea latifolia
leaves possess antioxidant activity. Leaves of Nauclea
latifolia have protective role against diabetes (Gidado et
al., 2005), hypertension (Nworgu et al., 2008), malaria
and typhoid fever (Oye, 1990; Boye, 1990). In view of
these attributes since no evidence has been tendered as
regards the remedial effects of the leaves of Nauclea
latifolia on liver malfunction, the burden at this work is
reposed on the in vivo determination of the effects of
ethanolic extract of the leaves of Nauclea latifolia on
some hepatocellular damage induced by Acetaminophen
in experimental animals.
Silymarin has been used for over 20 years in clinical
practice for the treatment of toxic liver diseases (Messner
and Brissot, 1990). Silymarin extract from the seeds of
the plant Silybum marianum, also called “milk thistle”, has
been described to be an antioxidant and exhibits
anticarcinogenic, antiinflammatory, hepatoprotection and
growth modulatory effects (Flora et al., 1998; Skottova et
al., 2008). In this study, silymarin was used as a positive
control against the acetaminophen-induced hepatic
damage in rats.
MATERIALS AND METHODS
Collection and preparation of plants’ extracts
Fresh but matured Nauclea latifolia leaves were obtained
from the Endocrine Research Farm of the University of
Calabar, Calabar. The plants were authenticated by Dr.
E. G. Amanke, of the Department of Botany, University of
Calabar and voucher specimens deposited in the
Department of Botany herbarium, University of Calabar.
The leaves were rinsed severally with clean tap water to
remove particles and debris and thereafter allowed to
completely drain. The ethanol extract was prepared using
the wet method of extraction; one kilogramme of the fresh
leaves of the plant were cut and chopped into pieces with
a knife on a chopping board, blended in 1.5 litres of
ethanol (96%) with an electric blender and transferred
into amber colored bottle and kept in cool dark
compartment for 72hours. It was then filtered using a
cheese cloth and thereafter with Whatman No 1 filter
paper to obtain a homogenous filtrate. The filtrate was
then concentrated in vacuo using a rotary evaporator at
o
37-40 C to about one tenth the original volume. The
concentrate was allowed open in a water bath (40oC) to
dryness yielding 78.95g of brown oily substances of NL.
It was dried completely in a desiccator containing a selfindicating silica gel and then refrigerated at 2-8OC until
used.
Phytochemical evaluation
NL leaves were subjected to quantitative analysis for
various phytoconstituent like cyanogenic glycosides
determined by the alkaline picrate colorimetric method
(Harborne, (1973)), saponins in which the method of
Obadoni and Ochuku (2001) as described by Okwu
(2004) was employed, phenols was done according to
Effiong et al. 57
the Folic- ciocaltean colorimetric method (AOAC, 1990),
tannins was determined by colorimetric method of Trease
and Evans (1996), flavonoid was done using the ethyl
acetate gravimetric method (Trease and Evans, 1996)
while alkaloid determination was done using the alkaloid
precipitation gravimetric method described by Harborne
(1973) and Okwu (2004).
1951) and antioxidants analysis using Spectrophotometer
assay kit from Cayman Chemical Company; for
superoxide dismutase (SOD) based on Nebot et al.,
(1993), catalase (CAT) based on Aebi (1984) and
glutathione peroxidase (GPx) based on Paglia and
Valentine (1967).
Histopathological examination
Animals
Thirty six Albino Wistar rats weighing 200-300g (males
only) were obtained from the animal house of the
Department of Pharmacology and Toxicology of the
University of Uyo, Nigeria. They were kept in well
ventilated clean cages (wooden bottom and wire mesh
top), maintained under standard laboratory conditions
o
(Temperature 25± 5 C, Relative humidity 50-60%, and a
12/12h light/dark cycle) and were allowed free access to
standard diet (Vital Feed from Grand Cereals and Oil
Mills Limited, Jos, Plateau State of Nigeria) and water ad
libitum. Animals were acclimatized for 14 days in the
animal house of the Department of Biochemistry,
University of Calabar, Nigeria, before the experiments.
Liver tissues were preserved in 10% formaldehyde
solution. The tissues processed and embedded in
paraffin wax. Sections of about 4‐6 microns were made
and stained with hematoxylin and eosin and
photographed.
Statistical analysis
The results were expressed as mean ± SEM and were
analyzed for statistically significant difference using
one‐way ANOVA followed by Bonferroni‟s multiple
comparison tests (BMCT) post hock test. The data were
analyzed with SPSS version 16 software (SPSS Inc.,
Chicago, USA). The difference showing a level of p <
0.05 was considered to be statistically significant.
Experimental Design
Hepatic injury or liver damage was induced orally with a
single dose of acetaminophen (2 g/kg, bw, p.o,) diluted
with sucrose solution (40% w/v). Animals were then
separated into groups as shown in Table 1; Control rats
received only normal diet and water while the test
animals were administered NL leaves extract or
Silymarin. At the end of the 21 days, food was withdrawn
from the rats and they were fasted overnight but had free
access to water. They were then euthanized under
chloroform vapour and sacrificed. Whole blood was
collected via cardiac puncture using sterile syringes and
needles, emptied into plain tubes and allowed to clot for
about two hours. The clotted blood was thereafter
centrifuged at 3,000rpm for 10 minutes to recover serum
from clotted cells. Serum was separated with sterile
syringes and needles and stored frozen until used. Prior
to dissection, the liver tissue was perfused in heparinized
saline (0.9% NaCl) to remove any red blood cells and
clots and thereafter blotted with blotting paper. The tissue
was then suspended in boun’s fluid for histologic
evaluation.
RESULT
Serum biochemical assays
Hepatic oxidative stress parameters
Serum was used for the estimation of various
biochemical parameters: ALT (Reitman, and Frankel,
1957), AST (Reitman, and Frankel, 1957), albumin
(Mallay and Evelyn, 1937), total protein (Lowry et al.,
The levels of GPx, CAT and SOD were significantly
(P<0.05) decreased in the untreated hepatotoxic
rats when compared to normal control. Administering
silymarin or NL leaves increased significantly
Preliminary phytochemical investigation
The quantitative phytochemical investigation of the NL
leaves showed moderate content of alkaloids,
polyphenols and flavonoids with high content of saponins
and hydrocyanic acid as shown on Table 2.
Serum biochemical parameters
The serum levels of aspartate aminotransferase (AST)
and alanine aminotransferase (ALT) were significantly
(P<0.05) increased and the levels of albumin and total
protein were significantly (P<0.001) decreased in
untreated hepatotoxic rats when compared to normal
control. Treating with silymarin or NL leaves reduced the
elevated levels of AST and ALT while the levels of
albumin and total protein was restored towards normalcy
when compared to the untreated hepatotoxic rats (Table 3).
58 Int. Res. J. Plant Sci.
Table 1. Experimental design
Group
1
No. of
Animals
6
2
6
3
6
Hepatotoxic Rats
Treatment
Non-Hepatotoxic Rats
No. of
Treatment
Animals
6
Placebo (Normal control)
Group
acetaminophen (2 g/kg, bw, p.o,)
diluted with sucrose solution (40%
w/v). ( Hepatotoxic control)
Acetaminophen(2 g/kg, bw, p.o,) +
NL (400mgkg-1b.w)
Acetaminophen(2g/kg,bw,p.o,)
+Silymarin commercial drug (100
mg/kg, bw, p.o,)
1
2
6
NL extract (400mgkg-1b.w)
3
6
Silymarin commercial drug
(100 mg/kg, bw, p.o,)
Table 2. Quantitative phytochemical composition of NL leaves
Flavonoids
(%)
1.52±0.02
Tannins (%)
Saponins (%)
0.24±0.00
17.52±0.02
Polyphenols
(%)
2.00 ± 0.01
Alkaloid (%)
7.07±0.02
Hydrocyanic
acid (HCN) (%)
13.2±0.02
Table 3. Effect of treatment on serum Biochemical indices in non Hepatotoxic and Hepatotoxic rats.
Group/Treatment
TP(mg/L)
Alb(mg/L)
AST(U/L)
ALT(U/L)
NLH
7.74±0.13a, b
42.71±0.08*,a
30.33±12.23*, a, b
14.33±1.38*, a, b
NLNH
10.31±2.03a
44.84±2.30a
27.33±8.34*, b
49.33±2.59*,a,b
ParacetamolH
6.73±0.06*,a
32.45±0.11*,a
33.33±2.11*
46.67±1.72*, a
ParacetamolNH
12.47±0.47*,a
57.50±8.99a
12.47±0.47*,a
57.50±8.99a
HC
5.98±0.34*
2.69±0.11*
70.67*±0.42*
69.33±18.09
NC
9.24±0.92
66.05±8.38
19.17±2.52
23.00±4.02
*P<0.05 vs NC; a = P<0.05 vs HC; b = P<0.05 vs Acetaminophen. Mean ±SE,=6,
H =Hepatotoxic rats, NH = Non- Hepatotoxic rats,
NC=normal control, HC= Hepatotoxic control
(P<0.05) the antioxidant levels (Table 4).
Histopathological examination
Liver sections from normal control rats showed normal
liver cytoplasmic changes, and no inflammation of cells
(Figure 1). Whereas untreated hepatotoxic rats showed
liver cells filled with uniformly distributed small dense fat
droplets, the nuclei are large and cells inflamed. There is
presence of necrosis and fibrosis (Figure 2). No
significant morphological changes were noted in livers of
animals treated with silymarin, as compared to that of
animals in the normal control (Figure 3). Treatment with
NL showed normal architecture with hepatic plates (HP),
conspicuous sinusoids(S) and central vein (CV)
hyperplasia with hardly ascertainable regenerative activity
as in acetaminophen-challenged animals (Figure 4).
DISCUSSION
Health is the subject of priority as far as life is concerned,
but despite effort to maintain good health, man and
Effiong et al. 59
Table 4. Effect of treatment on oxidative stress in non- Hepatotoxic rats and Hepatotoxic rats
Group/Treatment
GPx (U/g protein)
SOD (U/g protein)
CAT (U/g protein)
NLH
3307.26±343.69*, a, b
33.69±0.41*,a
475.20±50.13b1
NLNH
7976.98±73.23a
49.77±13.65a
441.47±130.95*, a
ParacetamolH
2648.67±12.47*
16.03±0.45*
884.55±7.85*, a,b
ParacetamolNH
8016.76±97.14a
54.03±14.64a
329.67±5.18
HC
2720.27±33.78*
13.52±0.21*
122.83±7.70*
NC
7992.09±338.28
52.56±3.96
337.50±8.32
*P<0.05 vs NC; a = P<0.05 vs HC; b = P<0.05 vs AcetaminophenNH, b1 =P<0.05vs; AcetaminophenH
Mean ± SE, n = 6, H = Hepatotoxic rats, NH = non- Hepatotoxic rats, HC=Hepatotoxic control,
NC=normal control.
Figure 1. Section in the liver of normal control
rat showing hepatic cells (h), Sinusoidal spaces
(s) with Kupffer cells (k) and central vein (CV)
Figure 2. Section in the liver of Acetaminopheninduced hepatotoxicity showing loss of the normal
architecture with distended central vein (CV)
60 Int. Res. J. Plant Sci.
Figure 3. Liver tissue of Acetaminopheninduced hepatotoxicity treated with silymarin
showing plates of hepatocytes (HP) radiating
from the central vein (CV) and showing
prominent sinusoids (S).
Figure 4. Liver tissue of hepatotoxic rat
treated with NL showing normal
architecture with hepatic plates (HP),
conspicuous sinusoids(S) and central vein
(CV) hyperplasia
animals alike still confront disease conditions which are
due to exposure to physiopathological agents (Lambo,
1979), such as noxious substances, etc in the
environment. Though the body system is made in such a
way that it tackles invading foreign substances in most
cases, the body system needs to be protected, enhanced
and activated (Murray et al., 1990). This ability to activate
the body defence mechanism or to protect the body
system has been found to be present in some nature
vegetation/herbal sources (Okafor, 1989; Lagnika et al.,
2011). And so it has become expedient to examine
scientifically the protective effects of these herbal plants.
The
phytochemical
constituents
quantitatively
demonstrated in the leaves of NL has been shown to
include tannins, alkaloids, flavonoids, polyphenols and
saponins which is a fundamental to the understanding of
the modes of action of medicinal plants in general. It is
the diverse composition of these components in plants
that places them at an advantaged position over and
above chemotherapy. The presence of saponins and
tannins in NL leave extract suggest why it has protective
effects on the liver, this is in consonance with the study of
Shimkim and Anderson, (1963) where they reported that
some medicinal plants possess hepatoprotective effects
Effiong et al. 61
because they contain some bioactive compounds.
The liver is a major target organ for toxicity of
xenobiotics and drugs, because most of the orally
ingested chemicals and drugs first go to liver where they
are metabolized into toxic intermediates. Various
pharmacological or chemical substances are known to
cause hepatic injuries such as acetaminophen, CCl4 and
dimethylnitrosamine. Excessive dose exposure to these
hepatotoxins may induce acute liver injury characterized
by abnormality of hepatic function and degeneration,
necrosis or apoptosis of hepatocytes (Higuchi and Gores,
2003). With the increasing ingestion of drugs or
exogenous chemicals, the possibilities of liver injury will
undoubtedly increase (Lee, 2003). At present, drug or
chemical-induced liver injury has become a major clinical
problem. In addition, the search for effective therapeutical
methods for the treatment of drug or chemical-induced
liver injury is also very important (James et al., 2003).
With respect to acetaminophen dependent hepatotoxicity
it is generally accepted that P450-dependent bioactivation
of acetaminophen is a main cause of potentially fulminant
hepatic necrosis upon administration or intake of lethal
dose of acetaminophen (Bailey et al., 2003; Masubuchi et
al., 2005). Acetaminophen hepatotoxicity is the result of a
cascade of interrelated biochemical events (Bartlett,
2004).
Hepatocellular necrosis or membrane damage leads
to very high levels of serum AST and ALT released from
liver to circulation which was in consonance with the
increased level of these enzymes in the Acetaminophen
intoxicated rats (group 2). The increased levels of serum
marker enzymes are indicative of cellular leakage and
loss of functional integrity of cellular membrane in liver
(Drotman and Lawhorn, 1978). In the present study,
Treatment with NL leaf (400 mg/kg, bw, p.o,) suppressed
the elevated serum levels of AST, ALT towards the
respective normal value this clearly indicates that the
plant extract has stabilizes the plasma membrane as well
as helped in healing of the hepatic tissue damage clearly
suggesting that the plant extract has the ability to heal
hepatic tissue damage.
Hypoalbuminaemia is most frequent in the presence
of advanced chronic liver diseases. Hence decline in total
protein content can be deemed as a useful index of the
severity of cellular dysfunction in chronic liver diseases.
Stimulation of protein synthesis has been advanced as a
contributory
hepatoprotective
mechanism,
which
accelerates the regeneration process and the production
of liver cells (Larsson et al., 1985). The lowered level of
total proteins recorded in the serum of untreated
hepatotoxic rats reveals the severity of hepatopathy.
However, protein synthesis was stimulated in the NL
treated groups as NL leaf restored in this study the
normalcy of albumin and total protein level in hepatotoxic
treated rats.
In the course of liver failure, oxidative stress
expressed by oxidant-antioxidant imbalance is profound
in liver tissue. A study with acetaminophen was found to
induce substantial mitochondrial oxidative stress and
peroxy nitrite formation (Knight et al., 2001). This
oxidative stress preceded cell injury by several hours
(Jaeschke, 2003) and free radical scavenger‟s
attenuated acetaminophen induced liver injury (Knight et
al., 2002). The ability of a hepatoprotective drug to
reduce the injurious effects or to preserve the normal
hepatic physiological mechanisms, which have been
disturbed by a hepatotoxin, is the index of its protective
effects.
Reduced glutathione is a substrate for
glutathione related enzymes, and a regenerator for alphatocopherol; therefore, it plays an important role in the
antioxidant defense system (Meister, 1991). It is well
known that a large dose of acetaminophen causes
hepatic GSH depletion because NAPQI reacts rapidly
with glutathione (Oz et al., 2004), which consequently
exacerbates oxidative stress in conjunction with
mitochondrial dysfunction. The GPx present in the cells
can catalyze this reaction. Cighetti et al., (1993) reported
that depletion of GSH below a threshold value was
associated with a significant conversion of xanthine
dehydrogenase to reversible xanthine oxidase, a
superoxide radical generation reaction catalyzing
enzyme. This tie in with the observed significantly
decreased GPx activity in hepatotoxic control rats
compared with the non-hepatotoxic control rats in
the present study. However, concomitant administration
with NL significantly prevented the acetaminophen
induced depletion of hepatic GPx, indicating the
antioxidant elevating status of NL in ameliorating
hepatotoxicity.
The significant decrease of hepatic CAT and SOD
activities observed in the hepatotoxic control rats in this
study may be due to increased free radical production
caused by administration of acetamoniphen (Bessems
and Vermeulen, 2001). Antioxidant enzymes such as
SOD and CAT are easily inactivated by lipid peroxides or
reactive oxygen species (offshoots of hepatotoxicity),
which results in decreased activities of these enzymes in
acetaminophen -induced liver toxicity in this study. NL is
rich in phytochemicals as shown on Table 2 and exhibits
antioxidant capacity against oxidative stress. This may
account for the reversal, in NL treated rat, of the
decrease in SOD and CAT activity observed in the
hepatotoxic control rats, and it agrees with the work of
Hunt et al, (1988) on hydroxyl radical production and
auto-oxidative glycosylation in diabetic models.
All
evidence, including serum ALT and AST activities, GPx,
SOD and CAT levels suggest that NL could decrease
acetaminophen-induced oxidative stress.
In the histopathological studies, paracetamol
intoxication was seen to produce extensive vascular
degenerative changes and centrilobular necrosis in liver
cells. Treatment with silymarin and NL extract produced
mild degenerative changes and absence of centrilobular
necrosis when compared with hepatotoxic control rats.
62 Int. Res. J. Plant Sci.
CONCLUSION
Conclusively, Nauclea latifolia in this study has
demonstrated a potent hepatoprotective action upon
acetaminophen-induced oxidative stress and liver toxicity
in rat. The hepatoprotective effect of Nauclea latifolia
could be correlated directly with its ability to reduce
activity of serum enzymes and enhance antioxidant
defense status. The findings of this study suggest that
Nauclea latifolia can be used as a safe, cheap, and
effective alternative chemopreventive and protective
agent in the management of liver diseases.
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