Anti-asthmatic and Anti-allergic Effects of Thymoquinone on Experimentally-Induced Hypersensitivity

Abstract

Nigella sativa L. has been used in folk medicine for treatment of many diseases. The oil and seed constituents, in particular thymoquinone (TQ), have shown promising medicinal properties in the treatment and prevention of various diseases. The present study aims to investigate the potential effect of TQ on airway-induced hypersensitivity. Ovalbumin (OVA) sensitization and challenge in guinea pig tracheal muscle preparation were used in order to investigate the anti-asthmatic activity of TQ. To study the effect of TQ on acute lung injury, lipopolysaccharide (LPS) induced lung injury method was used. In addition, rat peritoneal mast cells (RPMCs) were collected to investigate the histamine release from the mast cells. Furthermore, to study the anti-allergic activity of TQ, the systemic anaphylactic shock technique induced by compound 48/80 was performed. Pretreatment with TQ (3 mg/kg, i.p.) for 5 days prior to ovalbumin sensitization showed a marked decrease in the response of the tracheal spirals to acetylcholine and histamine, as spasmogens in a cumulative dose response-curve. TQ (8mg/kg, i.p.) prevented most of the pathological detrimental changes that occurred in response to the endotoxin LPS as the inflammatory cells infiltration, lipid peroxidation (LP), glutathione depletion (GSH), tumor necrosis factor-alpha (TNF- a) and interlukin-1 beta (IL-1ß) levels in both boronchoalevolar lavage fluid (BALF) and lung tissue homogenates. Sensitization of rats induced a significant increase in the histamine release from RPMCs which is inhibited by pretreatment with TQ (8 mg/kg, i.p.). Similarly, pretreatment of mice with TQ (50 and 100 mg/kg), 1hr prior to injection of compound 48/80 (8mg/kg, i.p.) significantly inhibited the % of mortality of mice following the systemic anaphylactic reaction. Considering the anti-asthmatic, anti-inflammatory, antioxidant and anti-allergic activities of TQ reported in this study, one can conclude that TQ could be of therapeutic potentials in treating various diseases associated with airway-induced hypersensitivity.

Full text

31
Original Article
Egyptian Journal of Basic and Clinical Pharmacology
Anti-asthmatic and Anti-allergic Effects of Thymoquinone on Experimentally- Induced
Hypersensitivity
Amira E. Abd El Aziz, Nesrine S. El Sayed and Laila G. Mahran
Department of Pharmacology and Toxicology, Faculty of Pharmacy and Biotechnology, German University in Cairo
A B S T R A C T
Nigella sativa L. has been used in folk medicine for treatment of many diseases. Thymoquinone (TQ), a main
constituent of its oil and seeds, has shown promising medicinal properties in the treatment and prevention
of various diseases. The present study aims to investigate the potential effect of TQ on airway-induced
hypersensitivity. Ovalbumin (OVA) sensitization and challenge in guinea pig tracheal muscle preparation
were used in order to investigate the anti-asthmatic activity of TQ. To study the effect of TQ on acute lung
injury, lipopolysaccharide (LPS) - induced lung injury method was used. In addition, rat peritoneal mast
cells (RPMCs) were collected to investigate the release of histamine from these cells. Furthermore, to study
the anti-allergic activity of TQ, the systemic anaphylactic shock technique induced by compound 48/80 was
performed.
Pretreatment with TQ (3 mg/kg, i.p.) for 5 days prior to ovalbumin sensitization showed a marked decrease
in the response of the tracheal spirals to acetylcholine and histamine, as spasmogens in a cumulative
dose response–curve. TQ (8mg/kg, i.p.) prevented most of the pathological detrimental changes that
occurred in response to the endotoxin LPS as the inammatory cells inltration, lipid peroxidation (LP),
glutathione depletion (GSH), tumor necrosis factor-alpha (TNF- α) and interlukin-1 beta (IL-1β) levels in
both boronchoalevolar lavage uid (BALF) and lung tissue homogenates. Sensitization of rats induced a
signicant increase in the histamine release from RPMCs which is inhibited by pretreatment with TQ (8 mg/
kg, i.p.). Similarly, pretreatment of mice with TQ (50 and 100 mg/kg), 1hr prior to injection of compound
48/80 (8mg/kg, i.p.) signicantly inhibited the % of mortality of mice following the systemic anaphylactic
reaction. Considering the anti-asthmatic, anti-inammatory, antioxidant and anti-allergic activities of TQ
reported in this study, one can conclude that TQ could be of therapeutic potentials in treating various diseases
associated with airway-induced hypersensitivity.
Key Words: Thymoquinone, hypersensitivity, anaphylaxis, allergy, antioxidants, trachea, cytokines.
Corresponding Author: Nesrine S. El Sayed Email: nesrine.elsayed@guc.edu.eg
1. INTRODUCTION
Asthma is a common chronic disorder of the airways
characterized by airow obstruction, bronchial hyper-
responsiveness, and an underlying inammation (Busse
and Lemanske, 2001). Inammation has a central role in
the pathophysiology of asthma. It involves an interaction of
many cell types and multiple mediators with the airways that
eventually results in the characteristic features of the disease
(O’Byrne, 2009). Many inammatory cells, including
eosinophils, mast cells, macrophages and neutrophils, are
involved in the pathogenesis of airway inammation in
asthma (Kelly et al., 1998). These cells produce more reactive
oxygen species (ROS) (Cluzel et al., 1987) that affect airway
smooth muscles and simulate histamine release from mast
cells (Adler et al., 1990).
Airway hypersensitivity is associated with the
pathophysiology of asthma, acute lung injury and
anaphylaxis.
Acute lung injury (ALI) and acute respiratory distress
syndrome (ARDS) are among the major causes of respiratory
failure and are associated with a high frequency of mortality
and morbidity (Ware and Matthay, 2000). Lipoplysaccharide
(LPS) induces intense lung inammation, with macrophage
activation and recruitment of neutrophils to the intersticium,
alveoli, and to the airways of guinea-pigs (Gordon et al., 1991),
rats (Ulich et al., 1994), and mice (Harmsen, 1988). Neutrophil
recruitment is accompanied by an augmented lung vascular
permeability. Since these are the characteristic features of ALI/
December 2011, Vol. 1, No. 1: 31-41
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32
Anti-asthmatic and Anti-allergic Effects of Thymoquinone
ARDS, LPS-induced lung inammation has been used as a
model for these syndromes (Wheeldon, et al., 1992; Ware and
Matthay, 2005). Tumor necrosis factor-alpha (TNF- α) and
interlukin-1 beta (IL-1β) have been identied as important
pathogenic mediators of LPS-induced ALI. During sepsis,
the alveolar compartmentalization is lost, allowing passage of
cytokines, which were released into the bloodstream and to the
pulmonary endothelium. These cytokines have important roles
in lung dysfunction (Simpson and Casey, 1989).
Anaphylaxis is an acute hypersensitivity reaction with
multi-organ-system involvement that rapidly progresses to,
a severe life-threatening reaction (Madaan and Maddox,
2003). Mast cells are the primary effector cells involved in an
allergic or immediate hypersensitivity response. Activation
of mast cells occurs in response to a challenge by a specic
antigen against which the surface immunoglobulin E (IgE)
is directed, or by other IgE-directed ligands. Activated
mast cells can produce histamine and a wide variety of
inammatory mediators (Kalesnikoff & Galli, 2008) which
result into various acute and chronic allergic responses.
Mast cell degranulation can also be elicited by the synthetic
compound 48/80 (Ennis et al., 1980). Compared with the
natural process, compound 48/80 induces histamine release
from mast cells and is so used as a direct and convenient
reagent to investigate the mechanisms of allergy and
anaphylaxis (Allansmith et al., 1989).
Among the promising medicinal plants used nowadays
to relieve the symptoms of allergy and asthma is the Nigella
sativa, in which many of the reported pharmacological
effects are due to thymoquinone (TQ); the major active
constituent of the volatile oil (El-Dakhakhany, 1982; Labib,
2005). TQ has been shown to exert anti-inammatory effects
in a number of diseases including bronchial asthma (Kalus
et al., 2003).
The present study was performed in order to investigate
the possible anti-asthmatic and anti-allergic activities of TQ
and to explore the underlying mechanisms of action against
airway-induced hypersensitivity. These effects were studied
in-vitro to study the effect of TQ on ovalbumin (OVA)-
sensitized trachea of guinea pigs and in-vivo in three animal
models namely; LPS-induced lung injury, histamine release
from rat peritoneal mast cells and the systemic anaphylaxis.
2. MATERIALS AND METHODS
2.1. Animals:
In the present investigation, male albino mice weighing
25-30 g, male Sprague Dawley albino rats weighing 120-180
g and guinea pigs weighing 250-350g were used. Animals
were obtained from the animal house of the National
Research Center, Giza, Egypt. They were fed a standard
pellet chow (El-Nasr Chemical Company, Cairo, Egypt) and
had free access to water. All animals were maintained on
a 12-h light, 12-h dark cycle and housed for 1 week before
experimentation. This study was conducted in accordance
with the ethical procedures and policies, approved by The
Animal Care and Use Committee Faculty of Pharmacy and
Biotechnology, German University in Cairo.
2.2. Chemicals:
TQ (2-isopropyl-5-methyl-1, 4-benzoquinone), available
in a yellow crystalline form, was purchased from Sigma-
Aldrich, Germany. TQ was dissolved in saline using water
bath kept at 60°C. It was administered intraperitoneally in
different doses; 3 mg /kg in guinea pigs in tracheal muscle
preparation (Gilani et al., 2001), 8 mg/kg in LPS-induced
lung injury method (El Gazzar et al., 2006) in mice, 8mg/
kg in rat peritoneal mast cells method (Chakravarty, 1993)
and 10 mg/kg, 50 mg/kg, and 100 mg/kg (Choi et al., 2006)
in systemic anaphylactic shock technique in mice. LPS
(Escherichia coli lipopolysaccharide serotype 0111:B4;
Sigma, St. Louis, MO) is dissolved in 0.3 ml saline.
Bordetella pertussis vaccine was purchased from the holding
company for biological products and vaccines (VACSERA).
All chemicals and reagents were purchased from Sigma (St.
Louis, MO, USA), unless otherwise specied.
2.3. Methods:
2.3.1. Tracheal Muscle Preparation:
This model was performed according to the method
described by Costantin et al.,(1965); and Burka and Saad
(1984). Guinea pigs were allocated randomly to three groups,
each of 8 animals. Group 1: Control group, in which animals
received 0.5 ml saline, i.p. for ve consecutive days. Group
2: Sensitized group, in which animals were sensitized using
200 mg of OVA given as 100 mg i.p. and 100 mg S.C. into
two sides on the back of the animal at days 0 and 14. Group
3: TQ treated group, in which the animals received a dose
of 3mg/kg, i.p., in 0.5 ml saline, daily for ve consecutive
days before OVA sensitization. Groups 2 and 3 were given
OVA. After 21 days, the animals were sacriced by stunning
and exsanguinations, the throat was opened, the trachea was
removed, cut spirally, and suspended in Krebs-Henseleit
solution (118 mM NaCl, 4.7 mM KCl, 1.2 mMMgSO4, 2.2
mM CaCl2, 24.9mM NaHCO3, 1.2 mM KHPO4, and 11.1
mM glucose; at pH 7.4) maintained at 370C and bubbled
with carbogen (95% O2 & 5% CO2). The responses were
displayed using Hugo Sachs Electronik-ACAD (HSE-
ACAD), version 1.1.1.180 (Germany). The inhibitory
effect of TQ on the tracheal muscle was investigated using
a cumulative concentration-response curve of acetylcholine
(Ach) and histamine - induced contraction of tracheal spirals
at doses ranging from 10-9 M to 10-4 M.
2.3.2. LPS-Induced Acute lung injury (ALI):
LPS, a bacterial cell wall component, is a stimulus for the
initiation of local acute inammation. It is used for induction
of a model for ALI. The method was performed according to
Wang et al., (2008). Mice were allocated randomly to three
groups, each of 8 animals. Group 1: Control group, in which
the animals received 0.3 ml saline i.p. Group 2: LPS-treated
group, in which animals received 1 mg/ kg LPS dissolved
in 0.3 ml saline i.p. Group 3: TQ treated group, in which
the animals received TQ (8mg/kg) i.p. in 0.3 ml saline, 30
minutes before LPS injection. One hour later, the animals
were anesthetized and bronchoalveolar lavage (BAL) was
performed to collect BAL uid (BALF) for measurement
of total and differential inammatory cell counts. BAL was
performed through a tracheal cannula using 1.0 ml aliquots
of ice-cold Ca2+/ Mg2+-free phosphate-buffered medium

33
Amira E. Abd El Aziz et al.
(145 mM NaCl, 5 mM KCl, 1.9 mM NaH2PO4, 9.35 mM
Na2HPO4, and 5.5 mM dextrose; at pH 7.4) for a total of 3
ml for each mouse. The recovered BALF was centrifuged
at 300 g for 10 min at 4°C. The total number of cells was
counted using a standard hemocytometer. Cell differentiation
was examined by counting at least 200 cells on a smear
prepared by using cytospin and Wright-Giemsa staining.
Then, mice were sacriced, chest opened, and lungs taken
out and dried. Lung tissues were homogenized as 10%w/v
in 1% KCl solution and stored at -80oC for measurements
of oxidative stress parameters and inammatory cytokines.
Measurements of TNF-α, IL-1 β were done in BALF and
lung homogenates while reduced glutathione (GSH) and
malondialdehyde (MDA) contents were measured in lung
homogenates.
2.3.2.1. Determination of TNF-α and IL-1β in BALF and
lung homogenates:
BALF was centrifuged at 1000 xg for 20 minutes. Then the
supernatant was kept at -80 ˚C for subsequent determination
of TNF-α and IL-1β.The activity of TNF-α and IL-1β
was later determined by enzyme-linked immunosorbant
assay (ELISA) in accordance with to the manufacturer’s
recommended protocols, using Quantikine rats TNF-α/
TNFSF1A and mouse IL-1β/IL-1β1F2 immunoassay
commercial kits provided by R & D systems, Inc., Germany.
2.3.2.2. Estimation of reduced glutathione (GSH) in lung
homogenates:
Glutathione content was estimated according to the
method described by Tietze, (1969).
Estimation of GSH contents was performed
spectrophotometrically at 412 nm, using Ellman’s reagent
and expressed as mg/g wet tissue.
2.3.2.3. Estimation of malondialdehyde (MDA) content in
lung homogenates:
The determination of MDA content was performed
according to Mihara and Uchiyama, (1978). The lipid
peroxidation products were estimated in lung homogenates
in ice cold saline by the determination of the level of
thiobarbituric acid reactive substances (TBARS) that were
measured as MDA. TBARS concentration was expressed as
nmol MDA/ g wet weight.
2.3.3. Rat peritoneal mast cells preparation (RPMCs):
This model was carried out following the method
described by Atkinson et al., (1979).
Male Sprague Dawley albino rats were actively sensitized
with egg albumin according to the method of Chakravarty,
(1980). Rats were randomly classied into 3 groups, each
of 6 animals. Group 1: Control group, in which animals
received 0.5 ml saline i.p. Group 2: Sensitized group, in
which animals were sensitized using a mixture of 0.25 ml
2% (w/v) egg albumin in 0.9% NaCl solution and 0.5 ml
Bordetella pertussis vaccine (2 x 1010 bacilli) s.c. on the
rst day, a mixture of 0.25 ml 2% egg albumin and 0.5 ml
Freund’s incomplete adjuvant on the second day and 0.25 ml
of the egg albumin solution only on the third day. Group 3:
TQ treated group, in which the sensitized animals received
a dose of TQ 8mg/kg (i.p) daily for 21 days starting from
the rst day of sensitization. Aliquots of cells (1ml) from
each of the 3 groups were allowed to equilibrate at 37°C for
5 minutes. A 100 µl of the solution of the releasing agent
(compound 48/80) at a concentration of 0.2 µg / ml was
added. Histamine release was allowed to proceed for a further
10 min and the reaction was terminated by the addition
of 2 ml ice-cold Tyrode solution. Cells and supernatants
were separated by centrifugation at 3000 rpm for 2 min
at 4°C .The cell pellets were re-suspended in 3 ml Tyrode
solution and allowed to stand in a boiling water bath for 10
min to release residual histamine. Individual supernatants
were treated similarly. Histamine was then determined
spectrouorimetrically (Atkinson et al., 1979) using Vector
multiple plate counter, ELISA reader. Histamine release was
expressed as percentage of the total cellular content of the
amine.
2.3.4. Systemic Anaphylactic Shock Induced by
Compound 48/80 in mice:
This model was carried out according to the method
described by Choi et al., (2006). Mice were randomly
classied into 3 groups each of 10 animals; Group 1: Control
group, in which animals received 0.5 ml saline i.p. Group 2:
TQ (10 mg/kg, i.p). Group 3: TQ (50 mg/kg, i.p).Group 4:
TQ (100 mg/kg, i.p). In groups 2, 3 &4, TQ was injected one
hour before the injection of compound 48/80 (8 mg/kg, i.p.).
Mortality rate was monitored for 1 h after the induction of
anaphylactic shock.
2.3.5. Statistical analysis:
Data were expressed as the mean ± standard error (S.E.M).
Statistical analysis was performed using prism software,
version 5 (Graph pad Software, Inc., San Diego, USA).
One-way analysis of variance (ANOVA) followed by Tukey-
Kramer was used for comparing means of different groups.
P values < 0.05 were considered statistically signicant.
3. RESULTS
3.1. Effect of TQ on the tracheal muscle preparation of
sensitized guinea pigs contracted with acetylcholine and
histamine.
Sensitization of guinea pigs with OVA produced a
signicant increase in the sensitivity of the tracheal spirals
contracted with Ach and histamine by about 3 folds and 2.5
folds, respectively, compared to the normal control group.
Five days prior to sensitization, animals were pretreated with
TQ (3mg/kg, i.p) daily. This produced a signicant reduction
in the sensitivity of the tracheal smooth muscle preparations
to Ach and histamine by 71% and 74%, respectively as
compared to the sensitized animals (Table 1; Figures 1 and 2)
3.2.1. Effect of LPS (1 mg/kg) and TQ (8 mg/kg) on the total
and differential inammatory cell counts in the BALF of mice.
LPS (1 mg/kg) produced a signicant increase in the
total count and the differential counts of inammatory cells
in BALF. LPS caused a signicant increase in the number

34
Anti-asthmatic and Anti-allergic Effects of Thymoquinone
of total as well as the eosinophils, neutrophils, macrophages
and lymphocytes, as compared to the normal control values.
Pretreatment with TQ (8 mg/kg) suppressed the number
of total as well as the differential cell counts to values
approaching the normal values (Table 2).
3.2.2. Effect of LPS (1 mg/kg) and TQ (8 mg/kg) on TNF-α
and IL-1β levels in BALF and lung tissue homogenates of
mice.
Treatment of mice with LPS (1 mg/kg, i.p.) signicantly
increased TNF-α and IL-1β in both BALF and lung
homogenates by about one to two fold, as compared to
the normal control value. Pretreatment with TQ (8 mg/kg,
i.p.) ameliorated both TNF-α, IL-1β in BALF and lung
homogenates to values that are signicantly lower than the
respective LPS-induced increments and approaching the
normal control values (Figures 3,4,5&6).
3.2.3. Effect of LPS (1mg/kg) and TQ (8 mg/kg) on MDA,
GSH oxidative stress indices in the lung homogenate of
mice.
Treatment with LPS (1 mg/kg, i.p.) signicantly
increased MDA level in tissue homogenate by about one-fold
compared to the normal control values. GSH content was
almost decreased by 54%, as compared to the normal control
value. Pretreatment with TQ (8 mg/kg, i.p.) suppressed the
LPS-induced increase in MDA by 51% compared to the LPS
treated mice. On the other hand, TQ ameliorated the LPS-
induced depletion of GSH content by 86%, as compared to
the LPS treated mice (Figures 7 &8).
3.3. Effect of TQ (8mg/kg, i.p.) on the percentage of
histamine release from RPMCs of sensitized rats.
Sensitization of rats increased the percentage of histamine
release from RPMCs by 94%, compared to the normal control
group. TQ (8mg/kg, i.p.) produced a signicant inhibition of
the percentage of histamine release by 41%, as compared to
the sensitized rats (Table 3).
3.4. Effect of TQ (10, 50, 100 mg/kg, i.p.) on the systemic
anaphylactic shock induced by compound 48/80(8 mg/kg,
i.p.) in mice.
Compound 48/80 (8 mg/kg, i.p.) produced a fatal
shock in all mice. Pretreatment of mice with TQ
(10, 50,100 mg/kg) for 1 h resulted in a dose-dependent
reduction in the percentage of mortality with compound
48/80, compared to control mice receiving compound
48/80 only.
Table 1: Effect of TQ (3 mg/kg) on the maximal responses of acetylcholine and histamine on tracheal muscle preparation of normal and sensitized guinea
pigs.
Treatment
Groups
Normal Control Sensitized (OVA) OVA +TQ
(3 mg/kg)
Acetylcholine (10-9-10-4M) 20.12 ± 0.39 81.20 ± 0.97* 23.34 ± 0.60@
(71%)
Histamine (10-9-10-4M) 25.10 ± 0.24 85.43 ± 1.03* 22.21 ± 0.24@
(74%)
TQ (3 mg/kg, i.p.) was administered to guinea pigs for ve consecutive days prior to OVA (200 mg given as 100 mg i.p. and 100mg S.C., at days 0 and 14).
Sensitization lasted for 21 days.
Percentage inhibitions from the respective sensitized value are given in parentheses.
Results are expressed as the mean ± S.E, n = 8. Statistical analysis was carried out using one-way ANOVA followed by Tukey- Kramer multiple comparisons
test.
*Signicant difference from control group at P < 0.05.
@Signicant difference from OVA sensitized group at P < 0.05.
Table 2: Effect of LPS and TQ on the total and differential inammatory (eosinophils, neutrophils, macrophages and lymphocytes) cell counts in
bronchoalveolar lavage uid (BALF).
Parameters
Groups
Normal control LPS
(1mg/kg)
LPS (1 mg/kg) +
TQ ( 8 mg/kg)
Total cell count (x106cells) 0.22 ± 0.011 0.56 ± 0.042* 0.26 ± 0.05@
Eosinophils (x106cells) 0.042 ± 0.006 0.201 ± 0.012* 0.089 ± 0.003@
Neutrophils (x106cells) o.847 ± 0.03 2.218 ± 0.13* 1.002 ± 0.03@
Macrophages (x106cells) 0.398 ± 0.02 0.861 ± 0.043* 0.412 ± 0.035@
Lymphocytes (x106cells) 0.016 ± 0.004 0.109 ± 0.02* 0.029 ± 0.006@
Saline (10 ml/kg, i.p.), TQ (8 mg/kg, i.p.) were administered to mice, 30 minutes prior to LPS (1 mg/kg, i.p.).
Results are expressed as the mean ± S.E, n=8. Statistical analysis was carried out using one-way ANOVA followed by Tukey- Kramer multiple comparisons
test.
*Signicant difference from normal control group at P < 0.05.
@Signicant difference from LPS-treated group at P < 0.05.

35
Amira E. Abd El Aziz et al.
Table 3: Effect of TQ (8 mg/kg) on compound 48/80-induced histamine release from RPMCs.
Groups
Normal control Sensitized Sensitized + TQ (8 mg/kg)
%Histamine release 42.78±0.19 83.17±0.13* 48.67±0.69 @
RPMCs from normal (10 ml/kg saline, i.p.), sensitized and sensitized rats, pretreated with TQ (8 mg/kg, i.p.), were incubated for 10 minutes at 37ºC with
compound 48/80 (8mg/kg). % of histamine release was determined spectrouorometrically.
Results were expressed as the mean ± S.E, n=6. Statistical analysis was carried out using one-way ANOVA followed by Tukey- Kramer multiple comparisons
test.
*Signicant difference from normal control group at P < 0.05.
@Signicant difference from sensitized group at P < 0.05.
Table 4: Effect of TQ (10-50-100 mg/kg) on compound 48/80-induced systemic anaphylaxis in mice.
Treatment Dose (mg/kg) i.p. Mortality %
at specied time in minutes
10 15 30 60
48/80 8 20 30 100 100
TQ 10 10 20 100 100
TQ 50 0 10 60 100
TQ 100 0 0 10 40
Groups of mice (n=10/group) were injected with saline (10 ml/kg, i.p.) or TQ (10-50-100 mg/kg, i.p.) 1 h before the injection of compound 48/80
(8 mg/kg, i.p.). Mortality (%) within 1 h following compound 48/80 injection was presented as the number of dead mice ×100/total number of experimental
mice.
Figure 1: Effect of TQ (3 mg/kg) on the cumulative concentration-
response curve of Ach- induced contraction on guinea pig tracheal
spiral pre-sensitized with OVA.
Results were expressed as percentage of the maximal response of
Ach.
TQ (3 mg/kg, i.p.) was administered to guinea pigs for ve
consecutive days prior to OVA (200 mg given as 100 mg i.p. and
100 mg S.C., at days 0 and 14). Sensitization lasted for 21 days.
Results are expressed as the mean ± S.E, n=8. Statistical analysis
was carried out using one-way ANOVA followed by Tukey- Kramer
multiple comparisons test.
*Signicant difference from normal control group at P < 0.05.
@Signicant difference from ovalbumin sensitized group at P < 0.05.
Figure 2: Effect of TQ (3 mg/kg) on the cumulative concentration-
response curve of histamine-induced contraction of guinea pig
tracheal spiral pre-sensitized with OVA.
Results are expressed as percentage of maximal response of
histamine.
TQ (3 mg/kg, i.p.) administered to guinea pigs for ve consecutive
days prior to OVA (200 mg given as 100 mg i.p. and 100 mg S.C.,
at days 0 and 14). Sensitization lasted for 21 days.
Results are expressed as the mean ± S.E, n=8. Statistical analysis
was carried out using one-way ANOVA followed by Tukey- Kramer
multiple comparisons test.
*Signicant difference from normal control group at P < 0.05.
@Signicant difference from ovalbumin sensitized group at P < 0.05.

36
Anti-asthmatic and Anti-allergic Effects of Thymoquinone
Figure 3: Effect of LPS (1 mg/kg) and TQ (8 mg/kg) on TNF- α
level in BALF of mice.
Saline (10 ml/Kg, i.p.) and TQ (10 mg/kg, i.p.) were administered
to mice, 30 minutes prior to LPS (1 mg/kg, i.p.).
Results are expressed as the mean ± S.E, n = 8. Statistical analysis
was carried out using one-way ANOVA followed by Tukey- Kramer
multiple comparisons test.
*Signicant difference from normal control group at P < 0.05.
@Signicant difference from LPS-treated group at P < 0.05.
Figure 4: Effect of LPS (1 mg/kg) and TQ (8 mg/kg) on IL-1β level
in BALF of mice.
Saline (10 ml/Kg, i.p.), and TQ (8 mg/kg, i.p.) were administered to
mice, 30 minutes prior to LPS (1 mg/kg, i.p.).
Results are expressed as the mean ± S.E, n = 8. Statistical analysis
was carried out using one-way ANOVA followed by Tukey- Kramer
multiple comparisons test.
*Signicant difference from normal control group at P < 0.05.
@Signicant difference from LPS-treated group at P < 0.05.
Figure 5: Effect of LPS (1 mg/kg) and TQ (8 mg/kg) on the TNF-α
level in the lung homogenates of mice.
Saline (10 ml/kg, i.p.) and TQ (8 mg/kg, i.p.) was administered to
mice, 30 minutes prior to LPS (1 mg/kg).
Results are expressed as the mean ± S.E, n = 8. Statistical analysis
was carried out using one-way ANOVA followed by Tukey- Kramer
multiple comparisons test.
*Signicant difference from normal control group at P < 0.05.
@Signicant difference from LPS-treated group at P < 0.05.
Figure 6: Effect of LPS (1 mg/kg) and TQ (8 mg/kg) on the IL-1β
level in the lung homogenates of mice.
Saline (10 ml/Kg, i.p.) and TQ (8 mg/kg, i.p.) was administered to
mice, 30 minutes prior to LPS (1 mg/kg).
Results are expressed as the mean ± S.E, n = 8. Statistical analysis
was carried out using one-way ANOVA followed by Tukey- Kramer
multiple comparisons test.
*Signicant difference from normal control group at P < 0.05
@Signicant difference from LPS-treated group at P < 0.05.
Figure 7: Effect of LPS (1 mg/kg) and TQ (8 mg/kg) on MDA
content in lung tissue of mice.
Saline (10 ml/kg, i.p.) and TQ (8 mg/kg, i.p.) were administered to
mice, 30 minutes prior to LPS (1 mg/kg).
Results are expressed as the mean ± S.E, n = 8. Statistical analysis
was carried out using one-way ANOVA followed by Tukey- Kramer
multiple comparisons test.
*Signicant difference from normal control group at P < 0.05.
@Signicant difference from LPS-treated group at P < 0.05.
Figure 8: Effect of LPS (1 mg/kg) and TQ (8 mg/kg) on reduced
glutathione (GSH) in lung tissue of mice.
Saline (10 ml/kg, i.p.) and TQ (8 mg/kg, i.p.) were administered to
mice, 30 minutes prior to LPS (1 mg/kg).
Results are expressed as the mean ± S.E, n = 8. Statistical analysis
was carried out using one-way ANOVA followed by Tukey- Kramer
multiple comparisons test.
*Signicant difference from normal control group at P < 0.05.
@Signicant difference from LPS- treated group at P < 0.05.

37
Amira E. Abd El Aziz et al.
4. DISCUSSION
In the present study, pretreatment of the sensitized guinea
pigs with TQ showed a signicant decrease in the response of
the tracheal spirals to histamine and acetylcholine compared
to that produced with sensitized animals. The traditional
use of Nigella sativa seeds and its active ingredients have a
substantial impact on the inammatory diseases mediated by
histamine (Kalus et al., 2003). These results were supported
by previous studies that investigated TQ’s effect on the guinea
pig isolated tracheal zig-zag preparation pre-contracted by
carbachol. TQ caused a concentration-dependent decrease in
the tension of the tracheal smooth muscle (Al-Majed et al.,
2001). The TQ - induced relaxation is probably mediated, at
least in part, by inhibition of lipoxygenase (LO) products of
arachidonic acid metabolism and possibly by non-selective
blocking of the histamine and serotonin receptors. This
relaxant effect of TQ, further support the traditional use
of black seeds to treat bronchial asthma (Al-Majed et al.,
2001). However, in an in vivo study, increasing respiratory
rate and intra tracheal pressure of guinea pigs due to i.v.
administration of volatile oil from Nigella sativa has been
demonstrated. These respiratory effects were mediated via
release of histamine with direct involvement of histaminergic
mechanisms and indirect activation of muscarinic
cholinergic mechanisms (El-Tahir et al., 1993). In previous
studies, the relaxant, anticholinergic (functional antagonism)
and antihistaminic, effects of Nigella sativa have been
demonstrated on guinea pig tracheal chains (Boskabady and
Shahabi, 1997; Boskabadi and Shiravi, 2000). This relaxant
effect is not attributed to the calcium channel blocking effect
of Nigella sativa extracts. However, a potassium channel
opening effect was suggested for its extracts (Boskabady
et al., 2004). Results of a recent study showed a preventive
effect of thymoquinone on tracheal responsiveness and
inammatory cells of lung lavage of sensitized guinea
pigs which was comparable or even greater than that of the
inhaled steroid (Keyhanmanesh et al., 2010). The reduction
in the response of the tracheal spirals to acetylcholine and
histamine could be also due to a direct inhibitory effect of
TQ on the release of mediators involved in chronic airway
inammation including histamine, serotonin and bradykinin.
Those mediators are known to be responsible for the
increased vascular permeability and blood ow observed
during the early stages of inammation (kumar et al., 2007).
In the present investigation, injection of LPS increased
inammatory total and differential cells count namely,
eosinophils, neutrophils, macrophages and lymphocytes both
in BALF and lung homogenates. It increased the oxidative
stress by increasing lipid peroxidation and decreasing GSH
levels. Similarly, it stimulated cytokines; TNF-α and IL-
1β production. Pretreatment with TQ (8 mg/kg) prevented
most of the pathological detrimental changes that occurred
in response to the endotoxin LPS. TQ reduced inammatory
cell inltration into respiratory airways, oxidative stress in
terms of lipid peroxidation and increased GSH levels and
decreased TNF-α and IL-1β production.
The main pathological feature of ALI/ARDS is pulmonary
edema brought about by aggregation of pulmonary
neutrophils and increased permeability of alveolar-capillary
membrane (Balibrea and Arias-Dlaz, 2003). In a previous
study, it was found that TQ improved oxygenation while
both TQ and steroids protect lung tissue from hazardous
effects of human gastric juice histopathologically in a rat
model of ALI/ARDS (Isik et al., 2005).
Oxidative stress has been shown to play a major role in
mediating lung injury both in animal models and ALI/ARDS
patients. Oxygen radicals were found to be responsible for
LPS-induced lung injury (Feng et al., 2004). Pulmonary
function was improved in ARDS patients in response to
N-acetylcysteine antioxidant therapy (Bernard, 1990). Asti
et al., (2000) have demonstrated that LPS treatment in mice
resulted in acute hemorrhagic lung injury with increased
neutrophil inltration and increased lung MPO activity.
Activation of the neutrophil NADPH oxidase leads to
liberation of reactive oxygen species (ROS) (Chanock et al.,
1994). ROS have been implicated in tissue injury associated
with inammation, organ ischemia and reperfusion, ARDS,
rheumatoid arthritis and asthma (Smith, 1994). O2•־
contributes to the inammatory response through several
mechanisms including lipid peroxidation, increase of
vascular permeability, cellular recruitment and tissue damage
(Boueiz and Hassoun, 2009). It was found to contribute to the
inammatory process via different pathways including lipid
peroxidation, enzymatic inactivation (Crow and Beckman,
1995), glutathione depletion (Phelps et al., 1995) and DNA
damage (Inoue and Kawanishi, 1995).
Recent study showed that LPS-administered rats with
TNF-α blocking peptide signicantly suppressed the levels of
pulmonary endothelin ( ET-1) and that differential alteration
in ET expression may be mediated by TNF-α and may, in
part, account for the pathogenesis of acute lung injury in
endotoxemia (Jesmin et al., 2011). TNF-α and IL-1β mediate
the neutrophil migration observed in several experimental
models and also in human inammatory disease (Gong et al.,
2009). TNF-α through induction of neutrophils adhesion and
their subsequent activation, mediates neutrophil-dependent
increase in vascular permeability (Lentsch and Ward, 2000).
El Gazzar et al. (2006) proved that TQ attenuated
pulmonary inammation in a mouse model of allergic
asthma by decreasing Th-2 cytokines and inammatory cell
inltration in the lung. Attenuation of cellular recruitment
observed with TQ can lead to subsequent reduction of
oxidative and/or nitrosative stresses and their accompanied
deleterious effects including increased vascular permeability,
lipid peroxidation, GSH depletion and tissue damage.
Moreover, TQ improved renal GSH depletion and lipid
peroxides accumulation in ifosfamide-induced renal damage
(Badary, 1999).
TQ was shown to inhibit cyclooxygenase (COX) and
5- lipooxygenase (5-LOX) pathways in rat peritoneal
leukocytes stimulated with calcium ionophore A23187
(Houghton et al., 1995). Another study showed that TQ
attenuates the inammatory response in activated mast cells
by blocking transcription and production of TNF-α. TQ
exerted its effects by targeting the nuclear transactivation of

38
Anti-asthmatic and Anti-allergic Effects of Thymoquinone
pro-inammatory transcription factor nuclear factor-kappa
B (NF-κB) (El Gazzar et al., 2007). Another study proved
that licorice avonoids effectively attenuate LPS-induced
acute pulmonary inammation in mice through inhibition
of inammatory cells inltration and inammatory mediator
release. This subsequently, reduces neutrophil recruitment
into lung and neutrophil-mediated oxidative injury. This may
be achieved through reduction of LPS-induced lung TNF-α
and IL-1β mRNA expression, increasing SOD activity (Xie
et al., 2009).
It was also reported that TQ tends to decrease the elevated
levels of LPS-induced TNF-α in macrophages from diabetic
rats (El-Mahmoudy et al., 2005).
The effect of TQ on TNF-α was previously investigated.
El Gazzar et al. (2007) showed that TQ attenuated the
proinammatory response in LPS-stimulated mast cells by
blocking transcription and production of TNF-α. Tekeoglu
et al. (2006) also reported that TQ exerted an inhibitory
effect on TNF-α production in rheumatoid arthritis, a well
established chronic inammation model in rats.
Adrenomedullin was shown to protect rats from LPS-
induced ALI. Similar to thymoquinone, adrenomedullin
decreased the total cells and neutrophils count and reduced
the TNF- α levels (Itoh et al., 2007). Additionally, statins were
shown to protect against LPS-induced lung injury through
reduction of inammatory cell inltration and decreased
cytokines production namely TNF-α and IL-6 (Jacobson
et al., 2005; Yao et al., 2006). Moreover, it was proved that
the free radical scavenger, edaravone, is able to attenuate
LPS-induced acute lung injury in mice via suppression of
pro-inammatory cytokine production by lung macrophages
(Tajima et al., 2008). The causal link between oxidative
stress and cytokine production was shown in vitro in alveolar
epithelium. Inhibition of GSH biosynthesis by buthionine
sulfoximine increased intracellular oxidative stress and
enhanced the production of IL-1β, IL-6, and TNF-α (Haddad
et al., 2001).
The anti-allergic effect of TQ was evaluated by its
reduction of the release of histamine from the rat peritoneal
mast cells and by decreasing the mortality of mice in the
systemic anaphylaxis reaction induced by compound 48/80.
Pretreatment with TQ inhibited the elicited increase in
histamine release compared to the sensitized group. These
results clearly indicated that TQ inhibited mast cell-mediated
immediate-type allergic reactions. It was reported that rats
pretreated with Nigella sativa oil before induction of ulcer
caused a signicant decrease in gastric mucosal histamine
content (El-Dakhakhny et al., 2000).
Previous reports showed that nigellone was very effective
in inhibiting histamine release induced by the secretagogues:
antigen in sensitized cells, compound 48/80, and the calcium
ionophore A23187 through decreasing intracellular calcium
by inhibiting its uptake and stimulating the efux, and by
an inhibition on protein kinase C. There is also indication
for a mild inhibition of oxidative energy metabolism
contributing to some inhibition of the release (Chakravarty,
1993). Moreover, Gilani et al., (2001) found that the crude
extract of Nigella sativa seeds exhibits spasmolytic and
bronchodilator activities mediated possibly through calcium
channel blockade. Recent studies done on curcumin and
α-lipoic acid showed that the inhibition of mediator release
from RPMCs may be due to inhibition of calcium uptake
and augmentation of intracellular cAMP levels (Choi et al.,
2010 a, b). These ndings together with the known anti-
oxidant properties of TQ suggest that it could be used in the
treatment of immediate-type allergic diseases.
It is well-recognized that compound 48/80 can induce a
mast cell-dependent, non-specic anaphylactoid reaction.
Compound 48/80 is known to activate mast cell secretory
processes by increasing the rate of guanosine triphosphate-
gamma S (GTP)γS binding to G-proteins (Palomäki and
Laitinen, 2006). This, in turn, triggers activation of PKC and
Ca2+ signaling and results in the release of histamine from
these cells. TQ potently suppressed histamine release probably
through the inhibition of the degranulation process following
a rise in intracellular Ca2+ levels, in accordance with previous
reports (Suzuki et al., 2005; Nugroho et al., 2009).
From the previous ndings, we can conclude that TQ
possesses marked anti-allergic and anti- asthmatic activity
and may have benecial effects in the prevention or treatment
of many allergic diseases.
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