research article hepatoprotective properties of

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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,

http://biosciencediscovery.com 45 ISSN: 2229-3469 (Print)

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).



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.



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).



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



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.

research article hepatoprotective properties of

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