up to 10 m high. The leaves are alternate, palmate lobed, with stipules. The
*Corresponding author (Email: [email protected]; phone: +2348030824063)
ROM. J. BIOCHEM., 49, 1, 3–12 (2012)
4 Oluseyi Adeboye Akinloye et al. 2
inflorescence consists of bright yellow flowers that are regular or slightly irregular
and borne in racemes or panicles. Fruits are elongated 3–5 valve capsules
containing seeds which are embedded in cotton foam (1). Its local/vernacular
names include "Oja Ikoko"/’Sewutu’ (Yoruba), ‘Obazi’/’Abanzi’ (Igbo) and
"Rawaya"/’Kyamba’ (Hausa). Decoctions of the whole roots have been reported to
be used as remedy for gonorrhoea, jaundice, gastrointestinal diseases, helminthes
and bilharzias infestations, as well as for the management of epilepsy (2–5). Togola
et al. (6) have also reported the antimicrobial properties of Cochlospermum
tinctorium. Recently, anticonvulsant properties and pharmacological evidence on
the folkloric use of Cochlospermum tinctorium was reported (7–9). To the best of
our knowledge, much of the published data focused on the properties of the
Cochlospermum tinctorium root bark extract. Thus, the objective of the present
study was designed to test the hepatoprotective activity of the methanolic extract of
plant leaf against carbon tetrachloride induced liver damage in rats.
MATERIALS AND METHODS
PLANT MATERIALS AND PREPARATION OF THE METHANOLIC EXTRACT
Leaves of Cochlospermum tinctorium were collected from a local garden in
Abeokuta, Nigeria. They were identified and authenticated by Dr. Aworinde D. O.
(a plant taxonomist/anatomist) of the Department of Biological Sciences,
University of Agriculture, Abeokuta, Nigeria. Some voucher specimen number was
submitted to the authority for future references.
The leaves were washed with water, then allowed to air dry for 5 days and
dried in an oven below 50oC, until a constant weight was obtained. The dried
leaves were pulverized with a blender into a fluffy mass (fine powder); 200 g of
powdered leaves were exhaustively extracted with 400 ml of 80% methanol
(MeOH) by simple percolation (cold extraction) for five days. The extraction was
repeated three times, to ensure that the extractable component has been fully
removed from the plant material. The extracts were pooled together, filtered and
concentrated in vacuum at 40oC, using a rotator evaporator, then about 12.5 g of
crude methanol extract was obtained and subsequently referred to as
Cochlospermum tinctorium methanolic leaf extract (CTMLE).
Forty white albino rats of either sex, weighing 150–180 g, purchased from
the animal house of the Department of Veterinary Anatomy, University of Ibadan,
Nigeria were used in this study. They were housed in iron cages under hygienic
and standard environmental conditions (28±2oC, humidity 60-70%, natural 12 hr
3 Hepatoprotective effect of Cochlospermum tinctorium on CCl4-induced toxicity 5
light/dark cycle). They were allowed free access to laboratory diet (Ladokun and
sons feeds, Nigeria Ltd) and water. They were allowed to acclimatize for two
weeks. The rats were handled with care, according to the Guide for the Care and
Use of Laboratory Animals Manual. All experimental protocols were approved by
the Departmental animal ethics committee.
CHEMICALS AND REAGENTS
The carbon tetrachloride used by us was manufactured by May and Baker
Ltd, Dagenham, England. Teco Diagnostic kits (Lakeview Ave. Anaheim, USA)
were used for the analysis of biochemical parameters. All the other chemicals were
of analytical grades.
The CTMLE was subjected to various phytochemical tests, in order to
identify the constituent secondary metabolites using standard methods, as described
by Harborne (10), Sofowora (11), and Trease and Evans (12).
EXPERIMENTAL DESIGN FOR HEPATOPROTECTIVE ACTIVITY
The rats were randomly divided into four groups of ten each. The
hepatoprotective activity of plant extracts was tested using the CCl4 model. Carbon
tetrachloride hepatotoxicity was induced in rats according to the method of Rao et al.
(13, 14), with slight modifications. Group I (normal control) received only food
and water. Group II (induction control) was given a single intraperitoneal dose of
2 mg/kg CCl4. Group III received CCl4 followed by oral administration of CTMLE
at the dose of 200 mg/kg b.wt as a fine suspension made by adding sorbitol. Group
IV received CCl4 and then prednisolone (standard anti-inflammatory drug).
The experiment was carried out as per the guidelines of committee for the
purpose of control and supervision of experiment on animal care and handling. The
protocol conforms to the guidelines of the National Institute of Health (NIH).
ASSESSMENT OF HEPATOPROTECTIVE ACTIVITY
In the present study, the hepatoprotective activity was estimated
biochemically and histopathologically. After a week administration/treatment, the
animals were dissected under diethylether anesthesia. Blood from each rat was
withdrawn by cardiac puncture into non-heparinized tubes, allowed to clot for 30
minutes at room temperature. Serum was separated by centrifugation at 3000 rpm
for 15 min. The separated sera were used for the estimation of some biochemical
6 Oluseyi Adeboye Akinloye et al. 4
The level of malondialdehyde (MDA) produced was estimated by the double
heating method of Draper and Hadely (15). Briefly, 1.0 ml of 100 g/L
trichloroacetic (TCA) solution was added to 0.2 ml serum, placed in a water bath
for 10 min. After cooling in tap water, the mixture was centrifuged at 1000 rpm for
10 min and 0.5 ml of the supernatant added to 0.5 ml of 6.7 g/L thiobarbituric acid
solution in a test tube and placed in a boiling water bath for 15 min. The solution
was then cooled under tap water and its absorbance measured at 532 nm (using a
Shimadzu UV-VIS 1610 Tokyo Spectrophotometer). The concentration of MDA
was calculated by the absorbance coefficient of MDA-TBA complex 1.56 x 105 cm-1m-1.
Serum alanine aminotransferase (ALT/SGPT) and aspartate aminotransferase
(AST/SGOT) were determined according to the method of Reitman and Frankel
(16), while cholesterol, bilirubin, urea and glucose levels were measured using the
Teco reagent diagnostic kit. Protein concentration was determined by the method
of Lowry et al. (17).
For histopathological studies, liver from each animal was removed after
dissection and preserved in Bouin fluid (picric acid+formalin+acetic acid). Then,
representative blocks of liver tissue from each lobe were taken and processed for
paraffin embedding, using the standard microtechnique (18). Section (5 μm) of
liver stained with hematoxylin and eosin was observed microscopically and
photographed (Olypus, CS21) for histopathological studies.
The results of biochemical analysis were expressed as mean ± standard error
of mean (Mean ± S.EM). The control and treatment groups were compared by
using one-way analysis of variance (ANOVA). Differences were detected by the
Turkey-Kramer multiple comparison test. The level of significance was taken at
probability less than 5%.
The present study attempted to show the potential hepatoprotective activity of
crude methanol leaf extract of Cochlospermum tinctorium in carbon tetrachloride
induced hepatotoxicity. The Cochlospermum tinctorium leaf extract was found to
contain saponins, flavonoids, tannins and alkaloids.
The results of CTMLE on some biochemical parameters used as basis for the
hepatoprotective index at a dose of 200 mg/kg on rats intoxicated with CCl4 are
resumed in Table 1.
5 Hepatoprotective effect of Cochlospermum tinctorium on CCl4-induced toxicity 7
Effects of methanolic extract of Cochlospermum tinctorium on various biochemical parameters in rats
with carbon tetrachloride induced hepatotoxicity
Values with different superscript along the same column are significantly
different at p<0.05. The table also shows a comparison of the CTMLE effects
among the untreated (normal control), carbon tetrachloride treated (induced
control), extract treated group and standard drug treated groups of rats. Data were
represented as Mean ± Standard Error of Mean (M ± SEM). The results were
analyzed statistically by one-way analysis of variance (ANOVA), followed by
Turkey’s test using SPSS 11.5 for window Software. P < 0.05 was regarded as
It was observed that the CCl4 group significantly increased the serum level of
SPGT (5.75%), SGOT (41.5%), bilirubin (42.4%) and cholesterol when compared
to the control group (group 1). However, the plant extract exhibited a significant
protection against CCl4 induced liver injury, as expressed by the reduction in toxin
mediated rise in SGPT, SGOT and cholesterol level of rats. There was no statistical
significant change in the blood glucose level of rats among all groups. The level of
MDA was significantly higher in CCl4 intoxicated rats by 32.7%, in comparison to
the normal control group; this is an indication for lipid peroxidation of hepatic
cells. Once administered to the CCl4 intoxicated rats, the extract improved the level
of peroxidation by reducing the amount of MDA. The results showed a significant
decrease in MDA levels by 39.7%, when compared to CCl4-intoxicated rats. The
value of MDA was close to that of the control group 1.
The results of histopathological studies also provided supportive evidence for
biochemical analysis. For instance, histology of liver section of control animal
(group 1) exhibited normal hepatic cells, each with well defined cytoplasm,
prominent nucleus and nucleolus with well revealed central vein (Plate 1), whereas
that of the CCl4 intoxicated group animal showed complete loss of hepatic
architecture with centrilobular hepatic necrosis fatty changes, vacuolization and
sinusoid congestion (Plate 2). Treatment with methanol extract of C. tinctorium
showed a moderate activity of protecting the liver cells against CCl4 injury. The
liver session of CTMLE treated group showed evidence of regeneration. The
severity of degenerative changes in tubules was lower than in CCl4 untreated group
(Plate 3). However, test results in the CCl4 + Predinsolone group were quite
comparable to the control (Plate 4).
The experimental induction of liver damage by CCl4 in this study was
adopted because CCl4 has been known to catabolise free radical-induced lipid
peroxidation, damaging the membranes of the liver cells and organelle, causing
swelling and necrosis of hepatocytes, and resulting in the release of cytosolic
enzymes (ALT, AST AP and GGT) into the circulating blood (19, 20). Also,
prednisolone was used as a standard/reference drug or positive control, because it
is known to be a hepatic curative agent through its modulatory anti-inflammatory
actions on hepatic disorders irrespective of the cause.
The study demonstrates that single dose of CCl4 injection produced elevated
levels of SGPT, SGOT and cholesterol; an increase in bilirubin and decrease in
protein was also found, which is in good agreement with the results of Etuk et al.
(8). Many authors have reported hepatoprotective properties of some medicinal
plants, such as Cassia tora (21), Phyllanthus amarus (22), Zizyphus Mauritian
(23). However, liver damage with CCl4 is a commonly used model for the
screening of hepatoprotective drugs (24). To the best of our knowledge, the
reviewed literature showed that no research has been reported on hepatoprotective
properties of leaves. The present biochemical and histopathological analysis of our
plant extract showed a good development in ameliorating carbon tetrachlorideinduced
damaged liver cells. The rise in serum levels of AST, ALT and cholesterol
have been attributed to the damaged structural integrity of the liver, because they
are located in the cytoplasm and are released into circulation after cellular
damages. This is in line with the reports of Sallie et al. (25), and Ashan et al. (26),
which show that, when administered to rats, chemicals or drugs often induce
hepatotoxicity by metabolic disturbance and activation. For instance, CCl4 is known
to be metabolically activated by the cytochrome P-450 dependent mixed oxidase in
the endoplasmic reticulum to form trichloromethyl radicals (CCl3), which
combined with cellular lipids and proteins in the presence of oxygen to induce lipid
peroxidation (27). Treatment with C. tinctorium methanol leaves extract recovered
the injured liver to normal after a week administration at a dose of 200 mg/kg body
weight, which indicates its potential as anti-hepatoprotective agent.
The ability of C. tinctorium extract to reduce the level of peroxidation could
be attributed to the presence of flavonoids, which have been reported to possess
anti-oxidant activity which is presumed to be responsible for the inhibitory effect
on several enzymes, including those involved in arachidonic acid metabolism (13, 14).
From the overall results, it could be inferred that C. tinctorium has
hepatoprotective activity and it was assumed that it confers hepatoprotection
probably as a result of the presence of both enzymic and non-enzymic antioxidants
9 Hepatoprotective effect of Cochlospermum tinctorium on CCl4-induced toxicity 11
that could bring about free radical suppressing activity. Meanwhile, work is in
progress on other possible protective mechanisms, such that it may lend
pharmacological/scientific credence to the ethno-medical claims of the use of this
plant in the management of ailments.