Liver Protective Activity of the Methanol Extract of Crinum jagus Bulb against Acetaminophen-induced Hepatic Damage in Wistar Rats
Chinaka O. Nwaehujor,
Florence C. Nwinyi
Julius O. Ode
Hepatotoxins constitute a serious health concern in both rural and urban population
globally. Despite advances in medical research, the discovery of an ideal hepatoprotective
agent remains a challenge. The present research sought to evaluate the hepatoprotective
activity of the crude methanol extract of Crinum jagus bulb as a step
towards further detailed study to isolate the bioactive principles. Wistar rats
were pre-challenged individually with a high dose of acetaminophen (paracetamol,
2000 mg kg-1) per os to induce hepatic damage prior to treatment.
The control group was given distilled water (10 mL kg-1, p.o.) while
one out of the other experimental rat groups was either treated with silymarin
(50 mg kg-1, p.o.) or with a dose of C. jagus bulb extract
(75, 150 and 300 mg kg-1). Pentobarbitone-induced sleeping time,
the mean relative liver weight of individual rats, biochemical assay and histopathological
lesions in the liver of the separate rat groups were assessed and compared to
determine the extent of hepatic damage. The prolonged paracetamol-induced pentobarbitone
sleeping time in untreated, control rats (145.2±1.4 min) was most remarkably
reduced to 122.5±2.1 and 109.5±0.4 min in rats which were treated
orally with 150 and 300 mg kg-1 of the extract respectively. The
acetaminophen-mediated decrease in the mean relative liver weight of intoxicated
rats was relatively reversed with 150 and 300 mg kg-1 of the extract.
C. jagus bulb extract also demonstrated significant (p<0.05) potency
at 150 and 300 mg kg-1 in reducing acetaminophen-induced increase
in the rat serum transaminases (AST, ALT and ALP) and total bilirubin but with
elevation in total serum protein values. Histopathology revealed that 2000 mg
kg-1 of paracetamol induced severe necrosis of hepatocytes in untreated
control rats. Treatment of the acetaminophen-challenged rats with silymarin
(50 mg kg-1, p.o.) and C. jagus bulb extract (150 and 300
mg kg-1, p.o.) gave a better protection with regeneration of hepatocytes
relative to the untreated control. Crinum jagus bulb extract seemed to
have multiplicity of effects in regenerating parenchymal cells, hepatic microsomal
enzymes with high antioxidant and anti-inflammatory activities. The bulb of
C. jagus could be a potential source of potent hepatoprotective agents.
June 29, 2012; Accepted: September 08, 2012;
Published: October 11, 2012
Liver is a vital internal organ of the body and part of the digestive system
(Karim et al., 2011). It is involved in first-pass
effect which essentially, is concerned with the metabolism of orally administered
drugs by gastrointestinal and hepatic enzymes, resulting in a significant reduction
of the amount of un-metabolized drug reaching systemic circulation (Kwan,
1997). Liver is also concerned with detoxification of drugs and food substances,
deamination of excess proteins, storage of iron, vitamins and glycogen, production
of bile, proteins and vital enzymes in the body. The strategic importance of
the liver could be a reason for some of its naturally endowed qualities. Liver
has a remarkable capacity to regenerate after injury and to adjust to size to
match its host following transplant. Experiments have shown that within a week
after partial hepatectomy which involves surgical removal of two-thirds of the
liver, hepatic mass regenerates back essentially to what it was prior to surgery
(Michalopoulos and DeFrowces, 1997). Partial hepatectomy
reportedly leads to proliferation of all population of cells within liver, including
hepatocytes, biliary epithelial cells and endothelial cells. DNA synthesis was
noted to be initiated in these cells within 10 to 12 h after surgery and essentially
ceases in about 3 days (Michalopoulos and DeFrowces, 1997).
The pivotal role of the liver in biotransformation however, makes it susceptible
to toxic assault by xenobiotics (Craig and Stitzel, 1994).
Liver could be damaged due to effects from medications e.g., acetaminophen,
Paracetamol® (Bartlett, 2004), alcohol
abuse (Bykov et al., 2004), hepatotoxins (Appiah
et al., 2009), autoimmune hepatitis, viral and microbial infections
(Ardanaz and Pagano, 2006). Fortunately, plants offer
recipe for many health challenges. It is on record that a large section of the
worlds population relies on herbal remedies to treat plethora of diseases
due to their low costs, easy access and reduced side effects (Marino-Betlolo,
1980) though pharmacological basis behind most herbal therapies however,
remains practically unknown.
Plants are also a source of novel anti-oxidant and hepatoprotective agents
since many industrial drugs are derived as a result of knowledge got from folklore
medicine (Brander et al., 1991). Some plants with
reported hepatoprotective properties are Garcinia kola Ker Gaul (Clusiaceae),
Tinospora cordifolia (A. Rich.), Ricinus cummunis Linn. (Euphorbiaceae),
Curcuma longa Linn. (Zingiberaceae), Enicostemma littorale Blume
(Gentianaceae), Flaveria trinervia Linn. (Asteraceae) and Boerhaavia
diffusa Linn. (Nyctaginaceae) (Devaki et al.,
2004; Umadevi et al., 2004; Vishwakarma
and Goyal, 2004). Most of the herbal preparations speed up the natural healing
processes of the liver (Senthilkumar et al., 2005).
Phellinus rimosus (Berk) Platt (Hymenochaetaceae), a mushroom has been
shown to protect the liver from acute and Chronic Carbon Tetrachloride (CCl4)-induced
hepatotoxicity in rats by restoring the liver anti-oxidant status, inhibiting
the phase I and enhancing the phase II enzyme activities (Ajith
et al., 2006).
C. jagus commonly called Harmattan lily, belongs to Amaryllidaceae,
a heterogenous family of 86 genera and about 1310 species (Lawrence,
1951). The plant is distributed worldwide in the tropics and subtropics.
C. jagus is locally called okonkilo inyi which literally
means elephants potato by the Igede people of Benue State who inhabit
the middle belt region of Nigeria. The plant is also called gadali
by the Hausa and Fulani tribes in Northern Nigeria (Dalziel,
1937). All the plant species are of ornamental value. In Sierra leone, it
is reported that a cold infusion of the fresh leaves is used to bathe young
children suffering from general body debility, rickets, etc., (Dalziel,
1937). A decoction is given as a vermifuge in Gold coast (Ghana). The bulbs
of several species are sold for various medicinal purposes in Lagos, Nigeria.
In East Africa, the decoction of C. jagus is used for treatment of sores
(Kokwaro, 1976). Ode and Asuzu (2006)
reported that the methanol extract of C. jagus bulb exhibited antivenom
effects when it completely inhibited the hemorrhagic activity of Echis ocellatus
venom (4.2 μg 1.5 μL-1) at various concentrations
(2.5, 5.0 and 10.0 μg 1.5 μL-1). The plant extract was
also found to possess greater antioxidant activities at increased concentrations
(50-400 μg mL-1) compared to the reference non-enzymatic antioxidant
(ascorbic acid) using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging
spectrophotometric assay (Ode et al., 2010).
The present study was undertaken to evaluate the hepatoprotective effects of
the methanol extract of C. jagus bulb against acetaminophen-challenged
hepatic injury in rats.
MATERIALS AND METHODS
Chemicals, reagents and drugs: Ethanol and methanol (Riedel-De Haen AG-hanover),
sodium chloride (BDH, England), silymarin (Legalon®
70, Chemical Industries, Madaus AG, Germany), 1,1-diphenyl-2-picrylhydrazyl
radical (DPPH), Ferric tripyridyltriazine (Fe (III)-TPTZ), Ascorbic acid and
acetaminophen i.e., Paracetamol (Sigma Aldrich Sigma Aldrich, Germany), Pentobarbitone
sodium (Abbot Laboratories Ltd., Kent, UK)., Kits for Serum alanine aminotransferase
(ALT), Aspartate aminotransferase (AST) and Alkaline phosphatase (ALP) (Randox
Laboratories Ltd, United Kingdom), Total bilirubin and Total protein laboratory
kits (Quimica Clinica Applicada S.A., Spain) were used in the study.
Animals: Adult Wistar rats (130-180 g) obtained from the Laboratory
Animal Unit of the Faculty of Veterinary Medicine, University of Nigeria, Nsukka
were used for the study. Animals were kept in stainless steel cages and had
access to feed (Vital feed®,
Nigeria Ltd.) and water ad libitum except in situations where fasting
was required. The rats were allowed 14 days to acclimatize before the experiments
were conducted according to the permission and prescribed guidelines of the
Institutional Animal Ethics Committee. A total number of 36 Wistar rats were
used for the experiments.
Plant materials and extraction: Fresh bulbs of C. jagus were
collected in August, 2011 from farm locations in Ochimode Village, Oju Local
government Area of Benue State, Nigeria. The plant materials were duly identified
by Mr. A.O. Ozioko of the Department of Botany, University of Nigeria, Nsukka.
C. jagus bulbs (CJB) were dismembered and sliced into small pieces. They
were air-dried for several weeks before being pulverized into coarse powder
using hammer mill. One kilogram of the powdered bulbs was extracted by cold
maceration with 80% methanol and intermittent vigorous shaking for 72 h. Concentration
of the extract in vacuo with rotary evaporator afforded 12.6% w/w dry
Effects of C. jagus bulb extract on acetaminophen-induced hepatotoxicity
in rats: A total of thirty-six Wistar rats (130-180 g) were randomly allocated
to 6 groups comprising of 6 animals in a group. All the rats except group 1
(normal) were fasted overnight and pretreated orally with a single dose (2000
mg kg-1) of acetaminophen. Following 12 h after challenge with acetaminophen,
group 2 (positive control) received distilled water (10 mL kg-1,
p.o.) only for 4 days. Group 3 rats were given silymarin (50 mg kg-1,
p.o.) for 4 days and this served as the reference drug for comparison. Groups
4-6 acetaminophen-challenged rats were treated orally with 75, 150 and 300 mg
kg-1 of the extract of C. jagus bulb respectively for 4 days.
All treatments were given by stomach intubation. Pentobarbitone-induced sleeping
time assay was carried out on day 4 by intraperitoneal administration of pentobarbitone
sodium (35 mg kg-1). The sleeping time was calculated as the interval
between the loss and recovery of the righting reflex (Shetty
and Anika, 1982).
On recovery, blood samples were collected and the rats were humanely sacrificed.
The blood samples were allowed to clot at room temperature. They were then centrifuged
at 2,500 rpm for 10 min to separate the serum which was used to determine the
serum ALT, AST, ALP, total bilirubin and total protein levels. All the rat livers
were weighed for determination of the relative liver weight for each group.
Tissue samples of the liver from each experimental group was collected and processed
for comparison of histopathologic lesions.
Biochemical assay: The serum levels of ALT, AST were determined using
the method of Reitman and Frankel (1957), serum ALP level
was assayed by the method of King and King (1954), total
bilirubin level by the method of Malloy and Evelyn (1937)
as modified by Tietz (1996) and total serum protein level
by the method of Johnson (1943).
Relative liver weight: Excess water from each of the rat liver samples
was absorbed with a piece of serviette before weights were taken on a weighing
balance (Metler, England). The individual rat liver weight was expressed in
relation to the total body weight to obtain the relative liver weight. The total
and the mean relative liver weight for each experimental group were determined
Histopathology: Tissue sample from the liver of rats in each group (1-6)
of the experiment was fixed in 10% formal-saline for a minimum of 24 h and then
dehydrated by washing in ascending grades of ethanol before clearing with xylene
and embedding in paraffin wax. The samples were sectioned with a microtome,
stained with Hematoxylin and Eosin (H and E) and mounted on Canada balsam. All
sections were examined under light microscope (10, 20 and x40) magnification.
Photographs of the lesions were taken with an Olympus photo microscope for observation
and comparison of histopathologic lesions.
Statistical analysis: All data collected were subjected to one-way Analysis
of Variance (ANOVA) and Duncans
New Multiple Range Test (DNMRT) was used as the post hoc test to separate the
treatment means. Differences at p<0.05 were considered significant.
Pentobarbitone-induced sleeping time: The pentobarbitone-induced sleeping
time was significantly (p<0.05) prolonged in acetaminophen-challenged, untreated
rats (145.2±1.4 min) compared to the normal rats, Group 1 (85.2±0.6
min). The effect of the extract of C. jagus bulb at 75 mg kg-1
on the pentobarbitone-induced sleeping time was not significantly (p>0.05)
different from that of the control, which were challenged with acetaminophen
(2000 mg kg-1, p.o.) without treatment. However, silymarin (50 mg
kg-1, p.o.) gave 114.2±0.4 min while the extract gave 122.5±2.1
and 109.5±0.4 min at 150 and 300 mg kg-1, p.o., respectively.
The methanol extract of C. jagus bulb was therefore able to produce significant
(p<0.05) decrease in pentobarbitone-induced sleeping time at 150 and 300
mg kg-1 when compared with the mean sleeping time value in acetaminophen-challenged,
untreated rats (Fig. 1).
The mean relative liver weights of acetaminophen-challenged rats: The
mean relative liver weight became significantly (p<0.05) reduced in untreated,
acetaminophen-intoxicated rats that were given only paracetamol (2000 mg kg-1,
p.o.) and also in rats that were treated with 75 mg kg-1 of C.
jagus bulb extract in contrast to the other groups (normal, silymarin, C.
jagus bulb extract at 150 and 300 mg kg-1). The mean relative
liver weight of the normal rats was 30.5x10-3±1.8 g but untreated
paracetamol-intoxicated rats had 20.8x10-3±1.3 g while rats
that were treated with silymarin (50 mg kg-1) had 27.6x10-3±2.6
g; 150 and 300 mg kg-1 of C. jagus bulb extract produced 28.2x10-3±1.4
and 29.1x10-3±0.5 g, respectively (Fig. 2).
There was no significant (p>0.05) difference between the mean relative liver
weight of the normal rats and those of intoxicated rats that were treated with
150 and 300 mg kg-1 of the crude extract of C. jagus bulb.
Acetaminophen-induced liver toxicity: The serum AST, ALT and ALP levels
were significantly (p<0.01) increased in acetaminophen-challenged, untreated
rats (136.8±3.6, 164.6±4.5, 54.0±1.6 μL-1)
respectively when compared to non-challenged normal group (54.6±1.4,
73.5±1.7, 25.2±1.4 μL-1). AST value (136.8±3.6
μL-1) and the total bilirubin (2.28±0.02 mg dL-1)
in the control, untreated rats were significantly (p<0.05) reduced to 118.9±1.3
μL-1 and 1.55±0.2 mg dL-1 respectively following
treatment with 75 mg kg-1, p.o. of the extract but the protein level
became significantly (p<0.05) increased comparable to the effects of the
other doses (150 and 300 mg kg-1) of the extract.
||The effect of methanol extract of C. jagus bulb and
Silymarin on sleeping time in rats intoxicated with acetaminophen, Parac:
Paracetamol, CJE: Crinum jagus bulb extract
||The effect of the crude methanol extract of C. jagus
bulb and silymarin on relative liver weights in acetaminophen-challenged
rats, Parac, Paracetamol, CJE: Crinum jagus bulb extract
||Effects of the methanol extract of C. jagus on serum
enzymes, bilirubin and protein of acetaminophen-induced hepatotoxicity in
|*Significant at p<0.05 when compared with control, ACT:
Acetaminophen, CJB: Crinum jagus bulb, AST: Aspartate aminotransferase,
ALT: Alanine aminotransferase, ALP: Alkaline phosphatase
The low dose (75 mg kg-1) of the extract did not however, induce
any significant (p>0.05) alteration on the serum levels of ALT and ALP in
acetaminophen-challenged rats. The bulb extract of C. jagus (150 and
300 mg kg-1, p.o.) significantly (p<0.05) decreased the elevated
levels of AST, ALT and ALP compared to the acetaminophen-challenged untreated
group. The reduction produced by the methanol extract of C. jagus on
ALT, AST and ALP was highest at 300 mg kg-1. Similarly, silymarin
(50 mg kg-1) and C. jagus extract (150 and 300 mg kg-1)
caused a significant (p<0.05) reduction in the total serum bilirubin level
but a significant (p<0.05) increase in the total protein level of test rats
when compared to acetaminophen-challenged, untreated group (Table
Histopathology of paracetamol-induced hepatotoxicity in rats:
||Normal rats, no acetaminophen intoxication: The rat
hepatocytes appeared normal without visible damage to the liver cells. Hepatocytes
were seen typically radiating from the central vein; the stellate macrophage
lined the endothelial cells. Some of the hepatocytes were mononucleated
while others were binucleated (Fig. 3).
||Control, given distilled water 10 mL kg-1, p.o.: There
was severe necrosis and vacuolation of hepatocytes. The normal histological
arrangement of hepatocytes in hepatic lobules and around the sinusoids was
greatly distorted (Fig. 4).
||Acetaminophen-challenged rats treated with silymarin, 50 mg kg-1,
p.o.: There was serious regeneration and repair in response to the injurious
effects of acetaminophen (2000 mg kg-1, p.o.) as evidenced from
the presence of megalocytes (mitotic cells) in the hepatic tissue (Fig.
||Acetaminophen-challenged rats treated orally with 75 mg kg-1
of C. jagus extract: There was mild coagulative necrosis even
though some normal hepatocytes could be seen (Fig. 6).
The liver damage caused a general disorganization of hepatocytes.
||Acetaminophen-challenged rats treated orally with 150 mg kg-1
of C. jagus extract: Megalocytes were present showing the possibility
of tissue repair taking place and some of the hepatocytes appeared normal
||Acetaminophen-challenged rats treated orally with 300 mg kg-1
of C. jagus extract: There was dissemination of a large number
of megalocytes, an indication of maximal regeneration within the hepatic
tissue (Fig. 8).
|| Normal rat liver, no acetaminophen toxicity, H and E x400,
K: Normal hepatocytes
||Micrograph of the liver from acetaminophen, (2000 mg kg¯1)
challenged rat, N: Severe necrosis of hepatocytes, H and E x400
|| Micrograph of the liver from acetaminophen-challenged rat
treated with silymarin (50 mg kg¯1, p.o.), Arrows point
to megalocytes, evidence of regeneration taking place, H and E x400
||A liver section from acetaminophen-challenged rat treated
with C. jagus extract (75 mg kg¯1, p.o.), N: Necrotic
hepatocytes, H: Normal hepatocytes, H and E x400
|| Liver section of acetaminophen-challenged rat treated with
C. jagus extract (150 mg kg¯1, p.o.), M: Megalocytes,
H: Normal hepatocyte, H and E x400
|| Micrograph of the liver of acetaminophen-challenged rat treated
with C. jagus extract (300 mg kg¯1, p.o.), M: Megalocytes
The prolonged pentobarbitone-induced sleeping time observed in the paracetamol-challenged,
untreated rats was found to be significantly (p<0.05) decreased when challenged
rats were treated with the extract of C. jagus bulb (150 and 300 mg kg-1).
This could be possible due to the protective effects of the extract on the liver
resulting in reduced destruction of hepatocytes which remained viable to carry
out biotransformation of the drug. The anaesthetic, pentobarbitone sodium was
metabolized by active liver cells and this culminated in the reduced duration
of anesthesia in the treated rats. Again, the paracetamol-intoxicated, untreated
control and rats that were treated with low dose (75 mg kg-1) of
C. jagus bulb extract had significantly (p<0.05) reduced mean relative
liver weight compared to others (normal rats and groups that were treated with
silymarin and the extract at 150 and 300 mg kg-1). The reduction
in the mean relative liver weight could be as a result of acute necrosis of
hepatocytes in the absence of adequate hepatoprotective agent. Liver is responsible
for biotransformation of toxic substances, including drugs and hormones (Banks,
1993). Increasingly large numbers of drugs, herbicides, food additives and
environmental carcinogenic hydrocarbons are found to stimulate their own metabolism
or the metabolism of other compounds by increasing the amount of drug metabolizing
enzymes in liver microsomes (Conney, 1967). The crude
extract of C. jagus bulb could have aided the rejuvenation of hepatic
microsomal enzymes which subsequently carried out accelerated metabolism of
pentobarbitone sodium with resultant decrease in duration and intensity of the
anaesthetic effect. The extract may contain or stimulate chemical mediators
in the local tissue microenvironment to enhance cell growth. Polypeptide growth
factors stimulate cellular proliferation and also mediate a wide variety of
other activities, including cell migration, differentiation and tissue remodeling
(Kumar et al., 1997). Crinum jagus bulb
extract may have aided liver parenchymal cell growth in acetaminophen-intoxicated
rats at 150 and 300 mg kg-1. Cell growth factors are involved in
various stages of wound healing.
The methanol extract of C. jagus bulb was also effective at 150 and
300 mg kg-1, p.o. in reducing the serum levels of AST, ALT and ALP
and also preserved the functional ability of the liver. This was revealed when
both doses of the extract produced significant (p<0.05) reduction in the
level of transaminases and the total bilirubin but increased total protein levels
relative to acetaminophen-challenged, untreated rats. The conjugating and synthesizing
ability of the liver was therefore intact. The extract did show minimal hepatoprotective
activity at 75 mg kg-1 when there was significant (p<0.05) decrease
in the mean serum AST and total bilirubin levels but elevated total protein
values relative to the control, untreated rats. The serum concentration of ALT
and ALP, however, remained high post treatment with 75 mg kg-1, p.o.
of the extract which was suggestive of a persisting hepatic tissue injury. Hepatic
cells contain higher concentrations of AST and ALT in the cytoplasm but AST
in particular exists in the mitochondria (Wells, 1988).
Damage to hepatic cells induces leakage into plasma leading to an increased
level of hepato-specific enzymes in serum (Tolman and Rej,
1999). The measurement of serum AST, ALT and ALP levels serve as a means
for indirect assessment of liver function.
Toxic injury to hepatocytes stimulates inflammatory reactions. The cell membrane
damage associated with inflammation results in leucocyte release of lysosomal
enzymes that can be injurious to nearby cells (Konturek
et al., 2000). Cell damage causes the release of arachidonic acid
and pro-inflammatory cytokines. Stimulation of neutrophils can lead to the production
of oxygen-derived free radicals that produce further cellular damage (Forman
and Torres, 2002). Crinum jagus bulb was found to possess a significantly
higher antioxidant activity compared to ascorbic acid (Ode
et al., 2010). The mechanism of the hepatoprotective activity of
the extract of C. jagus bulb in acetaminophen-challenged rats may be
derived from some anti-inflammatory and antioxidant principles in the extract.
The extract could also have caused accelerated regeneration of damaged liver
cells. Active regeneration of hepatocytes in the form of megalocytes (mitotic
liver cells) was prominently seen in the tissue section of the liver of acetaminophen-challenged
rats which were treated with 150 and 300 mg kg-1, p.o. of the extract.
Mitosis precedes regeneration and replacement of worn-out cells. During the
cell division, each chromosome made up of two chromatids attach randomly at
the centromere and later split. Daughter chromosomes begin to migrate to the
opposite poles in anaphase but in telophase, nuclear reconstitution and enlargement
occurs; cytokinesis ensues resulting in two identical daughter cells (Banks,
1993). Cell proliferation from mitosis is vital for tissue repair.
C. jagus bulb extract could have aided regeneration of parenchymal cells
and hepatic microsomal enzymes at 150 and 300 mg kg-1. The hepatoprotective
effect may also be due to the high antioxidant activity and some anti-inflammatory
principles in the extract.
The methanol extract of C. jagus bulb (150 and 300 mg kg-1,
p.o.) and silymarin (50 mg kg-1) demonstrated appreciable potency
at reducing serum levels of AST, ALT, ALP and total bilirubin but increased
total serum protein level in acetaminophen-induced liver damaged rats which
was suggestive of liver protection. The hepatoprotective activity of the plant
extract was also exhibited when the prolonged pentobarbitone-induced sleeping
time in the acetaminophen-challenged rats became significantly reduced relative
to the control, untreated rats. The mean relative liver weight of intoxicated
rats which were treated with 150 and 300 mg kg-1 of the extract became
comparable to the normal. The histopathology of the liver tissue from the experimental
animals showed signs of regeneration and more protected hepatocytes in the C.
jagus bulb extract-treated rats. Further studies to isolate the hepatoprotective
principles in C. jagus bulb and to determine the mechanism of action
are highly recommended.
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