Albeena Nisar et al.

/ Journal of Pharmacy Research 2012,5(8),4454-4460

Research Article Available online through
ISSN: 0974-6943 http://jprsolutions.info
Amelioration of carbon tetrachloride induced hepatic damage
by methanolic rhizome extract of Atropa accuminata in Wistar rats
Albeena Nisar 1a, Tabish Banday2b, Akbar Masood3a, Mohammed Afzal Zargar 4a #
a
Department of Biochemistry, University of Kashmir ,Srinagar-190006, J&K , India
b
Department of Clinical Biochemistry, University of Kashmir ,Srinagar-190006, J&K , India
Received on:11-05-2012; Revised on: 17-06-2012; Accepted on:20-07-2012

ABSTRACT
Ethnopharmacological relevance: Atropa acuminata has been widely used in folk medicine for the treatment of arthritis related inflammatory disorders,
muscle and joint pain, muscle spasms . Aerial parts of this plant have been used in traditional medicine to treat innumerable ailments such as acute infections
, asthma ,conjunctivitis , encephalitis , pancreatitis , pertonitis and neuorogical disorders. Aim of the study: The aim of the present study was to evaluate
the protective effect of ethanolic extract of rhizome of A. accuminata (AAEE) against hepatic damage in CCl4 intoxicated rat liver injuries. Materials and
methods: Protective action of MEAA was evaluated in an animal model of hepatotoxicity induced by CCl4. Liver damage was induced by intraperitoneal
administration of CCl4 for 14 days and methanolic extract of MEAA at doses of 100, 200, and 400 mg/kg were administered orally once daily for 7 days.
The extract was examined using biochemical parameters like AST , ALT,ALP ,BIL etc . In addition the effects on the levels of anti-oxidant enzyme system
were also examined. Results: The substantially elevated levels of marker enzymes ALT , AST , ALP, BIL ,etc were found to significantly get restored
towards normalization by methanolic ectract of A.Accuminata . In addition , MEAA significantly prevented the marked increase in TBARS levels as well as
that of SOD , GPX ,GR and GST in liver tissue homogenates. which showed that AAEE was hepatoprotective. The hepatoprotective effect of A.acuminata
was comparable with the standard drug, Silymarin. The effect of extract at 400 mg/kg was almost equal to that of standard drug. Conclusion: Our study
demonstrated that AAEE could ameliorate CCl4 induced hepatic damage which may be attributed to the individual or combined action of phytoconstituents
present in it.

Keywords: Atropa acuminata-carbon tetrachloride- hepatoprotective-Silymarin.

1.INTRODUCTION :
Inspite of the significant advances in modern medicine, no effective drugs et al.,1987), conjunctivitis, fever ,encephalitis (inflammation of the brain)
are available that provide protection to liver against damage or stimulate ,fever (Ceha et al.,1997;Duncan .,2003) muscle and joint pain, inflamma-
functions and regeneration of hepatic cells (Chattopadhyay et al.,2003). tion, pancreatitis ,peritonitis, scarlet fever, parkinsons disease and neuoro
Treatment with interferon ,colichine, penicillanmine and corticosteriods is disorders (Kahn et al.,1991 ;King et al.,1966). The roots of the plant have
accompanied with profound side effects and is inconsistent (Luper et also been used as sedative (Rhodes ,1978) and also against sore throat ,
al.,1998) . Hepatic dysfunction due to inhalation of hepatotoxins is increas- ulcerative colitis (Shanafelt 2002), whooping cough (Walach et al.,2001) .
ing worldwide (Reddy et al., 1993 , Preussmann et al.,1978 ). Therefore Chemically AA has been found to contain tropane alkaloids. Previously
there is an urgent need to develop alternative hepatoprotective drugs which these alkaloids have been found to have anti-cholinergic and anti-spas-
produce maximal therapeutic effects without any adverse side effects in modic activity( Tyler .,1988)
clinical experience. Of late natural remedies from medicinal plants have
been considered as safe effective alternatives for hepatotoxicity (Chatterjee, 2. MATERIALS AND METHODS:
2000). Many poly herbal formulations reported to have hepatoprotective
activity are already being commercially exploited and comprise about 100 2.1 Test material
Indian medicinal plants (Handa et al.,1986). The present study was envis- Atropa accuminata (rhizome) was collected from higher reaches of Ferozpur
aged to investigate the protective effects of the methanol extract of Atropa , Drang areas of Tangmarg in district Baramulla, Kashmir in the month of
acuminata against CCl4-induced hepatotoxicity in in Wistar albino rats May-June .The plant was correctly identified and authenticated as Atropa
and to elucidate the possible mechanism of action . accuminata (Solonaceae) by Centre of Biodiversity and Toxonomy , Uni-
versity of Kashmir, Srinagar, India. A voucher specimen is retained and
Atropa accuminata belongs to the family Solonaceae and is a tall perennial deposited at Central Herbarium, Department of Botany , University of
sub-alpine plant native to Asia .It is folklore Indian medicinal plant widely Kashmir, India . The material was ensued to be free from pathogens, afla-
distributed across north western Himalayas (Dhar and Kachroo,1983 ) In toxins, pesticidal residues and heavy metals (WHO,1998).
Kashmir rhizome of A.accuminata has been traditionally used since ages for
the treatment of arthritis related inflammatory disorders, muscle and joint 2.2. Preparation of extract (MEAA)
pain, muscle spasms (Kaul,1997; Manjunath ;1948). Aerial parts of this The authentically identified plant material was shade dried and then pow-
plant have been used in traditional medicine to treat innumerable ailments as dered. Powdered plant material (1 .6 kg) was subjected to Soxhlet extraction
acute infections , anxiety ,asthma ,chicken pox.(Kumar et al.,1997;Bergmans with absolute methanol and the solvent was removed under reduced pres-
sure on a vacuum rotary evaporator to get dark brownish MEAA extract
*Corresponding author. (yield: 32.2 %). The crude extract was stored at-20°C for experimental use.
MEAA was studied at doses ranging from 100 to 400 mg/kg/ b.w. in vivo
Albeena Nisar
and the test materials for experimentation were prepared as fresh suspen-
Department of Biochemistry, sions each time using 1% sterile gum acacia.
University of Kashmir ,
Srinagar-190006, J&K , India

Journal of Pharmacy Research Vol.5 Issue 8.August 2012 4454-4460

 Albeena Nisar et al. / Journal of Pharmacy Research 2012,5(8),4454-4460
2.3 Animals : thiobarbituric acid reactive substances (TBARS) by the method of Nichans
Studies were carried out using Adult male albino rats of Wister strain weigh- and Samuelson, 1968. In brief 0.1ml of tissue homogenate (PMS; Tris- HCl
ing 200- 250g . They were procured from IIIM, Jammu and kept in animal buffer, pH 7.5) was treated with 2ml of TBA-TCA-HCl reagent in 1:1:1
house, KU. The animals were grouped in polypropylene cages with not ratio (0.37% thiobarbituric acid, 0.25N HCl, and 15% TCA), placed in
more than six animals per cage and maintained under standard laboratory boiling water bath for 15min, cooled and centrifuged at room temperature
conditions (temperature 25±2°C, relative humidity 60% ±15% and with for 10min. The absorbance of the clear supernatant was measured against
dark & light circle 12hrs /14hrs).They were allowed free access to standard reference blank at 535nm.
dry pellet (Hindustan lever, Kolkata, India) and water ad libitum (CPCSEA,
2003). The rats were acclimatized to laboratory conditions before com- 2.6.2 Determination of total sulphydryl groups:
mencement of the experiment. All procedures described were reviewed and The acid soluble sulphydryl groups (non protein thiols) of which more
approved by the University animal ethical committee than 93% is reduced glutathione (GSH) forms a yellow colored complex
with DTNB that shows the absorption maximum at 412nm. The assay
2.4 Acute Toxicity Study: procedure followed was that of Moren et al., 1979. 500µl of homogenate
Acute toxicity studies were performed using male wistar rats. The animals precipitated with 100µl of 25% TCA, was then subjected to centrifugation
were fasted overnight prior to the experiment and maintained under stan- at 300xg for 10 minutes to settle the precipitate. 100µl of the supernatant
dard laboratory conditions MEAA was administrated orally in increasing obtained was added to the test tube containing the 2ml of 0.6mM DTNB
dose up to 2000 mg/kg .No signs of mortality were observed indicating and 0.9ml of 0.2mM sodium phosphate buffer (pH 7.4). The yellow color
A.acuminata to be safe for use . obtained was measured at 412nm against the reagent blank which contains
100µl of 25% TCA in place of the supernatant. Sulphydryl content was
2.5 Induction of hepatic injury : calculated using the DTNB molar extension coefficient of 13,100.
Experimental protocol was based on previously reported studies (Naveen
et al., 2005). Rats MEAA at graded doses 100 – 200 mg/ kg body weight 2.6.3 Glutathione peroxidase (GPx):
and standard hepatoprotective drug, Silymarin were prepared in 1% CMC GPx activity was assayed using the method of Sharma et al., 2001. The
.The extract was suspended in normal saline such that the final volume of assay mixture consists of 1.49ml of sodium phosphate buffer (0.1M p.H
extract at each dose was 1ml . Animals were were grouped into 6 groups 7.4), 0.1ml EDTA (1mM), 0.1ml sodium azide (1mM), 0.1ml 1mM GSH,
each containing 7 rats . 0.1ml of NADPH (0.02mM), 0.01ml of 1mM H2O2 and 0.1ml PMS in a
total volume of 2ml. Oxidation of NADPH was recorded spectrophoto-
ØGroup 1- Served as normal control and received olive oil only (vehicle) metrically at 340nm and the enzyme activity was calculated as nmoles
5.0ml/Kg. NADPH oxidized/min/mg of protein, using € of 6.22 x 103 M-1 cm-1.
ØGroup 2- Served as negative control and received CCl4 1.0 ml/Kg body
weight (suspended in olive oil). 2.6.4 Glutathione Reductase activity (GR):
ØGroup 3-Animals were administrated with Silymarin 200 mg/Kg body GR activity was assayed by the method of Sharma et al., 2001. The assay
weight suspended in normal saline . mixture consisted of 1.6ml of sodium phosphate buffer (0.1 M p.H 7.4),
ØGroup 4- Animals received 100 mg/Kg body weight of MEAA orally for 0.1ml EDTA (1mM), 0.1ml 1mM oxidized glutathione, 0.1ml of NADPH
all fifteen days. (0.02mM), 0.01ml of 1mM H 2O2 and 0.1ml PMS in a total volume of 2ml.
ØGroup 5- Animals received 200 mg/Kg body weight MEAA orally for all The enzyme activity was measured at 340nm and was calculated as nmoles
fifteen days. of NADPH oxidized/min/mg of protein using € of 6.22 x 103 M-1 cm-1.
ØGroup 6- Animals received 400 mg/Kg body weight MEAA orally for all
fifteen days. 2.6.5 Glutathione- S- transferase (GST) activity:
GST activity was assayed using the method of (Haque et al., 2003). The
On the thirteenth day, animals from all the groups were injected intra reaction mixture consisted of 1.67ml sodium phosphate buffer (0.1 M pH
peritoneally with CCl4 in olive oil vehicle at a dosage of 1 ml/ kg body 6.5), 0.2ml of 1mM GSH, 0.025ml of 1mM CDNB and 0.1ml of PMS in a
weight. After 48 hours livers were dissected out and used for preparation of total volume of 2ml. The change in absorbance was recorded at 340nm and
post mitochondrial supernatant (PMS), biochemical studies. Liver, iso- the enzyme activity was calculated as nmoles of CDNB conjugates formed/
lated from sacrificed animals were washed in ice cold 1.15% KCl and ho- min/mg protein using € of 9.6 x 103 M-1 cm-1.
mogenized in a homogenizing buffer (50mM Tris- HCl, 1.15% KCl pH 7.4)
using Teflon homogenizer. The homogenate was centrifuged at 9,000g for 2.6.6 Catalase activity (CAT):
20 minutes to remove debris. The supernatant so obtained was further CAT activity was assayed by the method of Claiborne (1985). The assay
centrifuged at 15,000 rpm for 20minutes at 4oC to get post mitochondrial mixture consisted of 1.95ml of phosphate buffer (0.05 M pH 7), 1.0ml
supernatant (PMS). H2O2 (0.019 M), 0.05ml of hepatic PMS in a final volume of 3ml. Change in
absorbance was recorded at 240nm. CAT activity was calculated in terms
2.6 Biochemical studies : of nmoles of H2O2 consumed/min/mg of protein.
Befor sacrificing the experimental animals , blood was collected from the
retro orbital plexus without the use of anticoagulant and allowed to clot 2.6.7 Super oxide dismutase activity (SOD):
.The blood was allowed to stand for 10 min before being centrifuged at SOD activity was estimated by Beauchamp and Fridovich, 1971. The reac-
2,000 rpm for 10 min to obtain serum for analysis . Alanine aminotrans- tion mixture consisted of 0.5ml of hepatic PMS, 1ml of 50 mM sodium
ferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase carbonate, 0.4 ml of 25µM NBT and 0.2ml of 0.1mM EDTA. The reaction
(ALP), direct bilirubin (Dbil), total bilirubin (Tbil) and total protein (TP) was initiated by addition of 0.4ml of 1mM hydroxylamine- hydrochloride.
were estimated using the assay kit (Lab Care Diagnostics (India) Pvt. Ltd) The change in absorbance was recorded at 560nm. The control was simul-
according to the manufacturers instructions. For the estimation of lipid taneously run without tissue homogenate. Units of SOD activity were
peroxidative indices, glutathione reduced (GSH), glutathione reductase (GR), expressed as the amount of enzyme required to inhibit the reduction of
glutathione peroxidases (GPx), glutathione-S- transferase (GST), catalase NBT by 50%.
(CAT) and superoxide dismutase (SOD) post mitochondrial supernatant
(PMS) is used. 2.9 Statistical analysis :
The data were expressed as mean±S.E.M. Statistical differences at P < 0.05
2.6.1 Estimation of lipid peroxidation : between the groups were analyzed by one-way ANOVA followed by
Lipid peroxidation in tissues was estimated by the formation of
Journal of Pharmacy Research Vol.5 Issue 8.August 2012 4454-4460
 Albeena Nisar et al. / Journal of Pharmacy Research 2012,5(8),4454-4460
Boneferroni multiple comparison test using SPSS 15.0 software. Figure 1 demostrates the effect of MEAA on GSH levels for all experimen-
tal groups. CCl4 adminstered reduced GSH levels in liver tissue homogenates
NOTE : from 98.64 (Normal control ) to 26.14 nmoles /mg protein (CCl4 treated
vThe free radical scavenging activity of the sample was calculated for all group). Pre treatment of Silymarin ( 200 mg/ kg body weight ) for 15 days to
in vitro assays by the following formula : rats enhanced the levels to 76.14 nmoles /mg protein .MEAA at graded
doeses of 100 , 200 and 400 mg /kg b.w elevelated the levels to 48.16, 62.8
Control absorbance - Sample absorbance
× 100 and 70.24 nmoles /mg protein indicating a significant dose dependent re-
Control absorbance sponse with effects comparable to Silymarin .

vThe percent inhibition of deoxyribose oxidation which was

calculated as : Figure 1: Effect on glutathione levels (GSH)
A- B 150
% inhibition = 100 ×
A

Reduced Glutatione (GSH)

(nmoles/mg protein)
where A = Malonialdehyde production and B was Malonialdehyde
100
production produced by AAEE / standard anti oxidant (Jung, et ω
ωns
al.,2006) by Fentons reaction ωδ

ωδ
3. RESULTS 50
ω
3.1 Acute toxicity studies
MEAA did not show any sign and symptoms of toxicity and mortality up to
2000 mg/kg dose. 0

0
0

0
20
10

40

in
il)

up

ar
3.2 Effects of MEAA on hepatic markers , AST, ALT, ALP, total bilirubin

eo

gro

ym
liv

Sil
and total protein

ed
(O

at
l
ro

tre
MBER
nt

4
Co

The result of hepatoprotective effect on CCl4 intoxication are shown in L

(mg/kg)
CC

TABLE 1. In CCl4 intoxicated rats serum AST , ALT , ALP , Dbil and Tbil
were increased compared to levels in Group 1 animals (normal control) . Pre Each value represents the mean ± SEM. of 6 animals. The data were presented
treatment with MEAA dramatically decreased the levels of these parameters as means ± SEM.of six parallel measures .Statistical significance was evalu-
in a significant dose dependent manner with maximal effect for highest dose ated by one way ANOVA followed by the Bonferroni multiple comparison test
of MEAA, 400 mg/ kg body weight .The levels of AST , ALT , ALP, Dbil and to detect inter group differences. Differences were considered to be statistically
significant if P < 0.05., r; P <0 .001, as compared with normal control group.,
Tbil were reduced maximally from 287 to 112 IU/L , 215 to 68.7 IU/L, 318.8
©; P < 0.001 as compared with CCl4 group., d; P < 0.001 as compared with
to 108.2 IU/L , 1.14 to 0.62 mg/dl and 4.18 to 1.9 mg/dl respectively as Silymarin group., , ns; non significant as compared with Silymarin group.
compared to CCl4 treated animals. Total protein levels were significantly
increased at all graded does (P value < 0.001). In case of CCl4 treated animals
the levels of total protein were reduced (5.27 to 8.3 mg/dl). For Silymarin 3.4 Effect of extract on glutathione reductase (GR) :
treated group there was s similar decrease in the levels of enzymes with 95 Effect of MEAA on GR levels in all groups is as shown in Figure 2. After
IU/L (AST) , 60 IU/L (ALT ), 98.7 IU/L (ALP),0.54 mg/dl (Dbil) and 1.72 CCl4 administered group GR acfivity was lowered to 4.17 mg / g protein in
mg/dl (Tbil) . In view of these values it could be indicated that MEAA comparison to activity in normal control group (35 GSSG / minute / mg
showed protective effect on hepatic damage caused by CCl4 and recovered protein). Pretreatment of MEAA for 15 days at oral doses ranging from (100
the hepatic markers . The effects were significantly close to that of silymarin. to 400 mg/ kg body weight) enchanced the levels in dose dependent manner
3.3. Effect on glutathione levels (GSH) : . In case of animals tereated with MEAA at 400 mg/ kg body weight the
glutathione levels ( 28.2 GSSG / minute / mg protein mg/g ) were significantly
increased comparable to that of animals treated with Silymarin ( 26.8 GSSG
/ minute / mg protein ).

Table 1: Effect of Atropa accuminata methanol extract on liver functional tests in CCl4 induced hepatotoxicity in albino rats.
Dose(mg/kg) AST(IU/L) ALT(IU/L) ALP(IU/L) DBil(mg/dl) TBil(mg/dl) TP(mg/dl)

Control(Normal) - 80.46 ± 4.67 53± 3.48 102.6 ± 5.50 0.5 ± 0.08 1.6 ± 0.04 9.20 ± 0.19
CCl4 Control 0.5 ml/kg 287 ± 3.45ø 215 ± 2.68 ø 318.8 ± 4.12 ø 1.14 ± 0.04 ø 4.8 ± 0.02 ø 5.27 ± 0.09 ø§
Silymarin 25 mg/kg 95± 5.15© 60 ± 4.70 © 98.7 ± 3.74 © 0.54± 0.08 © 1.72 ± 0.04© 8.41±0.12 ø©
MEAA 100 mg/kg 190.2± 3.28 ø©§ 158 ± 4.15 ø©§ 223.7 ± 3.10 ø©§ 29.8 ± 0.05 ø©§ 3.24 ± 0.06 ø©§ 6.8 ± 0.05 ø©§
MEAA 200 mg/kg 160± 2.90ø©§ 118.7± 3.33 ø©§ 170.2± 5.46 ø©§ 0.76± 0.03 ø©§ 2.8 ±0.05 ø©§ 7.39 ± 0.06 ø©§
MEAA 400 mg/kg 112 ± 3.12ø©ns 68.7 ± 3.64 ©ns 108.2 ± 6.30 ©ns 0.62 ± 0.04 ©ns 1.9 ± 0.03 ø©? 8.3 ± 0.14 ø©ns

Each value represents the mean ± SEM. of 6 animals. The data were presented as means ±SEM.of six parallel measures .Statistical significance
was evaluated by one way ANOVA followed by the Bonferroni multiple comparison test to detect inter group differences. Differences were
considered to be statistically significant if P < 0.05., ø; P <0 .001, as compared with normal control group, ©; P < 0.001 as compared with CCl4
group., §; P < 0.001 as compared with Silymarin group., ,r; P < .05 as compared with Silymarin group., ns; non significant as compared with
Silymarin group. MEAA.,methanolic extract of Atropa accuminata.

Journal of Pharmacy Research Vol.5 Issue 8.August 2012 4454-4460

 Albeena Nisar et al. / Journal of Pharmacy Research 2012,5(8),4454-4460
Figure 2 : Effect of extract on glutathione reductase (GR) : mg protein) . This result was close to that of Silymarin group which depicts
stong protective effects on all anti-oxidant enzymes. (Figure 4)
(µgGSSG utilized/minute/mg protein)

50
Figure 4 : Effect of glutathione -S- transferase activity (GST)
40
Glutathone Reductase

20

(nmoles of CDNB conjugated/min/mg protein)

ε ns

Glutathione S-transferase Activity

30 ω

ωΦ 15
20 ωδ  ε
ω ns

10 10 ωΦδ
ω

0 ωδ
5
0

0
10

20

40

in
l)

ar
oi

ou

ym
ive

gr

Sil
Ol

ed

0
l(

at

M EAA
ro

tre
nt

4
L

0
(mg/kg)

0
Co

CC

20
10

40

in
l)

ar
oi

ou

m
e

gr
iv

y
Each value represents the mean ± SEM. of 6 animals. The data were presented

Sil
d
Ol

te
M EAA

l(

ea
ro
as means ± SEM.of six parallel measures .Statistical significance was evalu-

tr
4
(m g / k g )

nt

L
CC
Co
ated by one way ANOVA followed by the Bonferroni multiple comparison test
to detect inter group differences. Differences were considered to be statistically
significant if P < 0.05., r ; P <0 .001, as compared with normal control Each value represents the mean ± SEM. of 6 animals. The data were presented
group., ©; P < 0.001 as compared with CCl4 group., δ; P < 0.001 as compared as means ± SEM.of six parallel measures .Statistical significance was evalu-
with Silymarin group., r;P < 0.05 as compared with Silymarin group ., ns; ated by one way ANOVA followed by the Bonferroni multiple comparison test
to detect inter group differences. Differences were considered to be statistically
non significant as compared with Silymarin group.,
significant if P < 0.05., r ; P <0 .001, as compared with normal control
3.5 Effect on glutathione peroxidase (GPX) : group.,e; P < .05 as compared with normal control group ., ©; P < 0.001 as
Figure 3 demostrated effect of MEAA on GPX levels. The effect of MEAA compared with CCl4 group., ,r; P < .05 as compared with CCl4 group., δ; P <
at the doses of 100 , 200 and 400 mg per kg body weight resulted in a dose 0.001 as compared with Silymarin group., ns; non significant as compared with
dependent increase of GPX and the values were significantly comparable to Silymarin group.
that of silymarin. 3.7 Effect on SOD activity :
The activity of SOD in tissue homogenates of all experimental rats are as
Figure 3 : Effect on glutathione peroxidase (GPX) shown in Figure .5. In animals treated with vehicle , olive oil only the levels
50 of SOD were 7.24 U/mg protein. Comparatively the levels of SOD in liver
(µg GSH utilized /minute/mg protein)

sections from groups treated with Silymarin ( 100 mg / kg body weight ) and
MEAA 400 mg / kg body weight were 6.52 and 6.2 U/mg protein respec-
Gluatathione Peroxidase

40
tively. MEAA thus demonstrates a potent dose dependent pattern on en-
ω hancement of SOD levels in liver tissue .
30 ωδ
Figure 5 : Effect on SOD activity
ωδ
20
10
ωδ
10 8
Superoxide Dismutase

ωδ ε ns 
(U/mg protein)

6 ωΦ
0 ωδ
0

4
0

ωδ
20
10

40

in
p
il)

ar
ou
eo

ym
gr
liv

Sil

2
ed
(O

at
l
ro

MEAA
tre
nt

4
Co

0
CC

0
0

0
20
10

40

in
p
l)

ar
ou
oi

Each value represents the mean ± SEM. of 6 animals. The data were presented
ym
ive

gr

Sil
ed
Ol

as means ± SEM.of six parallel measures .Statistical significance was evalu-

at
l(

M EAA
ro

tre

ated by one way ANOVA followed by the Bonferroni multiple comparison test
4
nt

L
CC
Co

to detect inter group differences. Differences were considered to be statistically

Each value represents the mean ± SEM. of 6 animals. The data were presented
significant if P < 0.05.,r; P <0 .001, as compared with normal control group.,©; as means ± SEM.of six parallel measures .Statistical significance was evaluated
P < 0.001 as compared with CCl4 group., δ; P < 0.001 as compared with by one way ANOVA followed by the Bonferroni multiple comparison test to
Silymarin group. detect inter group differences. Differences were considered to be statistically
significant if P < 0.05., r ; P <0 .001, as compared with normal control
3.6 Effect of glutathione -S- transferase activity (GST) :
group.,e; P < .05 as compared with normal control group ., ©; P < 0.001 as
The GST activity in animals administered with MEAA at oral doses of 100 compared with CCl4 group., δ; P < 0.001 as compared with Silymarin group.,
, 200 and 400 mg per kg body weight increased the levels to 7.9 , 9.8 and 11.6 ,r; P < .05 as compared with Silymarin group., ns; non significant as compared
nmoles of CDNB/min/mg protein in comparison to CCl4 group (5.08 nmoles/ with Silymarin group.

Journal of Pharmacy Research Vol.5 Issue 8.August 2012 4454-4460

 Albeena Nisar et al. / Journal of Pharmacy Research 2012,5(8),4454-4460
3.8 Effect on Catalase (CAT) : 4.DISCUSSION:
The CAT activity of CCl4 treated rats was considerably low as compared to In the absence of reliable liver protective drugs in allopathic medical prac-
normal .In groups treated with AAEE , prior to CCl4 adminstration CAT tices, herbs have been reported to play a role in the management of various
activity was restored at all concentrations. Maximal CAT activity was seen liver disorders. Traditional medicine has been established over the centuries
at a dose of 400mg / kg (60.7 U/mg protein ) and was significantly compa- and for these reasons developing drugs from herbs with protective roles has
rable to that of Silymarin control (69 U/mg protein) .In case of tissues the emerged as a vital therapeutic target . In the absence of reliable liver protec-
CAT activity was lower in CCl4 treated rats but administration with MEAA tive drugs in allopathic medical practices, herbs have been reported to play a
at all tested concentrations restored its activity ( Figure 6 ). role in the management of various liver disorders. Such natural remedies have
Figure 6 : Effect on catalase (CAT) activity been reported to speed up the natural healing processes of liver by stimulat-
100 ing liver functions and in the regeneration of hepatic cells . To the best of the
authors knowledge no systematic study has been carried out yet on the
80 protective efficacy of methanolic extract of rhizome of Atropa accuminata to
ω
(U/mg protein)

ω n s treat hepatic dysfunction, herein we report A.accuminata to have potent

60 ωδ
Catalase

ωδ protective effects against CCl4 induced rat liver injuries.

ωδ

40
CCl4 produces an experimental damage that historically resembles very closely
to viral hepatitis (James et al., 1976, Johnson et al., 1998) . It is well accepted
20
that tissue toxicity caused by CCl4 is due to trichloro methyl radical (.CCl3)
0
which is produced by reductive metabolism of hepatic cytochrome p450 this
free radical in turn reacts with radicals and targets biological components
0

0
0

20

40
10

in

such as membrane lipids , protein and nucleic acids (Nomura et al.,1999,H

p
l)

ar
ou
oi

ym
ive

gr

De Groot et al., 1989). .CCl3 .reacts with molecular oxygen to produce

Sil
ed
Ol

at
l(

M EAA
Cl3OO- which inturn causes peroxidation of PUFA in membrane lipids lead-
ro

tre
nt

4
L
CC
Co

Each value represents the mean ± SEM. of 6 animals. The data were presented
ing to necrosis of hepatocytes (Brattin et al.,1985, Shi GF et al., 2005) .
as means ± SEM.of six parallel measures .Statistical significance was evalu- Immediately after CCl4 administration toxicity begins with changes in ER
ated by one way ANOVA followed by the Bonferroni multiple comparison test resulting in loss of metabolic enzymes localized in intracellular structures
to detect inter group differences. Differences were considered to be statistically (Recknagel et al., 1989). High exposure to CCl4 can result in kidney, lung,
significant if P < 0.05.,r; P <0 .001, as compared with normal control group., liver and CNS damage. It has been reported that liver being the primary site
©; P < 0.001 as compared with CCl4 group., δ; P < 0.001 as compared with of metabolism in the body , it is most sensitive to CCl4 induced damage
Silymarin group., , ns; non significant as compared with Silymarin group. (Sakata et al., 1987). Much of the hepatic injury is attributed to free radical
3.9 Effect on Lipid Peroxidation (LPO): generation in hepatic microsomes and is characterized by leakage of cell
From our results ( Figure ) it was found that MEAA could significantly enzymes into the blood stream and by necrosis and fibrosis (Meyer et al.,
decrease the formation of MDA. In CCl4 treated rats after CCl4 administra- 2001). Various in vitro and in vivo studies demonstrate that CCl4 reduces
tion the liver MDA levels significantly increased from 3.12 (normal control ) kidney and liver lung microsomal NADPH cytochrome p450 and reduced /
to 13.64 nmol/mg protein. Oral administration of MEAA at graded doses oxidized glutathione ratio,GSH / GSSG (Rungby et al.,1992) .
100, 200 and 400 mg / kg b.w decreased the levels in dose depenedant
manner. In case of Silymarin group MDA levels were dound to decrease to It has been documented that several herbal extracts could protect organs
4.07 nmoles/mg protein. This depicted significant inhibitory effect of MEAA against CCl4 oxidative stress by upregulation of anti-oxidant enzymes such
on LPO under in vivo conditions (Figure 7). as SOD, CAT ,GST as well as decreased levels of reduced glutathione (GSH)
(Rajesh et al., 2004).
Figure 7 : Effect of Atropa accuminata methanolic extract and silymarin
on liver homogenate lipid peroxidation of CCl 4 -treated rats in vivo.
20 Lipidperoxide degradation of biomembranes is hence one of the principle
causes of CCl4 induced hepatotoxicity (Kaplowitz,et al., 1986). MDA is
15
ω known to be one of the main end products of lipid peroxidation and oxidative
(nmol/mg protein)

reaeactions and involved in the pathogenesis of some diseases (Kurata et al.,

MDA level

ωδ 1993, Hagighara et al., 1984 ). Peroxidation of lipids results in dramatic

10
change in properties of membrane and disease ensues (Aleynk et al., 1997).En-
ωΦ
hanced lipid peroxidation which is expressed as TBARS indicates the extent
 ns 
5 of membrane damage and change in structure and function of cell membranes
(Halliwell et al., 1995). In groups treated with CCl4 alone , levels of LPO
0 increased. Increase in MDA levels suggests enhanced membreane damage and
failure of anti oxidant defence system to inhibit free radical generation. Inter-
0
0

0
20
10

40

in
l)

ar
oi

ou

estingly pre treatment with MEAA and silymarin significantly reveals the
m
e

gr
iv

y
Sil
d
Ol

te

decreased changes in LPO levels. The effect was significantly dose depen-
l(

M EAA
ea
ro

tr
4
nt

(m g / k g ) dent and comparable to known standard , silymarin.

CC
Co

Each value represents the mean ± SEM. of 6 animals. The data were presented
as means ± SEM.of six parallel measures .Statistical significance was evalu-
ated by one way ANOVA followed by the Bonferroni multiple comparison test to Elevated levels of serum exzymes AST, ALT which are cytoplasmic in loca-
detect inter group differences. Differences were considered to be statistically tion and released into circulation after cellular damages are good indicators of
significant if P < 0.05., r; P <0 .001, as compared with normal control group., cellular leakage and loss of functional integrity of cell memebrane in liver
©; P < 0.001 as compared with CCl4 group., δ; P < 0.001 as compared with (Drotman et al.,1978) . Treatment with MEAA significantly decreased the
Silymarin group., r; P < .05 as compared with Silymarin group., ns; non serum levels of ALT & AST towards the normal values. This suggests stabi-
significant as compared with Silymarin group.
Journal of Pharmacy Research Vol.5 Issue 8.August 2012 4454-4460
 Albeena Nisar et al. / Journal of Pharmacy Research 2012,5(8),4454-4460
lization of plasma membrane and repair of hepatic tissue damage initiated by (Ketterer et al., 1967).A significant dose dependent increase was observed
CCl4 by MEAA. This effect is in agreement with the commonly accepted for all treatment groups of MEAA. ,
view that the serum levels of transaminases return to normal with healing of
parenchyma regeneration of hepatocytes .Also ALP levels were significantly Glutathione is considered to be the master antioxidant of the body and is
downregulated along with bilirubin levels after pretreatment with MEAA.. found in almost all living cells .It removes free radical species such H2O2 and
ALP is an indicator of pathologic alterations in biliary flow (Plao et al., SO2 and also maintains membrane proteins thiols. In addition it is a susbtrate
1989). MEAA. induced suppression of increased serum ALP along with for GPx and decreased levels of GSH are associated with enhanced LPO in
depletion of serum bilirubin. This indicates the ability of extracts to stabilize H2O2 treated animals ( Kidd, 1997). Pretreatment of MEAA. significantly
biliary dysfunction caused by CCl4. Hence the administration of MEAA. increased levels of glutathione in a dose dependent manner.
revealed potent hepatoprotective activities of MEAA. against toxic effect of
CCl4.There findings on biochemical parameters were further supported by Overall the study suggests that MEAA. extract has significant potency to
histopathologic studies. protect kidney and lung tisues against oxidative damages and hence could be
used as an effective protection against CCl4 induced kidney and lung dam-
Administration of MEAA. was found to cause a significant increase in activties ages. Further investigations are in process to fully characterize the active
of th anti-oxidant enzymes. Reduced GSH levels were important in several principles present in the plant responsible for these actions.
defence processes against oxidative damage. It protects the cells against free
radicals peroxides and other toxic compounds (Seis et al., 1999) .It is well REFRENCES:
known that deficiency of GSH within the living organism can lead to tissue 1. Aleynik SI, Leo MA, Ma X, Aleynik MK and Lieber CS.
disorder and injury (Limon et al., 2007). The maintenance of cellular GSH Polyenylphosphatidylcholine prevents carbon tetrachloride-in-
levels is dependent activities of GR and NADH (Meister et al., 1983). CCl4 duced lipid peroxidation while it attenuates liver fibrosis. J Hepatol.
induces a significant reduction in GSH / GSSG and also a decrease in GR 27,1997 , 554-61.
activity in comparison to normal. Pretreatment with MEAA. demonstrated 2. Bandyopadhyay U, Das D and Banerjee ,RK. Reactive oxygen
it to be capable of restricting the GSH / GSSG ratio and also the GR activity species-oxidative damage and pathogenesis Current Science. 77,
to normal. This anti-oxidant effect is likely to be mediated by upregulation 1999, 658-666.
of SOD ,GPX and GST activities in rat tissues. GPX, GR, GST , SOD and 3. Beauchamp C, Fridovich I . Superoxide dismutase: Improved as-
CAT all connect a system of defence against ROS (Bandhopadhya et al., says and an assay applicable to acrylamide gel. Analytical Bio-
1999). Treatment with MEAA. significantly restricted the CCl4 induced chemistry 44, 1971,276-287.
decreased levels of SOD GPX and GST activities in kidney and lung tissues. 4. Bergmans M, Merkus J, Corbey R. Effect of Bellergal Retard on
These enzymes are the major free radical scavenging enzymes and are re- climacteric complaints: a double-blind, placebo-controlled study.
duced in several pathologic conditions (Cohen et al., 1974). Activation of Maturitas, 9, 1987,227-234.
such enzymes by treatment with MEAA. signifies the hepatoprotective 5. Brattin WJ, Glende Jr, EA. Recknagel, RO, 1985. Pathological
effects are mediated by free radical scavenging activities. These results clearly mechanisms in carbon tetrachloride hepatotoxicity. Journal of Free
demonstrated that MEAA.could exert a beneficial action against pathological Radical Biology in Medicine 1, 27–38.
alterations caused by the presence of SO2, H2O2 and OH radicals. 6. Ceha, LJ, Presperin C, Young E. Anticholinergic toxicity from
nightshade berry poisoning responsive to physostigmine. The Jour-
nal of Emergency Medicine 15, 1997, 65-69.
SOD removes superoxide (O2) by converting it to H2O2, which can be rap-
7. Chatterjee, TK. Medicinal Plants with Hepatoprotective Proper-
idly converted to water by CAT and GPx (Halliwell and Gutteridge, et al
ties in Herbal Opinions, vol. III. Books and Allied (P) Ltd., Calcutta,
1992). Catalase localized in peroxisomes is responsible for decomposition of 2000,135.
H2O2 and protects tissues from highly reactive OH radicals . It could be 8. Chattopadhyay RR.. Possible mechanism of hepatoprotective ac-
speculated that MEAA. may ameliorate the levels of H2 O2 and SO2 thereby tivity of Azadirachta indica leaf extract. Part II. Journal of
consequently restoring CAT levels in a dose dependent manner. Ethnopharmacology 89, 2003 ,217–219.
9. Claiborne A . Catalase activity. In: Greenwald, R. A. (Ed), CRC
GPx catalyzes the reaction of hydroperoxides with reduced glutathione to Handbook of methods of oxygen radicals research. CRC Press,
form glutathione disulphide (GSSG) and the reduction product of the hydro- Boca Raton, FL, 1985,283-284.
peroxides. GPx activity was significantly decreased in CCl4 receiving group 10. Cohen G, Heikkila RE. The generation of hydrogen peroxide, su-
as compared to normal control. Groups fed with MEAA. showed increased peroxide radical, and hydroxyl radical by 6-hydroxydopamine,
levels of GPx in liver in a dose dependent manner and it is probably due to dialuric acid, and related cytotoxic agents. J Biol Chem ,249, 1974,
strengthening of antioxidant activity by scavenging the endogenous meta- 2447-2452.
11. CPCSEA, Government of India Committee for the purpose of
bolic peroxides generated by CCl4.
control and supervision of experiments on animals (CPCSEA)
guidelines for laboratory animal facility. Indian Journal of Pharma-
Glutathione reductase is concerned with the maintenance of cellular level of cology, 35 ,2003, 257–274.
GSH especially in the reduced state by effecting fast reduction of oxidized 12. Dhar U, Kachroo. Alpine flora of Kashmir Himalayas. Scientific
glutathione to reduced form. The activity of GR may reflect the physiologi- Publishers, Jodhpur. 1983
cal needs of the cell (Dunbar et al., 1984). Administration of methanolic 13. Drotman, RB, Lawhorn, GT. Serum enzymes are indicators of
extract of MEAA. restored the GR activity in a dose dependent manner dose chemical induced liver damage. Drug and Chemical Toxicology 1,
dependently and comparable to standard , Silymarin . GST is known to play 1978,163-71.
an essential role in liver as it is involved in elimination of toxic compounds 14. Dunbar JR, Lucia De and Bryant LR. Glutathione status of iso-
by conjugation with glutathione. Substrates for GST including carcinogens lated rabbit lungs. Effects of nitrofurantoin and paraquat perfusion
and their metabolites ,pesticides ,leukotrienes and cancer chemotherapeutic with normoxic and hyperoxic ventilation, Biochem. Pharmacol.
agents (Singh et al., 1991). It has been reported that GST provides protec- 33, 1984,1343.
15. Duncan G, Collison DJ,.Role of the non-neuronal cholinergic sys-
tion to the tissues by removal such compounds through “suicidal” binding

Journal of Pharmacy Research Vol.5 Issue 8.August 2012 4454-4460

 Albeena Nisar et al. / Journal of Pharmacy Research 2012,5(8),4454-4460
tem in the eye: a review. Life Sciences 72, 2003,2013-2019. 37. Moren MA, Depierre JW and Mannervick, B . Levels of glu-
16. Groot H. De and Sies H. Cytochrome P-450, reductive metabo- tathione, glutathione reductase and glutathione S- transferase ac-
lism, and cell injury, Drug Metabolism Reviews,20, 1989, 2–4 tivities in rat lung and liver. Biochem Biophys Acta, 582, 1979,67-
17. Hagihara M, Nishigaki I, Maseki M, Yagi K . Age dependent changes 78.
in lipid peroxide levels in the lipoprotein fractions of human se- 38. Naveen T, Sangeeta, P, Anurag K., et al.. Hesperidin, a citrus biofla-
rum. J. Gerontol, 39, 1984,269-272. vonoid, decreases the oxidative stress produced by carbon tetra-
18. Halliwell B, Gutteridge JMC and Cross CE . Free radicals, antioxi- chloride in rat liver and kidney. BMC Pharmacology 5, 2005, 2
dants and human diseases: where are we now? Journal of labora- 39. Nichans, WG and Samuelson D. Formation of malondialdehyde
tory and clinical medicine. 119, 1992, 598-620. from phospholipid arachidonate during microsomal lipid
19. Halliwell B, Aeschbach R, Löliger J, Aruoma OI. The character- peroxidation. Eur J Biochem 6, 1968,126.
ization of antioxidants. Food Chem Toxicol. ,33,1995 601-17. 40. Nomura TN, Yamaoka K . Low-dose ?RAY irradiation
20. Handa SS, Sharma A, Chakraborti KK Natural products and plants reducesoxidative damage induced by CCl4 in mouse liver. Free
as liver protecting drugs. Filoterapia ,1986,57, 307-45 Radic. Biol. Med, 27, 1999,1324-1333.
21. Haque R, Bin-Hafeez B, Parvez S, Pandey S, Sayeed I, Ali M and 41. Ozbek HS, Ugras I, Bayram IU and Erdogan E. Hepatoprotective
Raisuddin S . Aqueous extract of walnut (Juglans regia L.) protects effect Foeniculum vulgare essential oil: A carbon tetrachloride in-
mice against cyclophosphamide induced biochemical toxicity, Hum. duced liver fibrosis model in rats. Scand. J. Anim. Sci, 31,2004, 9-
Exp. Toxicol. 22, 2003, 473-480 17.
22. James, GWL, Pickering RW. The protective effect of a novel com- 42. Ploa GL, Hewitt In: W.R., Wallace Hyes, A., Editor, Principle and
pound RU-18492 on galactosamine induced hepaptotoxicity in Methods of Toxicology vol. II, Raven Press, New York. 1989,pp.
rats, Drug Research 26, 1976,2197–2199. 399.
23. Johnson D E and Kroening CMechanism of early Carbon tetra- 43. Preussmann R; Hepato carcinogens as potential risk for human
chloride toxicity in cultured rat hepatocytes, Pharmacol Toxicol,83, liver cancer. In: Remmer H, Bolt HM, Bannasch P, eds. Primary
1998,231-9 17. liver tumors. Lancaster, MTP press, 1978;11-29.
24. Kahn A, Rebuffat, E, Sottiaux M..Prevention of airway obstruc- 44. Rajesh MG, Latha MS: Protective activity of Glycyrrhiza glabra
tions during sleep in infants with breath- holding spells by means Linn. On Carbon tetrachloride-induced peroxidative damage. In-
of oral belladonna: a prospective double-blind crossover evalua- dian J Pharmacol, 36, 2004, 284-287.
tion. Sleep 14, 1991432-438. 45. Recknagel, RO, Glender, EA, Walter, RL. Mechanism of
25. Kaplowitz N, Aw TY, Simon FR and Stolz A.. Drug induced hepa- Carbontetrachloride toxicity. Pharmal ther., 43,1989,139-54.
totoxicity. Ann Intern Med. 104, 1986, 826–839. 46. Reddy P B, Reddy C K, Rambhau D et al. Indian J Pharm Sci 1993;
26. Kaul,M.K.,. Medicinal plants of Kashmir and Ladakh. Indus pub- 55:134.
lications, New Delhi. 1997 47. Rhodes JB, Abrams JH, Manning RT. Controlled clinical trial of
27. Ketterer B, Ross-Mansell P and Whitehead JK . The isolation of sedative-anticholinergic drugs in patients with the irritable bowel
carcinogen-binding protein from livers of rats given 4- syndrome. Journal of Clinical Pharmacology 18, 1978,340-345.
dimethylaminoazobenzene, Biochem. J., 103, 1967, 316. 48. Rungby J, Ernst E, Experimentally induced lipid peroxidation af-
28. Kidd PM . Glutathione: systemic protectant against oxidative and ter exposure to chromium, mercury or silver. Interactions with
free radical damage. Altern Med Rev, 1, 1997,155–76. carbon tetrachloride, Pharmacol. Toxicol. 70 ,1992, 205–207.
29. King JC.. Anisotropine methylbromide for relief of gastrointesti- 49. Sakata T, Watanabe N, Hobara N, and Nagashima H.. Chronic liver
nal spasm: double- blind crossover comparison study with bella- injury in rats by carbon tetrachloride inhalation. Bull. Environ.
donna alkaloids and phenobarbital. Current Ther Res Clin Exp 8, Contam. Toxicol, 38,1987, 959-961.
1966, 535-541. 50. Shanafelt, TD, Barton DL, Adjei AA, 2002. Pathophysiology and
30. Kumar S, Shukla YN, Lavania UC, Sharma A, Singh AK,.Medicinal treatment of hot flashes. Mayo Clin Proc 77, 1207-1218.
and Aromatic Plants: Prospects for India. Journal of Medicinal 51. Sharma N, Trikha P, Athar M and Raisuddin S . Inhibition of
and Aromatic Plants . 19 , 1997,361-365. benzo [a] pyrene and cyclophosphamide induced mutagenicity by
31. Kurata M, Suzuki M, Agar NS . Antioxidant systems and eryth- cinnamomum cassia, Mutat. Res., 480-481, 2001,179-188.
rocyte life-span in mammals. Comp. Biochem. Physiol. B., 106, 52. Shi G F, Q Li .Effects of oxymetrine on experimental hepatic
1993,477-487. fibrosis and its mechanism in-vivo. World J Gastroenterol ,11,
32. Limon-Pacheco JH, Hernandez NA, Fanjul-Moles ML, Gonsebatt 2005, 268-271.
ME: Glutathione depletion activates mitogen activated protein 53. Sies H. Glutathione and its role in cellular functions. Free Radic
kinase (MAPK) pathways that display organ-specific responses Biol Med 1999, 27:916-921
and brain protection in mice. Free Radic Biol Med, 43,2007,1335- 54. Singh SV, Roberts B, Gudi VA, Ruiz P and Awasthi YC . Immuno
1347. histochemical localization, purification, and characterization of
33. Luper S. A review of plants used in the treatment of liver disease: human urinary bladder glutathione S-transferases, Biochim.
part 1. Altern Med Rev. 3, 1998,410-21. Biophys. Acta, 1074, 1991,363.
34. Manjunath BL. The Wealth of India, Council of Scientific and 55. Tyler VE, Brady LR, Robbers JE . Pharmacognosy, 9th ed. Lea and
Industrial Research, Delhi 1, 1948, 42 Febiger , Philadelphia. 1988
35. Meister A, Anderson ME. Glutathione. Annu Rev 56. Walach H, Koster H, Hennig T .The effects of homeopathic bella-
Biochem. 52,1983,711-60. donna 30CH in healthy volunteers — a randomized, double-blind
36. Meyer SA, Kulkarni AP . Hepatotoxicity. In: Introduction to bio- experiment. J Psychosom Res 50, 2001, 155-160.
chemical toxicology. 3 rd Edn. Newyork: John Wiley & Sons:
2001,p. 487.

Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.5 Issue 8.August 2012 4454-4460