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Contents lists available at ScienceDirect
Experimental and Toxicologic Pathology
journal homepage: www.elsevier.de/etp
Hepatoprotective potential of aqueous extract of Butea monosperma
against CCl4 induced damage in rats
Neetu Sharma, Sangeeta Shukla ∗
School of Studies in Zoology, Jiwaji University, Gwalior 474011, MP, India
a r t i c l e
i n f o
Article history:
Received 22 January 2010
Accepted 19 May 2010
Keywords:
Hepatoprotective
CCl4
Liver diseases
Butea monosperma
a b s t r a c t
Aqueous extract of flowers of Butea monosperma (Fabaceae) was evaluated at different dose levels (200,
400, 800 mg/kg, p.o.) for its protective efficacy against CCl4 (1.5 ml/kg i.p.) induced acute liver injury to validate its use in traditional medicines. The CCl4 administration altered various biochemical parameters,
including serum transaminases, protein, albumin, hepatic lipid peroxidation, reduced glutathione and
total protein levels, which were restored towards control by therapy of B. monosperma Adenosine triphosphatase and glucose-6-phosphatase activity in the liver were decreased significantly in CCl4 treated
animals. Therapy of B. monosperma showed its protective effect on biochemical and histopathological
alterations at all the three doses in dose dependent manner. B. monosperma extract possess modulatory
effect on drug metabolizing enzymes as it significantly decreased the hexobarbitone induced sleep time
and increased excretory capacity of liver which was measured by BSP retention. Histological studies also
supported the biochemical finding and maximum improvement in the histoarchitecture was seen at
higher dose of BM extract.
© 2010 Elsevier GmbH. All rights reserved.
1. Introduction
Herbs have recently attracted attention as health beneficial food
and as source materials for drug development. They offer a potential
natural health care approach that focuses on protecting and restoring the health. Recently herbal medicines are being increasingly
utilized to treat a wide variety of clinical diseases, including liver
diseases (Gupta et al., 2007) with relatively little knowledge regarding their modes of action (Jeong et al., 2002). India is sitting on a gold
mine of well-recorded and traditionally well-practiced knowledge
of herbal medicines, therefore, any scientific data on such plant
derivatives could be of clinical importance (Singanan et al., 2007).
There are number of medicinal preparations in Ayurveda that are
recommended for the treatment of liver disorders (Samudram et al.,
2008), however, no scientific evidence is available for their clinical
usage.
Butea monosperma Lam. is commonly known as Palash in Hindi
and widely disturbed in India. Its flowers are used to treat leprosy,
gout, skin and eye diseases and has been reported to be associated
with various remedial properties such as anti-hepatotoxic (Wagner
et al., 1986), antistress (Kasture et al., 2002), antiestrogenic (Shah
and Bakxi, 1990) and chemopreventive (Sehrawat et al., 2006).
Its flowers contain various flavanoids like butein, butin, isobutrin,
isomonospermoside and steroids (Kasture et al., 2002; Lavhale and
Mishra, 2007).
Investigation of hepatoprotective herbal drug as a major indicator of the general screening systems can trigger the safety
evaluation in the early phase of drug discovery because most of the
toxic compounds are metabolized in liver. Therefore, to justify the
traditional claim of B. monosperma as hepatoprotective agent, we
assessed its hepatoprotective potential against CCl4 induced liver
damage in rats.
2. Materials and methods
2.1. Preparation of plant extract
Flowers of B. monosperma were generously obtained from
Central Council for Research in Unani Medicine, India. Flowers
were dried in shade, powdered and extracted with distilled water
(250 g/4 l) for 18 h with concomitant shaking. Filtrate was evaporated in vacuum to yield a yellow powder (BM extract), which was
administered orally according to body weight of animals. Silymarin
(50 mg/kg, p.o.) was used as a positive control during experimental
regimen (Chandan et al., 2007).
2.2. Chemicals
∗ Corresponding author. Tel.: +91 751 4016750; fax: +91 751 2341450.
E-mail addresses: [email protected], [email protected]
(S. Shukla).
All chemicals were procured from Sigma–Aldrich (USA), EMerck (Germany), Ranbaxy Pvt. Ltd. and BDH Company (India).
0940-2993/$ – see front matter © 2010 Elsevier GmbH. All rights reserved.
doi:10.1016/j.etp.2010.05.009
Please cite this article in press as: Sharma N, Shukla S. Hepatoprotective potential of aqueous extract of Butea monosperma against CCl4
induced damage in rats. Exp Toxicol Pathol (2010), doi:10.1016/j.etp.2010.05.009
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2.3. Maintenance of animals and their feeding
2.9. Hexobarbitone induced sleep time
Adult female albino Swiss mice (30 ± 5 g body weight) and
albino rat of Sprague–Dawley strain (160 ± 10 g body weight)
were randomly selected from departmental animal facility where
they were housed in polypropylene cages under uniform husbandry conditions of light (14 h) and dark (10 h) with temperature
(25 ± 2 ◦ C) and relative humidity (60–70%). Animals were fed
on commercially available standard animal diet (Pranav Agro
Industries Ltd., New Delhi, India) and drinking water ad libitum.
Experimental protocol was approved by Institutional Ethics Committee following guidelines set by Committee for the Purpose of
Control and Supervision of Experiments on Animals, India.
Hexobarbitone induced sleep time was measured according
to Fujimoto et al. (1960) and separate set of mice were divided
into four groups of six each. Group 1 served as normal control
and received vehicles only. Groups 2–4 were administered CCl4
(1.5 ml/kg, i.p.) and group 2 served as experimental control. Groups
3 and 4 received BM extract (800 mg/kg) and silymarin (50 mg/kg)
respectively after 48 h of toxicant administration. Hexobarbitone
(60 mg/kg, i.p.) was administered to all the groups after 48 h of last
administration. Time of onset of loss of reflex up to the recovery
was taken in min as duration of sleep and protective activity of BM
extract and silymarin was calculated by given formula:
2.4. Preparation of doses and treatments
% protection = 1 −
The CCl4 was administered at a dose of 1.5 ml/kg, i.p. with vehicle
(olive oil) (Bhadauria et al., 2008). An aqueous suspension of extract
was prepared in distilled water and different doses of BM extract
(200 mg, 400 mg and 800 mg/kg) and silymarin (50 mg/kg) were
administered to the animals orally.
where T is sleep time; c, d and n are CCl4 , drug (BM extract and
silymarin) and normal groups respectively.
2.5. Experiment design
Thirty-six adult female rats were divided into six groups of six
animals each. The animals were administered CCl4 at a dose of
1.5 ml/kg, i.p. once only followed by different doses of BM extract
after 48 h of toxicant administration once only. All the animals were
euthanized after 48 h of last treatment and various blood and hepatic biochemical parameters were performed:
Group 1: Control (Vehicle only).
Group 2: CCl4 (1.5 ml/kg, i.p.) once only.
Group 3: CCl4 (as in group 2) + BM (200 mg/kg, p.o.).
Group 4: CCl4 (as in group 2) + BM (400 mg/kg, p.o.).
Group 5: CCl4 (as in group 2) + BM (800 mg/kg, p.o.).
Group 6: CCl4 (as in group 2) + silymarin (50 mg/kg, p.o.).
Td − Tn
× 100
Tc − Tn
2.10. Bromosulphalein (BSP) retention
BSP retention was estimated as described by Kutob and Plaa
(1962) using another set of mice divided into four groups of six
each. Group 1 served as normal control and was given vehicles only.
Groups 2–4 were administered CCl4 (1.5 ml/kg, i.p.) and groups 2
served as experimental control. BM extract (800 mg/kg) and silymarin (50 mg/kg) were administered orally to groups 3 and 4
respectively after 48 h of toxicant administration. BSP (100 mg/kg,
i.v.) was injected to the entire four groups after 48 h of last treatment. Blood was collected in heparinized tubes exactly after 30 min,
centrifuged for plasma isolation and BSP concentration was estimated in it. Percent retention of dye was used as excreting capacity
by following formula:
% protection = 1 −
Rd − Rn
× 100
Rc − Rn
where R is retention of BSP in plasma; c, d and n are CCl4 , drug (B.
monosperma and silymarin) and normal groups respectively.
2.6. Blood biochemical assay
Blood was drawn by puncturing retro-orbital venous sinus,
centrifuged and serum was isolated to determine aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (Reitman and
Frankel, 1957) and protein (Lowry et al., 1951). Blood sugar (Kit No.
FBCER0017) and albumin (Kit No. 1118275) were assessed by kit
methods as per instructions provided by the company (E-Merck,
Germany).
2.7. Tissue biochemical assay
Lipid peroxidation (LPO) was determined by measuring thiobarbituric acid reactive substances (TBARS) (Sharma and Krishna
Murty, 1968). Reduced glutathione (GSH) level was determined by
dithionitrobenzoic acid (DTNB) (Brehe and Burch, 1976). Activities
of adenosine triphosphatase (ATPase) (Seth and Tangari, 1966) and
glucose-6-phosphatase (G-6-Pase) (Baginski et al., 1974) were also
determined in liver.
2.8. Histological observations
Liver samples were fixed in Bouin’s fixative and processed to
obtain 5 ␮m thick paraffin sections and stained with hematoxylin
and eosine (H&E) for histological observations.
2.11. Statistical analysis
Results are presented as mean ± S.E.M. of six animals used in
each group. Data were subjected to statistical analysis through oneway analysis of variance (ANOVA) taking significant at 5% level of
probability followed by Student’s t-test taking significant at P ≤ 0.05
(Snedecor and Cochran, 1989).
3. Results
3.1. Blood biochemical assay
The results of blood biochemical parameters are presented in
Table 1. Administration of CCl4 induced significant increase in the
enzymatic activities of ALT and AST (P ≤ 0.05) as compared to the
control group. Oral administration of extract at different doses
(200, 400 and 800 mg/kg) showed significant recoupment in a dose
dependent manner (P ≤ 0.05). Significant increase was observed in
albumin, blood sugar and serum protein after CCl4 intoxication.
The BM extract significantly decreased the elevated levels towards
control. No significant change was observed in serum protein level.
The 800 mg/kg dose of BM extract revealed more significant therapeutic effectiveness (P ≤ 0.05). The 200 and 400 mg/kg showed less
effective significant changes.
Please cite this article in press as: Sharma N, Shukla S. Hepatoprotective potential of aqueous extract of Butea monosperma against CCl4
induced damage in rats. Exp Toxicol Pathol (2010), doi:10.1016/j.etp.2010.05.009
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Table 1
Effectiveness of Butea monosperma against carbon tetrachloride induced blood biochemical alterations.
Treatments
AST (IU/L)
ALT (IU/L)
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
68.0 ± 3.51
188 ± 14.8#
140 ± 9.90*
106 ± 8.17*
90.0 ± 4.93*
88.0 ± 6.52*
50.0 ± 4.28
425 ± 22.8#
130 ± 11.4*
124 ± 10.7*
93.0 ± 6.72*
55.0 ± 3.72*
34.2@
ANOVA F-value
167@
Albumin (g/dl)
3.50 ± 0.33
4.60 ± 0.34#
3.50 ± 0.32*
3.65 ± 0.24*
3.60 ± 0.20*
3.50 ± 0.34*
13.1@
Blood sugar (mg/dl)
Serum protein (mg/100 ml)
75.0 ± 5.62
145 ± 9.16#
76.8 ± 5.84*
79.0 ± 5.27*
81.0 ± 6.87*
80.0 ± 6.48*
48.3± 3.39
138 ± 3.58#
43.5 ± 2.44
42.1 ± 3.83
42.1 ± 3.21
40.0 ± 2.48
20.1@
1.39
Data are mean ± S.E.M., n = 6.
ANOVA (F values at 5% level).
#
P ≤ 0.05 vs. Control.
*
P ≤ 0.05 vs. CCl4 .
@
Significant.
Table 2
Effectiveness of B. monosperma against carbon tetrachloride treated animals in tissue biochemical estimations.
Treatments
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
ANOVA F-value
LPO (n mole of TBARS/mg protein)
0.28 ± 0.01
1.25 ± 0.10#
0.59 ± 0.03*
0.42 ± 0.03*
0.34 ± 0.02*
0.30 ± 0.02*
71.4@
GSH (␮mole/g)
7.8 ± 0.48
4.2 ± 0.37#
6.8 ± 0.37*
6.9 ± 0.40*
7.2 ± 0.40*
7.2 ± 0.41*
11.6@
ATPase (mg Pi/100g/min)
G-6-Pase (␮mole Pi/min/g liver)
2000 ± 100
930.0 ± 47.4#
1784 ± 96.0*
1757 ± 91.3*
1846 ± 99.2*
1910 ± 99.8*
21.9@
7.0 ± 0.37
3.1 ± 0.18#
6.1 ± 0.40*
6.3 ± 0.38*
6.3 ± 0.39*
6.5 ± 0.35*
18.2@
Tissue protein (mg/100 mg)
15.0 ± 0.75
19.5 ± 0.87#
17.0 ± 0.85*
15.7 ± 0.81*
15.5 ± 0.85*
15.1 ± 0.77*
8.17@
Data are mean ± S.E.M., n = 6.
ANOVA (F values at 5% level).
#
P ≤ 0.05 vs. Control.
*
P ≤ 0.05 vs. CCl4 .
@
Significant.
3.2. Tissue biochemical assay
Various biochemical parameters were performed in liver tissues which are presented in Table 2. A significant increase was
observed in the level of LPO in liver after 48 h of CCl4 intoxication
when compared with the control group (P ≤ 0.05). Treatment with
different doses of crude extract reversed the oxidative stress significantly towards control by inhibiting LPO in dose dependant manner
(P ≤ 0.05). Reduced glutathione is presumed to be an important
endogenous defense against peroxidative destruction of cellular
membranes. In the present study, significant decline was seen in the
reduced glutathione level (P ≤ 0.05). Post treatment of extract was
very effective in restoring the glutathione content which had been
substantially decreased by CCl4 (Table 2). All three doses of extract
improved the GSH level in liver, however, therapy at 800 mg/kg
was very effective. In our study, decline in the activities of G-6Pase and ATPase in CCl4 administered animals were also observed,
which were significantly reversed towards control with the highest
dose of BM extract (P ≤ 0.05). Total protein level was significantly
increased in CCl4 intoxicated animals that was brought towards
control by all the doses of BM extract significantly, however, maximum reversal was noticed at 800 mg/kg dose (P ≤ 0.05).
3.3. Histological observation
The light microscopy examination of the transverse section of
control rat liver clearly illustrates complete hepatic lobules with
well formed hepatocytes with distinct portal triads. Hepatic cells
were arranged in cord like fashion, which are separated by sinusoids and central vein was seen clear (Fig. 1a). The liver sections
of CCl4 intoxicated rats showed massive fatty changes, necrosis,
ballooning and degeneration in hepatic plates and loss of cellular boundaries (Fig. 1b). Treatment with the aqueous extract was
effective in restoring the CCl4 induced histopathological lesions
when compared to CCl4 per se, however highest dose was found
to be more effective. The histological architecture of liver sections
showed mild degree of degeneration and necrosis at lower doses
(Fig. 1c–d). Therapy with BM extract at 800 mg/kg dose, the micrographs exhibited an almost normal architecture (Fig. 1e). Silymarin
treated group depicted symmetrically arranged well formed hepatocytes separated by sinusoids and maintained cord arrangement
(Fig. 1f).
3.4. Hexobarbitone induced sleep time
Fig. 2 depicts the effects of hexobarbitone induced sleep
time. CCl4 administration significantly prolonged the barbiturate
induced sleep time (P ≤ 0.05) when compared to normal. Significant prolongation in sleeping time proved the event of hepatic
damage by free radicals, which was shortened significantly by the
BM extract. This can well be compared to silymarin group.
3.5. Bromosulphalein retention
BSP retention after 30 min of its injection in normal animals was
significantly increased (P ≤ 0.05) by carbon tetrachloride administration. Treatment of BM extract significantly reduced the BSP
retention (P ≤ 0.05) indicating improved excretory capacity of liver.
Similar results were observed by silymarin (Fig. 3).
4. Discussion
In the Indian traditional medicinal systems, B. monosperma is
used as anti-hepatotoxic agent. Hepatic cells participate in a variety of metabolic activities and contain a host of enzymes. The liver
functions in a coordinated way with various systems of the body
and any disease involving this organ have serious and far reaching
effects not only on the liver itself but also on other organs and sys-
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Fig. 1. (a) Control group show normal lobular architecture with clear central vein (X-100). (b) CCl4 induced degeneration and ballooning of hepatocytes (X-100). (c):Aqueous
extract of BM extract (200 mg/kg) show mild cytoplasmic vacuolation and granulation in hepatocytes (X-100). (d) Treatment of BM extract (400 mg/kg) show mild improvement in chord arrangement but perinuclear vacuolation was visible (X-100). (e) 800 mg/kg of BM extract show normal cellular architecture with distinct hepatic cells
sinusoidal spaces (X-100). (f) Treatment with silymarin exhibit almost normal histology (X-100).
tems. In the assessment of liver damage, the determination of liver
function tests enzyme levels such as AST and ALT is largely used
by CCl4 (Shukla et al., 2007; Lynch and Price, 2007). In the present
study, exposure to CCl4 resulted in a significant hepatic damage
as elicited by the elevated level of serum marker enzymes, AST
and ALT. These marker enzymes are cytoplasmic in origin and are
released into the circulation after cellular damage (Lin et al., 2000).
The rise in the enzyme AST is usually accompanied by an elevation
in the levels of ALT, which plays a vital role in the conversion of
amino acids to keto acids (Salie et al., 1999).
Albumin, which is produced only in the liver is the major plasma
protein that circulates in the bloodstream. It is involved in scav-
enging of oxygen free radicals. It is also very important in the
transportation of many substances such as drugs, lipids, hormones,
and toxins that are bound to albumin in the bloodstream. Hence,
increase in total protein content can be deemed as a useful index of
the severity of cellular dysfunction in liver diseases as clearly shown
in our studies. The elevated level of albumin test is indicative of
cellular leakages and loss of functional integrity of cell membrane
in liver. Stimulation of protein synthesis has been advanced as a
contributory hepatoprotective mechanism, which accelerates the
regeneration process and the production of liver (Awang, 1993).
Treatment with BM extract prevented to a large extent the membrane lesion with concomitant decrease in the albumin and protein
Please cite this article in press as: Sharma N, Shukla S. Hepatoprotective potential of aqueous extract of Butea monosperma against CCl4
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Figs. 2 and 3. Hexobarbitone induced sleep time and bromosulphalein retention of control (Cnt), CCl4 (C), CCl4 + Butea monosperma (C + BM 800), CCl4 + silymarin (C + S50).
n = 6 for each group. # P ≤ 0.05 vs. Control, *P ≤ 0.05 vs. CCl4 .
concentration leakage in the serum. The level of blood sugar was
maintained towards control after BM extract therapy which might
be due to its modulatory effect on glucose metabolism in liver.
The increased TBARS, as seen in the present study, is due to tissue
injury and failure of antioxidant defense mechanism thus increased
the LPO. There is an intimate relationship between the normal cellular functions of GSH, cellular redox status and the generation of
ATP in mitochondria (Manibusan et al., 2007). Treatment with BM
extract significantly reversed these changes through attenuation
of LPO and decreased production of free radical derivatives, as is
evident from the decreased level of TBARS. Antioxidant effect of flavanoids enhanced the process of regeneration. This might be due to
destruction of free radicals, supplying a competitive substrate for
unsaturated lipids in the membrane and/or accelerating the repair
mechanism of damaged cell membrane. Silymarin also reduced LPO
significantly due to its free radical scavenging activity (Basosio et
al., 1992). The non-enzymatic antioxidant, GSH is one of the most
abundant tripeptides, widely distributed in liver cells. Its functions
are mainly concerned with the removal of free radical species such
as H2 O2 , superoxide radicals, alkoxy radicals and maintenance of
membrane protein thiols (Fang et al., 2003). Explanations of the
possible mechanism underlying the hepatoprotective properties of
drugs include the prevention of GSH depletion and destruction of
free radicals these two factors are believed to attribute to the hepatoprotective properties of BM extract. Aloe barbadensis (Chandan
et al., 2007) and Cytisus scorparius (Raja et al., 2007) also substantiated these findings.
The G-6-Pase is a crucial enzyme of glucose homeostasis and
plays an important role in the regulation of the blood glucose
level. Administration of BM extract restored this enzyme activity
of G-6-Pase due to membrane stabilization and improvement in
metabolism. Our findings substantiated the therapeutic effect of
Rhoicissus tridentate (Opoku et al., 2007). ATPase is a mitochondrial enzyme and is known to be intimately associated with the
intracellular iron regulation and transport. Uncoupling of oxidative
phosphorylation leads to fall in activity of ATPase after CCl4 exposure. The BM extract at highest dose (800 mg/kg) up regulated the
activity of ATPase enzyme and could be explained on the basis of
action of. Emblica officinalis (Tasduq et al., 2005). This is supported
by the view that enzyme level returns to normal with the healing of
hepatic parenchyma and regeneration of hepatocytes. Histological
observations basically support the results obtained from biochemical assays. These histological appearances have nearly returned to
normal by therapy with extract at the highest dose.
Transport, conjugation and excretory ability of the liver cells
were examined by BSP retention that is an important and sensitive test for estimating functional integrity of liver (Chandan et al.,
1991). In the present study, significant increase in BSP retention
after CCl4 administration clearly indicates the fall or unavailability
of microsomal drug metabolizing enzymes (MDMEs). Reduction in
BSP retention by B. monosperma extract indicated improvement in
the capacity of damaged liver to perform its normal function probably due to increased regenerative process in liver and antioxidant
nature of BM extract. These findings are very similar to propolis
extract treatment (Bhadauria et al., 2007a).
Hexobarbitone is metabolized by hepatic MDMEs and duration
of hexobarbitone induced sleep in intact animals is considered as
a reliable index for the activity of hepatic MDMEs. Prolongation in
hexobarbitone induced sleep time after CCl4 toxicity (Singh et al.,
2001) substantiated decreased availability of CYP2E1 contents. BM
extract shortened this prolongation of hexobarbitone sleep time
suggesting its protective effect on CYP2E1 system. Other investigators also advocated the protection against liver damage by extract
of propolis (Bhadauria et al., 2007b) and Eclipta alba (Singh et al.,
1993), which possess strong antioxidant potential against free radicals.
The current investigation verified the hepatoprotective effects
of B. monosperma against model hepatotoxicant CCl4. The hepatoprotective action is likely related to its potent antioxidative
activity. Neutralizing reactive oxygen species by non-enzymatic
mechanism and enhancing the activity of original natural hepatic
antioxidants enzymes may be the main mechanisms against injury.
These data provide a scientific explanation for the folklore usage of
B. monosperma in the treatments of hepatic disorders. The findings
Please cite this article in press as: Sharma N, Shukla S. Hepatoprotective potential of aqueous extract of Butea monosperma against CCl4
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provide a rationale for further studies on pharmacological evaluation.
Acknowledgments
Authors are thankful to Jiwaji University, Gwalior (MP) and
CCRUM, New Delhi for financial assistance.
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Please cite this article in press as: Sharma N, Shukla S. Hepatoprotective potential of aqueous extract of Butea monosperma against CCl4
induced damage in rats. Exp Toxicol Pathol (2010), doi:10.1016/j.etp.2010.05.009