Protective Effects of Ethanolic Extract of Nigella sativa Seed in
Paracetamol Induced Acute Hepatotoxicity In vivo
Paracetamol overdose causes serious liver necrosis. Hepatoprotective
activity of ethanolic extract of Nigella sativa in Paracetamol induced
acute hepatotoxicity was investigated in rats. Fasted male Wistar rats were
orally treated with Nigella sativa extract in graded doses for 5 days
followed by Nigella sativa extract and paracetamol 3 g kg-1
on 6 and 7th day. Circulatory liver markers and reduced glutathione (GSH) levels
were estimated and histopathological study of liver performed. Paracetamol caused
a significant increase in serum alkaline phosphatase, glutamic oxaloacetic transaminase,
glutamic pyruvic transaminase and total Bilirubin and a significant decrease
in GSH compared to control. Nigella sativa pretreatment significantly
prevented the increase in liver enzymes and total bilirubin and decrease in
GSH level as compared to paracetamol group. Liver histopathology showed marked
reduction in sinusoidal dilatation, midzonal necrosis, portal triaditis and
occasional apoptosis in Nigella sativa extract treated groups as compared
to group receiving only paracetamol. Nigella sativa extract possesses
hepatoprotective action against paracetamol induced acute hepatoxicity. Further
research is needed to advocate its prophylactic use for drug induced hepatotoxicity.
Received: March 11, 2013;
Accepted: May 22, 2013;
Published: November 26, 2013
Traditional NSAIDs like paracetamol (PCM) are one of the most commonly used
drugs throughout the world (Al-Turki et al., 2010).
PCM, safe at therapeutic doses, can lead to centrilobular hepatic necrosis in
overdose which can be fatal (Najafzadeh et al., 2011).
The US Food and Drug Administration (FDA) asked drug manufacturers to limit
the strength of PCM in prescription drug products and a Boxed Warning was added
to the label of all prescription products containing PCM highlighting the potential
for severe liver injury (FDA, 2011).
In absence of reliable hepatoprotective agents there is an urgent need for
searching an agent for prevention of PCM induced hepatotoxicity. Herbal drugs
play a role in folk medicine due to their hepatoprotective activity and several
plants have been scientifically evaluated for hepatoprotective activity (Iweala
et al., 2011; Zamani-Moghaddam et al.,
2012). Among the promising medicinal plants, Nigella sativa, commonly
known as Kalonji in India and Pakistan, has been used traditionally for various
ailments (Qidwai et al., 2009). It is one of
the important medicines of Tibbe Nabawi, i.e., Prophetic Medicine (Ahmad
et al., 2009) in which it is considered as a healing for all diseases
except death. Al-Bukhari (1976a, b)
Nigella sativa has been thoroughly studied scientifically and has been shown
to have analgesic (Abdel-Fattah et al., 2000),
reproductive efficiency improving (Bashandy, 2007),
nephroprotective (Abdelaziz and Kandeel, 2011) diuretic
and antihypertensive (Zaoui et al., 2000), bronchodilator
and calcium antagonist (Gilani et al., 2001),
hepatoprotective (Kushwah et al., 2012), anthelmintic
(Akhtar and Riffat, 1991), antimicrobial (Abu-Al-Basal,
2011; Salman et al., 2009) and anticancer
activities (Worthen et al., 1998).
Since, oxidative stress plays an important role in Paracetamol induced hepatotoxicity
(Jaeschke et al., 2003; James
et al., 2003) and Nigella sativa possesses strong anti-oxidative
activity (Farrag et al., 2007), we hypothesized
that Nigella sativa Extract (NSE) could protect against Paracetamol induced
hepatotoxicity by improving Glutathione levels. This study has investigated
whether or not pretreatment of oral NSE ameliorates Paracetamol induced acute
MATERIALS AND METHODS
Plant material: Nigella sativa (Black Cumin) seeds were purchased
from the local market and authenticated by a botanist at National Botanical
Research Institute, Lucknow. A voucher specimen of the seeds was kept in the
museum of Department of Pharmacology, Eras
Lucknow Medical College, Lucknow. Seeds were grounded to powder with the help
of mortar and pestle and 150 mg of powder was soaked in 250 mL of 99% ethanol
in a closed container at room temperature for 7 days with periodic stirring
with a sterile glass rod. After 7 days it was filtered with Wattmans
filter paper No.1 and extract was concentrated by rotary vacuum evaporator and
kept in a vacuum desiccator for complete removal of solvent. The extract so
obtained was stored at 4°C till further use.
Animals: Male Wistar rats, weighing 225-250 g, were purchased from Central
Drug And Research Institute (CDRI), Lucknow, India, housed in a temperature
controlled room (21±1°C) with a 12 h light-12 h dark cycle and allowed
free access to a standard rat chow and filtered tap water for at least 5 days
for acclimatization. Solid food but not the water was removed 12 h prior to
an experiment. The study received the approval of the Institutional Animal ethics
Committee of Eras Lucknow medical
college and hospital. Animals were cared for in accordance with the internationally
accepted principles for laboratory animal use and care and the procedures followed
were in accordance with the standards set forth in the Guide for the Care and
Use of Laboratory Animals (published by the National Academy of Science, National
Academy Press and Washington, D.C).
Treatment and samples: Rats were randomly divided into groups of 8 each
and treated as follows:
||Control group received normal saline for 7 days
||PCM group received normal saline for 7 days followed by PCM 3 g kg-1
on day 6 and 7
||NSE groups received NSE 250 or 500 mg kg-1 for 7 days followed
by PCM 3 g kg-1 on day 6 and 7
||NSE alone group received NSE 500 mg kg-1 for 7 days
Administration of all doses was done with the help of oral feeding tube. At
the end of the study (on day 8), the rats were sacrificed and dissected. Blood
and liver tissue samples were taken for biochemical and histopathological investigations.
Biochemical study: Serum Alkaline phosphatase (ALP), Serum Glutamic
Oxaloacetic Transaminase (SGOT) and Serum Glutamic Pyruvic Transaminase (SGPT)
and total bilirubin was determined using semi auto analyzer.
Biochemical assay: Serum was de-protonized by adding 1.0 mL of 10% TCA
and centrifuged at 6000xg for 5 min. A 0.5 mL aliquot from clear supernatant
was mixed with 0.5 mL double distilled water. Thereafter, 2 mL of 0.4 M tris
buffer and 0.1 mL DTNB were added to it with proper stirring. The absorbance
was read at 412 nm within 5 min of the addition of GSH (200-1600 μmoles).
GSH reduced in the sample were calculated using the standard curve and the results
were expressed as μmoles g-1 tissue.
Histopathological study: Liver tissues collected from the distal portion
of the left lateral lobe of each rats were stored in 10% formalin solution for
48 h or until processing. For block preparation, livers were processed using
a graded ethanol series and embedded in paraffin. Four micrometer paraffin sections
(5 μm) were cut and stained with haematoxylin and eosin for light microscopic
Statistical analysis: The different groups were compared using ANOVA
followed by post hoc dunnetts
test. All tests were performed using SPSS (17.0 versions). The p<0.05 was
Administration of PCM caused a significant increase in ALP, SGOT, SGPT and
total bilirubin and a significant decrease in GSH as compared to control. The
rise in liver enzymes due to paracetamol was highest in case of SGOT (144.6
IU L-1 serum, 95% CI-106.25-182.94) and SGPT (170.85 IU L-1
serum, 95% CI-123.3-218.3) Pre-treatment with NSE significantly prevented the
increase in liver enzymes and total bilirubin and the decrease in GSH level
as compared to PCM group (p>0.05 as compared to control in all parameters)
in a dose dependent manner. Most significant difference from paracetamol group
was seen in SGOT (49.47 IU L-1 serum, 95% CI-30.77-68.92), SGPT (49.75
IU L-1 serum, 95% CI-31.50-68) and GSH (31.51 μmol g-1
tissue, 95% CI-27.13-35.88) levels in group pretreated with 500 mg kg-1
All the parameters were found to be not significantly different from control
group in NSE pre-treated groups (p>0.05 as compared to control in all parameters).
Level of liver enzymes, total bilirubin and GSH was not significantly different
from control group in animals who received NSE alone (Table 1).
|| Effect of Nigella sativa extract on liver enzymes
level in Paracetamol induced acute hepatotoxicity in rats
|N = 8, Values are expressed as mean (C.I.); NSE 1-N. sativa
extract (250 mg kg-1); NSE 2-N. sativa extract (500 mg
kg-1); PCM-Paracetamol, *significantly different from control,
*p<0.05, **p<0.01, ***p<0.001, #significantly different
from Paracetamol treatment group, #p<0.05, ##p<0.01,
||Photomicrograph from control rat (H and E, 100x), showing
normal liver parenchyma and normal architecture of hepatocytes and vasculature,
distinct hepatic cords and central vain
||Photomicrograph from paracetamol treated rats showing extensive
necrosis, fraying of cell margins and portal triditis with mononuclear lymphoplasmocytic
Histopathologic changes: In control group animals, normal liver parenchyma
and normal architecture of hepatocytes and vasculature, distinct hepatic cords
and central vain was seen (Fig. 1).
||Photomicrograph from paracetamol treated rats showing Strip
focal necrosis, feathery degeneration (in the mid zone) involving several
hepatocyte, necrosis of several hepatocyte extended in band like fashion
in the mid zonal area and occasional apoptotic bodies
||Liver section from rat orally treated NSE 500 mg kg-1,
showing protective effects of plant extract
However, animals treated with PCM (3 g kg-1 p.o.) showed extensive
necrosis, fraying of cell margins, portal triditis with mononuclear lymphoplasmocytic
inflammatory infiltration (Fig. 2, 3). This
effect was significantly decreased in animals pretreated with NSE in both doses
(Fig. 4). NSE pretreatment caused resumption of normal liver
architecture with decrease in lymphocytic infiltration.
These results show that N. sativa protects against paracetamol induced
hepatotoxicity possibly by preventing reduction of glutathione, levels and lymphocytic
infiltration and may lead to further research in prevention of drug induced
Our results suggest that oral administration of NSE might protect the liver
against PCM induced acute toxicity. PCM induced hepatotoxicity manifested biochemically
by significant elevation of serum levels of liver enzymes such as ALP, SGOT
and SGPT coupled with decrease in GSH level. Marked destruction of hepatic structure
further evidenced the liver cell damage. Pretreatment with NSE (250, 500 mg
kg-1 p.o.) significantly ameliorated the elevated level of these
enzymes which were comparable to control group.
PCM is readily detoxified by hepatic phase 2 drug metabolizing systems in the
liver by glucuronidation and sulfation (Henderson et
al., 2000), with a small portion undergoing a cytochrome P-450-mediated
conversion to a highly reactive electrophilic arylating intermediate N-acetyl-p-benzoquinoneimine
(NAPQI) (Dahlin et al., 1984). NAPQI is detoxified
principally by conjugation with reduced glutathione (GSH) spontaneously or via.,
glutathione transferase (GST)-mediated reactions to the 3-glutathione-S-yl-PCM
conjugate (Henderson et al., 2000).
If there is an overdose of PCM, the high levels of NAPQI produced eventually
exhaust GSH, UDP-glucuronic acid and inorganic sulfate, inhibit GSH synthesis
(Hazelton et al., 1986; Lauterburg
and Mitchell, 1982) and decrease cytosolic GST activity (Yonamine
et al., 1996). NAPQI also causes hepatocellular damage and centrilobular
hepatic necrosis (James et al., 2003). Since,
GSH plays a key role in the detoxification of reactive toxic metabolites of
PCM, liver necrosis begins when GSH stores are markedly depleted (Mitchell
et al., 1973). As expected PCM treatment caused remarkable depletion
of liver GSH levels in our study. These results agree with other reports pertaining
to PCM-induced GSH depletion.
Nigella sativa has antioxidant properties and can elevate reduced glutathione
level in oxidative stress (Abdel-Sater, 2009). Present
data shows that Nigella sativa improves GSH level which is in conformity
to results of previous studies (Abdel-Sater, 2009; Neveen
and Iman, 2010). In order to find weather NSE alone is increasing GSH levels
or pretreatment with NSE blocking the decrease in GSH levels, we administered
NSE alone in one group. No significant difference between this group and control
group shows that pretreatment with NSE blocks the depletion of GSH by Paracetamol
toxicity. Another mechanism of PCM induced hepatotoxicity is the formation of
reactive oxygen species such as superoxide anion, hydrogen peroxide, hydroxyl
radical, reactive nitrogen species such as nitric oxide and peroxynitrite and
peroxidation reaction products (James et al., 2003;
Reid et al., 2005; Bessems
and Vermeulen, 2011). Burits and Bucar (2000) tested
the essential oil of Nigella sativa L., for antioxidant activity and
showed that thymoquinone, active constituent of Nigella sativa and the
components carvacrol, t-anethole and 4-terpineol demonstrated high free radical
Several studies have reported hepatoprotective activity of Nigella sativa
and its active constituent Thymoquinone against CCL4 induced liver
damage (Oyagbemi and Odetola, 2010). In the present
study we induced liver cell damage by PCM, a common over the counter drug. Further
research is required before Nigella sativa can be used in humans.
Further research is warranted to explore individual effects of its active components
and effect of N. sativa in hepatic damage caused by other drugs where
oxidative damage or lymphocytic infiltration is involved.
A limitation of the study is that the only two doses of extract have been used
as for establishing dose-response relationship three or more doses are required.
Furthermore in the present study NSE was supplemented only before PCM administration
and another study is required to find out if the plant will be curative when
given after PCM-induced hepatotoxicity.
Nigella sativa is effective in protecting against PCM induced hepatotoxicity
possibly via protecting depletion of GSH and cellular injury by Paracetamol.
This research received no specific grant from any funding agency in the public,
commercial, or not-for-profit sectors. Authors have no conflict of interest
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