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Hepatoprotective Activity of Ocimum americanum L Leaves against

Paracetamol − Induced Liver Damage in Rats

Article  in  American Journal of Life Sciences · January 2013

DOI: 10.11648/j.ajls.20130102.13

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American Journal of Life Sciences
2013; 1(2) : 37-42
Published online April 10, 2013 (http://www.sciencepublishinggroup.com/j/ajls)
doi: 10.11648/j.ajls.20130102.13

Hepatoprotective activity of Ocimum americanum L

leaves against paracetamol – induced liver damage in
rats
B. T. Aluko1, 2, O. I. Oloyede1, A. J. Afolayan2,*
1
Department of Biochemistry, Ekiti State University, Ado Ekiti, Nigeria
2
Department of Botany, University of Fort Hare, Alice, South Africa

E mail address:
[email protected](A. J. Afolayan)

To cite this article

B. T. Aluko, O. I. Oloyede, A. J. Afolayan. Hepatoprotective Activity of Ocimum americanum L Leaves against Paracetamol – Induced
Liver Damage in Rats. American Journal of Life Sciences. Vol. 1, No. 2, 2013, pp. 37-42. doi: 10.11648/j.ajls.20130102.13

Abstract: This study was designed to investigate the hepatoprotective activity of aqueous extract of Ocimum ameri-
canum leaves against paracetamol – induced liver damage in rats. Hepatic damage was induced by paracetamol. Thereafter,
the levels of some serum biochemical parameters such as alanine trasaminase (ALT), aspartate transaminase (AST), alka-
line phosphatase (ALP), albumin, total bilirubin (TBIL) and total protein (TP) were investigated. The activities of ALP,
AST, ALT and histological changes in the liver of rats were also determined. Silymarin was used as the standard hepatopro-
tective drug. The pre – treatment of rats with aqueous extract of O. americanum leaves caused a significant increase in the
serum levels of TP and albumin. There was a significant decrease in the serum levels of ALP, AST, ALT and TBIL with a
corresponding increase in the activities of ALP, AST and ALT in the liver of extract treated rats. The hepatoprotection was
confirmed by histological examinations of liver sections of normal and treated rats. Furthermore, rats intoxicated with para-
cetamol alone had their serum ALP, AST, ALT and TBIL levels significantly increased, while TP and albumin concentra-
tions decreased when compared with the normal rats. The aqueous extract of Ocimum americanum leaves at doses of 200
and 400 mg /kg p.o. have significant hepatoprotective ability against paracetamol – induced hepatic damage in rats.

Keywords: Ocimum americanum, Hepatoprotective Activity, Paracetamol, Biochemical Parameters

retic drug that is known to be safe at recommended doses.

1. Introduction However, it produces acute liver damage at higher doses
The liver is the chemical factory which regulates, syn- [4].
thesizes, stores and secretes important macromolecules in Ocimum americanum L commonly known as African ba-
the body. It has a strategic anatomical location and large sil belongs to the genus Lamiaceae. It is a wild herb with a
capacity for metabolic transformation of drugs and other distinct mint flavor, hairy leaves and scented flowers that is
toxins entering from the gastrointestinal tract. As a result of native to tropical Africa [5]. It is popularly called "Efinrin
this, the healthy functioning of the liver determines the elewe dudu" in south-western Nigeria. The leaf has been
health status of an individual [1]. Liver diseases are a glob- used in traditional folk medicine in Ghana to treat diabetes
al problem and the synthetic drugs available for the treat- [6]. It is used by traditional healers in Nigeria for the treat-
ment of liver disorders are believed to have serious adverse ment of constipation, diarrhoea, piles, dysentery and as
effects on biological systems [2]. Due to these facts, atten- insect repellent. The leaf is rich in essential oils of thera-
tion has been given to finding suitable curative agents for peutic importance and mostly used as a condiment for the
the treatment of liver diseases from natural product of plant preparation of delicious local soup because of its aromatic
origin [3]. Acetaminophen – induced liver injury in living properties [7]. The acetone extract of this plant has been
systems has been well documented. Hepatotoxicity of ace- reported to inhibit neurotoxins - induced brain damage in
taminophen has been attributed to the formation of N – rats [8]. Our survey of literature reveals that the hepatopro-
acetyl – p – benzo quinoneimine (NAPQI) a toxic metabo- tective activity of Ocimum americanum leaves has not been
lite that causes glutathione depletion and oxidative stress. scientifically investigated. The aim of this research there-
Acetaminophen (Paracetamol) is an analgesic and antipy- fore, was to assess the protective effects of aqueous extract
38 B. T. Aluko et al.: Hepatoprotective activity of Ocimum americanum L leaves
against paracetamol – induced liver damage in rats

O. americanum leaves in rat model of acetaminophen – 4000 mg/kg, p.o. of the extract were given to 4 groups con-
induced liver injury. taining 4 animals in each group. Single dose of the extract
was administered orally to each animal. The animals were
2. Materials and Methods observed individually during the first 30 min and thereafter
24 hourly for a period of 14 days. Signs of toxicity, body
2.1. Collection and Identification of Plant Material weight, feed and water intake for each group was observed
everyday for 14 days. The extract was devoid of toxicity
The leaves of Ocimum americanum were obtained from even at a dose of 4000 mg/kg by oral route in rats. Hence,
a local farmland near Orin Ekiti South-Western Nigeria in 200 and 400 mg/kg doses of the extract were selected for
the month of May, 2011. The plant was identified and au- further experiment.
thenticated by Mr Omotayo (herbarium curator) at the De-
partment of Plant Science, University of Ado Ekiti, Nigeria 2.6 . Acetaminophen – Induced Hepatotoxicity in Rats
where the voucher specimen (Aluko 09) was deposited.
The method of Nirmala et al [10] was used to evaluate
2.2. Reagents the hepatoprotective activity of aqueous extract of O. amer-
icanum leaves with some modifications. The animals were
Silymarin used in this study was purchased from Sigma- divided into five groups of six animals each. Animals were
Aldrich Gmbh, Sternheim, Germany. Assay kits for the treated for a period of seven days as follows:
determination of ALT, AST and ALP were purchased from Group 1 (normal control) received 1ml of distilled water
Randox laboratories Ltd. Ardmore, United Kingdom. All p.o. for 7 days
other chemicals used were of high analytical grade. Group 2 (toxic group) received 1ml of distilled water p.o.
2.3. Sample Extraction for 7 days and paracetamol (2 g/kg b.w., p.o.) on the 4th
and 5th day.
The leaves of O. americanum were air dried for 10 days Group 3 received aqueous extract (200 mg/kg b.w. p.o.)
and then ground into fine powder using an electric blender. for 7 days and paracetamol (2 g/kg b.w., p.o.) on the 4th
Fifty grams of the powdered sample was extracted in 1 liter and 5th day, 30 min after extract administration.
of cold sterile distilled water maintained on a mechanical Group 4 received aqueous extract (400 mg/kg b.w. p.o.)
shaker (Stuart Scientific Orbital Shaker SO1, Essex, UK) for 7 days and paracetamol (2 g/kg b.w., p.o.) on the 4th
for 24 h. The extract was filtered using a Buchner funnel and 5th day, 30 min after extract administration.
with Whatman’s No 1 filter paper. The filtrate was frozen at Group 5 (standard group) received silymarin (100 mg/kg
-40oC and dried for 72 h using a freeze drier (Savant Refri- b.w. p.o.) for 7 days and paracetamol (2 g/kg b.w., p.o.) on
gerated vapor Trap, RVT 41404, USA) to give a percentage the 4th and 5th day 30 min after the administration of sily-
yield of 14.2% w/w. The resulting extract was reconstituted marin.
in cold distilled water to give doses of 200 and 400 mg/kg No mortality was observed at the end of the experimental
body weight. period (7 days). Four animals per group were sacrificed
under light ether anesthesia 24 h after their respective doses.
2.4. Experimental Animals Blood samples were collected into EDTA bottles by cardiac
Male Wistar rats (Rattus norvegicus) weighing 140 – 180 puncture while the liver was quickly removed into ice cold
g were obtained from the animal house of the laboratory of 0.25 M sucrose solution. Thereafter, the liver was blotted
School of Biological Sciences, University of Fort Hare with clean tissue paper and homogenized in Tris Hcl buffer
Alice 5700 South Africa. They were kept in clean cages (0.1 M pH 7.4 1: 10 w/v). The homogenates were kept fro-
placed in a well ventilated house condition (temperature: zen overnight before being used for various enzyme assays.
22 ± 2oC; photoperiod: 12 h light and 12 h dark cycle; hu- 2.7. Determination of Biochemical Parameters
midity: 40 – 45%). The animals were fed ad libtum with rat
pellets and tap water freed of contaminants. The experi- The biochemical parameters such as alanine trasaminase
ment was carried out in line with the guidelines of Ethics (ALT), aspartate transaminase (AST), alkaline phosphatase
Committee on the use and care of Experimental Animals of (ALP), albumin, total bilirubin (TBIL) and total protein
the University of Fort Hare Alice, South Africa. (TP) in the serum were analyzed using liver panel plus
reagent discs on an automated chemistry analyzer (Piccolo
2.5. Acute Toxicity Studies (LD50) Blood Chemistry Analyzer, Abaxis, Union City, CA, USA.
Aqueous extract of O. americanum leaves was studied [11] The liver homogenate was assayed for ALT, AST and
for acute oral toxicity as per Organization for Economic ALP according to the manufacturer’s instructions on the
Co-operation and Development (OECD) guidelines number assay kits.
423 [9]. The modified method of Nirmala et al [10] was 2.8. Histopathological Studies
used to assess the acute oral toxicity of O. americanum
leaves. Healthy Wistar rats weighing (140 – 180 g) were The liver specimen from the control and test groups were
used for this purpose. Four doses of 500, 1000, 2000 and fixed in 10% buffered formalin for 48 h. The formalin fixed
 American Journal of Life Sciences 2013, 1(2) : 37-42 39

samples were stained with haematoxylin – eosin. The sec- Results were expressed as mean ± SD and analyzed by
tions were examined microscopically for changes in histo- one – way analysis of variance (ANOVA) followed by
pathological architecture. Duncan multiple range test. The results obtained from both
the extract and standard drug were compared with that of
2.9. Statistical Analysis the normal rats. P values < 05 were considered significant.
Table 1. Effect of aqueous extract of O. americanum leaves on some serum biochemical parameters of paracetamol intoxicated rats.

Treatment Albumin (g/dl) ALP (U/L) ALT (U/L) AST (U/L) TP (g/dl) TBIL (mg/dl)

Normal rats 4.07 ± 0.42a 264.67 ± 8.08 a 45.00 ± 4.58 a 116.33± 5.13 a 6.37 ± 0.05 a 0.27 ± 0.06 a

PARA (2 g/kg) 1.80 ±0.20 c 323.66± 14.50c 92.31 ± 5.86 c 188.00 ± 9.00 c 3.40 ±0.20 c 1.27 ± 0.35 c

PARA + AOA (200 mg /kg) 2.40 ±0.20 b 280.33 ± 6.51 b 65.00 ± 2.65 b 138.33 ± 3.51 b 5.37 ±0.25 b 0.67 ± 0.05 b

PARA + AOA (400 mg /kg) 2.83 ±0.45 b 259.67 ± 9.71 a 44.30 ± 6.03 a 120.67 ± 5.86 a 6.13 ± 0.06 a 0.30 ± 0.10 a

PARA + SILY (100 mg /kg) 3.73 ±0.31 a 258.00 ± 4.36 a 45.33 ± 2.89 a 117.00 ± 1.73 a 6.30 ± 0.10 a 0.33 ± 0.06 a

Data represent mean ± SD values of triplicate determination for each animal. Test values carrying superscripts (b - c) are significantly different (P < 05)
from the normal control (a) for each parameter. PARA: paracetamol ; AOA: aqueous extract of O. americanum leaves; SILY: silymarin.

3. Results Table 2. Effect of aqueous extract of O. americanum leaves on the activity

of some enzymes in the liver of paracetamol intoxicated rats.
3.1. Acute Toxicity

The aqueous extract of O. americanum leaves was found Treatment ALP (U/L) ALT (U/L) AST (U/L)
to be safe. No mortality was observed for 14 days of treat-
ment with a limit dose of 4000 mg/kg body weight. All the
Normal rats 365.03 ± 13.51a 127.83 ± 6.22 a 141.83± 8.47 a
rats tolerated the extract with no signs of toxicity.

3.2. Effect of Aqueous Extract of O. Americanum Leaves

PARA (2 g/kg) 201.60 ± 6.68 c 73.90 ± 6.18c 76.56 ± 5.86 d
Against Paracetamol – Induced Hepatotoxicity in
Rats
PARA + AOA
The effect of aqueous extract of O. americanum leaves 224.83 ± 12.48c 78.86 ± 4.09 bc 100.47 ± 10.11 c
(200 mg /kg)
on some serum biochemical parameters is presented in Ta-
ble 1 The administration of paracetamol (2000 mg/kg b.w.)
PARA + AOA
induced a marked significant increase in the levels of blood 317.19 ± 12.35b 126.19± 8.22 a 130.41 ± 11.42 b
(400 mg /kg)
ALT, AST, ALP and TBIL as compared to normal control.
However, the pretreatment of rats with aqueous extract of
PARA +SILY
O. americanum leaves prior to paracetamol administration 367.30 ± 25.95a 127.92 ± 5.66 a 137.04 ± 3.04 b
(100 mg /kg)
caused a significant reduction in the levels of these markers
in a dose dependent manner. The administration of the ex- Data are mean ± SD values of triplicate determination for each animal.
tract and silymarin (400 and 100 mg/kg b. w. respectively) Test values carrying superscripts (b - c) are significantly different (P < 05)
restored the levels of these parameters to normal levels in from the normal control (a) for each parameter. PARA: paracetamol ; AOA:
aqueous extract of O. americanum leaves; SILY: silymarin.
the blood. In the case of albumin and total protein there
was a remarkable reduction in their levels in the blood of 3.3. Histopathological Studies
paracetamol intoxicated rats indicating hepatocellular in-
jury. Treatment with O. americanum extract caused a sig- The hepatoprotective potential of O. americanum leaf
nificant rise in their levels to values that were comparable extract was confirmed by histological examination of nor-
with those of silymarin treated group and normal control. mal and treated rats. The histological profile of normal rats
Also, there was a significant decrease in the levels of showed normal cellular architecture (Fig 1 a). In paraceta-
ALT, AST and ALP in the liver of paracetamol treated. This mol intoxicated animals, there were drastic alterations in
was an indication of liver damage due to leakage of these the histological architecture of the liver. Histological ex-
enzymes from hepatic cells into the extracellular compart- amination showed distende hepatocytes, fatty degeneration
ment. However, a dose dependent increase in the levels of and area of necrosis (Fig 1 b). The administration of O.
these enzymes was noticed in the rats pretreated with O. americanum extract and silymarin brought about significant
americanum extract and silymarin which was reversed to recovery. There was less degeneration of hepatocytes as a
the level of normal control (Table 2). result of marked regeneration activity. (Fig 1 c - e).
40 B. T. Aluko et al.:
al. Hepatoprotective activity of Ocimum americanum L leaves
a
against paracetamol – induced liver damage in rats

4. Discussion
The liver plays a pivotal role of detoxification of xeno-
xen
biotics and drugs in biological systems. Hepatic damage
has been reported to upset the normal metabolic
met processes
in the body [12]. Paracetamol is metabolized in the liver to
excretable glucuronide conjugates [13]. However the over-
dose of this drug results in the accumulation of a toxic me-
m
a
tabolite known as N – acetyl – p – benzo quinoneimine
(NAPQI) [14].. This metabolite induces toxicity
t by binding
to proteins and DNA to produce protein adducts which are
responsible for the dysfunction and death of hepatocytes
leading to liver necrosis [15].]. Protection against paraceta-
mol – induced liver damage has been used to assess the
hepatoprotective potential of plant extracts [16,
[ 17, 18].
In this study, the evidence of paracetamol – induced liver
injury was the elevation of blood levels of cellular markers
(AST, ALT, ALP and TBIL) and decreased levels of albu- alb
b min and total proteins. Increased levels of enzyme in the
blood have been attributed to disintegration and turnover of
tissues [19]. Therefore, the increased levels of these mark-
mar
ers in the blood of intoxicated animals imply that damage
has been inflicted on the plasma membrane of the hepatic
cells leading to their leakage into the extracellular fluid
[20]. This is confirmed by the reduction in the level of
marker enzymes in the liverver of paracetamol – induced rats.
Bilirubin on the other hand, is a product of enzymatic
c breakdown of heme within the reticuloendothelia system.
Its elevation in the blood stream can be adduced to over
production, increased hemolysis, decreased conjugation
conjugatio or
impaired bilirubin transport [21].
[21 Bilirubin is an index that
is used to assess the normal functioning of the liver instead
of the extent of hepatocellular injury. The increase in the
level of TBIL in the blood is a pointer to the impaired func-
fun
tion of the liver. Our
ur findings revealed that the hepatopro-
rection exhibited by the extract of O. americanum leaves
was dose dependent. The highest dose of the extract (400
d mg/kg b.w.) and the standard
standar drug (silymarin 100 mg/kg
b.w.) caused the reduction of elevated blood levels of AST,
ALT, ALP and TBIL to normal values. There was also an
increase in the levels of albumin and total protein in extract
and silymarin pretreated groups. Therefore, it could be de-
duced that the aqueous extract of O. americanum leaves
provided protection to the liver against the toxic effect of
paracetamol by preserving the functionality of this organ.
Consequently, the beneficial effect of the extract and stan-
sta
e dard was supported by significant increase in the levels of
marker enzymes of the liver (AST, ALT and ALP) of
Figure 1. Histological micrograph of liver section excised from the nor-
no treated groups in an attempt to recover from the assault
mal rat (a, X 40) showed normal structure of the hepatocytes. Histological inflicted on the tissue by paracetamol. Reduction in the
section of paracetamol intoxicated rat (b, X40) showed damage to the
liver structure as indicated by infiltration of monocytes and the presence
concentrations of these enzymes in the liver implies com-co
of necrotic cells. Treatment with 200 mg/kg b. w.of aqueous extract of O. promised plasma membrane integrity which resulted into
americanum leaves normalized the damaged hepatocytes with a great the leakage of these markers into the blood circulation [22].
[2
reduction in the quantity of necrotic cells (c, X 40). When the concentra- The extent of damage by paracetamol was further sup- su
tion of the extract was increased to 400 mg/kg b. w. the liver micrograph ported by histological changes in the liver of experimental
showed recovery of hepatocytes into normal with very mild monocyte
infiltration (d, X 40). Upon treatment with silymarin, the structure of the
animals. Histological examination of the liver sections
hepatocytes reverted to normal (e, X 40). showed that the normal liverver architecture was disturbed by
 American Journal of Life Sciences 2013, 1(2) : 37-42 41

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