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Research Article
Hepatoprotective Properties of Caesalpinia bonducella against CCl4
induced in Rats
Santosh Kumar S R1, Venkatesh1±, Krishna Venkatarangaiah1*, Pradeepa Krishnappa2, Sudhesh L
Shastri1
1 P.G. Department of Studies and Research in Biotechnology and Bioinformatics, Kuvempu University,
Shankaraghatta – 577451, Karnataka, India. 2 Department of Biotechnology, MS Ramaiah Institute of Technology, Bengaluru – 560 054, India. ± Present address: Department of Biochemistry, Indian Institute of Science, Bengaluru - 560012
Article Info
Abstract
Received: 16-09-2017,
Revised: 23-11-2017,
Accepted: 01-12-2017
To investigate the hepatoprotective property of leaves, stem bark extract and the
phytoconstituent of Caesalpinia bonducella on CCl4 induced rat models. The
Wistar albino rats of either sex, weighing about 180–200 g were used to induce
the hepatotoxicity using CCl4. Animals were treated with different leaves, stem
bark extracts (300 mg/kg b.wt.) and the isolated phytoconstituent (70 mg/kg
b.wt.) orally once in day for a period of 15 days. All the groups received the
intraperitoneal dose of 50% CCl4 after every 72h except vehicle control (1%
(v/v) DMSO, 1ml/kg body weight, p.o). At the end of the experimental period,
animals were sacrificed by cervical decapitation. The serum and liver samples
were used for biochemical marker analysis. The animals treated with stem bark
chloroform extract showed significant amelioration effect by elevating the
decreased levels of SOD, CAT, GPx and GST levels with increased level of
MDA. The isolated phytoconstituent SC2 (Methyl (4E)-5-{2-[(1E)-buta-1,3-
dien-1-yl]-4,6-dihydroxyphenyl}pent-4-enoate) was more significant by
reducing the serum markers level similar to standard drug silymarin. Normal
hepatic architecture, absence of necrosis and few fatty lobules were noticed in
the liver sections of the animals treated with stem bark chloroform extract and its
isolated compound SC2 against the toxicant CCl4. The C. bunucella extract and
the isolated phytoconstituent has potent hepatoprotective property.
Keywords:
C. bunucella, acute toxicity,
Stem bark chloroform
extract, phytoconstituent,
Hepatoprotective activity.
INTRODUCTION
Traditional medical inheritance in India is
unique its journey from several millennia, it saw the
progression of valuable indigenous systems in Folk
and Ethnomedicine, Ayurveda, Sidda, Unani etc.
Plants have been one of the important cradle of
medicines since the dawn of human evolution.
Many herbs were predominantly used to treat
cardiovascular problems, liver disorders, central
nervous system, digestive and metabolic disorders
(Lalitharani et al., 2013; Anand and Mohan, 2014).
Given their potential to produce significant
therapeutic effect, they can be useful as drug or
supplement in the treatment of various diseases
(Fawazi Mahomoodally, 2013).
Caesalpinia bonducella is belonging to the
family Fabaceae. The plant is distributed in tropical
regions of the world especially in India, Sri Lanka,
Brazil, Madagaskar Islands etc. (Asolkar et al.,
1992; White, 2008).
In India, this species is found particularly
along the seacoast throughout the hotter regions of
the Western Ghats and Eastern Himalayan forests.
It is also present in deltaic regions of Western,
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Santosh Kumar et al.,
Eastern and Southern India (Kirtikar and Basu,
1975). In the Western Ghats of Karnataka C.
bonducella is very sparsely distributing along the
banks of rivers and gullies in the forest ranges of
Bhadra Wild Life Sanctuary. Numerous
phytochemicals investigation and pharmacological
activities such as antidiabetic, abortifacient,
antioxidant, analgesic, anti-inflammatory,
antifilarial, anticonvulsive, antibacterial,
antidiarrhoeal, antimalarial, antipyretic, antifungal,
antispermatogenic, antitumor, antipsoriatic,
immunomodulatory, anticataract, anthelmintic,
antiulcer and anticancer has been reported and
summarized in review paper (Nirmala et al., 2016).
Reports are very scarce for the
hepatoprotective property of C. bonducella
(Sambath et al., 2010). The traditional practitioners
residing in the vicinity of the Western Ghats of
Karnataka are using the leaves and stem bark to
cure jaundice and liver disorders. Hence the present
study evaluate the on the medicinal uses of the
leaves and stem bark extract of this species.
MATERIALS AND METHOD
Plant collection and extract preparation
Detail report for plant collection, deposition
at Kuvempu University (voucher number KUAB
301) preparation of plant extract, quantitative
analysis of extract, isolation and characterization of
phytoconstituent from leaves and stem bark has
been described in our previous paper (Santosh
Kumar et al., 2017).
Acute toxicity study
The experimental animals Wistar albino
rats of either sex, weighing about 180–200 g were
procured from the Central Animal House, National
College of Pharmacy, Shivamogga. These animals
were maintained at standard housing conditions
(temperature 271C; relative humidity 60 5%)
and were fed with commercial diet (Hindustan
Lever Ltd., Bangalore) and water ad libitum, during
the experiment. The institutional animal ethical
committee (Ref No. NCP/IAEC/CL/10/12/ 2010-
11. Dated: 28-11-2012) approved the study. Acute
toxicity study was conducted for the leaves and
stem bark extracts by the Up and Down procedure
(Adeneye et al., 2006). DMSO (1% v/v) was used
as a vehicle to suspend the extracts and
administered orally. Animals were observed
individually at least once during the first 30 min
after dosing, periodically during the first 24 h (with
special attention given during the first 4 h), and
daily thereafter, for a total of 14 days for changes in
their behavioural pattern and mortality
Determination of hepatoprotective Activity
Grouping of animals
Rats were divided into 9 groups consisting
of six animals in each group. Group-I served as
vehicle control and received 1% (v/v) DMSO
(1ml/kg body weight, p.o); Group-II (Toxic control)
received 50% CCl4 in olive oil (1ml/kg b.wt., i.p);
Group-III received leaves chloroform extract (300
mg/kg b.wt.); Groups-IV received leaves ethanol
extract (300 mg/kg b.wt.); Group-V received stem
bark chloroform extract (300 mg/kg b.wt.); Group-
VI received stem bark ethanol extract (300 mg/kg
b.wt.); Group-VII received SC2 (70 mg/kg b.wt.);
Group-VIII received standard drug silymarin (25
mg/kg b.wt.) once in a day. Treatment duration was
15 days and all the groups received the
intraperitoneal dose of 50% CCl4 after every 72h
(Khadeer Ahamed et al., 2010). At the end of the
experimental period, animals were sacrificed by
cervical decapitation. Blood was collected and
serum was separated. The liver tissue was excised,
part of the excised liver was homogenized in ice-
cold saline and utilized for biochemical analysis.
Estimation of liver function enzyme activities in
serum
Liver damage was assessed by estimating
serum marker enzymes such as ALT, AST and ALP
using commercially available test kits. The results
were expressed as units/litre (U/l). Also, the levels
of cholesterol, triglycerides (TG), total bilirubin and
total protein were estimated in the serum of
experimental animals using assay kits and semi
auto-analyzer, which were obtained from the
Robonik India Pvt. Ltd., New Mumbai.
Estimation of oxidative enzyme activities in liver
homogenate
Hepatic tissues were homogenized (10%) in
frozen normal saline and centrifuged at 4000 rpm
for 5 min. The supernatant was used for the
measurement of Superoixde dismutase (SOD)
(Beauchamp and Fridovich, 1971) expressed as
units (U) of SOD mg-1 protein, Catalase (CAT)
(Aebi, 1984), Glutathione peroxidase GPx)
(Mohandas et al., 1984) and Glutathione S-
transferase (GST) (Margareta et al., 1987). The
activities of these enzymes are expressed as nmol
min-1 mg-1 of protein. Malondialdehyde (MDA) is
one of lipid peroxidation products determined by
the method (Ohkawa et al., 1979).
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Bioscience Discovery, 9(1): 44-52, Jan - 2018
Histopathology of liver tissue
The liver tissue was washed with normal
saline and kept in 10% formaldehyde buffer for
18h. The tissues were dehydrated in graded (50–100
%) ethanol, followed by clearing in xylene. Paraffin
(56–58oC) embedding was done at 58 ± 1oC for 4 h,
followed by paraffin block preparation. Paraffin
sections of 5 μm were taken using a rotary
microtome. The sections were de-paraffinised with
alcohol xylene series, stained with haematoxylin–
eosin, mounted in DPX with a cover slip and
histological changes were observed under
microscope (Galigher et al., 1971).
Statistical analysis
Results are expressed as mean ± S.E.M. The
statistical analysis was carried out using one way
ANOVA followed by Tukey’s t-test. The
differences in values at p < 0.05 or p < 0.01 were
considered as statistically significant. Statistical
analysis was performed by ezANOVA 0.98.
RESULTS AND DISCUSSION
The leaves and stem bark extracts of
chloroform showed the presence of high phenolic
compounds and flavonoids. The isolation and
characterized the Methyl (4E)-5-{2-[(1E)-buta-1,3-
dien-1-yl]-4,6-dihydroxy phenyl}pent-4-enoate
from stem bark chloroform extract has been isolated
and characterized in our previous reported paper.
Acute toxic studies
The earlier investigators Sagar and
Vidyasagar, (2010) has reported the acute and sub-
acute toxicity studies of ethyl acetate extract of C.
bonducella leaves in albino mice was higher than
2000 mg/kg. Sambath Kumar et al., (2010) reported
similar results in the methanol extracts of C.
bonducella at low and median doses (100 and 200
mg/kg). In our study acute toxicity results revealed
that the LD50 value of chloroform and ethanol
extracts of leaves and stem bark has found to be
3,000 mg/kg b. wt. similarly, LD50 value of the
isolated constituents SC2: (Methyl (4E)-5-{2-
[(1E)-buta-1,3-dien-1-yl]-4,6-
dihydroxyphenyl}pent-4-enoate) were 180 mg/kg
b.wt respectively. One tenth of these LD50 doses has
considered as safer dose for oral drug
administration in the pharmacological models.
Hepatoprotective activity
Carbon tetrachloride can also induce
cellular hypomethylation, leading to inhibition of
protein synthesis (possibly through ribosomal RNA
hypomethylation) and defects in lipid and protein
metabolism (Weber, 2013). Thus in the present
study, significantly reduced serum total protein
level was observed in the animals treated with CCl4.
The leaves, stem bark chloroform extract and the
isolated constituents viz., SC2 have elevated the
decreased level of total proteins and albumin in the
serum, which indicates their hepatoprotective
activity. The intoxication of CCl4 to the rats had
resulted in a marked increase in the levels of liver
function serum markers viz., AST (489. 97 ± 4.16
U/l), ALT (171.0 ± 2.12), ALP (420.30 ± 6.01 U/l),
total bilirubin (0.735 ± 0.14 mg/dl), total cholesterol
(270.00 ± 2.50 mg/dl) and with the decrease in total
proteins level (7.80 ± 0.09 g/dl) as compared to the
control group treated with only vehicle (1% DMSO)
viz. AST (191.30 ± 2.14), ALT (73.04 ± 4.1),
ALP(254.2 ± 5.85), total bilirubin (0.038 ± 0.1),
total cholesterol (150.00 ± 2.00) and total proteins
(14.30 ± 0.15). On the contrary, the increased levels
of these liver function markers were brought down
nearer to normalcy due to the amelioration effect of
the leaves and stem bark extracts. Significant
hepatoprotective activity was noticed in the animals
treated with the stem bark chloroform extract viz.,
ALP (309.17 ± 3.02 U/l), AST (269.43 ± 1.63 U/l),
ALT (119.24 ± 1.18 U/l), total bilirubin (0.107 ±
0.01 mg/dl), total cholesterol (198.24 ± 2.30 mg/dl),
and total protein (10.09 ± 0.05 mg/dl). In animals
treated with the leaves chloroform extract, reduction
of toxic effect was noticed in moderate level. The
hepatoprotective effect of leaves and stem bark
extracts, and isolated phytochemicals were
comparatively evaluated with the standard drug
Silymarin is shown in the Table 1.
Amelioration effect of the isolated
phytoconstituents has been observed. Among the
isolated phytochemicals, SC2: (Methyl (4E)-5-{2-
[(1E)-buta-1,3-dien-1-yl]-4,6-
dihydroxyphenyl}pent-4-enoate) was more
significant when compared to CCl4 intoxicated and
their hepatoprotective activity was evaluated by
reducing serum markers level towards normalcy
viz., ALP (309.08± 0.60 U/l), AST (301.04 ± 0.17
U/l), ALT (98.37 ± 0.12 U/l), total bilirubin (0.098
± 0.01 mg/dl), total cholesterol (192.16 ± 0.70
mg/dl) and total protein (10.28 ± 0.22 mg/dl). The
hepatoprotective effect of the compound SC2:
(methyl (4E)-5-{2-[(1E)-buta-1,3-dien-1-yl]-4,6-
dihydroxyphenyl}pent-4-enoate) was almost similar
to standard reference Silymarin.
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Santosh Kumar et al.,
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Santosh Kumar et al.,
Estimation of oxidative stress markers
Oxidative stress marker enzymes were used
as tools to assess the intensity of liver injury.
Several studies have demonstrated that the
pathological effects of CCl4 are mediated by
induction of oxidative stress (Premkumar et al.,
2003; Manibusan et al., 2007). Among the various
mechanisms involved in the hepatotoxic effect of
carbon tetrachloride, one is oxidative damage
through free radical generation and propagation of
by-products of lipid peroxidation such as highly
reactive aldehydes, melanodialdedyde (MDA).
These by products can form protein and DNA
adducts and may contribute to hepatotoxicity.
The oxidative stress markers estimation of
the liver homogenates revealed that intoxication of
rats with CCl4, significantly decreased the activities
of oxidative stress marker enzymes in liver like
SOD (5.26 ± 052 U/mg), CAT (214.6 ±
0.7nmol/min/ mg), GPx (78.87 ± 0.35nmol
NADPH/min/mg) and GST (209.58 ±
1.0nmol/min/mg) as compared to the control group
10.37 ± 0.2 U/mg; 456.12 ± 1.07nmol/min/mg;
146.34 ± 1.0nmol/min/mg and 387.2 ±
3.9nmol/min/mg respectively (Table 2). In addition,
two fold increases in the levels of MDA was also
noticed in CCl4 intoxicated rat liver (1.01 ±
0.01nmol/mg) as compared with the liver
homogenates of control animals with MDA level of
0.58 ± 0.02nmol/mg. Among the four extracts
tested for in vivo antioxidant activity, the liver
homogenates of animals administered with stem
bark chloroform extract showed significant
amelioration effect by elevating the decreased
levels of SOD, CAT, GPx and GST levels viz., 7.41
± 0.2 U/mg; 351.15 ± 3.45nmol/min/mg, 104.14 ±
3.44nmol NADPH/min/mg and 337.15 ±
0.45nmol/min/mg respectively. The increased level
of MDA was also restored (0.81 ± 0.01nmol/mg).
These results clearly indicate a weakening or failure
of the antioxidant defence system in the hepatic
cells, which in turn would affect oxidative stress in
overall milieu of cells (Beddowes et al., 2003). The
restoration of levels of oxidative stress marker
enzymes was also significant in the animals treated
with the compound SC2: (methyl (4E)-5-{2-[(1E)-
buta-1,3-dien-1-yl]-4,6-dihydroxyphenyl}pent-4-
enoate) among tested phytochemicals. The levels of
these oxidative stress enzyme markers were nearly
equal to that of the standard drug Silymarin treated
group.The chronic sub lethal doses of CCl4 elevated
the levels of MDA content in liver, whereas the
various extracts of C. bonducella specifically stem
bark chloroform extract and leaves chloroform
extract and among phytoconstituents SC2 markedly
reversed these effects. It is conceivable that the
effect of tested compounds may be due to a
reduction in hepatic peroxidative activities thereby
leading to the restoration of the glutathione content
in CCl4-induced hepatotoxicity. Treatment with the
stem bark chloroform extract, the compound SC2 as
well as the standard drug silymarin significantly
reversed changes caused by CCl4. Hence, it is
possible to know that the hepatoprotection of the
extract and constituents is due to their antioxidant
effect.
Histopathological examination of liver tissue
The histological profile of liver sections of
the control animals showed normal hepatic
architecture with well-preserved cytoplasm,
prominent nucleus, central vein, compact
arrangement of hepatocytes and without fatty
lobulation (Fig. 1A). The liver sections of CCl4
treated animals showed hydropic changes in
centrilobular hepatocytes with cell necrosis
surrounded by neutrophils. Congestion of the
central vein and sinusoids were seen with acute and
chronic inflammatory cells infiltrating sinusoids
mainly in the central zone. The midzonal and
periportal hepatocytes showed vacuolization and
fatty change (steatosis) which includes the
intracellular accumulation of neutral fat (Fig. 1B).
Animals administered with leaves chloroform
extract exhibited significant liver protection against
CCl4 induced liver damage, as evident by the
presence of normal hepatic cords with moderate
fatty change, necrosis and few inflammatory cells
(Fig. 1C). In the liver sections of leaves ethanol
extract administered group showed mild fatty
changes and mild sinusoidal congestion (Fig. 1D).
The hepatocytes are distended with fat vacuoles due
to increased deposition of intracellular lipids in liver
section of stem bark chloroform extracts
administered animals (Fig. 1E). The sections of
liver taken from the animals treated with stem bark
ethanol extract (Fig. 1F) showed the normal hepatic
architecture and its hepatoprotectivity is nearly
similar to the standard reference Silymarin treated
animals. In the liver sections of SC2: methyl (4E)-
5-{2-[(1E)-buta-1,3-dien-1-yl]-4,6
dihydroxyphenyl}pent-4-enoate(Fig.2A) showed
the hepatocytes are distended with fat vacuoles due
to increased deposition of intracellular lipids,
hepatic architecture and its hepatoprotectivity is
similar to the standard reference Silymarin treated
animals (Fig. 2B).
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Bioscience Discovery, 9(1): 44-52, Jan - 2018
Fig. 1: Liver histology of Rat showing hepatoprotective effect of extracts and the isolated constituents
of C. bonducella against CCl4 induced hepatotoxicity
(A) Control; (B) CCl4 treated; (C) CCl4+ Leaves chloroform extract treated; (D) CCl4+ Leaves ethanol
extract treated; (E) CCl4+Stembark chloroform extract treated; (F) CCl4+ Stembark ethanol treated. (1-
cellular necrosis; 2-vacuolization; 3- Ballooning degeneration). Stain: haematoxylin–eosin, magnification:
100×.
A B
C D
E F
1
3
2
2
3
1
1
2
3
3
1 2 3
1
2
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Santosh Kumar et al.,
Fig. 2: Liver histology of Rat showing hepatoprotective effect of extracts and the isolated constituents
of C. bonducella against CCl4 induced hepatotoxicity
(G) CCl4+ (Methyl (4E)-5-{2-[(1E)-buta-1,3-dien-1-yl]-4,6-dihydroxyphenyl}pent-4-enoate treated; (H)
CCl4+ Silymarin treated. (1-cellular necrosis; 2-vacuolization;3- Ballooning degeneration). Stain: haemato-
xylin–eosin, magnification: 100×.
CONCLUSION:
The present investigation also supported the
traditional claim of C. bonducella as a
hepatoprotective plant. The significant
hepatoprotective effect of stem bark chloroform
extract of C. bonducella is due to the presence of
the phytochemicals SC2: (methyl (4E)-5-{2-[(1E)-
buta-1,3-dien-1-yl]-4,6-dihydroxyphenyl}pent-4-
enoate), and its liver damage amelioration effect
was similar with that of the standard drug silymarin.
ACKNOWLEDGEMENT:
The authors are thankful to DBT, New
Delhi, India for providing financial support through
DBT-BUILDER program (Order No.
BT/PR9128/INF/22/190/2013, Dated: 30/06/2015)
and the Kuvempu University administrative
authority for providing the facility to carry out the
work.
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How to cite this article
Santosh Kumar S R, Venkatesh, Krishna Venkatarangaiah, Pradeepa Krishnappa, Sudhesh L
Shastri, 2018. Hepatoprotective Properties of Caesalpinia bonducella against CCl4 induced in Rats.
Bioscience Discovery, 9(1): 44-52.