A Bushy Savannah Plant

Published Date: 02 Nov 2017

commonly found in fallow farms across northern Nigeria. It is a shrub that grows

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).

ANIMALS

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.

PHYTOCHEMICAL SCREENING

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

parameters.

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.

STATISTICAL ANALYSIS

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%.

RESULTS

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

Table 1

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

statistically significant.

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).

DISCUSSION

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).

CONCLUSIONS

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.