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Experimental and Toxicologic Pathology 63 (2011) 671– 676

Contents lists available at ScienceDirect

Experimental and Toxicologic Pathology

jo u rn al h omepage: www.elsev ier .de /e tp

epatoprotective potential of aqueous extract of Butea monospermagainst CCl4 induced damage in rats

eetu Sharma, Sangeeta Shukla ∗

chool of Studies in Zoology, Jiwaji University, Gwalior 474011, MP, India

r t i c l e i n f o

rticle history:eceived 22 January 2010ccepted 19 May 2010

eywords:epatoprotectiveCl4

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 val-idate its use in traditional medicines. The CCl4 administration altered various biochemical parameters,including serum transaminases, protein, albumin, hepatic lipid peroxidation, reduced glutathione andtotal protein levels, which were restored towards control by therapy of B. monosperma Adenosine triphos-phatase and glucose-6-phosphatase activity in the liver were decreased significantly in CCl4 treated

iver diseasesutea monosperma

animals. Therapy of B. monosperma showed its protective effect on biochemical and histopathologicalalterations at all the three doses in dose dependent manner. B. monosperma extract possess modulatoryeffect on drug metabolizing enzymes as it significantly decreased the hexobarbitone induced sleep timeand increased excretory capacity of liver which was measured by BSP retention. Histological studies alsosupported the biochemical finding and maximum improvement in the histoarchitecture was seen athigher dose of BM extract.

. Introduction

Herbs have recently attracted attention as health beneficial foodnd as source materials for drug development. They offer a potentialatural health care approach that focuses on protecting and restor-

ng the health. Recently herbal medicines are being increasinglytilized to treat a wide variety of clinical diseases, including liveriseases (Gupta et al., 2007) with relatively little knowledge regard-

ng their modes of action (Jeong et al., 2002). India is sitting on a goldine of well-recorded and traditionally well-practiced knowledge

f herbal medicines, therefore, any scientific data on such planterivatives could be of clinical importance (Singanan et al., 2007).here are number of medicinal preparations in Ayurveda that areecommended for the treatment of liver disorders (Samudram et al.,008), however, no scientific evidence is available for their clinicalsage.

Butea monosperma Lam. is commonly known as Palash in Hindind widely disturbed in India. Its flowers are used to treat leprosy,out, skin and eye diseases and has been reported to be associated

ith various remedial properties such as anti-hepatotoxic (Wagner

t al., 1986), antistress (Kasture et al., 2002), antiestrogenic (Shahnd Bakxi, 1990) and chemopreventive (Sehrawat et al., 2006).

∗ Corresponding author. Tel.: +91 751 4016750; fax: +91 751 2341450.E-mail addresses: neetupandit2000@gmail.com, profsshukla@gmail.com

S. Shukla).

940-2993/$ – see front matter © 2010 Elsevier GmbH. All rights reserved.oi:10.1016/j.etp.2010.05.009

© 2010 Elsevier GmbH. All rights reserved.

Its flowers contain various flavanoids like butein, butin, isobutrin,isomonospermoside and steroids (Kasture et al., 2002; Lavhale andMishra, 2007).

Investigation of hepatoprotective herbal drug as a major indi-cator of the general screening systems can trigger the safetyevaluation in the early phase of drug discovery because most of thetoxic compounds are metabolized in liver. Therefore, to justify thetraditional claim of B. monosperma as hepatoprotective agent, weassessed its hepatoprotective potential against CCl4 induced liverdamage in rats.

2. Materials and methods

2.1. Preparation of plant extract

Flowers of B. monosperma were generously obtained fromCentral Council for Research in Unani Medicine, India. Flowerswere dried in shade, powdered and extracted with distilled water(250 g/4 l) for 18 h with concomitant shaking. Filtrate was evapo-rated in vacuum to yield a yellow powder (BM extract), which wasadministered orally according to body weight of animals. Silymarin(50 mg/kg, p.o.) was used as a positive control during experimentalregimen (Chandan et al., 2007).

2.2. Chemicals

All chemicals were procured from Sigma–Aldrich (USA), E-Merck (Germany), Ranbaxy Pvt. Ltd. and BDH Company (India).

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.3. Maintenance of animals and their feeding

Adult female albino Swiss mice (30 ± 5 g body weight) andlbino rat of Sprague–Dawley strain (160 ± 10 g body weight)ere randomly selected from departmental animal facility where

hey were housed in polypropylene cages under uniform hus-andry conditions of light (14 h) and dark (10 h) with temperature25 ± 2 ◦C) and relative humidity (60–70%). Animals were fedn commercially available standard animal diet (Pranav Agrondustries Ltd., New Delhi, India) and drinking water ad libitum.xperimental protocol was approved by Institutional Ethics Com-ittee following guidelines set by Committee for the Purpose of

ontrol and Supervision of Experiments on Animals, India.

.4. Preparation of doses and treatments

The CCl4 was administered at a dose of 1.5 ml/kg, i.p. with vehicleolive oil) (Bhadauria et al., 2008). An aqueous suspension of extractas prepared in distilled water and different doses of BM extract

200 mg, 400 mg and 800 mg/kg) and silymarin (50 mg/kg) weredministered to the animals orally.

.5. Experiment design

Thirty-six adult female rats were divided into six groups of sixnimals each. The animals were administered CCl4 at a dose of.5 ml/kg, i.p. once only followed by different doses of BM extractfter 48 h of toxicant administration once only. All the animals wereuthanized after 48 h of last treatment and various blood and hep-tic 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.).

.6. Blood biochemical assay

Blood was drawn by puncturing retro-orbital venous sinus,entrifuged and serum was isolated to determine aspartate amino-ransferase (AST) and alanine aminotransferase (ALT) (Reitman andrankel, 1957) and protein (Lowry et al., 1951). Blood sugar (Kit No.BCER0017) and albumin (Kit No. 1118275) were assessed by kitethods as per instructions provided by the company (E-Merck,ermany).

.7. Tissue biochemical assay

Lipid peroxidation (LPO) was determined by measuring thio-arbituric acid reactive substances (TBARS) (Sharma and Krishnaurty, 1968). Reduced glutathione (GSH) level was determined by

ithionitrobenzoic acid (DTNB) (Brehe and Burch, 1976). Activitiesf adenosine triphosphatase (ATPase) (Seth and Tangari, 1966) andlucose-6-phosphatase (G-6-Pase) (Baginski et al., 1974) were alsoetermined in liver.

.8. Histological observations

Liver samples were fixed in Bouin’s fixative and processed tobtain 5 �m thick paraffin sections and stained with hematoxylinnd eosine (H&E) for histological observations.

icologic Pathology 63 (2011) 671– 676

2.9. Hexobarbitone induced sleep time

Hexobarbitone induced sleep time was measured accordingto Fujimoto et al. (1960) and separate set of mice were dividedinto four groups of six each. Group 1 served as normal controland received vehicles only. Groups 2–4 were administered CCl4(1.5 ml/kg, i.p.) and group 2 served as experimental control. Groups3 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 lastadministration. Time of onset of loss of reflex up to the recoverywas taken in min as duration of sleep and protective activity of BMextract and silymarin was calculated by given formula:

% protection = 1 − Td − Tn

Tc − Tn× 100

where T is sleep time; c, d and n are CCl4, drug (BM extract andsilymarin) and normal groups respectively.

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 sixeach. 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 2served as experimental control. BM extract (800 mg/kg) and sily-marin (50 mg/kg) were administered orally to groups 3 and 4respectively after 48 h of toxicant administration. BSP (100 mg/kg,i.v.) was injected to the entire four groups after 48 h of last treat-ment. Blood was collected in heparinized tubes exactly after 30 min,centrifuged for plasma isolation and BSP concentration was esti-mated in it. Percent retention of dye was used as excreting capacityby following formula:

% protection = 1 − Rd − Rn

Rc − Rn× 100

where R is retention of BSP in plasma; c, d and n are CCl4, drug (B.monosperma and silymarin) and normal groups respectively.

2.11. Statistical analysis

Results are presented as mean ± S.E.M. of six animals used ineach group. Data were subjected to statistical analysis through one-way analysis of variance (ANOVA) taking significant at 5% level ofprobability 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 inTable 1. Administration of CCl4 induced significant increase in theenzymatic activities of ALT and AST (P ≤ 0.05) as compared to thecontrol group. Oral administration of extract at different doses(200, 400 and 800 mg/kg) showed significant recoupment in a dosedependent manner (P ≤ 0.05). Significant increase was observed inalbumin, 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 thera-peutic effectiveness (P ≤ 0.05). The 200 and 400 mg/kg showed lesseffective significant changes.

N. Sharma, S. Shukla / Experimental and Toxicologic Pathology 63 (2011) 671– 676 673

Table 1Effectiveness of Butea monosperma against carbon tetrachloride induced blood biochemical alterations.

Treatments AST (IU/L) ALT (IU/L) Albumin (g/dl) Blood sugar (mg/dl) Serum protein (mg/100 ml)

Group 1 68.0 ± 3.51 50.0 ± 4.28 3.50 ± 0.33 75.0 ± 5.62 48.3± 3.39Group 2 188 ± 14.8# 425 ± 22.8# 4.60 ± 0.34# 145 ± 9.16# 138 ± 3.58#

Group 3 140 ± 9.90* 130 ± 11.4* 3.50 ± 0.32* 76.8 ± 5.84* 43.5 ± 2.44Group 4 106 ± 8.17* 124 ± 10.7* 3.65 ± 0.24* 79.0 ± 5.27* 42.1 ± 3.83Group 5 90.0 ± 4.93* 93.0 ± 6.72* 3.60 ± 0.20* 81.0 ± 6.87* 42.1 ± 3.21Group 6 88.0 ± 6.52* 55.0 ± 3.72* 3.50 ± 0.34* 80.0 ± 6.48* 40.0 ± 2.48

ANOVA F-value 34.2@ 167@ 13.1@ 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 2Effectiveness of B. monosperma against carbon tetrachloride treated animals in tissue biochemical estimations.

Treatments LPO (n mole of TBARS/mg protein) GSH (�mole/g) ATPase (mg Pi/100g/min) G-6-Pase (�mole Pi/min/g liver) Tissue protein (mg/100 mg)

Group 1 0.28 ± 0.01 7.8 ± 0.48 2000 ± 100 7.0 ± 0.37 15.0 ± 0.75Group 2 1.25 ± 0.10# 4.2 ± 0.37# 930.0 ± 47.4# 3.1 ± 0.18# 19.5 ± 0.87#

Group 3 0.59 ± 0.03* 6.8 ± 0.37* 1784 ± 96.0* 6.1 ± 0.40* 17.0 ± 0.85*

Group 4 0.42 ± 0.03* 6.9 ± 0.40* 1757 ± 91.3* 6.3 ± 0.38* 15.7 ± 0.81*

Group 5 0.34 ± 0.02* 7.2 ± 0.40* 1846 ± 99.2* 6.3 ± 0.39* 15.5 ± 0.85*

Group 6 0.30 ± 0.02* 7.2 ± 0.41* 1910 ± 99.8* 6.5 ± 0.35* 15.1 ± 0.77*

ANOVA F-value 71.4@ 11.6@ 21.9@ 18.2@ 8.17@

Data are mean ± S.E.M., n = 6.ANOVA (F values at 5% level).

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# P ≤ 0.05 vs. Control.* P ≤ 0.05 vs. CCl4.

@ Significant.

.2. Tissue biochemical assay

Various biochemical parameters were performed in liver tis-ues which are presented in Table 2. A significant increase wasbserved in the level of LPO in liver after 48 h of CCl4 intoxicationhen compared with the control group (P ≤ 0.05). Treatment withifferent doses of crude extract reversed the oxidative stress signifi-antly towards control by inhibiting LPO in dose dependant mannerP ≤ 0.05). Reduced glutathione is presumed to be an importantndogenous defense against peroxidative destruction of cellularembranes. In the present study, significant decline was seen in the

educed glutathione level (P ≤ 0.05). Post treatment of extract wasery effective in restoring the glutathione content which had beenubstantially decreased by CCl4 (Table 2). All three doses of extractmproved the GSH level in liver, however, therapy at 800 mg/kg

as very effective. In our study, decline in the activities of G-6-ase and ATPase in CCl4 administered animals were also observed,hich were significantly reversed towards control with the highestose of BM extract (P ≤ 0.05). Total protein level was significantly

ncreased in CCl4 intoxicated animals that was brought towardsontrol by all the doses of BM extract significantly, however, max-mum reversal was noticed at 800 mg/kg dose (P ≤ 0.05).

.3. Histological observation

The light microscopy examination of the transverse section ofontrol rat liver clearly illustrates complete hepatic lobules withell formed hepatocytes with distinct portal triads. Hepatic cellsere arranged in cord like fashion, which are separated by sinu-

oids and central vein was seen clear (Fig. 1a). The liver sections

f CCl4 intoxicated rats showed massive fatty changes, necrosis,allooning and degeneration in hepatic plates and loss of cellu-

ar boundaries (Fig. 1b). Treatment with the aqueous extract wasffective in restoring the CCl4 induced histopathological lesions

when compared to CCl4 per se, however highest dose was foundto be more effective. The histological architecture of liver sectionsshowed mild degree of degeneration and necrosis at lower doses(Fig. 1c–d). Therapy with BM extract at 800 mg/kg dose, the micro-graphs exhibited an almost normal architecture (Fig. 1e). Silymarintreated group depicted symmetrically arranged well formed hepa-tocytes separated by sinusoids and maintained cord arrangement(Fig. 1f).

3.4. Hexobarbitone induced sleep time

Fig. 2 depicts the effects of hexobarbitone induced sleeptime. CCl4 administration significantly prolonged the barbiturateinduced sleep time (P ≤ 0.05) when compared to normal. Signif-icant prolongation in sleeping time proved the event of hepaticdamage by free radicals, which was shortened significantly by theBM extract. This can well be compared to silymarin group.

3.5. Bromosulphalein retention

BSP retention after 30 min of its injection in normal animals wassignificantly increased (P ≤ 0.05) by carbon tetrachloride admin-istration. Treatment of BM extract significantly reduced the BSPretention (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 isused as anti-hepatotoxic agent. Hepatic cells participate in a vari-

ety of metabolic activities and contain a host of enzymes. The liverfunctions in a coordinated way with various systems of the bodyand any disease involving this organ have serious and far reachingeffects not only on the liver itself but also on other organs and sys-

674 N. Sharma, S. Shukla / Experimental and Toxicologic Pathology 63 (2011) 671– 676

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):Aqueousextract 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 improve-m 800 ms logy (

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ent in chord arrangement but perinuclear vacuolation was visible (X-100). (e)

inusoidal spaces (X-100). (f) Treatment with silymarin exhibit almost normal histo

ems. In the assessment of liver damage, the determination of liverunction tests enzyme levels such as AST and ALT is largely usedy CCl4 (Shukla et al., 2007; Lynch and Price, 2007). In the presenttudy, exposure to CCl4 resulted in a significant hepatic damages elicited by the elevated level of serum marker enzymes, ASTnd ALT. These marker enzymes are cytoplasmic in origin and areeleased into the circulation after cellular damage (Lin et al., 2000).he rise in the enzyme AST is usually accompanied by an elevation

n the levels of ALT, which plays a vital role in the conversion ofmino acids to keto acids (Salie et al., 1999).

Albumin, which is produced only in the liver is the major plasmarotein that circulates in the bloodstream. It is involved in scav-

g/kg of BM extract show normal cellular architecture with distinct hepatic cellsX-100).

enging of oxygen free radicals. It is also very important in thetransportation 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 ofthe severity of cellular dysfunction in liver diseases as clearly shownin our studies. The elevated level of albumin test is indicative ofcellular leakages and loss of functional integrity of cell membranein liver. Stimulation of protein synthesis has been advanced as a

contributory hepatoprotective mechanism, which accelerates theregeneration process and the production of liver (Awang, 1993).Treatment with BM extract prevented to a large extent the mem-brane lesion with concomitant decrease in the albumin and protein

N. Sharma, S. Shukla / Experimental and Toxicologic Pathology 63 (2011) 671– 676 675

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oncentration leakage in the serum. The level of blood sugar wasaintained towards control after BM extract therapy which might

e due to its modulatory effect on glucose metabolism in liver.The increased TBARS, as seen in the present study, is due to tissue

njury and failure of antioxidant defense mechanism thus increasedhe LPO. There is an intimate relationship between the normal cel-ular functions of GSH, cellular redox status and the generation ofTP in mitochondria (Manibusan et al., 2007). Treatment with BMxtract significantly reversed these changes through attenuationf LPO and decreased production of free radical derivatives, as isvident from the decreased level of TBARS. Antioxidant effect of fla-anoids enhanced the process of regeneration. This might be due toestruction of free radicals, supplying a competitive substrate fornsaturated lipids in the membrane and/or accelerating the repairechanism of damaged cell membrane. Silymarin also reduced LPO

ignificantly due to its free radical scavenging activity (Basosio etl., 1992). The non-enzymatic antioxidant, GSH is one of the mostbundant tripeptides, widely distributed in liver cells. Its functionsre mainly concerned with the removal of free radical species suchs H2O2, superoxide radicals, alkoxy radicals and maintenance ofembrane protein thiols (Fang et al., 2003). Explanations of the

ossible mechanism underlying the hepatoprotective properties ofrugs include the prevention of GSH depletion and destruction ofree radicals these two factors are believed to attribute to the hep-toprotective properties of BM extract. Aloe barbadensis (Chandant al., 2007) and Cytisus scorparius (Raja et al., 2007) also substan-iated these findings.

The G-6-Pase is a crucial enzyme of glucose homeostasis andlays an important role in the regulation of the blood glucose

evel. Administration of BM extract restored this enzyme activityf G-6-Pase due to membrane stabilization and improvement inetabolism. Our findings substantiated the therapeutic effect of

hoicissus tridentate (Opoku et al., 2007). ATPase is a mitochon-

rial enzyme and is known to be intimately associated with the

ntracellular iron regulation and transport. Uncoupling of oxidativehosphorylation leads to fall in activity of ATPase after CCl4 expo-ure. The BM extract at highest dose (800 mg/kg) up regulated the

rol (Cnt), CCl4 (C), CCl4 + Butea monosperma (C + BM 800), CCl4 + silymarin (C + S50).

activity of ATPase enzyme and could be explained on the basis ofaction of. Emblica officinalis (Tasduq et al., 2005). This is supportedby the view that enzyme level returns to normal with the healing ofhepatic parenchyma and regeneration of hepatocytes. Histologicalobservations basically support the results obtained from biochem-ical assays. These histological appearances have nearly returned tonormal by therapy with extract at the highest dose.

Transport, conjugation and excretory ability of the liver cellswere examined by BSP retention that is an important and sensi-tive test for estimating functional integrity of liver (Chandan et al.,1991). In the present study, significant increase in BSP retentionafter CCl4 administration clearly indicates the fall or unavailabilityof microsomal drug metabolizing enzymes (MDMEs). Reduction inBSP retention by B. monosperma extract indicated improvement inthe capacity of damaged liver to perform its normal function prob-ably due to increased regenerative process in liver and antioxidantnature of BM extract. These findings are very similar to propolisextract treatment (Bhadauria et al., 2007a).

Hexobarbitone is metabolized by hepatic MDMEs and durationof hexobarbitone induced sleep in intact animals is considered asa reliable index for the activity of hepatic MDMEs. Prolongation inhexobarbitone induced sleep time after CCl4 toxicity (Singh et al.,2001) substantiated decreased availability of CYP2E1 contents. BMextract shortened this prolongation of hexobarbitone sleep timesuggesting its protective effect on CYP2E1 system. Other investiga-tors also advocated the protection against liver damage by extractof propolis (Bhadauria et al., 2007b) and Eclipta alba (Singh et al.,1993), which possess strong antioxidant potential against free rad-icals.

The current investigation verified the hepatoprotective effectsof B. monosperma against model hepatotoxicant CCl4. The hep-atoprotective action is likely related to its potent antioxidativeactivity. Neutralizing reactive oxygen species by non-enzymatic

mechanism and enhancing the activity of original natural hepaticantioxidants enzymes may be the main mechanisms against injury.These data provide a scientific explanation for the folklore usage ofB. monosperma in the treatments of hepatic disorders. The findings

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76 N. Sharma, S. Shukla / Experimental an

rovide a rationale for further studies on pharmacological evalua-ion.

cknowledgments

Authors are thankful to Jiwaji University, Gwalior (MP) andCRUM, New Delhi for financial assistance.

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