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NSHM Journal of Pharmacy and Healthcare Management
Vol. 03, February (2012) pp. 57-65
57
Hepatoprotective activity of ethanolic extract of Aerva sanguinolenta (Amaranthaceae)
against paracetamol induced liver toxicity on Wistar Rats
Asif Lalee, Bolay Bhattacharaya, Mousumi Das, Debmalya Mitra, Sudipta Kaity, Samit Bera,
Amalesh Samanta*
Division of Microbiology, Department of Pharmaceutical Technology, Jadavpur University,
Kolkata-700032, India.
*Corresponding author: asamanta61@yahoo.co.in
ABSTRACT The aim of this study is to investigate the Hepatoprotective effects of
ethanolic extract of Aerva sanguinolenta (Family: Amaranthaceae) by oral route to
adult male Wistar albino rats weighing 160-180gm.The protocol started with oral
feeding of 200 and 400mg/kg body weight of extract and 25mg/kg body weight of
Silymarin. After treatment of sixteen consecutive days and after 24-hours of last dose
and 18-hours fasting, all animals in each group were sacrificed by cervical
dislocation. The blood and liver were collected for biochemical estimation and
histopathological observation. From the result it was found out that the ethanolic
extract of the plant has hepatoprotective activity and that is comparable to that of
Silymarin. Here hepatoprotective activity of ethanolic extract of Aerva sanguinolenta
leaves may be due to the presence of polyphenolic compounds. Besides Aerva
sanguinolenta contains flavonoid and tannin which are also known as natural
antioxidants due to their electron donating property which either scavenge the
principal propagating radicals or halt the radical chain.
Key words: Aerva sanguinolenta; silymarin; hepatoprotective; antioxidants
Introduction
The plant Aerva sanguinolenta (family-
Amaranthaceae) is a perennial herb. The
plant is available in tropical countries of Asia
such as Bhutan, India, Nepal, Pakistan and
also in China, Malaysia and Indo-China
regions. In China it is known as Bai-hua-mi,
in Maharashtra as Burval, in Uttarakhand as
Sufedphulia and in Assam as Soru-araksan.
In folklore medication leaf and flower of the
plant were used as wound healing and anti
inflammatory for injuries from falls,
rheumatic arthritis and pain in muscles [1],
the whole plant was used as diuretic and
demulcent [2], tender shoot of the plant used
as decoction form for galactogue to nursing
mother [3] and decoction of whole plant was
taken twice a day to expel intestinal worms
[4]. Leaves and root of the plant have been
used traditionally for body pain and the
paste of leaf and root is applied to affected
area [5].The plant extract showed significant
wound healing property [6].
Liver is one of the largest organs in
human body and the chief site for intense
metabolism and excretion. So it has a
surprising role in the maintenance,
performance and regulating homeostasis of
the body. It is involved with almost all the
biochemical pathways to growth, fight
against disease, nutrient supply, energy
provision and reproduction [7].
Liver diseases are some of the fatal
disease in the world today. They pose a
serious challenge to international public
health. Modern medicines have little to offer
for alleviation of hepatic diseases and it is
chiefly the plant based preparations which
are employed for their treatment of liver
disorders. But there is not much drug
available for the treatment of liver disorders
[8, 9].
The experimental intoxication induced
by Paracetamol (640mg/kg body wt.) is
NSHM J Pharm Healthcare Manage 03 (2012) 57-65
58
widely used for modeling liver injury in rats.
Hepatotoxicity is connected with severe
impairment of cell protection mechanisms.
The location of liver injury is defined mainly
by the biotransformation of paracetamol,
which is cytochrome P-450 dependent. Free
radicals initiate the process of lipid
peroxidation, which is generally caused by
inhibition of enzyme activity. Damage to the
liver is not due to the drug itself but to a
toxic metabolite (N-acetyl-p-benzoquinone
imine NAPQI) which is produced by
cytochrome P450 enzymes in the liver. In
normal circumstances this metabolite is
detoxified by conjugating with glutathione in
phase 2 reaction. In overdose large amount
of NAPQI [10], is generated which overwhelm
the detoxification process and lead to
damage to liver cells.
Liver diseases remain one of the serious
health problems [11]. However, we do not
have satisfactory liver protective drugs in
allopathic medical practices for serious liver
disorders. Herbal drugs play a role in the
management of various liver disorders most
of them speed up the natural healing
processes of liver. Numerous medicinal
plants and their formulations are used for
liver disorder in ethno medical practices as
well as traditional system of medicine in
India. More than 15 of these plants were
evaluated for their hepatoprotective action
in light of modern medicine.
Realizing the fact, the present
investigation was carried out to evaluate the
hepatoprotective activity of ethanolic extract
of Aerva sanguinolenta leaves in
paracetamol induced liver damage in rats.
A number of pharmacological and
chemical agents act as hepatotoxin and
produce variety of liver ailments. Carbon
tetrachloride (CCl4) [12], Paracetamol are
amongst them. Here Paracetamol
intoxicated rat liver is used as model.
Experimental Design
Animals
Adult male Wistar albino rats weighing 160-
180g were used for the present
investigation. They were housed in clean
polypropylene cages and fed with standard
pellet diet (Hindustan Lever, Kolkata, India)
and water ad libitum. The animals were
acclimatized to laboratory condition for one
week prior to experiment. The animals were
housed and used accordance with the
guidelines of the Institutional Animal Ethical
Committee. Vide Reg. No.
{(0367/01/C/CPCSEA) India}. All proce-
dures described were reviewed and
approved by the university animal ethical
committee.
Treatment protocol
Male Wistar albino rats (160-180 gm) were
divided into five groups (n=6). Group I
served as saline control (0.9% w/v sodium
chloride, orally). Group II-V received
Paracetamol suspension [13], (640mg/kg
suspended in 1% methyl cellulose; orally)
once and group II served as paracetamol
control. After administration of paracetamol
suspension, group III and IV received
ethanolic extract of Aerva sanguinolenta
200 and 400 (mg/kg body wt. orally)
respectively daily for 16 days. Group V
received standard drug Silymarin (25 mg/kg
body wt. orally) daily for 16 days [14]. After
24-h of last dose and 18-h of fasting all
animals were sacrificed by cervical
dislocation. The blood and liver were
collected for biochemical estimation and
histopathological observation.
Biochemical Estimation
Serum glutamine oxaloacetate trans-
aminase (SGOT), serum glutamine pyruvate
transaminase (SGPT), serum alkaline phos-
phatase (ALP) and total bilirubin content
were estimated by using commercially
available kits (Span Diagnostic Ltd., Surat,
India). Serum total protein (TP) is estimated
according to the method of Lowry et al. [15].
Serum Glutamate – Oxaloacetate
Transaminase (SGOT)
The method used for estimation of SGOT is
[16], which is a colorimetric method.
NSHM J Pharm Healthcare Manage 03 (2012) 57-65
59
GOT catalyses the reaction:
α-Ketoglutaric acid + aspartic acid ←→
Oxaloacetic acid + Glutamic acid
SGOT (AST) catalyzes the transfer of
amino group from aspartic acid to 2-
oxoglutarate to form oxaloacetate and L-
glutamate. The oxaloacetic acid formed in
the above reaction is decarboxylated
spontaneously to pyruvic acid which is
measured by rection with 2,4- dinitrophenyl
hydrazine to form corresponding
hydrazone,a brownish red colour complex in
alkaline medium.
Serum glutamate pyruvate transaminase
(SGPT)
The colorimetric method is used for
estimation of SGPT according to Reitman et
al. [16].
SGPT catalyses the following reaction:
α-Ketoglutaric acid + Alanine ↔ Pyruvic
acid + Glutamic acid
The pyruvic acid produced by SGPT reacts
with 2, 4-Dinitrophenyl hydrazine solution in
alkaline medium which is measured at 520
nm.
Serum alkaline phosphatase (SALP)
Paranitrophenyl phosphate used as the
substrate for the determination of alkaline
phosphatase activity. The enzymatic
product liberated is paranitrophenol which,
in the presence of sodium hydroxide forms
a yellow anion. Estimation of SALP is done
by using colorimetric method as per the
scientist Kind et al. [17].
Alkaline phosphatase from serum converts
phenyl phosphate to inorganic phosphate
and phenol at pH-10. Phenol so formed
reacts in an alkaline medium with 4-amino
antipyrine in presence of oxidizing agent
potassium ferricyanide and form an orange
red colour complex, which can be measured
colourimetrically at 510 nm.
Reaction can be presented as:
Serum Bilirubin
Total serum bilirubin consists of conjugated
(mostly with glucuronic acid) and free form.
Conjugated and total serum bilirubin
concentration was measured. The
unconjugated bilirubin content was
determined by subtracting conjugated
bilirubin from total bilirubin. In this method,
serum is diluted with water and methanol
added in an amount insufficient to
precipitate the proteins, yet sufficient to
ensure that all the bilirubin reacts with the
diazo reagent.
The method used for estimation of SGPT is
“Malloy and Evelyn method” [18].
Direct: Bilirubin couples with diazotized
sulfanilic acid, forming azobilirubin, a red
purple colour product in acidic medium.
Indirect (Unconjugated): Bilirubin is
diazotized only in presence of its dissolving
solvent (methanol). Thus red purple
coloured azobilirubin produced in presence
of methanol originates from both direct and
indirect fraction and thus represents total
bilirubin concentration. The difference of
total and direct bilirubin gives indirect
(Unconjugated bilirubin). The intensity of red
purple colour so developed is measured
colorimetrically at 540 nm and it is
proportional to the concentration of the
appropriate fraction of bilirubin. This
reaction can be represented as:
In-vivo antioxidant activity
After sacrificing the animal, liver tissue was
collected, washed with normal saline and
soaked in filter paper. One gram of the liver
tissue was homogenized in 10 ml of 0.15M
Tris buffer PH-7 to7.4 and centrifuged at
3000rpm at 4°C for 30 minutes. Supernatant
was collected and following biochemical
assays were performed using the following
formula:
• Lipid Peroxidation [19]
• Reduced Glutathione (GSH) [20]
• Catalase (CAT) [21]
NSHM J Pharm Healthcare Manage 03 (2012) 57-65
60
Lipid Peroxidation (LPO)
Lipid peroxidation (LPO) was assayed
according to the method of [19]. 1ml of
supernatant (liver tissue homogenate), 1ml
of normal saline and 2ml of 10% TCA were
mixed thoroughly and centrifuged at
3000rpm for 10 minutes at room
temperature. 2ml of supernatant was taken,
mixed with 5 ml of 0.1% TBA and heated at
95°C for 60 minutes, after appearance of
pink color the OD of the samples was
measured at 532nm using Beckman DU 64
spectrophotometer. The level of lipid
peroxides were expressed as nM of
MDA/mg wet tissue using extinction co-
efficient of 1.56x105 M-1cm-1.
Reduced Glutathione (GSH)
0.1ml of supernatant (liver tissue
homogenate) with 2.4ml of 0.02M EDTA
solution was mixed and kept in ice bath for
10 minutes. Then 2ml of distilled water and
0.5 ml of 50%TCA were added and kept in
ice bath for another 10 minutes and then
centrifuged at 3000rpm for 15 minutes. 1ml
of supernatant was mixed with 2ml of Tris
buffer and 0.05ml of DTNB solution
(Ellman’s reagent). OD of the sample was
measured at 412nm in spectrophotometer
against the blank. Appropriate standards
were run simultaneously.
Catalase (CAT)
Catalase activity was measured based on
the ability of the enzyme to break down
H2O2. 10μl of supernatant (liver tissue
homogenate) was mixed with 3ml of H2O2 in
Phosphate buffer. Time required for 0.05
optical density changes was observed at
240nm against a blank containing the
enzyme source in H2O2 free phosphate
buffer (0.16ml H2O2 is 30% w/v was diluted
to 100ml of phosphate buffer). The
absorbance was noted at 240nm and after
the addition of enzyme, ∆t was noted till
0.45. If ∆t was longer than 60seconds, the
procedure was repeated with more
concentrated enzyme sample. Reading was
taken at every 3second interval. A unit
catalase activity is the amount of enzyme
that liberates half the peroxide oxygen from
H2O2 solution of any concentration in
100seconds at 25°C.
CAT activity expressed as follows:
Moles of H2O2 consumed/min (Units/mg) =
2.3/∆t x log (E initial /E final x 1.63x10-3).
E=optical density at 240nm
2.3=factor to convert into log.
∆t=time required for a decrease in the
absorbance.
Histopathological Observation
After sacrificing the rats by cervical
dislocation, liver tissue was collected and
washed in normal saline. Then slides were
prepared to find out the histological
structure of liver tissue.
Statistical analysis
All data are expressed as mean ± S.E.M
(n=6 rats per group). Statistical significance
(P) calculated by one-way ANOVA between
the treated groups and the Paracetamol
control group followed by Dunnett’s post-
hoc test of significance where <0.05 and
<0.01 considered to be significant and
highly significant respectively.
Table 1.Effect of ethanolic extract of Aerva sanguinolenta on serum enzymes (SGOT, SGPT
and ALP), bilirubin and total protein in paracetamol-intoxicated rats.
Treatment groups
SGOT
(U/L)
SGPT
(U/L)
ALP
(U/L)
Total Bilirubin
(mg/dl)
Total protein
(mg/dl)
Normal saline control (1ml/100g)
59.41±0.77**
49.57±0.72**
28.30±0.28*
0.44±0.03*
7.51±0.55**
Paracetamol control (640mg/kg)
127.65±0.24**
131.41±0.19*
68.25±0.51*
3.12±0.04**
5.03±0.09*
Ethanolic extract (200mg/kg)
81.36±0.33**
92.09±0.57*
47.62±0.29*
1.14±0.01*
5.98±0.94**
Ethanolic extract (400mg/kg)
66.73±0.54*
60.23±73**
39.01±0.99**
0.54±0.05*
6.20±0.48**
Standard (Silymarin 25mg/kg)
61.95±0.69*
53.8±0.78*
34.94±0.06**
0.50±0.17*
7.12±0.31**
Values are mean ± S.E.M (n=6); * P < 0.001; ** P < 0.05
NSHM J Pharm Healthcare Manage 03 (2012) 57-65
61
Table 2. Effect of ethanolic extract of Aerva sanguinolenta on liver enzymes on GSH, LPO &
CAT.
Values are mean ± S.E.M (n=6); * P < 0.001, Control group compared with normal group; ** P < 0.05,
Experimental groups compared with paracetamol control groups.
Results
The effect of ethanolic extract of Aerva
sanguinolenta on biochemical parameters
such as SGOT, SGPT, ALP, total bilirubin,
and total protein is summarized in Table (1)
and the effect of the ethanolic extract of
Aerva sanguinolenta on antioxidant
enzymes such as glutathione (GSH), (CAT)
and lipid peroxidation levels are
summarized in Table (2). There was a
significant increase in SGOT, SGPT, ALP
and total bilirubin levels and decrease in
protein level in paracetamol control group
when compared with that of saline control
group. The ethanolic extract of Aerva
sanguinolenta significantly restored the all
altered biochemical parameters which are
comparable with that of standard drug
silymarin treated group.
Histopathological study of livers of
saline control group showed a normal
hepatic architecture (Fig.1). Livers
challenged with paracetamol showed
disarrangement of normal hepatic cells with
massive centrilobular necrosis,
inflammatory infiltration of lymphocytes and
fatty changes (Fig.2). Moderate protection
was observed in case of ethanolic extract
200mg/kg body weight group animals
(Fig.3).The ethanolic extract 400mg/kg body
weight treated rats exhibited significant
protection against paracetamol intoxication
as evident by presence of normal hepatic
cords and absence of necrosis with minimal
inflammatory conditions around the central
vein (Fig.4). The last figure represents the
standard group which treated with Silymarin
(Fig. 5).
Figure 1. Normal control liver Slide
Figure 2. Negative control liver Slide
Treatment groups
GSH
LPO
CAT
Normal saline control (1ml/100g)
5.91±0.57**
0.93±0.43**
342.03±0.14**
Paracetamol control (640mg/kg)
1.72±0.36*
3.03±0.88**
287.47±0.39*
Ethanolic extract (200mg/kg)
3.32±0.26*
2.03±0.75*
299.38±0.43*
Ethanolic extract (400mg/kg)
4.98±0.78**
1.35±0.41*
327.00±0.68**
Standard (Silymarin 25mg/kg)
5.03±0.55*
1.04±0.97**
333.16±0.46**
NSHM J Pharm Healthcare Manage 03 (2012) 57-65
62
Discussion and conclusion
Acetaminophen (paracetamol, N-acetyl-p-
aminophenol, 4-hydroxyacetanilide) is a
widely used analgesic and antipyretic.
Adverse effects from acetaminophen are
rare at therapeutic doses of 500-1000 mg
three to four times daily [22] in humans. Yet
even with a high margin of safety,
Acetaminophen toxicity remains the leading
cause of drug-induced liver failure in the
United States [23]. Single doses of
Acetaminophen in humans around 15 g
carry a great risk of hepatotoxicity [24],
although doses of as little as 6.2 g may
result in liver damage [25]. Overexposure to
Acetaminophen results in fulminating
centrilobular hepatic necrosis, which can
bridge to the periportal regions of the liver
lobule [26]. Acetaminophen is primarily
metabolized by conjugation with sulfate and
glucuronic acid [27], while a small percentage
of the dose undergoes bioactivation by
cytochrome P450 enzymes to the reactive
intermediate N-acetyl p-benzoquinoneimine
(NAPQI) [28]. At non-toxic doses, NAPQI is
eliminated from the liver after conjugation
with reduced glutathione (GSH) [29].
However, with toxic doses, the two main
conjugation pathways for Acetaminophen
become saturated, resulting in increased
formation of NAPQI. Consequently,
detoxification of NAPQI is compromised
when existing stores of GSH have been
depleted and NAPQI then binds to cellular
macromolecules, initiating cell death
pathways [30, 31]. Protection against
paracetamol-induced toxicity has been used
as a test for potential hepatoprotective
activity by several investigators [32, 33, 34]. An
obvious sign of hepatic injury is leakage of
cellular enzyme into plasma [35, 36, 37]. When
the liver cell plasma membrane is damaged,
a variety of enzymes normally located in the
cytosol are released into blood stream.
Their estimation in the serum is a useful
quantitative marker for the extent and type
of hepatocellular damage [38]. The ethanolic
extract of Aerva sanguinolenta. used in the
study preserved the structural integrity of
the hepatocellular membrane in a dose
dependent manner as evident from the
protection provided as compared to the
enzyme level in the hepatotoxin treated rats.
Figure 3. Treated group 200 mg/kg
Figure 4.Treated group 400 mg/kg
Figure 5. Standard control
NSHM J Pharm Healthcare Manage 03 (2012) 57-65
63
The ethanolic extract of Aerva
sanguinolenta decreased the elevated
serum enzyme levels and bilirubin level in
the paracetamol fed rats which are
comparable to that of the saline control
group. Preliminary phytochemical analysis
of ethanolic extract of Aerva sanguinolenta
indicates the presence of flavonoid and
tannin and these types of polyphenols are
well known natural antioxidants due to their
electron donating property which either
scavenge the principal propagating radicals
or halt the radical chain [39, 40]. Thus the
hepatoprotective activity of ethanolic extract
of Aerva sanguinolenta leaves may be due
to the presence of polyphenolic compounds.
However, this claim demands for further
research to isolate hepatoprotective
principle, since the present study was
preliminary investigation.
Acknowledgement
We express thankful appreciation to
“Botanical Survey of India, Shibpur, West
Bengal, India” for botanical identification
and authentication of the plant (Vide Ref.
no.-CNH/I-I/44/2009/Tech.II/124, dated 04th
November 2009).
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