Food and Chemical Toxicology 46 (2008) 34173421

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Food and Chemical Toxicology

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Hepatoprotective activity of Amaranthus spinosus in experimental animals

Hussain Zeashan a, G. Amresh a,*, Satyawan Singh b, Chandana Venkateswara Rao a
a
Pharmacognosy and Ethnopharmacology Division, National Botanical Research Institute (Council of Scientic and Industrial Research), Rana Pratap Marg, Post Box No. 436,
Lucknow 226 001, Uttar Pradesh, India
b
Department of Pharmacy, Saroj Institute of Technology and Management, Ahimamau, Lucknow, Uttar Pradesh, India

a r t i c l e i n f o a b s t r a c t

Article history: The hepatoprotective and antioxidant activity of 50% ethanolic extract of whole plant of Amaranthus
Received 10 December 2007 spinosus (ASE) was evaluated against carbon tetrachloride (CCl4) induced hepatic damage in rats. The
Accepted 14 August 2008 ASE at dose of 100, 200 and 400 mg/kg were administered orally once daily for fourteen days. The sub-
stantially elevated serum enzymatic levels of serum glutamate oxaloacetate transaminase (AST), serum
glutamate pyruvate transaminase (ALT), serum alkaline phosphatase (SALP) and total bilirubin were
Keywords: restored towards normalization signicantly by the ASE in a dose dependent manner. Higher dose exhib-
Amaranthus spinosus
ited signicant hepatoprotective activity against carbon tetrachloride induced hepatotoxicity in rats. The
Amaranthaceae
Carbon tetrachloride
biochemical observations were supplemented with histopathological examination of rat liver sections.
Hepatoprotective Meanwhile, in vivo antioxidant activities as malondialdehyde (MDA), hydroperoxides, reduced glutathi-
one (GSH), superoxide dismutase (SOD) and catalase (CAT) were also screened which were also found sig-
nicantly positive in a dose dependent manner. The results of this study strongly indicate that whole
plants of A. spinosus have potent hepatoprotective activity against carbon tetrachloride induced hepatic
damage in experimental animals. This study suggests that possible mechanism of this activity may be due
to the presence of avonoids and phenolics compound in the ASE which may be responsible to hepato-
protective activity.
2008 Elsevier Ltd. All rights reserved.

1. Introduction recommended in the Indian traditional system of medicine

Amaranthus spinosus Linn. (Family: Amaranthaceae) is com-
monly known as Kate Wali Chaulai (Kanatabhajii) in Hindi, also
used as vegetable and cultivated throughout in India, Sri Lanka and
many tropical countries (Kirtikar and Basu, 2001). The juice of A.
spinosus is used by tribal of Kerala, India to prevent swelling
around stomach while the leaves are boiled without salt and
consumed for 23 days to cure jaundice (Hema et al., 2006). Plant
as one of the vegetable have high concentration of antioxidant
components (Odhav et al., 2007; Cao et al., 1996; Gil et al., 1999;
Vinson et al., 1998) and high nutritive values due to presence of
bre, proteins and high concentration of essential amino acids,
especially lysine (Teutonico and Knorr, 1985).
The liver regulates several important metabolic functions and
the hepatic injury is associated with distortion of these metabolic
functions (Wolf, 1999). Thus, liver diseases remain one of the seri-
ous health problems. In spite of tremendous strides in the modern
medicine, there are not much drugs available for the treatment of
liver diseases. There are a number of medicinal preparations
* Corresponding author. Tel.: +91 941 5520130/522 2391902/522 2205831
35x352; fax: +91 522 2205836/39.
E-mail address: amreshgupta@gmail.com (G. Amresh).
Ayurveda for the treatment of liver diseases. There are scientic
claims to offer signicant relief as hepatoprotective (Rao et al.,
2006).
A. spinosus is also used as antiinammatory, antimalarial,
antibacterial, antimicrobial, antidiuretic, antiviral and in hepatic
disorders (Olajide et al., 2004; Stintzing et al., 2004; Van Dunen,
1985). Water extract of plant showed signicant immunostimulat-
ing activity (Lin et al., 2005) and stem extract showed antimalarial
activities (Hilou et al., 2006). A. spinosus have several active
constituents like alkaloids, avonoids, glycosides, phenolic acids,
steroids, amino acids, terpenoids, lipids, saponins, betalains,
b-sitosterol, stigmasterol, linoleic acid, rutin, catechuic tannins
and carotenoids. The betalains in stem bark of A. spinosus were
identied as amaranthine, isoamaranthine, hydroxycinnamates,
quercetin and kaempferol glycosides (Srinivasan et al., 2005;
Ibewuike et al., 1997; Rastogi and Mehrotra, 1999; Stintzing
et al., 2004; Hilou et al., 2006). It also contains amaranthoside, a
lignan glycoside, amaricin, a coumaroyl adenosine along with
stigmasterol glycoside, betaine such as glycinebetaine and trigo-
nelline (Blunden et al., 1999; Azhar-ul-Haq et al., 2006). Betalains
are well known for their antioxidant, anticancer, antiviral and
antiparasitosis properties (Kapadia et al., 1995, 1996; Patkai
et al., 1997). Many betalain containing species are used as popular
medicinal plants to treat various kinds of ailments such as hepatic

0278-6915/$ - see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2008.08.013
3418 H. Zeashan et al. / Food and Chemical Toxicology 46 (2008) 34173421
disorders, malaria, jaundice and scanty urine or to cure wounds
(Berghofer and Schoenlechner, 2002).
The whole plants of A. spinosus are used for the treatment of
jaundice in traditional system of medicine. However, there is lack
of scientic report regarding the hepatoprotective activity of A.
spinosus. The present investigation is an endeavor to validate the
scientic use of 50% ethanolic extract of whole plants of A. spinosus
(ASE), against carbon tetrachloride induced hepatic damage in
experimental animals.
2. Materials and methods
2.1. Plant collection and preparation of the extracts
Whole plant of A. spinosus Linn. (Family: Amaranthaceae) were freshly collected
in the botanical garden of National Botanical Research Institute, India in October
2006. The plant material was identied and authenticated taxonomically at Na-
tional Botanical Research Institute, Lucknow. A voucher specimen (NAB 75006) of
the collected sample was deposited in the departmental herbarium for future refer-
ence. Plant materials were washed with double distilled water to remove dirt and
shade dried. The dried materials were powdered and passed through a 10-mesh
sieve. The coarsely powdered material (1 kg) was extracted with petroleum ether
thrice to remove the fatty material and further marc was extracted thrice with eth-
anol (50%, v/v). The extracts were ltered, pooled and concentrated at reduced tem-
perature (5 C) on a rotary evaporator (Buchi, USA) and then freeze dried (Freezone
4.5, Labconco, USA) at high vacuum (133  103 mBar) and at temperature
40 2 C (yield 6.12%, w/w). Preliminary qualitative phytochemical screening of
ASE has given the positive testes for, alkaloids, glycosides, avonoids, saponins, car-
bohydrates, protein, amino acids, lipids, steroids, phenolic acid and tannins (Trease
and Evans, 1983). For the pharmacological tests the ASE was suspended in double
distilled water containing surfactant CMC (carboxymethyl cellulose, 1%, w/v).
2.2. Animals
SpragueDawley rats weighing (140 20 g) of either sex were procured from
the National Laboratory Animal Centre (NLAC), Central Drug Research Institute, Luc-
know. They were kept in departmental animal house in well cross ventilated room
at 27 2 C, and relative humidity 4456%, light and dark cycles of 10 and 14 h,
respectively, for 1 week before and during the experiments. Animals were provided
with standard rodent pellet diet (Amrut, India) and the food was withdrawn 18
24 h before the experiment though water was given ad libitum. All studies were per-
formed in accordance with the guide for the care and use of laboratory animals, as
adopted and promulgated by the Institutional Animal Care Committee, CPCSEA, In-
dia (Reg. No. 222/2000/CPCSEA). All the chemicals used were of the analytical grade
from standard companies and the water used was always the double distilled water.
A standard orogastric cannula was used for oral drug administration (self made).
2.3. CCl4 induced hepatotoxicity
The animals were divided into ve groups, each group had six animals. Group
rst served as control animals were received a single daily dose of carboxymethyl
cellulose (1 ml of 1%, w/v, p.o. body weight). Group second received carbon tetra-
chloride (1 ml/kg body weight, i.p., 1:1 v/v mixture of CCl4 and olive oil) alone while
group third, fourth and fth received orally 100, 200 and 400 mg/kg body weight of
ASE in (1%, w/v, CMC) respectively along with carbon tetrachloride as in group sec-
ond. The ASE was given daily while carbon tetrachloride was given for every 72 h
for 14 days. Animals were sacriced 48 h after the last dose of the drug. The liver
samples were dissected and blood was collected (Rao et al., 2006).
2.4. Assessment of hepatoprotective activity
The collected blood was allowed to clot and serum was separated at 2500 rpm
for 15 min and the biochemical parameters like serum enzymes: aspartate amino-
transferase (AST, U/L), serum glutamate pyruvate transaminase (ALT, U/L) (Reitman
and Frankel, 1957), serum alkaline phosphatase (ALP, IU/L) (King, 1965) and total
bilirubin (mg/dL) (Malloy and Evelyn, 1937) were assayed using assay kits.
2.5. Assessment of antioxidant parameters
2.5.1. Assessment of lipid peroxidation (LPO)
The dissected out liver samples were washed immediately with ice cold saline to
remove as much blood as possible. Liver homogenized (5%) in ice cold 0.9% NaCl with a
Potter-Elvenhjem glass homogenizer. The homogenate was centrifuged at 800g for
10 min and the supernatant was again centrifuged at 12,000g for 15 min and the ob-
tained mitochondrial fraction was used for the estimation of LPO (Das and Banerjee,
1993). A volume of the homogenate (0.20 ml) was transferred to a vial and was mixed
with 0.2 ml of a 8.1% (w/v) sodium dodecyl sulphate solution, 1.50 ml of a 20% acetic
acid solution (adjusted to pH 3.5 with NaOH) and 1.50 ml of a 0.8% (w/v) solution of
TBA and the nal volume was adjusted to 4.0 ml with distilled water. Each vial was
tightly capped and heated in a boiling water bath for 60 min. The vials were then
cooled under running water. Equal volumes of tissue blank or test samples and 10%
trichloroacetic acid were transferred into a centrifuge tube and centrifuged at
1000g for 10 min. The absorbance of the supernatant fraction was measured at
532 nm (Beckman DU 650 spectrometer). Control experiment was processed using
the same experimental procedure except the TBA solution was replaced with distilled
water (Jamall and Smith, 1985). Malonyldialdehyde (MDA) is an end product of lipid
peroxidation, which reacts with thiobarbituric acid to form pink chromogen thiobar-
bituric acid reactive substance. 1,1,3,3-Tetraethoxypropan was used as standard for
calibration of the curve and is expressed as nmole/mg protein.
A volume of the homogenate (0.1 ml) was treated with 0.9 ml of Fox reagent
(88 mg of BHT, 7.6 mg of xylenol orange, and 0.8 mg of ammonium iron sulphate
were added to 90 ml of methanol and 10 ml of 250 mM sulphuric acid) and incu-
bated at 37 C for 30 min. The color that developed was read at 560 nm (Jaing
et al., 1992). Hydroperoxides were expressed as mmol per hundred gram.
2.5.2. Assessment of catalase and superoxide
The liver tissue was homogenized (5%) and mitochondrial fraction was prepared
as described above. Decomposition of H2O2 in presence of catalase (CAT) was fol-
lowed at 240 nm (Aebi, 1984). One unit (U) of catalase was dened as the amount
of enzyme required to decompose 1 lmol of H2O2 per min, at 25 C and pH 7.0. Re-
sults are expressed as units (U) of CAT activity/mg protein. Superoxide dismutase
(SOD) activity was estimated by the inhibition of nicotinamide adenine dinucleo-
tide (reduced)-phenazine methosulphatenitrobluetetrazolium reaction system as
described by Nishikimi et al. (1972) and as adapted by Kakkar et al. (1972). One unit
of the enzyme is equivalent to 50% inhibition in the formazan formation in 1 min at
room temperature (25 2 C) and the results have been expressed as units (U) of
SOD activity/mg protein.
2.5.3. Assessment of reduced glutathione (GSH) activity
The concentration of GSH was determined by the method of Anderson (1985)
based on the development of a yellow color when 5,50 -dithiobis(2-nitrobenzoic
acid) is added to compounds containing sulfhydryl groups. The reaction mixture
contained equal volumes of 4% sulfosalicylic acid and tissue samples homogenized
in 4 vol. of ice cold 0.1 m/l phosphate buffer (pH 7.4). The method used for estimat-
ing GSH in this study also measures non-protein sulfhydryl concentration inclusive
of GSH. However, 8090% of the non-protein sulfhydryl content of the cell repre-
sents free endogenous GSH. Enzyme activity was expressed as milligram per hun-
dred gram (Amresh et al., 2007a).
2.6. Histopathological studies
For histologic studies, the liver tissues were xed with 10% phosphate buffered
neutral formalin, dehydrated in graded (50100%) alcohol and embedded in paraf-
n. Thin sections (5 lM) were cut and stained with routine hematoxylin and eosin
(H & E) stain for photo microscopic assessment. The initial examination was qual-
itative, with the purpose of determining histopathological lesions in liver tissue
(Amresh et al., 2007c).
2.7. Statistical test
All the data were presented as mean SEM and analysed by SPSS for Windows,
version 9 (SPSS, Chicago, IL, USA) for the possible signicant interrelation between
the various groups, the independent samples t-test. A value of P < 0.05 was consid-
ered statistically signicant.
3. Results
3.1. Effect of ASE on AST, ALT, ALP and total bilirubin
The effect various doses of ASE were studied on serum marker
enzymes and total bilirubin in CCl4 intoxicated animal. Hepatic in-
jury induced by CCl4 caused signicant changed in marker enzyme
as AST by 396.78%, ALT by 368.90%, ALP by 121.42% and total bili-
rubin by 355.81% compared to control group. The percentage chan-
ged in marker enzyme of treated group at 100, 200 mg/kg as AST
355.81 (P < 0.05), 303.4 (P < 0.001), ALT 327.04 (P < 0.001), 249.14
(P < 0.001), ALP 101.30 (P < 0.05), 41.48 (P < 0.001) and total biliru-
bin 310.92 (P < 0.01), 250.42 (P < 0.001) compared to CCl4 group
while maximum percentage protection in marker enzyme at the
dose of 400 mg/kg body weight as AST 168.11 (P < 0.001), ALT
11.63 (P < 0.001), ALP 5.23 (P < 0.001) and total bilirubin 164.70
(P < 0.01) (Figs. 1 and 2).
 H. Zeashan et al. / Food and Chemical Toxicology 46 (2008) 34173421 3419

500 icantly prevented this heave in levels and the percentage protec-
*** AST tion in MDA were 86.27 (P < 0.001), 69.58 (P < 0.001), 20.29
*** ALT (P < 0.01) and hydroperoxides 22.96 (P < 0.001), 15.83 (P < 0.01)
400 ALP and 12.12 (P < 0.05), respectively (Figs. 2 and 3).
*** The GSH, SOD and CAT content had signicantly increased in
300 *** ASE treated groups whereas CCl4 intoxicated group had shown
*** * signicant decrease in these parameters compared to control
* group. The percentage changed of GSH, SOD and CAT in CCl4
*** ***
200 *** intoxicated group were as 56.36 (P < 0.001), 49.04 (P < 0.001)
*** and 38.12 (P < 0.001), respectively. The percentage protection in
***
GSH as 40.71(P < 0.001), 24.42 (P < 0.01), 16.91 (P < 0.01) and
100
SOD 38.73 (P < 0.001), 26.21 (P < 0.05), 23.12 (P < 0.01) while in
CAT 37.59 (P < 0.001), 27.17 (P < 0.001), 13.85 (P < 0.01) at the
0 doses levels 100, 200 and 400 mg/kg, respectively. In different
Control Carbon tetrachloride 100mg/kg 200mg/kg 400mg/kg doses level of ASE, 400 mg/kg has shown maximum protection
(Figs. 2 and 3).
Fig. 1. Effect of 50% ethanolic extract of Amaranthus spinosus (ASE) on biochemical
parameters. 100 mg/kg, 200 mg/kg, 400 mg/kg represent the dose of ASE. Value
expressed as S.E.M., n = 6. *P < 0.05, **P < 0.01, ***P < 0.001 compared to CCl4. 3.3. Histopathological observations
Group CCl4 compared to control group. Group 100, 200 and 400 mg/kg compared to
CCl4 group. Histopathology of liver section in normal control rats showed
central vein surrounded by hepatic cord of cells while CCl4 treated
rats liver section showed patches of liver cell necrosis with inam-
3.2. Estimation of MDA, hydroperoxides, GSH, SOD and CAT matory collections around central vein. Whereas the ASE treated
groups showed absence of cell necrosis, but with minimal inam-
The results also showed clear signicant percentage protection matory conditions around the central vein. The ASE 400 mg/kg, p.o.
in the levels of MDA and hydroperoxides in CCl4 intoxicated rats as treated group showed minimal inammatory conditions with near
133.57 (P < 0.001) and 41.51 (P < 0.01) compared to control group. normal liver architecture possessing higher hepatoprotective
Treatment with ASE at the doses of 100, 200 and 400 mg/kg signif- action (Fig. 4).

8 Total bilirubin (mg/dL) 500

*** SOD (U/mg)
7 MDA (nmol/mg)
400
6 ***
*** *** **
*
5 300
** *** ***
4 **
***
.***
3 200

2
100
1

0 0
Control Carbon tetrachloride 100 mg/kg 200 mg/kg 400 mg/kg

Fig. 2. Effect of 50% ethanolic extract of Amaranthus spinosus (ASE) on total bilirubin, SOD and MDA. 100 mg/kg, 200 mg/kg, 400 mg/kg represent the dose of ASE. Value
expressed as S.E.M., n = 6. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control group.

Hydroperoxides
120
GSH
**
100 CAT
***
** *
80

60

40 ** *** ** **
*** ***
*** ***
20

0
Control Carbon tetrachloride 100 mg/kg 200 mg/kg 400 mg/kg

Fig. 3. Effect of 50% ethanolic extract of Amaranthus spinosus (ASE) on hydroperoxides, GSH and CAT. 100 mg/kg, 200 mg/kg, 400 mg/kg represent the dose of ASE. Value
expressed as S.E.M., n = 6. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control group.
3420 H. Zeashan et al. / Food and Chemical Toxicology 46 (2008) 34173421

Fig. 4. Histopathology of liver tissues. (A) Liver section of normal control rats showing: section shows central vein surrounded by hepatic cord of cells (normal architecture).
(B) Liver section of CCl4 treated rats showing: massive fatty changes, necrosis, ballooning degeneration, and broad inltration of the lymphocytes and the loss of cellular
boundaries. (C) Liver section of rats treated CCl4 and 100 mg/kg of ASE showing: inammatory collections around central vein and focal necrosis with sinusoidal dilatation. (D)
Liver section of rats treated CCl4 and 200 mg/kg of ASE showing: less inammatory cells around central vein, absence of necrosis. (E) Liver section of rats treated CCl4 and
400 mg/kg of ASE showing: minimal inammatory cellular inltration. Almost near normal liver architecture. Regeneration of hepatocytes around central vein.

4. Discussion and conclusion
In the present study, ASE was evaluated for the hepatoprotec-
tive activity using CCl4 induced hepatotoxicity in rat. CCl4 is being
used extensively to investigate hepatoprotective activity on vari-
ous experimental animals (Rao et al., 2003; Bhathal et al., 1983).
The changes associated with CCl4 induced liver damage are similar
to that of acute viral hepatitis (James and Pickering, 1976; Suja
et al., 2004), due to preferred as the experimental model.
The ability of a hepatoprotective drug to reduce the injurious
effects or to preserve the normal hepatic physiological mecha-
nisms that have been disturbed by a hepatotoxin is the index of
its protective effects (Yadav and Dixit, 2003). The hepatotoxicity
induced by CCl4 is due to its metabolite CCl3, a free radical that
alkylates cellular proteins and other macromolecules with a
simultaneous attack on polyunsaturated fatty acids, in the pres-
ence of oxygen, to produce lipid peroxides, leading to liver dam-
age (Bishayee et al., 1995). Hepatocellular necrosis leads to
elevation of the serum marker enzymes, which are released from
the liver into blood (Ashok Shenoy et al., 2001). The increased
levels of AST, ALT, ALP and serum bilirubin are conventional indi-
cators of liver injury (Achliya et al., 2004; Thabrew et al., 1987).
The present study revealed a signicant increase in the activities
of AST, ALT, ALP and serum bilirubin levels on exposure to CCl4,
indicating considerable hepatocellular injury. Administration of
ASE at different dose levels (100, 200 and 400 mg/kg) attenuated
the increased levels of the serum enzymes, produced by CCl4 and
caused a subsequent recovery towards normalization comparable
to the control groups animals. The hepatoprotective effect of the
ASE was further accomplished by the histopathological examina-
tions. ASE at different dose levels offers hepatoprotection, but
400 mg/kg is more effective than all other lower doses. It has
been hypothesized that one of the principal causes of CCl4 in-
duced liver injury is formation of lipid peroxides by free radical
derivatives of CCl4 (CCl3). Thus, the antioxidant activity or the
inhibition of the generation of free radicals is important in the
protection against CCl4 induced hepatopathy (Castro et al.,
1974). The body has an effective defense mechanism to prevent
and neutralize the free radical induced damage. This is procient
by a set of endogenous antioxidant enzymes such as SOD, and
catalase. These enzymes constitute a mutually supportive team
of defense against ROS (Amresh et al., 2007c). In CCl4 induced
hepatotoxicity, the balance between ROS production and these
antioxidant defenses may be lost, oxidative stress results, which
through a series of events deregulates the cellular functions lead-
ing to hepatic necrosis. The reduced activities of SOD and catalase
observed point out the hepatic damage in the rats administered
with CCl4 but the treated with 100, 200 and 400 mg/kg of ASE
groups showed signicant increase in the level of these enzymes,
which indicates the antioxidant activity of the A. spinosus. Regard-
ing non enzymic antioxidants, GSH is a critical determinant of tis-
sue susceptibility to oxidative damage and the depletion of
hepatic GSH has been shown to be associated with an enhanced
toxicity to chemicals, including CCl4 (Hewawasam et al., 2003).
In the present study, a decrease in hepatic tissue GSH level was
observed in the CCl4 treated groups. The increase in hepatic
GSH level in the rats treated with 100, 200 and 400 mg/kg of
ASE may be due to de novo GSH synthesis or GSH regeneration.
 H. Zeashan et al. / Food and Chemical Toxicology 46 (2008) 34173421
The level of lipid peroxide (MDA) is a measure of membrane
damage and alterations in structure and function of cellular mem-
branes. In the present study, elevation of lipid peroxidation and
hydroperoxides in the liver of rats treated with CCl4 was observed.
The increase in MDA and hydroperoxides levels in liver suggests
enhanced lipid peroxidation leading to tissue damage and failure
of antioxidant defense mechanisms to prevent the formation of
excessive free radicals (Amresh et al., 2007b). Treatment with
ASE signicantly reversed these changes. Hence, it is possible that
the mechanism of hepatoprotection of whole plant of A. spinosus
may be due to its antioxidant activity.
On phytochemical screening, ASE revealed the presence of
avonoids, phenolics, triterpenes, steroids and saponins as major
compounds. These antioxidant phytochemicals might contribute
to the hepatoprotective and antioxidant activities of the whole
plant of A. spinosus. From the results it is clear that the ASE has
shown dose dependent activity among which at the dose level of
400 mg/kg, p.o. shows greater activity which is comparable with
the control group.
Conict of interest statement
The authors declare that there are no conicts of interest.
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