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205
Research Article
HEPATO-PROTECTIVE ROLE OF THE AQUEOUS AND N-HEXANE EXTRACTS OF NIGELLA
SATIVA LINN. IN EXPERIMENTAL LIVER DAMAGE IN RATS
FARIDA YESMIN
1
, ZAIDA RAHMAN
2
, JESMIN FOUZIA DEWAN
3
, ASADUL MAZID HELALI
4
, NOR IZA A RAHMAN
5
,
AHMED G. ALATTRAQCHI
6
, AREFUDDIN AHMED
7
, RABEYA YOUSUF
8
, ABDUS SALAM
9
, MAINUL HAQUE
10
1
Assistant Professor, Department of Pharmacology & Therapeutics, Gonoshashthaya Samajvittik Medical College (GSSVMC), Savar, Dhaka,
Bangladesh;
2
Associate Professor, Department of Pharmacology & Therapeutics, Enam Medical College & Hospital, Savar, Dhaka,
Bangladesh;
3
Professor, Department of Pharmacology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Shahbag Dhaka,
Bangladesh;
4
Assistant Professor, Department of Pharmacology & Therapeutics, GSSVMC, Dhaka, Bangladesh;
5
Medical Lecturer, Faculty of
Medicine and Health Sciences (FPSK), Universiti Sultan Zainal Abidin (UniSZA), Terengganu, Malaysia;
6
Medical Lecturer, FPSK, UniSZA,
Terengganu, Malaysia;
7
Senior Lecturer, Medical Radiation Programme, School of Health Sciences, Universiti Sains Malaysia, Malaysia;
8
Medical Officer, Blood Bank Unit, Department of Pathology, Universiti Kebangsaan Malaysia (UKM) Medical Centre, Kuala Lumpur,
Malaysia;
9
Associate Professor, Department of Medical Education, UKM Medical Centre, Kuala Lumpur, Malaysia;
10
Professor, FPSK,
UniSZA, Terengganu, Malaysia.
10
Email: runurono@gmail.com
Received: 13 June 2013, Revised and Accepted: 3 July 2013
ABSTRACT
Objective: Liver disease is associated with the formation of oxygen derived free radicals. Reactive oxygen species (ROS) as well as nitrogen species
are responsible for nuclear DNA fragmentation and cell death. The active principle of thymoquinone (TQ) of Nigella sativa acts as a scavenger of
superoxide anion. The current study was conducted to evaluate the hepatoprotective effect of Nigella sativa on rats.
Methods: The study was carried out at prime postgraduate medical University of Bangladesh. Liver damage and oxidative stress were evaluated by
measuring serum alanine amino transferase (ALT), hepatic malondialdehyde (MDA) and hepatic Glutathione (GSH) levels. Aqueous extract of
Nigella sativa and n-hexane extract of Nigella sativa were administered orally into two groups of rat through intra-gastric tube for 28 days. Both the
groups received paracetamol intra-peritoneally on day 28
th
and were sacrificed on day 30
th
. Subsequently, the following parameters were studied:
Serum ALT, hepatic MDA, and hepatic GSH.
Results: Hepatic damage was evaluated by significant increases in serum ALT (p<0.001) and hepatic MDA (p<0.001) concentration with depleted
hepatic GSH (p<0.001) in paracetamol treated group. Pre-treated with aqueous extract of Nigella sativa significantly reduced serum ALT (p<0.001)
and hepatic MDA (p<0.001) levels and also significantly associated with the increase in hepatic GSH (p<0.01). Pretreatment with n-hexane extracts
of Nigella sativa decreased serum ALT (p<0.001), hepatic MDA (p<0.001) and increased hepatic GSH (p<0.001).
Conclusion: Hepatoprotective properties of Nigella sativa in liver damage of experimental rats by reducing oxidative stress are evident. The
protection afforded by the n-hexane extract of Nigella Sativa in pre-treated group has also been validated.
Keywords: Hepatoprotective, Liver-damage, Nigella sativa Linn.
INTRODUCTION
Liver diseases are always dealt very seriously by the graduate
physicians due to their potentiality to cause morbidity and mortality.
The prevalence rate of liver disease in Bangladesh is the highest in
the world [1]. Liver is the main organ involved in the metabolism of
biological toxins and medicinal agents [2]. Hence; metabolism is
always associated with the disturbance of hepatocyte biochemistry
and generation of ROS [3, 4, 5]. ROS are involved in liver damage
induced by several conditions such as viral hepatitis [6], alcohol
abuse [3], cirrhosis of liver [7], hepatocellular carcinoma [8] and
paracetamol-induced liver damage [9].
Paracetamol (acetaminophen) is a safe and effective analgesic and
antipyretic drug when used at therapeutic dose [10]. However, an
overdose can produce fatal hepatic necrosis in man [11] and other
animals [10]. It has been stated that paracetamol overdose is one of
the most frequent causes of drug induced liver failure in the United
States and in the Great Britain [12].
Previously, researchers studying the toxic mechanism of
paracetamol focused on the metabolic activation of the drug by
cytochrome P450 enzymes to a reactive metabolite that depleted
GSH and covalently bound to protein. Reduced amount of GSH leads
covalent binding of the reactive metabolite N-acetyl-p-benzoquinone
imine (NAPQI) with cellular protein resulting in hepatic cell death
[13, 10]. Current drug for the management of high-dose
paracetamol-induced toxicity includes N-acetylcysteine and
methionine. They provide protection after paracetamol overdose
primarily by replenishment of hepatic GSH stores and direct
detoxification of NAPQI. Although these antidotes have been
available for more than two decades, they possess certain limitations
and hepatic damage and deaths are still frequently seen, largely
because of late presentation [14]. Therefore, experiments are being
carried out in search of more effective, non-toxic, inexpensive
agents.
Ample researches have been carried out to obtain appropriate
therapy for paracetamol-induced hepatotoxicity as well as different
approaches of preventing and treating liver diseases. Antioxidant
therapy used in different liver diseases is GSH [13], l-ascorbic acid
[15, 16], Andrographis paniculata (kalamegh) [17], Spirulina [18],
Cajunus indica (arhar) [19], Phyllanthus niruri (bhuiamla) [20, 21,
22], Silymarin [23], vitamin E [24] and selenium (Se) [25].
The black cumin is an important spice, also known as black seed,
fennel flower, nutmeg flower, Roman coriander, or black caraway. N.
sativa is a common spice that grows once a year and a member of the
Ranunculaceae family [26]. The seeds have traditionally been used
in South Asia and Middle Eastern folk medicine as a natural remedy
for various diseases as well as spice for over 2000 years.
The seeds of N. sativa have been subjected to a range of
pharmacological, phytochemical and nutritional investigations.
Human studies and laboratory studies on the seeds and oil have
been subjected to scientific experiments and have been reported to
be effective for immune stimulation and treatment of rheumatism,
[27], diabetes [28,29], cancer and inflammatory diseases [30]. The
extracts of the black seeds have many therapeutic effects such as
antibacterial [31], antifungal [32], anthelmintic [33], analgesic [34],
Vol 6, Suppl 3, 2013 ISSN - 0974-2441
Mainul Haque et al.
Asian J Pharm Clin Res, Vol 6, Suppl 3, 2013, 205-209
206
antiulcer [35], diuretic and antihypertensive [36, 37, 38],
bronchodilator [39], antioxidant and hepatoprotective activities [23,
40, 41, 42,].
The black seed is composed of fixed oil, volatile oil, alkaloid,
saponins, sterols and quinines [40]. The n-hexane extract of N. sativa
contains TQ, tocopherols and carotenoids [43]. TQ has an
antioxidant potential [44] and possesses superoxide anion radical-
scavenging ability in vitro and vivo [45]. It was reported that TQ
protects isolated rat hepatocytes against CCl
4
-induced hepatotoxicity
by preventing the depletion of intracellular GSH and thus maintain
the integrity of cell membrane [40, 46]. The aqueous extract of N.
sativa has been suggested to possess antioxidant property and
reduces the hepatotoxicity against CCl
4
-induced liver damage [47].
Therefore, the present study was designed to evaluate the
hepatoprotective promises of the aqueous and the n-hexane extracts
of N. sativa, in experimental liver injury in rats.
MATERIALS AND METHODS
This experimental study was carried out in the laboratory of the
Department of Pharmacology, BSMMU, Dhaka, Bangladesh during
the period from October, 2008 to July, 2010.
The study was carried out upon 30 adult rats of the Long-Evans
Norwegian strain, aged between 3-4 months (weighing between
160-210 gm). They were obtained from the animal house of BSMMU.
The rats were divided into five groups with six animals in each.
Group I (V) or vehicle treated group received a single dose of vehicle
for paracetamol (propylene glycol) (1 mL intra-peritoneal (i.p) on
the day 1 and were sacrificed on 3
rd
day (48 hours after a single
dose). This propylene glycol treated group was designated as the
control group of the present study. Group II (P
I
) or paracetamol-
control group received a single dose of paracetamol solution in
propylene glycol at a dose of 800 mg/Kg body weight (b.w).
Paracetamol was given i.p on the day 1 and were sacrificed on the
3rd day (48 hours after a single dose). Group III (P
II
) or
paracetamol- treated group received a single dose of paracetamol
solution (in propylene glycol) at a dose of 800 mg/Kg b.w.
Paracetamol was given i.p on the day 1 and were sacrificed on 30
th
day. Group IV (A +P
I
) or (aqueous extract + paracetamol) treated
group received aqueous extract of N. sativa at a dose of 500 mg/kg
b.w, orally through a Ryle’s tube from day 1-28 and paracetamol was
administered on day 28
th
and were sacrificed on 30
th
day. Group V
(H+P
I
) or (n-hexane extract + paracetamol) treated group received
n-hexane extract of N. sativa at a dose of 5 mL/kg b.w, orally through
Ryle
’
s tube from day 1-28 and paracetamol was administered on day
28 and were sacrificed on 30
th
day. Animals were sacrificed under
anesthesia by cutting the carotid artery with the blade and blood
samples were taken for investigation. Liver damage and oxidative
stress were evaluated by measuring serum ALT, MDA and hepatic
GSH levels.
Estimation of serum ALT concentration
Principle
ALT or glutamate pyruvate transaminase (GPT) catalyses the
reversible transfer of an amino group from L-alanine to 2-
oxoglutarate forming L-glutamate and pyruvate. The pyruvate
produced was reduced to lactate by LDH and NADH. The Serum ALT
level was estimated by kinetic method [48] according to the
recommendation of the expert panel of the International Federation
of Clinical Chemistry and Laboratory Medicine. Absorbance and the
concentration of enzyme were measured in a spectrophotometer.
Estimation of serum GSH Concentration
Principle
The simple spectrophotometric procedure for GSH estimation in
tissue is based on the method of Ellman, who reported that 5, 5-
dithiobis-2-nitrobenzoic acids reduced by SH group to form 1 mole
of 2-nitro-5-mercaptobenzoic acid per mole of SH. The
nitromercaptobenzoic acid anion has an intense yellow color and
can be used to measure SH groups. The optimal condition for colour
development and preparation of homogeneous has been studied
with respect to precision, reproducibility and specificity of the
estimation [49]. This color intensity was measured by the
spectrophotometer (UV-VIS spectrophotometer) at 412 nm
wavelength.
Method for estimation of MDA level [50]
Principle: MDA is formed as a result of lipid peroxidation and reacts
with thiobarbituric acid (TBA) at 90- 100
0
C temperature and in
acidic condition. The reaction yields a pink MDA-TBA adduct the
product of two moles of TBA plus 1 mole of MDA. The colored
complex can be measured by spectrophotometer using wave length
532 NM. The extent of lipid peroxidation was estimated by using the
TBA method to determine the level of MDA, which served as the
index of lipid peroxidation.
Statistical analysis
Data obtained from the findings of the above experiments have been
expressed as mean Standard deviation (mean SD). Statistical
analysis was done by SPSS version 16, using Bonferroni t-test and
one way analysis of variance (ANOVA) followed post-hoc analysis.
The differences between groups were considered highly significant
at P< 0.001, moderately significant at p< 0.01 and significant at P
<0.05.
RESULTS
Serum ALT levels (U/L) (mean ± SD)
The mean values of serum ALT level in group I (V), group II (P
I
),
group III (P
II
), group IV (A +P
I
), group V (H+P
I
) were 22.34± 4.69
U/L, 67.21± 5.39 U/L, 46.91± 5.99 U/L, 33.97± 4.38 U/L, 30.59± 4.52
U/L respectively (Table 1). The mean ± SD of serum ALT in
paracetamol-treated group (group II) was significantly higher
(p<0.001) when compared to those of control (group I). So, pre-
treatment with aqueous extract and n-hexane extract of N. sativa
decreased the serum ALT concentration significantly and
percentages of reduction were 49.46%, 54.49% respectively (Table
1). Serum ALT level among these groups were compared and
significant difference was found [p<0.001] (Table 2). There were
highly significant difference (p<0.001) observed between group I
and II, group II and IV, group II and V respectively.
Table 1: Serum ALT levels (U/L) (mean ± SD) in pre-treatment
groups
Group
(n = 6)
ALT (U/L)
(mean ± SD)
Reduction
(%)
p-value
I (V)
II (P
I
)
III (P
II
)
IV
(A+P
I
)
V
(H+P
I
)
22.34 ± 4.69
67.21±5.39
46.91± 5.99
33.97 ±4.38
30.59 ± 4.52
49.46%,
54.49%,
35.23%
26.69%
54.49%
<0.001***
*** Significant difference (p<0.001) between group I and II
Table 2: Comparison of serum ALT levels (U/L) (mean ± SD)
between groups using Bonferroni‘t’ test
Comparing groups
Compared groups
Level of significance
I (V)
II (P
I
)
III (P
II
)
IV (A+P
I
)
V (H+ P
I
)
0.000***
0.000***
0.015*
0.266
NS
II(P
I
)
III (P
II
)
IV (A+P
I
)
V (H+P
I
)
0.000***
0.000***
0 .000***
III (P
II
)
IV (A+P
I
)
V (H+ P
I
)
0.005**
0.000***
IV(A+P
I
)
V (H+ P
I
)
1.000
NS
P<0.001= ***, P<0.01= **, P<0.05= *NS = no significant
difference (p>0.05) between groups.
Mainul Haque et al.
Asian J Pharm Clin Res, Vol 6, Suppl 3, 2013, 205-209
207
Hepatic GSH concentrations (mg/gm) (mean ± SD)
The mean GSH concentrations in liver in group I (V), group II (P
I
),
group III (P
II
), group IV (A +P
I
), group V (H+P
I
) were 5.24±0.42
mg/gm, 2.20±0.56 mg/gm, 3.05±0.18 mg/gm, 3.08 ±0.27 mg/gm,
3.51±0.61 mg/gm respectively (Table 3). The mean ± SD of GSH in
liver in paracetamol-treated group (group II) was significantly lower
(p<0.001) when compared to those of control (group I). So, pre-
treatment with aqueous extract and n-hexane extract of N. Sativa
significantly increased the GSH concentration in the liver and by
40%, 59.54% respectively (Table 3). GSH concentrations among
these groups were compared and significant difference was found [
p<0.001] (Table 4).
There were significant difference (p<0.001) between group I and II,
group I and IV, group I and V respectively. The significant difference
(p<0.001) was also observed between group II and V, Significant
difference (p<0.01) was observed between group II and IV. No
significant difference (P>0.05) between group IV and V.
Table 3: Hepatic GSH concentrations (mg/gm) (mean ± SD) in
pre-treatment groups
Group
(n = 6)
Hepatic GSH
(mg/gm)
Increased
(%)
p-value
I (V)
II (P
I
)
III (P
II
)
IV (A+P
I
)
V (H+P
I
)
5.24±0.42
2.20±0.56
3.05±0.19
3.08±0.27
3.51± 0.61
40%
59.54%
<0.001***
Data were expressed as mean ± SD. The statistical significance of
difference among the groups was evaluated by using one way
ANOVA test between group I and II
Table 4: Comparison of hepatic GSH concentrations (mg/gm)
between groups using Bonferroni‘t’ test
Comparing
groups
Compared
groups
Level of
significance
I (V)
II (P
I
)
III (P
II
)
IV (A+P
I
)
V (H + P
I
)
0.000***
0.000***
0.000***
0.000***
II (P
I
)
III (P
II
)
IV (A+P
I
)
V (H +P
I
)
0.011*
0.007**
0.000***
III (P
II
)
IV (A+P
I
)
V (H +P
I
)
1.000
NS
0.930
NS
IV
(A+P
I
)
V (H+P
I
)
1.000
NS
P<0.001= ***, P<0.01= **, P<0.05= *NS = no significant difference
(p>0.05) between groups
Hepatic MDA concentrations (nmol/mg of protein) (mean ±SD)
The mean MDA concentrations in liver in group I (V), group II (P
I
),
group III (P
II
), group IV (A +P
I
), group V (H+P
I
) (S+P
I
) were 70.90
±16.72, 208.95 ±14.30, 143.23 ± 8.19, 134.62 ± 7.80, 131.38 ± 6.02
(Table 5). MDA concentrations among these groups were compared
and significant difference was found [p<0.001] (Table 4). The mean
± SD of MDA in the liver in paracetamol-treated group (group II) was
significantly increased (p<0.001) when compared to those of control
(group I). So, pre-treatment with aqueous extract and n-hexane
extract of N. sativa significantly decreased the MDA concentration in
liver and percentages of reduction were 35.57%, 37.12% (Table 5).
There were significant difference (p<0.001) between group I and II,
group II and IV, group II and V. The study could not detect significant
differences (P>0.05) between group IV and V.
Table 5: Hepatic MDA concentrations (nmol/mg of protein)
(mean ± SD) in pre-treatment groups
Group
(n = 6)
MDA
(nmol/mg)
Reduction (%)
p-value
I (V)
II (P
I
)
III (P
II
)
IV (A+P
I
)
V(H +P
I
)
70.89 ±16.72
208.95 ± 14.30
143.23 ± 8.19
134.62 ±7.80
131.38 ± 6.08
35.57%
37.12%
<0.001***
*** indicates significant difference (p<0.001) between group I and II
Table 6: Comparison of hepatic MDA concentrations (nmol/mg)
between groups using Bonferroni‘t’ test
Comparing
groups
Compared
groups
Level of
significance
I(V)
II(P
I
)
III(P
II
)
IV(A+P
I
)
V(H+P
I
)
0.000***
0.000***
0.000***
0.000***
II(P
I
)
III(P
II
)
IV(A+P
I
)
V(H+P
I
)
0.000***
0.000***
0 .000***
III(P
II
)
V(A+P
I
)
V(H+P
I
)
1.000
NS
1.000
NS
IV(A+P
I
)
V(H+P
I
)
1.000
NS
P<0.001 = ***, NS = no significant difference (p>0.05) between
groups.
DISCUSSION
The pre-treated with the aqueous extract of N. sativa of paracetamol-
treated group shows decreased the elevated levels of serum ALT and
hepatic MDA and hepatic GSH concentrations were significantly
higher. Another study [51] have reported that the treatment of CCl
4
exposed rats with N. Saliva was able to protect the liver from
damage by decreased MDA and increased GSH (which indicates less
lipid peroxidation and less oxidative stress) levels in their study.
These findings are in accordance with the finding of the present
study where the aqueous extract of N. sativa administration suggests
less lipid peroxidation or less oxidative stress. Similar work was
reported [47] that pretreatment with the aqueous suspension of N.
sativa reduced the CCl
4
-induced liver damage by decreasing elevated
levels of serum enzymes (ALT, AST) and demonstrating almost
normal hepatic architecture. Similar improvement also reported in
hepatic damage [42] induced by CCl
4
in their experimental animals
following N. sativa seed administration. One more research [52]
claimed that 6% N. sativa seed diet was able to alleviate
paracetamol-induced hepatotoxicity. The antioxidant effects of the
N. sativa seed or its extracts were probably responsible for this
alleviation.
The pre-treatment of paracetamol-treated group with the n-hexane
extract of N. sativa decreased the elevated levels of serum ALT and
hepatic MDA while hepatic GSH concentrations were increased.
Another work [41] observed that the essential oil of N. sativa
possessed antioxidant activities and free radical scavenging activity.
A number of studies [46, 53] have in a similar way, reported that TQ,
(an ingredient of N. sativa oil) exhibits hepatoprotective activity
(possibly by its antioxidant effect). It also reported that
pretreatment of mice with TQ ameliorate the CCl
4
induced
hepatotoxicity, that is further evidenced by a significant change in
the elevated levels of serum ALT, AST, ALP and hepatic MDA
concentration along with a significant rise in hepatic sulfhydryl
concentration [53]. To sum up, all these findings in the n-hexane
Mainul Haque et al.
Asian J Pharm Clin Res, Vol 6, Suppl 3, 2013, 205-209
208
extract pre-treated group suggesting the TQ present in N. sativa oil
was probably responsible for the better alleviation of the n-hexane
extract pre-treatment compared to the aqueous extract pre-
treatment.
CONCLUSION
This study concludes that n-hexane extract and the aqueous extract
of N. sativa has a worthy hepatoprotective outcome. The protective
effect was higher in the n-hexane extract of N. sativa pre-treated
group than the aqueous extract pre-treated group. Well-designed
prospective study is suggested to formulate more cheaper and
indigenous treatment to ensure improved health care for common
Bangladeshi people.
DESCLAMER
This study obtained no financial assistance. This publication is
supported by Universiti Sains Malaysia Incentive Grant No
2012/0712. Authors do not have any conflict of interest.
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