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INTERNATIONAL JOURNAL OF PHARMACOLOGY AND THERAPEUTICS
ISSN 2249 – 6467
Research Paper
SYNERGISTIC HEPATOTOXIC POTENTIAL OF MANIHOT ESCULENTA
CRANTZ LEAF EXTRACT ON PARACETAMOL-INDUCED LIVER
DAMAGE IN RATS
Awe Emmanuel O*1 , Kolawole Timothy O1, Olaniran Olutayo. B2.
1
Department of Pharmacology & Therapeutics, College of Health Sciences, Ladoke
Akintola University of Technology, Osogbo Campus.
2
Department of Biomedical Sciences, College of Health Sciences, Ladoke Akintola
University of Technology, Osogbo Campus.
Abstract
Paracetamol is an analgesic, antipyretic drug available as an over the counter (OTC) medication which causes
hepatotoxicity at high doses. The effect of Manihot esculenta (100, 200 and 400mg/kg body weight,
administered for 7days) on paracetamol-induced acute hepatic damage was studied by investigating the effects
on liver enzymes, bilirubin, albumin, total protein and urea. Acute hepatotoxicity was induced by administering
2g/kg body weight of PCM orally on the eighth day. All the rats were sacrificed 24hours after the
administration of PCM. The results shows that PCM at a dose of 2g/kg body weight induced acute
hepatotoxicity 24 hours after oral administration as evident by the increase plasma alanine aminotransferase
(ALT), aspertate aminotransferase (AST) and alkaline phoshatase(ALP) activities. The aqueous leaf extract of
Manihot esculenta dose dependently potentiate acute hepatotoxicity induced by PCM by increasing the
activities of liver enzymes but not statistically significant. In conclusion, Manihot esculenta leaf extract did not
show significant hepatotoxic activities on paracetamol liver damage in rats but tends to potentiate the effect of
paracetamol-induced hepatotoxicity. It was suggested that the potentiation may be due to the presence of
cyanogenic glycosides.
Keywords: Synergy, Manihot esculenta, paracitamol, hepatotoxicity, cyanogenic glycosides.
INTRODUCTION
Manihot esculenta is a woody shrub of
Euphorbiaceae (spurge family) native to
South America, is extensively cultivated
as an annual crop in tropical and
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subtropical regions for its edible starchy
tuberous root. It’s a major source of
carbohydrates [1].
Cassava is one of the most forgiving and
adaptable plants. It grows well in tropical
humid conditions but can also withstand
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draught. It does well in poor soil where
little else will grow and require little care.
Tubers of cassava or ‘Yuka’ as the plant is
commonly called in South America, are
extremely rich in starch. It is the richest
source of starch of any food plant (it
contains up to 10 times as much as starch
as corn and twice as much as potatoes).
However the entire plant is poisonous if
consumed raw due to its linamarin content,
a precursor of cyanide glycosides. Even a
relatively small content can be fatal [2].
Thus, the roots have to be rendered edible
by a ritualized process of grating, washing,
pulp and squeezing out of the harmful
juices. Heating also renders the substances
harmless [3]. There are several different
species of cassava, differentiated as sweet
and bitter varieties. The sweet varieties
contain considerably less linamarin than
the bitter types [4].
Phytochemically, the plant contains a
number of oxidant compounds namely
anthocyanins (flavonoids), saponions,
steroids, tannins, alkaloids, anthraquinone,
3-rutinoside of kaempferol and quercetin;
the cyanogenic glycosides, lotaustralin and
linamarin [5-7]. The leaves can be used as
a stypic, while starch mixed with rum has
been used for skin problems, especially for
children [8].
Cassava is used in folk remedies for
various ailments such as cancer, abscesses,
boils, conjuctivitis, diarrhea, dysentery,
flu, hernia, inflammation, marasmus,
prostatitis, sore, snake bite, fever,
headache, rheumatism and hemorrhoids
[9-11].In Nigeria, cassava leaves are also
used in the treatment of ringworms,
conjuctivitis, sores and abscesses [8].
Some ethno medicinal uses of Manihot
esculenta plant have been validated
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scientifically, and the plant´s leaf extract
was reported to produce dose-related,
sustained and significant reduction in fresh
egg albumin-induced acute inflammation
in rat paw oedema [12]. Chloroform
extract of leaf of Manihot esculenta has
been reported to have activity against
bacteria such as V. cholera, Shigella
flexneri, S. thyphi, P. auruginosa [13]. In
addition, the crude extract of Manihot
esculenta
showed
dose-dependent
inhibitory effect on the production of lipid
peroxides in rats [14]. Anti helmintic and
antipyretic activities of the leaf extract
have also been reported [15-16].
It has been shown that liver damage is
caused by chemicals such as paracitamol
and carbon tetrachloride in large doses
[17-18], therefore, this study was designed
to
evaluate
possible
synergistic
hepatotoxic potential of aqueous Manihot
esculenta leaf extract on paracitamolinduced liver damage in rat.
MATERIALS AND METHODS
Ethical consideration
Experimental procedures and protocols
used in this study were approved by the
Ethics committee of the Ladoke Akintola
University of Technology, Nigeria and
conform to the “Guide to the care and use
of animals in research and teaching” (NIH
publications number 85-93 revised in
1985)
Plant collection
Cassava leaves were collected at a farm in
Dada Estate, Egbedore Local Government,
Osogbo, Osun State. The leaves were
identified by a taxonomist, Mr. Odewo, at
the herbarium of the Federal Institute of
Forest Research Ibadan where voucher
specimen was deposited.
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Extraction
Mannihot esculenta fresh leaves (1kg)
were air-dried at room temperature. The
air-dried leaves of the plant were milled
into fine powder in a Waring commercial
blender. The powdered leaves were
macerated in 3 liters of water at room
temperature (27± 1 C) and extracted for 48
hours with occasional shaking. The
combined water extract was filtered and
concentrated to dryness under reduced
pressure at 60 ± 1o C in rotary evaporator.
Freeze-drying and solvent elimination of
the resulting aqueous extract was weighed
and finally gave 40.78 g. The percentage
yield was calculated using this formula:
weight of extract/original weight x 100
giving 4.0.35% yields) of brown powdery
crude Manihot esculenta leaf extract
(MEE). A stock solution of 10mg/ml was
prepared by dissolving 100mg of leaf
extract in 10ml of water. Different doses
were calculated from the stock solution.
Animals
Healthy, male and female healthy young
adult, Wistar rats (Rattus norvegicus)
weighing 220–250 g, were used. The
animals were kept and maintained under
laboratory conditions of temperature and
humidity with a 12 h light/dark cycles.
They were allowed free access to food
(standard pellet diet) and drinking tap
water ad libitum.
Determinations of Acute Oral Toxicity
(LD50)
Acute oral toxicity (AOT) of methanol
extract was determined as per OECD
guidelines. The animals were fasted 3 h
prior to the experiment and as per up and
down procedure (OECD 2001). Animals
were administered with single dose of
aqueous extract and observed for its
mortality for 48 h study period (short
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term) toxicity. Based on short-term profile
of drug, the dose of next animals was
determined as per as OECD guideline 425.
All the animals were also observed for
long term toxicity (14 Days).
HEPATOTOXIC ACTIVITIES
The animals were divided into five groups
of six animals in each group. The extract
was given to the animals for seven days.
On the eighth day, paracetamol was given
to cause liver damage.
Group I:
Negative
control
i.e.
untreated
Group II:
Positive control. Received
2000mg/kg of paracetamol on the 8th day
Group III:
Received 100mg/kg of
plant extract for 7 days and 2000mg/kg of
paracetamol on the 8th day
Group IV:
Received 200mg/kg of
plant extract for 7 days and 2000mg/kg of
paracetamol on the 8th day
Group V:
Received 400mg/kg of
plant extract for 7 days and 2000mg/kg of
paracetamol on the 8th day
Collection of blood sample
Heparinized and EDTA blood samples
were obtained under light anesthesia from
the orbital sinus using microhaematocrit
tubes [19-20] for analysis of biochemical
and
hematological
parameters
respectively. All animals were sacrificed
by exsanguinations after light ether
inhalation anesthesia at the end of the
experiment. The animals were subjected to
post-mortem examination with collection
of sample from the liver.
Histopathological examination
The liver tissues obtained from sacrificed
animals were fixed in 10% neutral
buffered formalin for at least 48 h. Liver
sections (4-5 µm) paraffin wax were
processed and stained with haematoxylin
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and eosin for microscopic examination
using standard protocol of Carleton,
(1980) [21].
Statistical analysis
The data are presented as mean± SEM and
analyzed by one-way ANOVA, followed
by independent’s test. P values < 0.05
were considered significant
RESULTS
PCM at a dose of 2g/kg body weight
induced acute hepatotoxicity 24hours after
oral administration as evident by
significant increase (P<0.05) in plasma
ALT, AST and ALP (37.20±3.42,
Table 1: Effects of
hepatoxicity in rats
80.20±6.22 and 18.50±2.51) values
compared to normal control (23.40±3.44,
53.00±4.64 and 10.28±1.47). The aqueous
leaf extract of Manihot esculenta dose
dependently and significantly increase
(P<0.05) the values of ALT, AST and
ALP (36.60±2.70, 36.60±2.70 and
38.20±5.59),
but
not
statistically
significant (P>0.05) when compared to
PCM-induced group. Table 1. There were
dose-dependent increase in liver tissue
degenerations compared to the normal
control and PCM-induced liver damaged
(untreated) control (Fig 1A, B, D and E).
on biochemical parameters in paracitamol-induced
M. esculenta
GROUP
ALT(IU/L)
AST
(IU/L)
ALP
(IU/L)
BILIRUBIN
(mg/dl)
ALB
(g/l)
TP
(g/l)
BUN(mmo
l/l)
NEGATIVE
CONTROL
23.40±3.44
53.00±4.64
10.28±1.47
10.10±1.26
34.80±2.86
72.40±3.97
11.18±2.20
POSITIVE
CONTROL
37.20±3.42*
80.20±6.22
*
18.50±2.51*
11.44±2.21
32.06±2.00
72.20±2.59
12.06±0.81
36.60±2.70*
81.60±7.83
*
19.78±3.39*
11.08±1.99
29.40±1.14
70.80±2.49
12.46±0.61
38.20±5.59*
83.00±7.52
*
19.38±3.67*
12.04±1.82
26.80±2.39
68.20±4.27
10.84±0.88
40.40±3.36*
84.80±5.89
*
22.38±2.47*
12.94±2.06
27.40±3.13
69.60±6.11
11.10±3.53
100mg/kg
EXTRACT
+2000mg/kg
PARACETA
MOL
200mg/kg
EXTRACT +
2000mg/kg
PARACETA
MOL
400mg/kg
EXTRACT
+2000mg/kg
PARACETA
MOL
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D
A
B
E
C
Fig 1: Histopathological changes in the liver
A: control showing normal hepatocytes, central veins and biliary tracts
B: Liver showing fewer vessels with inflammatory lesions ((PCM alone)
C: Liver section showing bloated hepatocytes with many inflammatory cells
D: Liver section showing loss of tissue architecture and inflammatory lesions ((200mg/kg of
extract + 2g/kg of PCM)
E showing loss of tissue architecture with mass degeneration of hepatocytes suggestive of hepatic
failure (400mg/kg of extract + 2g/kg of PC
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DISCUSSION
Paracetamol toxicity is due to the
formation of toxic metabolites when a part
of it is metabolized by cytochrome P450.
Introduction of cytochrome or depletion of
hepatic glutathione is a prerequisite for
paracitamol-induced hepatotoxicity [22].
So in the present study, paracitamol was
employed as toxic agent and the
hepatotoxic effect of aqueous Manhot
esculenta leaf extract against the
paracetamol induced hepatotoxicity was
studied. The extent of toxicity was
estimated by histopathological studies and
biochemical markers like, ALT, AST,
ALP, bilirubin, total protein, albumin and
BUN levels.
The present study reports the potential
hepatotoxic activity of Manihot esculenta
leaf extract against hepatic injury
produced by paracetamol in rats.
Paracitamol is known as antipyretic and
analgesic agent, which is safe in
therapeutic doses, but can produce fatal
hepatic necrosis with high dose. It is
employed as an experimental hepatotoxic
agent [23].
The hepatic cytochrome P450 enzyme
system metabolizes paracitamol, forming a
minor yet significant alkylating metabolite
known as NAPQI. NAPQI is then
irreversibly conjugated with the sulfhydryl
groups of glutathione [24].
NAPQI depletes glutathione and initiates
covalent binding to cellular proteins.
These events lead to the disruption of
calcium
homeostasis,
mitochondrial
dysfunction, and oxidative stress and may
eventually culminate in cellular damage
and death [24].
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Reinforcing the above stated mechanisms,
biochemical parameters (liver enzymes)
demonstrate significant increase in groups
that received toxic dose of paracitamol in
the present study.
Histopathological profile also reveals a
major damage in the same groups. Thus, it
clearly shows that, toxicity is due to either
of the above mechanisms such as
depletion of glutathione store or free
radical generation or lipid peroxidation.
Normally, AST and ALT are present in
high concentration in the liver cells and
are released from the cells due to
hepatocyte necrosis, resulting to their
increase in the blood. ALT is a sensitive
indicator of acute liver damage and
elevation of this enzyme is a common
feature in hepatic damage. ALT is more
selectively a liver parenchymal enzyme
than AST [25]. Liver function is assessed
by estimating the activities of plasma
ALT, AST, ALP, bilirubin, albumin and
total protein. In liver damage, these
enzymes leak into the blood stream in
conformity with the extent of damage [26].
The elevated levels of these enzymes
(ALT, AST and ALP) observed in group 2
(paracitamol treated rats) in this present
study corresponds to the extensive liver
damage induced by paracetamol. The
increase in the concentrations of the liver
enzymes observed in groups III–V,
although not significant, might probably
be due to M .esculenta leaf extract
toxicity.
Plasma ALP and total bilirubin levels are
also related to the status and function of
hepatic cells. High serum level of ALP is
due to its increase synthesis and biliary
pressure [27]. In this present study, it was
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observed that M. esculenta leaf extract
(100, 200, 400 mg/kg), slightly increase
plasma ALP and bilirubin levels though
not statistically significant (p>0.05) when
compared to paracetamol control group.
This clearly shows that the plant extract
shows no hepatoprotective activity, but
tends to potentiate the damage caused by
paracitamol toxicity.
Hypoalbuminaemia is most frequent in
chronic liver disease hence decrease in
total protein content is an indicator and
useful index of the severity of cellular
dysfunction in chronic liver damage[27].
The lowered level of albumin recorded in
plasma of rats that received 2000 mg/kg of
paracitamol reveals the severity of hepatic
damage. There was significant decrease
(p>0.05) in albumin level of rats that
received M. esculenta leaf extract at doses
of 100, 200, 400 mg/kg compared to
paracitamol control group. This shows that
M. esculenta leaf extract possess no
hepatoprotective activity, but potentiates
liver damage as a result of paracitamolinduced liver toxicity.
Blood urea nitrogen of the hepatotoxic
groups is normal when compared to the
normal control group (p>0.05). This
indicates that no injury was caused to the
kidney as a result of M. esculenta leaf
extract and paracitamol-induced toxicity.
M. esculenta leaf extract toxicity may be
as a result of the presence of cyanogenic
glucosides.
The
presence
of
cyanoglycosides,
linamarin
and
lotaustralin pose potential toxic effect.
Linamarin is hydrolysed by intestinal
luminal bacterial beta glucosidase to
release hydrogen cyanide which can cause
acute poisoning [28-30]. It has been
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reported that cassava leaf contains a
number of antioxidant compounds namely
α-carotene, vitamin C, vitamin A,
anthocyanin (flavonoid), saponins and
steroids [31] but they do not offer any
protective activity to the liver cells.
CONCLUSION
The results obtained from the present
study suggest that M. esculenta leaf extract
did not show hepatoprotective activity, but
potentiates liver damage induced by
paracitamol.
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