EFFECT OF AQUEOUS EXTRACT OF BITTER LEAF (Vernonia
amygdalina del) ON CARBON TETRA-CHLORIDE (CCl4) INDUCED
LIVER DAMAGE IN ALBINO WISTAR RATS.
BY
Arhoghro, E.M., Anosike, E.O. and Ibeh, G.O.
Department of Biochemistry,
University of Port Harcourt,
P.M.B. 5323,
Port Harcourt
Nigeria
ABSTRACT
The effect of aqueous extract of the leaves of Vernonia amygdalina
del on CCl4-induced liver damage was investigated in experimental rats.
Separate groups of rats were administered orally with 2.5 ml/kg body weight
of 5, 10 and 15% aqueous leaf extracts of V. amygdalina for 21 days after
treatment with CCl4. Another group of rats were administered (5, 10 and
15%) extract before treatment with CCl4.
The latter showed less
hepatotoxcity, than the former, as measured by serum alkaline phosphatase
(ALP), alanine aminotransferase (ALT) and aspartate aminotransferase
activities. The difference was significant (P < 0.05). Rats administered
extract after treatment with CCl4 showed decrease in ALP, ALT and AST
activities. The effect of the extract was dose-dependent, Histopathological
study also showed significant reduction and even reversal of liver damage in
the rats. The results of this study show that aqueous leaf extract of V.
amygdalina might have a potent hepatoprotective action against CCl4induced liver damage in rats.
Key words:
Vernonia amygdalina (v.a), aqueous extract, carbon
tetrachloride, Hepatoprotective action, histopathological study.

Corresponding author (c/o uwadik@yahoo.com).
1
INTRODUCTION
The use of herbal products for medicinal benefits has played an
important role in nearly every culture on earth.
Herbal medicine was
practised by ancient people of Africa, Asia, Europe and the Americas
(Wargovich et al, 2001). Over 50% of all modern clinical drugs are of
natural product origin and natural products play an important role in drug
development programs of the pharmaceutical industry.
The consumption of a variety of local herbs and vegetables by man is
believed to contribute significantly to the improvement of human health, in
terms of prevention, and or cure of diseases because plants have long served
as a useful and rational source of therapeutic agents (Roberts and Tyler,
1999).
Vernonia amygdalina is a widely used local plant in Nigeria for both
therapeutic and nutritional purposes.
V. amygdalina grows in a range of ecological zones in Africa and
produces large mass forage and is drought tolerant (Bonsi et al, 1995). The
bitter taste is due to anti-nutritional factors such as alkaloids, saponins
tannins and glycosides (Butter and Bailey, 1973). The macerated leaves of
the plants are consumed as vegetables and condiments while the water
extract serves as tonic for the prevention of certain illnesses.
2
V. amygdalina has been reported for its use by wild chimpanzee for
the treatment of parasite related diseases in Tanzania (Huffman and Seifu,
1989).
Philipson et al, (1993) reported the antiplasmodial effects of
sesquiterpene and steroidal constituents of V. amygdalina and some were
also effective against plasmodium falciparium in vitro.
Uhegbu and
Ogbuehi (2004) reported the antidiabetic effects of aqueous extract of leaves
of Vernonia amygdalina.
Recently, Izevbigie (2003) isolated some peptides (edotides) from the
aqueous extract of V. amygdalina. The peptides were shown to be potent
inhibitor of nitrogen activated protein kinases (NAPK) which are crucial for
breast tumour growth and also represents a key regulatory point for tumour
growth. The anti-estrogen breast cancer drug (Tamoxiten) has also been
shown to modulate MAPK activity (Atanaskova et al, 2002; Mandelekar and
Kong 2001), this indicates that edotides from V. amygdalina may be
considered as alternative to tamoxiten.
Teas containing bitter leaf (vernonia amygdalina) are also used
throughout West Africa for the management of diabetes and other metabolic
diseases associated with the liver. The liver is an organ of paramount
importance, which plays an essential role in the metabolism of foreign
compounds entering the body.
Human beings are exposed to these
3
compounds through environmental exposure, consumption of contaminated
food or during exposure to chemical substances in the occupational
environment. Liver disease is a world wide problem. Conventional drugs
used in the treatment of liver diseases are sometimes inadequate and can
have serious adverse effects.
It is therefore necessary to search for
alternative drugs for the treatment of liver disease to replace currently used
drugs of doubtful efficacy and safety. In this publication, we report the
effects of aqueous leaf extracts of vernonia amygdalina on carbon
tetrachloride-induced liver damage in rats.
MATERIALS AND METHODS
Animals
Wistar Albino rats (100-150g) of both sexes about three months old
bred in the animal house of Biochemistry department, University of Port
Harcourt were used in this study. They were randomly selected and kept in
6 groups of not less than three rats per group. Each group was kept in a
separate cage. All animals were fed with commercially formulated rat feed
and water was given ad libitum. Their cages were cleaned daily and food
and water changed daily. The animals were allowed to acclimatize for 2
weeks.
4
Chemicals
All chemicals used were of analytical grade.
Preparation of Aqueous Extract:
The leaves of Vernonia amygdalina were collected from Sagbama in
Bayelsa State and were identified at the Department of Plant Science and
Biotechnology, University of Port Harcourt, Nigeria. The leaves were sun
dried, pulverized and then sieved. Twenty-five (25) grams of the powdered
leaf was weighed out and dissolved in 250ml of distilled water to make 10%
extract. From the 10% stock of bitter leaf extract 5%, 10% and 15% (v/v)
solutions in distilled water were prepared.
Experimental procedure:
In the experiment, a total of 19 rats (13 CCl4-induced liver damaged
rats, 3 normal rats, 3 vegetable oil treated rats) were used. The rats were
assigned to one of 6 groups each of not less than 3 rats. The first group was
not treated with CCl4 and served as normal control (C). The second group
was treated with 0.5ml veg. oil/kg rat. CCl4 was dissolved in vegetable oil
and given intraperitonally (ip) to rats in group 3, (disease control) and those
in groups 4, 5 and 6 (test) at a dose of 0.5ml CCl 4/kg body weight in not
5
more than 0.5ml of vegetable oil. 5, 10 and 15% (v/v) bitter leaf extracts
were given 2.5ml/kg body weight orally by intubation to rats in groups 4, 5
and 6 respectively.
At the end of treatment at 7, 14 and 21 days
respectively, blood sample were collected by direct cardial puncture and the
serum was used for the assay of marker enzymes, aspartate aminotransferase
(AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP).
The liver was removed and part of the right lobe was sliced, fixed in 10%
buffered formaldehyde solution and used for histological examination.
Assessment of Liver Function
Biochemical analysis of the serum enzymes for ALT and AST was by
the method of Reitman and Frankel, (1957). ALP was assayed according to
the method of REC (1972).
Statistical Analysis
Results of Biochemical estimations are reported as mean + SD and
statistical analysis was performed using students t-test and P < 0.05 being
considered as statistically significant.
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RESULTS
The results of the effect of aqueous leaf extract of Vernonia
amygdalina (V.A.) on CCl4-treated rats are shown in Table 1. In the CCl4treated group, serum ALT, AST and ALP levels were quite high.
In
contrast, the CCl4 group treated with leaf extracts of V.A. had significantly
(P < 0.05) lower levels of ALT, AST and ALT when compared with the
CCl4 control group. The reduction of the ALT, AST and ALP activities by
V.A. extract though not statistically significant (P < 0.05) was dose
dependent. The effect of time of administration of V.A. extracts on ALT,
AST and ALP activities was statistically significant (P < 0.05). (Table 2)
Table 1: The effect of aqueous leaf extract of Veronica amygdalina (V.A.)
on activities of ALT, AST and ALP on rats treated with CCl4.
Groups
ALT (U/L)
AST (U/L)
ALP (U/L)
1 normal
35.10 + 3.77b
120.17+ 2.91b
247.30+ 3.56b
35.30 +5.42b
122.58 + 3.25b
214.24 + 6.43ab
68.95 + 21.38a
165.65 +17.75a
364.65 + 37.75a
40.24 +4.03b
123.40 + 9.38ab
213.44 + 17.52ab
n = 3
2 veg oil
n =3
3 ccl4
n =4
4 CCl4 +
5% V.A n = 3
7
Groups
ALT (U/L)
AST (U/L)
ALP (U/L)
5 CCl4 +
37.38 +4.63b
115.77 + 18.17ab
201.14 + 20.23ab
39.31 + 3.30b
116.90 + 1.75ab
207.35 + 5.51ab
10% V.A n = 3
6 CCl4 +
15% V.A n = 3
Data are mean + SD
a – significantly different from normal control group
b – Significantly different from CCl4 treated group.
Table 2: The effect of time (duration) of administration of aqueous leaf
extract on ALT, AST and ALP activities on rats treated with CCl4
Group
Period
ALT (U/L)
AST (U/L0
ALT (U/L)
3 CCl4
7 days
80.60 + 0.59a
177.50 + 0.54a
375.36 + 0.94a
N
14 days
57.70 + 0.29b
157.00 + 1.30b
381.92 + 0.26b
21 days
48.80 + 0.40c
145.00 + 0.10c
309.00 + 0.90c
4 CCl4 + 5%
7 days
44.30 + 1.20d
133.40 + 0.70d
222.76 + 0.88d
V.A.
14 days
36.26 + 0.94e
114.80 + 0.26e
194.92 + 0.44e
21 days
40.17 + 0.18f
122.00 + 2.25f
215.64 + 0.86f
5 CCl4 + 10%
7 days
36.00 + 0.53e
122.00 + 1.93f
210.00 + 0.61g
V.A.
14 days
42.55 + 0.67g
130.00 + 0.21g
215.43 + 0.40f
21 days
33.60 + 0.50h
95.30 + 2.95h
178.00 + 1.47g
6 CCl4 + 15%
7 days
42.60 + 0.37g
124.90 + 0.38f
201.20 + 1.20h
V.A.
14 days
36.00 + 0.52e
116.80 + 0.40g
211.84 + 0.83g
21 days
39.33 + 0.77f
109.00 + 0.46j
209.00 + 1.05g
Mean with different superscripts are significantly different from each other
at (P < 0.05).
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DISCUSSION
Carbon tetrachloride (CCl4) is one common hepatotoxin used in the
experimental study of liver diseases (Obi et al, 1998, Ulicina et al., 2003,
Yan-Jun luo et al., 2004). CCl4 treatment generates free radicals that trigger
a cascade of events resulting in hepatic fibrosis. In this study, when treated
with CCl4, the liver exhibited drastic alterations, extensive fatty change,
distended hepatocytes vacuolated cytoplasm, compressed sinusoids, fatty
degeneration, area of necrosis and infiltration by inflammatory cells. In
addition, serum levels of ALT, AST and ALP were elevated.
This is in agreement with the report by previous workers (Rienke et
al, 1988, Obi et al, 1998, Ulicna et al, 2003, Yan Jun luo et al, 2004).
A primary consideration in the assessment of the efficacy of a
potential therapeutic agent for hepatic injury (damage) is its effect on liver
histology. Those livers that were treated with CCl4 (Group 3) had a high
degree of fatty change, compression of sinusoid and distention of
hepatocytes. V.A. extract in the dosage range administered to liver damage
rats apparently accelerated the reversion of the liver damage and lowered the
high levels of serum ALT, AST and ALP activities when compared to rats
treated with CCl4 alone. The effect was time and dose dependent.
9
Akah and Okafor (1992), reported that the leaves of Vernonia
amygdalina contain saponins, sesquiterpenes, lactone, steroid glycosides,
alkaloids, tannins and flavonoids.
Flavonoids are reported to exhibit
antioxidant activity (Ramanthan, et al, 1989) and are effective scavengers of
superoxide anions (Robak and Gygkwski, 1988). The aqueous leaf extract
of Vernonia amygdalina may have exhibited hepatoprotective activity due to
its antioxidant property attributable to flavonoids.
In conclusion, our results indicate that treatment with V.A. extracts
after establishment of CCl4 – induced liver damage significantly reduced and
even reversed the damage in rats. Hence the leaf extracts of Vernonia
amygdalina might be effective hepatoprotectors in the diets of patients with
hepatopathies since the model of CCl4 – induced liver damage in rats
simulates many of the features of human liver fibrosis (damage).
10
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