Hepatoprotective and antioxidant potential of Withania somnifera against Paracetamol-induced liver damage in rats

Abstract

Objective: The aim of this study was to evaluate the hepatoprotective and antioxidant effects of Withania somnifera against Paracetamol-induced liver injury in rats. Methods: In the present study, the protective effect of Withania somnifera was investigated against Paracetamol-induced hepatotoxicity and compared with Silymarin, a standard hepatoprotective reference drug. The rats received a single dose of paracetamol (900 mg/kg body weight, i.p.); Withania somnifera (500 mg/kg body weight and 1000mg/kg body weight, p.o.) and Silymarin (25 mg/kg body weight, p.o.) were administered 30 min after the injection of paracetamol. Liver marker enzymes (Aspartate Transaminase, Alanine Transaminase and Alkaline Phosphatase), Total Protein content, Bilirubin, Antioxidant status (Reduced Glutathione, Superoxide Dismutase, Catalase and Glutathione-S-Transferase) were evaluated and histopathological analysis was done for the control and experimental rats. Results: Paracetamol treatment leads to elevated levels of liver marker enzymes and bilirubin and there was deterioration in total protein content, histological observations and antioxidant status. However, treatment with Withania somnifera significantly reversed (p < 0.05) the above changes compared to the control group as observed in the paracetamol-challenged rats. Conclusion: The results clearly demonstrate that Withania somnifera possesses promising hepatoprotective effects through its antioxidant activity and hence suggests its use as a potential therapeutic agent for protection from paracetamol overdose.

Full text

Research Article
HEPATOPROTECTIVE AND ANTIOXIDANT POTENTIAL OF WITHANIA SOMNIFERA AGAINST
PARACETAMOL-INDUCED LIVER DAMAGE IN RATS
*EVAN PRINCE SABINA, MAHABOOBKHAN RASOOL, MAHIMA VEDI, DHANALAKSHMI NAVANEETHAN,
MEENAKSHI RAVICHANDER, POORNIMA PARTHASARTHY, SARAH RACHEL THELLA
SBST, VIT University, Vellore-632014, Tamil Nadu, India. Email: eps674@gmail.com
Received: 01 Mar 2013, Revised and Accepted: 11 Apr 2013
ABSTRACT
Objective: The aim of this study was to evaluate the hepatoprotective and antioxidant effects of Withania somnifera against Paracetamol-induced
liver injury in rats.
Methods: In the present study, the protective effect of Withania somnifera was investigated against Paracetamol-induced hepatotoxicity and
compared with Silymarin, a standard hepatoprotective reference drug. The rats received a single dose of paracetamol (900 mg/kg body weight, i.p.);
Withania somnifera (500 mg/kg body weight and 1000mg/kg body weight, p.o.) and Silymarin (25 mg/kg body weight, p.o.) were administered 30
min after the injection of paracetamol. Liver marker enzymes (Aspartate Transaminase, Alanine Transaminase and Alkaline Phosphatase), Total
Protein content, Bilirubin, Antioxidant status (Reduced Glutathione, Superoxide Dismutase, Catalase and Glutathione-S-Transferase) were evaluated
and histopathological analysis was done for the control and experimental rats.
Results: Paracetamol treatment leads to elevated levels of liver marker enzymes and bilirubin and there was deterioration in total protein content,
histological observations and antioxidant status. However, treatment with Withania somnifera significantly reversed (p < 0.05) the above changes
compared to the control group as observed in the paracetamol-challenged rats.
Conclusion: The results clearly demonstrate that Withania somnifera possesses promising hepatoprotective effects through its antioxidant activity
and hence suggests its use as a potential therapeutic agent for protection from paracetamol overdose.
Keywords: Hepatotoxicity, Paracetamol, Withania somnifera.
INTRODUCTION
Acetaminophen (Paracetamol or AAP) is a commonly available drug
well known for its analgesic and antipyretic effects. At therapeutic
doses, AAP is considered a safe drug and is safely bio transformed
and eliminated as non-toxic conjugates of sulfate and glucuronic
acid, and a small portion is converted to NAPQI (N-acetyl-p-
benzoquinone imine) which is detoxified by glutathione (GSH) and
eventually eliminated in the urine or bile [1].
However, during
overdose of AAP, the glucuronidation and sulfation routes become
saturated and rapid depletion of hepatic GSH levels occurs which
causes oxidative stress and the NAPQI thus formed binds covalently
to liver proteins
[2]. Hepatotoxicity induced by acetaminophen
results in prominent elevations of liver marker enzymes and
reactive oxygen species (ROS) which further aggravates oxidative
stress and are involved in a number disease processes, including
heart disease, diabetes, liver injury, cancer, cardiovascular
dysfunctions and aging [3-7]. Therefore, new potential therapeutics
for AAP overdose is being routinely investigated in preclinical
studies.
Liver is an important organ in the body as it provides protection
from potentially injurious exogenous and endogenous compounds
and in this process it gets affected [8]. Thus protective mechanisms
for liver are of special concern. Conventional medicines used for
treatment of liver diseases have adverse side effects and are costlier.
So, there is a need to evaluate natural compounds as an effective
alternative which are safer and cost effective.
Withania somnifera also known as Ashwagandha or Indian Ginseng
[9], dunal Solanaceae, is cultivated in drier parts of India and in
Nepal. It is a well known medicine in Ayurveda and has proved to
exhibit anti-inflammatory [10], immunomodulatory [11], anti-
arthritic [12] and antiageing properties [13]. W. somnifera is known
to alter the oxidative stress markers of the body. The root extract
has found to significantly reduce the lipid peroxidation [14] and
increase the superoxide dismutase (SOD) and catalase activities,
thus carrying free radical scavenging property [15]. It is been proved
to have hepatoprotective effect against radiation induced [16] and
iron induced toxicity [17]. However, protective activity of Withania
somnifera has not been scientifically investigated against
paracetamol induced hepatotoxicity. Hence, an attempt was made to
investigate the effects of aqueous extract of Withania somnifera
against hepatic injury induced by acetaminophen hepatotoxicity.
MATERIALS AND METHODS
Drugs and chemicals
Commercially available Ashwagandha (W.somnifera) powder was
obtained from Indian Medical Practitioners Co-operative Stores and
Society, Mylapore, Chennai, Tamilnadu, India. Its aqueous
suspension at dose 500mg/kg body weight and 1000mg/ kg body
weight was injected intraperitoneally. Silymarin and Paracetamol
were obtained from Natural remedies private limited, Bangalore.
Paracetamol was dissolved in double distilled water and injected
intravenously. All other reagents used were standard laboratory
reagents of analytical grade.
Experimental Animals
The study was performed using rats of either sex, having a mean
weight of 190 grams, procured from VIT Animal house, VIT
University, Vellore, Tamilnadu, India. The rats were fed commercial
pelleted feed from Hindustan Lever Ltd. (Mumbai, India) and water
ad libitum. The animals were well treated and cared for in
accordance of the guidelines recommended by the Committee for the
Purpose of Control and Supervision of Experiments on Animals,
Ministry of Culture, Govt. of India, Chennai, India.
Evaluation of hepatoprotective activity
Animals were divided into 5 groups. All animals were made to fast
24 hours before the commencement of the study. Group I or the
control group, received saline (0.89 % NaCl); In Group II,
Paracetamol induced test group, hepatotoxicity was induced by
single dose of paracetamol (900 mg/kg body weight i.p. dissolved in
distilled water); Group III i.e. drug treated group, were given
Ashwagandha suspended in distilled water (500 mg/ kg/ body
weight /day, orally) administered 30 minutes after the single
injection of Paracetamol (900 mg/kg body weight, i.p.); Group IV
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491 Vol 5, Suppl 2, 2013
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Sabina et al.
Int J Pharm Pharm Sci, Vol 5, Suppl 2, 648-651
649
received Ashwagandha (1000mg/ kg body weight, orally)
administered 30 minutes after the single injection of Paracetamol
(900 mg/kg body weight, i.p). Group V was administered Silymarin
(25 mg/kg body weight, i.p.) 30 minutes after the single injection of
Paracetamol (900 mg/kg body weight, i.p.). Rats were decapitated
after 4 hours of Paracetamol injection; blood was collected from the
trunk, serum was separated and stored at -70º C. Tissue samples
from the liver were processed for biochemical and histological
analysis.
The activities of AST [18], ALT [18], Alkaline Phosphatase [19], and
Bilirubin [20] were determined in serum of control and
experimental rats. Superoxide dismutase was assayed according to
Marklund and Marklund [21] and the unit of enzyme activity was
defined by the enzyme required to give 50 % inhibition of pyrogallol
autoxidation. Catalase [22], Glutathione-S-transferase [23] and
Reduced Glutathione [24] were evaluated and Total Protein was
estimated using Lowry’s method [25] using bovine serum albumin
as standard.
Histopathological Studies
Immediately after sacrifice, a portion of the liver was fixed in 10%
formalin, then washed, dehydrated in descending grades of
isopropanol and finally rinsed with xylene. The tissues were then
embedded in molten paraffin wax. Sections were cut at 5 mm
thickness, stained with haematoxylin and eosin was observed
microscopically for histopathological changes.
Statistical Analysis
Results were expressed as mean± SD and statistical analysis was
performed using ANOVA, to determine the significant differences
between the groups, followed by Student Newman-Keul’s test.
p<0.05 implied significance.
Table 1: Effect of W.somnifera on liver marker enzymes and protein content in serum of control and Paracetamol - intoxicated rats
Parameters
Group 1
Group 2
Group 3
Group 4
Group 5
Alanine transaminase (U/dl)
86.52±4.57
180.90± 6.21a
*
95.52± 4.98a
*
b
*
88.32±4.78 a
*
c
*
88.44± 4.62a
*
d
*
Aspartate transaminase (U/dl)
73.38±5.86
230.48±6.16a
*
123.28±6.57 a
*
b
*
100.27±6.21 a
*
c
*
98.32± 6.37a
*
Alkaline phosphatase (U/dl)
99.10±5.08
279.22±7.44a
*
143.86±5.28a
*
b
*
131.55±4.89a*c*
134.07±5.21 a
*
Total bilirubin (mg/dL)
0.57± 0.72
5.68± 1.03a
*
2.02± 0.98a
*
b
*
1.81± 0.98a
*
c
*
1.97± 0.83 a
*
Total protein (mg/dL)
6.86± 0.04
3.22±0.78 a
*
6.73±0.42 a
*
b
*
6.68±0.13a
*
c
*
5.58±0.37 a
*
For each group n═6, the values are mean ± SD. Comparisons indicated by lowercase letters were made as follows: a—group I vs. groups II, III, IV and
V; b—group II vs. group III; c—group II vs. group IV. Statistical analysis was calculated by one way ANOVA followed by Student's Newman–Keul's
test. The symbols represent statistical significance at: * p < 0.05
Table 2: Effect of W. somnifera on antioxidant status in serum of control and Paracetamol - intoxicated rats
Parameter
Group 1
Group 2
Group 3
Group 4
Group 5
Superoxide dismutase
242.40±13.00
135.67±6.80 a
*
227.17±16.90 a
*
b
*
204.83±19.70 a
*
c*
220.50±17.9 a
*
Catalase
78.31±1.70
45.31±3.62 a
*
69.96 ±8.35 a
*
b
*
63.16±7.39 a
*
c*
70.33±7.41 a
*
Glutathione-S-transferase
2.73±0.53
0.81±0.14 a
*
2.21±0.27 a
*
b
*
2.38±0.39 a
*
c*
2.80 ±0.21 a
*
Reduced glutathione
24.46±2.29
11.70±1.21 a
*
21.30±2.61 a
*
b
*
19.40±2.38 a
*
c*
22.01±1.64 a
*
For each group n═6, the values are mean ± SD. Comparisons indicated by lowercase letters were made as follows: a—group I vs. groups II, III, IV and
V; b—group II vs. group III; c—group II vs. group IV. Statistical analysis was calculated by one way ANOVA followed by Student's Newman–Keul's
test. Units: lipid peroxidation—nanomoles of MDA formed/milligram protein, CAT—micromoles of H2O2 consumed/minute/ milligram protein,
SOD—units/milligram protein (1 U= amount of enzyme that inhibits the autoxidation of pyrogallol by 50 %), GST—nanomoles of 1-chloro-2, 4-
dinitrobenzene–GSH conjugate formed/minute/milligram protein, reduced glutathione—nanomoles/milligram/protein *p<0.05 (statistically
significant)

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Int J Pharm Pharm Sci, Vol 5, Suppl 2, 648-651
650
Fig. 1: Histopathalogical monograph of extract and standard. a : control; b: Paracetamol (900 mg/kg) alone; c: Paracetamol + Ashwagandha (900
mg/kg +500 mg/kg); d: Paracetamol + Ashwagandha(900 mg/kg +1000 mg/kg); e: Paracetamol + Silymarin(25 mg/kg+ 900 mg/kg).
RESULTS
Hepatoprotective activity
The activities of Aspartate aminotransferase(AST), Alanine
aminotransferase (ALT) and Alkaline Phosphatase(ALP) in serum
were significantly increased (p < 0.05) in the acetaminophen-treated
group as compared with the normal control group[Table 1]. The
increase in the levels of these liver marker enzymes clearly indicated
the damage of the hepatic cells. However, treatment with Withania
somnifera prevented the alteration in the above to levels similar to
those in control rats. The levels of serum bilirubin was also
increased significantly (p < 0.05) as compared to the normal rats
which was brought to normal after treatment with Withania
somnifera. In acetaminophen treated rats, the total protein content
was also decreased significantly (p < 0.05) as compared to the
normal rats [Table 1]. On the other hand, Withania somnifera
reversed the effect of Acetaminophen toxicity and thus protein level
was brought to normal as that of control rats.
Antioxidant activity
In rats treated with acetaminophen, a significant decrease in
antioxidant enzymes (superoxide dismutase, catalase and
glutathione-S-transferase) and total reduced glutathione was
observed [Table 2]. However, treatment of W.somnifera significantly
increased the antioxidant status as compared with the control group.
Histopathological results
The hepatoprotective effect of Ashwagandha was confirmed by
histopathological examination of the liver tissue of control and
treated animals [Figure 1]. The histological architecture of control
rats was found to be normal with distinct hepatic cells and
sinusoidal space. In group II i.e. the paracetamol treated liver
sections showed congestion, mild centrilobular degeneration of
hepatocytes, mild bile duct hyperplasia and multifocal cell
infiltration. The histopathological profile of the Group III rats
showed mild degeneration of hepatocytes and in group IV rats no
visible changes were observed confirming the safety of the extract at
selected dose. In group V,rats treated with silymarin, intoxicated
with paracetamol showed less disarrangement and degeneration of
hepatocytes.
DISCUSSION
Paracetamol-induced hepatic failure is the second leading cause of
liver transplantation and accounts for considerable levels of
morbidity and mortality. The advantage of this model is that being a
dose-dependent toxicant, the experiments are technically easy to
perform and, most importantly, it is a clinically relevant. The
estimation of enzymes in the serum is a useful quantitative marker
of the extent and type of hepatocellular damage. The rise in serum
AST and ALT levels has been attributed to the damaged structural
integrity of the liver because these are cytoplasmic in location and

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are released into circulation after cellular damage. In this study,
significant elevation in the liver marker enzymes namely aspartate
transaminase, alanine transaminase, alkaline phosphatase, and total
bilirubin in serum was caused by a single dose of paracetamol
(900mg/kg bodyweight, i.p.) treatment as compared to the control
group. The increased serum transaminases, alkaline phosphatase,
and total bilirubin levels in paracetamol-intoxicated rats indicate a
deterioration of the hepatic functions due to liver membrane
damage resulting from acetaminophen toxicity which leads to the
escape of these enzymes in serum (circulation) [26]. However, we
observed that administration of Ashwagandha (Withania somnifera)
after paracetamol intoxication resulted in subsidence of the
increased activities of liver markers indicating that Ashwagandha
treatment may enhance recuperation of liver from paracetamol
induced damage in paracetamol intoxicated rats [Table 1]. The
prevention of the leakage of intracellular enzymes in serum of
acetaminophen-intoxicated mice might be due to the membrane
stabilizing activity of Withania somnifera.
Bilirubin is a yellow pigment produced when heme is catabolized.
Hepatocytes render bilirubin water-soluble and therefore easily
excretable by conjugating it with glucuronic acid prior to secreting it
into bile by active transport. Hyperbilirubinemia may result from the
production of more bilirubin than the liver can process, damage to
the liver impairing its ability to excrete normal amounts of bilirubin
or obstruction of excretory ducts of the liver. Serum bilirubin is
considered as one of the true test of liver functions since it reflects
the ability of the liver to take up and process bilirubin into bile [6].
Withania somnifera helped in decreasing the significantly altered
levels of Bilirubin thus bringing liver to function normally [Table 1].
Production of ROS and glutathione depletion are key players in AAP
induced toxicity [27]. This is evident from the reduction of
antioxidant status (Superoxide dismutase, Catalase, Reduced
glutathione and Glutathione-S- Transferase) of paracetamol
intoxicated rats. W.somnifera was able to restore the levels of
Antioxidants suggesting its protective role in AAP mediated liver
injury. This was confirmed by the histopathological observations
[Figure 1]. In conclusion, the results obtained in the present study
suggest that Ashwagandha possesses a promising hepatoprotective
and antioxidant effect in acetaminophen-intoxicated rats probably
due to its antioxidant effects. However, further pharmacological
evidence at the molecular level is required to establish the actual
mechanism of the action of the drug and research into this area is
underway.
REFERENCES
1. Coles B, Wilson I, Wardmon P, Hinson JA, Nelson SD, Ketterer B.
The spontaneous and enzymatic reaction of N-acetyl-p-
benzoquinonimine with glutathione: a stopped-flow kinetic
study. Archives of Biochemistry and Biophysics 1988; 264:
253–260.
2. Dai G, He L, Chou N, Wan YJ. Acetaminophen metabolism does
not contribute to gender difference in its hepatotoxicity in
mouse. Toxicol Sci 2006;92 Suppl1:33–41.
3. Anusuya N, Manian S.Antioxidant and free radical scavenging
potential of different solvent extracts of Indigofera tinctoria l.
leaves. International journal of pharmacy and pharmaceutical
sciences 2013;5 Suppl 1.
4. Jaeschke H. Reactive oxygen and mechanisms of inflammatory
liver injury. Journal of Gastroenterology and Hepatology 2000;
15 Suppl 7 :718–724.
5. Klaunig JE, Kamendulis LM. The role of oxidative stress in
carcinogenesis. Annual Review of Pharmacology and
Toxicology 2004; 44: 239–267.
6. Jaeschke H, Knight TR, Bajt ML. The role of oxidant stress and
reactive nitrogen species in acetaminophen hepatotoxicity.
Toxicology Letters 2003; 144 Suppl 3 :279–288.
7. Bokov A, Chaudhuri A, Richardson A. The role of oxidative
damage and stress in aging. Mechanisms of Ageing and
Development 2004;125 Suppl 10-11:811–826.
8. Ghosh D, Firdaus Sb, Mitra E, Dey M, Chattopadhyay A, Pattari
Sk et al.Hepatoprotective activity of aqueous leaf extract of
Murraya koenigii against lead-induced hepatotoxicity in male
wistar rat. International Journal of Pharmacy and
Pharmaceutical Sciences 2013 ;491 Suppl 5.
9. Singh A, Naidu PS, Gupta S, Kulkarni SK. Effect of natural and
synthetic antioxidants in a mouse model of chronic fatigue
syndrome. J Med Food 2002;52:11–20.
10. Rolo AP , Palmeira CM. Diabetes and mitochondrial function:
role of hyperglycemia and oxidative stress. Toxicology and
Applied Pharmacology 2006;212 Suppl 2:167–178.
11. Rasool M, Varalakshmi P.Immunomodulatory role of Withania
somnifera root powder on experimental induced inflammation:
An in vivo and in vitro study. Vascular Pharmacology 2006;
44:406–410.
12. Sethi PD, Thiagarajan AR, Subrahamanian SS.Studies on the
anti-inflammatory and anti-arthritic activity of withaferin-A.
Indian Journal of Pharmacology 1990; 12: 165-172.
13. Bhattacharya SK, Satyan KS, Ghosal S. Antioxidant activity of
glycowithanolides from Withania somnifera. Indian J Exp Biol
1997 ;35: 236–9.
14. Dhuley JN. Effect of ashwagandha on lipid peroxidation in
stress-induced animals. J Ethnopharmacol 1998;60:173–8.
15. Panda S, Kar A. Evidence for free radical scavenging activity of
Ashwagandha root powder in mice. Indian J Physiol Pharmacol
1997;41:424–6.
16. Mansour HH, Hafez HF. Protective effect of Withania somnifera
against radiation-induced hepatotoxicity in rats. Ecotoxicology
and Environmental Safety 2012; 80:14–19.
17. Bhattacharya A, Ramanathan M, Ghosal S, Bhattacharya S
K.Effect of Withania somnifera Glycowithanolides on Iron-
induced Hepatotoxicity in Rats. Phytother Res 2000; 14: 568–
570.
18. King J. The transferases-alanine and aspartate transaminases.
In: Van D. Practical clinical enzymology. Nostrand Company
Limited: London; 1965a. p. 121-138.
19. King J.The hydrolases acid and alkaline phosphatases. In: Van
D.Practical clinical enzymology. Nostrand Company Limited:
London; 1965b, p. 191-208.
20. Jendrassik L, Vereinfachte GP. Photometrische Methoden zu r
Bestimmun g des Blubilirubins. Biochem A 1938 ;297: 81-89.
21. Marklund SL, Marklund G. Involvement of superoxide anion radical
in the autooxidation of pyrogallol and a convenient assay for
superoxide dismutase.Eur J Biochem 1974; 47: 469-474.
22. Sinha AK. Colorimetric assay of Catalase. Anal Biochem 1972;
47: 389-394.
23. William H Habig, Michael J Pabst, William B Jakoby. Glutathione S-
Transferases the first enzymatic step in mercapturic acid
formation.The Journal of Biological Chemistry 1974;249: 7130-
7139.
24. Moron MS, Depierre JW, Mannervik B. Levels of glutathione,
glutathione reductase and glutathione-s-transferase activities
in rat lung and liver. Biochim Biophys Acta 1979; 582: 67-78.
25. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein
Measurement with the Folin Phenol Reagent. J Biol Chem 1951;
193:265-275.
26. Sallie RW, Tredger JM, Williams R. Extrahepatic production of
the lignocaine metabolite monoethylglycinexylidide
(MEGX). Biopharm Drug Dispos 1992;13 Suppl 7:555–558.
27. Olaleye MT, Akinmoladun AC, Ogunboye AA, Akindahunsi AA.
Antioxidant activity and hepatoprotective property of leaf
extracts of Boerhaavia diffusa Linn against acetaminophen-
induced liver damage in rats. Food and Chemical Toxicology
2010; 48: 2200–2205.