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Review
Immunomodulatory and anti-inflammatory action of Nigella sativa and
thymoquinone: A comprehensive review
Amin F. Majdalawieh ⁎,MuneeraW.Fayyad
Department of Biology, Chemistry and Environmental Sciences, College of Arts and Sciences, American University of Sharjah, Sharjah, P.O. Box 26666, United Arab Emirates
abstractarticle info
Article history:
Received 9 April 2015
Received in revised form 17 June 2015
Accepted 22 June 2015
Available online xxxx
Keywords:
Nigella sativa
Thymoquinone (TQ)
Immunomodulation
Adaptive immunity
Inflammation
Many herbal products are now used as remedies to treat various infectious and non-infectious conditions. Even
though the use of herbs and natural products is much more evident in the Eastern world, their use in Western
culturesis continuously increasing. Although the immunomodulatoryeffects of some herbs have beenextensive-
ly studied, research related to possible immunomodulatory effects of many herbs and various spices is relatively
scarce. Here, we provide a comprehensive review of the immunomodulatory and anti-inflammatory properties
of Nigella sativa, also known as black seed or black cumin, and its major active ingredient, thymoquinone (TQ).
This review article focuses on analyzing in vitro and in vivo experimental findings thatwere reported with regard
to the ability of N. sativa and TQ to modulate inflammation, cellular and humoral adaptive immune responses,
and Th1/Th2 paradigm. The reported capability of N. sativa to augment the cytotoxic activity of natural killer
(NK) cells against cancer cells is also emphasized. The molecular and cellular mechanisms underlying such im-
munomodulatory and anti-inflammatory effects of N. sativa and TQ are highlighted. Moreover, the signal trans-
duction pathways implicated in the immunoregulatory functions of N. sativa and TQ are underscored.
Experimental evidence suggests that N. sativa extracts and TQ can potentially be employed in the development
of effective therapeutic agents towards the regulation of immune reactions implicated in various infectious
and non-infectious conditions including different types of allergy, autoimmunity, and cancer.
© 2015 Elsevier B.V. All rights reserved.
Contents
1. Introduction.............................................................. 296
2. Effects on inflammation......................................................... 296
2.1. Effects of N. sativa on inflammation................................................ 296
2.2. Effects of TQ on inflammation .................................................. 298
3. Effectsoncellularimmunity....................................................... 298
3.1. Effects of N. sativa oncellularimmunity.............................................. 298
3.2. EffectsofTQoncellularimmunity................................................. 299
4. Effectsonhumoralimmunity...................................................... 299
4.1. Effects of N. sativa onhumoralimmunity ............................................. 299
4.2. EffectsofTQonhumoralimmunity................................................ 300
5. EffectsonTh1/Th2paradigm ...................................................... 300
5.1. Effects of N. sativa onTh1/Th2paradigm.............................................. 300
5.2. EffectsofTQonTh1/Th2paradigm ................................................ 300
6. EffectsonNKcytotoxicactivity ..................................................... 300
6.1. Effects of N. sativa onNKcytotoxicactivity............................................. 300
6.2. EffectsofTQonNKcytotoxicactivity ............................................... 301
7. Signaling pathways underlying the immunomodulatory and anti-inflammatoryeffectsofTQ ......................... 301
8. Conclusion .............................................................. 303
References................................................................. 303
International Immunopharmacology 28 (2015) 295–304
⁎Corresponding author.
E-mail address: amajdalawieh@aus.edu (A.F. Majdalawieh).
http://dx.doi.org/10.1016/j.intimp.2015.06.023
1567-5769/© 2015 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
International Immunopharmacology
journal homepage: www.elsevier.com/locate/intimp
1. Introduction
The medicinal use of natural herbs and spices is deeply rooted in the
history and folklore of human beings, and many herbs and spices have
been incorporated into the traditional medicine of virtually all human
cultures. Throughout history, people have used a variety of herbs and
plants to prevent or treat diseases. In a study done by Barrett and col-
leagues, it was found that as many as 50% of Americans use herbal rem-
edies in a given year [1], indicating that the use of herbal medicine is
both widespread and growing dramatically. It is the gentle, nourishing,
and synergistic actions of herbal remediesthat make them an excellent
treatment choice [2]. Whether a treatment is approached using natural
herbs or chemical drugs, whose synthesis is based on properties and ac-
tions of those herbs, medicinal herbs have played a major role in the de-
velopment of modern, conventionalmedicine, and they will remain, like
a historical treasure, as a source of therapy. Immunomodulation in-
volves immunostimulation or immunoinhibition of certain cellular
and/or humoral immune responses. This suggests that the immune sys-
tem functions as an open integrated system, rather than functioning in a
strictly autarchic manner. Many herbs and food additives have been
shown to exert immunomodulatory effects by stimulating various
branches and components of the immune system. For example, black
pepper (Piper nigrum) and cardamom (Elettaria cardamomum)have
been shown to possess potent immunomodulatory effects [3].Some
food products have been shown to trigger certain molecular and cellular
mechanisms that lead to food allergies and oral tolerance including
immunomodulation [4].“Immunonutrition”is a term that has emerged
during the last decade, which has been adopted to describe diets that
have certain chemicals in the form of arginine, glutamine, fish oil, and
nucleotides alone or in combination [5,6]. Although the link between
many herbal products and various aspects of immunity is well-
established and although the immune-related therapeutic potential of
such herbal products cannot be underestimated, the exact molecular
pathways and cellular mechanisms by which these products manifest
their immunomodulatory effects are not fully understood.
Nigella sativa, commonly known as black seed or black cumin, is a di-
cotyledonous flowering plant that belongs to the botanical family
Ranunculaceae [7,8]. Although it originated in South and Southwest
Asia, it is widely grown in the Middle East, Northern Africa, and South-
ern Europe [9,10]. Morphologically, the plant is 20–90 cm tall and pro-
duces 5–10 petal-bearing flowers that are typically white, pale blue,
pale purple, or in some cases, dark blue [11].N. sativa plant and its
seeds are shown in Fig. 1. The black seed reproduces asexually, whereby
the fruit forms with its encapsulated whiteseeds. Once ripen, the encap-
sulated white seeds break open, become exposed to the air, and turn
black in color, and hence their name [11].N. sativa seeds are traditional-
ly used as a food preservative, additive, or a spice in various cultures.
However, many cultures use the seeds or their oil for their various med-
ical benefits. Several therapeutic effects have been attributed to N. sativa
seeds in their purified as well as crude components. Thymoquinone
(TQ) (2-isopropyl-5-methyl-1,4-benzoquinone), which has the chemi-
cal formula C
10
H
12
O
2
and a molecular weight of 164.2 g/mol, is a
major phytochemical bioactive ingredient in N. sativa oil and extracts
[12–14]. The chemical structure of TQ is shown in Fig. 2.TQmakes
about 30–48% of N. sativa seeds [11], and since its isolation and charac-
terization in 1963 [15], it has been extensively studied by many re-
searchers worldwide. Some of the medical benefits of N. sativa and TQ
are due to their anti-histaminic, anti-inflammatory, anti-hypertensive,
hypoglycemic, anti-cancer, and immunity-boosting effects [7–14,
16–19]. There is a growing research interest in evaluating TQ as a ther-
apeutic agent against various in vitro and in vivo disease models.
In this review, the molecular and cellular mechanisms utilized by
N. sativa and TQ to modulate inflammation, cellular and humoral adap-
tive immune responses, and Th1/Th2 differentiation are summarized.
The signal transduction pathways involved in mediating the immuno-
modulatory and anti-inflammatory effects of N. sativa and TQ are also
reviewed. Very recent in vitro and in vivo experimental findings related
to the immunoregulatory roles of N. sativa and TQ and their pathophys-
iological significance are discussed.
2. Effects on inflammation
2.1. Effects of N. sativa on inflammation
Inflammatory reactions are protective biological processes carried
out by endogenous mediators to eliminate harmful stimuli. The most
common inflammatory mediators include eicosanoids, oxidants, cyto-
kines, chemokines, and lytic enzymes. These are often secreted by mac-
rophages and neutrophils, but could also be produced by the injured
tissue itself [20,21]. Moreover, cyclooxygenase (COX) and lipoxygenase
(LO) enzymes are key factors in the biosynthesis of prostaglandins
(PGs) and leukotrienes (LTs), which are critically involved in inflamma-
tory responses [22,23]. Despite being a protective biological process, in-
flammation can have detrimental outcomes in the affected tissue
leading toits damage, especially if the inflammatory reaction is accom-
panied by the production of reactive oxygen species (ROS). Nitric oxide
(NO) is a highly reactive free radical that could trigger toxic oxidative
reactions leading to inflammation and tissue damage.
Houghton and colleagues have shown that N. sativa fixed oil signifi-
cantly inhibited cyclooxygenase (COX) and 5-lipoxygenase (5-LO)
pathways of arachidonate metabolism in rat peritoneal leukocytes due
to inhibited formation of thromboxane B2 (TXB2) and leukotriene
B4 (LTB4) metabolites [24]. Similarly, N. sativa oil treatment was accom-
panied by inhibited production of 5-LO products and 5-hydroxyl-
eicosa-tetra-enoic acid (5-HETE) in rat polymorphonuclear leukocytes
at half maximal inhibitory concentration (IC
50
)of25±1μg/ml and
24 ± 1 μg/ml, respectively [25]. Mutabagani and El-Mahdy evaluated
the anti-inflammatory effects of N. sativa seeds oil by assessing
carrageenan-induced hind paw edema and cotton seed pellet granulo-
ma formation in rats [26]. Intraperitoneal injection of N. sativa oil
(0.66 ml and 1.55 ml/kg) led to a significant dose-dependent suppres-
sion of hind paw edema by 64.1% and 96.3%, respectively [26]. More-
over, intraperitoneal injection of N. sativa oil (0.33 ml and 0.66 ml/kg)
significantly reduced granuloma weightby 17.6% and 46.9%, respective-
ly [26]. These effects were comparable to those caused by indomethacin
(3 mg/kg), which inhibited edema by 46.9% and granuloma formation
by 34.4% [26]. The researchers suggested that such potent anti-
inflammatory effects of N. sativa oil could be attributed to the inhibited
generation of eicosanoids and lipid peroxidation [26]. Very similar find-
ings using N. sativa seeds oil were reported in mice [27]. Moreover, Al-
Ghamdi demonstrated that an aqueous extract of N. sativa (500 mg/kg)
significantly reduced carrageenan-induced inflammation (paw edema)
in Albino Wistar rats and Albino Swiss mice [28]. The anti-
inflammatory effects of N. sativa were comparable to 100 mg/kg aspirin
[28]. Similar anti-inflammatory and analgesic effects of N. sativa seeds
Fig. 1. N. sativa plant and its seeds.
296 A.F. Majdalawieh, M.W. Fayyad / International Immunopharmacology 28 (2015) 295–304
polyphenols were reported by Ghannadi and colleagues [29].In vivo
studies have demonstrated that N. sativa exerts anti-inflammatory ef-
fects that may be effective in ameliorating different inflammatory condi-
tions including rheumatoid arthritis (RA) [7], experimental allergic
encephalomyelitis (EAE) [30,31], and acetic acid-induced ulcerative coli-
tis [32]. A recent study by Nikakhlagh and colleagues investigated the
anti-inflammatory role of N. sativa oil in patients with allergic rhinitis.
In that study, patients consumed placebo capsules or capsules containing
0.5 ml of N. sativa oil on a daily basis for 30 days [33].Itwasdemonstrat-
ed that N. sativa oil treatment could significantly suppress nasal mucosal
congestion, nasal itching, sneezing attacks, turbinate hypertrophy, and
mucosal pallor [33]. The study did not test the potential effects of specific
components of N. sativa oil nor traced the biochemical pathways under-
lying the protective effects of N. sativa oil against allergic rhinitis. Rather,
the results were mainly based on comparisons between concentrations
of eosinophils and IgE in nasal mucosa. Moreover, oral administration
of hexanic extract of N. sativa significantly reduced the clinical symptoms
(plasma concentration of mouse mast cell protease-1 (MMCP-1) and in-
testinal mast cell numbers) associated with ovalbumin-induced allergic
diarrhea in mice, with no effects on total immunoglobulin E (IgE) levels,
ovalbumin-specific IgE levels, or interleukin (IL)-4, IL-5, IL-10, and inter-
feron γ(IFNγ) release from mesenteric lymphocytes following ex vivo
re-stimulation with ovalbumin [34].
Yousefiand colleagues evaluated the physiological significance of the
anti-inflammatory properties of N. sativa in treating hand eczema [35].
That study represented a clinical trial comparing the potential of
N. sativa to that of betamethasone and eucerin, potent glucocorticoids
steroid with anti-inflammatory and immunosuppressive properties, to
alleviate hand eczema. The results indicated that N. sativa seems to be
as efficacious as betamenthasone in improving the clinical condition
and life quality of eczema patients and decreasing the severity of the con-
dition [35]. In a recent study, Abdel-Aziz and colleagues suggested that
N. sativa fixed oil reduces peripheral blood eosinophil count and im-
proves lung inflammation in a murine model of allergic asthma [36].In
another study, a hydroethanolic extract of N. sativa seed shows preven-
tive effects on tracheal responsiveness in a guinea pig model of chronic
obstructive pulmonary disease (COPD) [37]. In a very recent clinical
trial, Hadi and colleagues investigated the anti-oxidant and anti-
inflammatory effects of N. sativa oil in patients with RA [38]. Oral admin-
istration of N. sativa oil (1 g/patient/day) in the form of capsules for
8 weeks was associated with significantly elevated IL-10, but not tumor
necrosis factor α(TNFα), serum levels, suggesting that N. sativa oil or
other extracts can be potentially used in combination with RA medica-
tions to alleviate the inflammatory responses in RA patients [38].In
addition, an in vitro study showed that treatment of lipopolysaccharide
(LPS)- and IFNγ-challenged thioglycollate-elicited peritoneal macro-
phages with N. sativa aqueous extract (25–100 μg/ml) led to a dose-
dependent suppressed expression of IL-6, TNFα, and NO; key pro-
inflammatory mediators [39].Thesefindings are consistent with those
reported by Mahmood and colleagues, who demonstrated that an aque-
ous extract of N. sativa exhibited an inhibitory effect on NO production in
murine macrophages and argued that the active component is non-
proteininnature[40].
In an in vivo study, it was demonstrated that whole body irradiation
of Wistar rats caused a significant reduction in hemolysin antibodies ti-
ters, plasma total protein concentration, and globulin concentration as
well as delayed type hypersensitivity (DTH), leukopenia, depletion of
lymphoid follicles of the spleen and the thymus gland [41]. Moreover,
irradiation led to a significant increase in malondialdehyde concentra-
tion with a significantdecrease in the activity of plasmaglutathione per-
oxidase, catalase, and erythrocyte superoxide dismutase [41].Oral
administration of N. sativa oil (1 ml/kg/day for 5 days/week) prior to ir-
radiation considerably attenuated the irradiation-related biochemical,
immunological, and pathological effects [41]. Along the same lines,
Rastogi and colleagues demonstrated that an ex vivo treatment of
mouse splenic lymphocytes with an ethanolic extract of N. sativa 1h
prior to irradiation prevented that the formation of lipid peroxides
and intracellular ROS; factors that mediate radiation-induced DNA dam-
age and apoptosis [42]. Furthermore, oral administration of an ethanolic
extract of N. sativa (100 mg/kg/day for 5 days) protected irradiated
Swiss albino mice against oxidative injury to the spleen and liver tissues
in as measured by the activity of anti-oxidant enzymes and lipid perox-
idation [42]. These radio-protectiveeffects were attributed to the radical
scavenging potential of N. sativa [42]. Consistently, Velho-Pereira and
colleagues demonstrated that oral administration of a macerated ex-
tract of N. sativa seeds (100 mg/kg) protected the healthy liver, spleen,
brain, and intestinal tissues of fibrosarcoma-bearing mice as well as in
tumor-free mice [43]. Protection was measured through estimates of
biochemical and blood parameters such as the levels of anti-oxidant en-
zymes, protein oxidation in organs, and total leukocyte count in blood.
The researchers attributed such radio-protective effects to the ability
of the N. sativa extract constituents to scavenge free radicals and pre-
vent oxidative stress [43]. Very recently, a clinical trial revealed that pa-
tients with RA who received a daily dose of 1 g N. sativa oil for 8 weeks
displayed significantly lower serum levels of NO and malondialdehyde
(MDA), a biomarker of lipid peroxidation, with no significant effect on
the activity of superoxide dismutase (SOD) or catalase (CAT); anti-
oxidant enzymes [38]. This clinical study suggests that N. sativa can
Fig. 2. The chemical structures of TQ and other chemical constituents found in N. sativa seed extracts and oil.
297A.F. Majdalawieh, M.W. Fayyad / International Immunopharmacology 28 (2015) 295–304
potentially reduce certain aspects of oxidative stress, and hence, it can
be used as an adjuvant therapy to manage oxidative stress in RA
patients [38].
2.2. Effects of TQ on inflammation
About two decades ago, TQ was shown to exert anti-inflammatory
effects by inhibiting COX and 5-LO biosynthesis in rat peritoneal leuko-
cytes due to inhibited formation of TXB2 and LTB4 metabolites [24].
Similarly, El-Dakhakhny and colleagues demonstrated that TQ signifi-
cantly inhibited the production of 5-LO products and5-HETE in rat poly-
morphonuclear leukocytes at IC
50
= 0.26 ± 0.02 μg/ml and IC
50
=
0.36 ± 0.02 μg/ml, respectively [25]. Further experimental investigation
by Mansourand Tornhamre revealed that TQ strongly inhibits LT forma-
tion in human blood cells [44]. The inhibition of LT formation is caused
by a significant reduction in the activity of both 5-LO and LTC4 synthase
in a time- and dose-dependent manner [44]. This study also revealed
that TQ could inhibit the formation of LTC4 and LTB4 from endogenous
substrates in human granulocyte suspensions at IC
50
=1.8μMand
IC
50
= 2.3 μM, respectively, and within a time period of 15 min [44].
TQ was shown to significantly inhibit 5-LO activity at IC
50
=3μMas
judged by suppressed conversion of exogenous arachidonic acid into
5-HETE in sonicated polymorphonuclear cell suspensions [44].
Moreover, TQ significantly suppressed LTC4 synthase activity at
IC
50
=10μM, leading to inhibited conversion of exogenous LTA4 into
LTC4 in sonicated polymorphonuclear cell suspensions [44]. The activity
of LTC4 synthase was inhibited by TQ even in the presence of
staurosporine, a non-selective protein kinase inhibitor, indicative of
the potent inhibitory action of TQ against LTC4 synthase activity [44].
Likewise, TQ was also shown to significantly down-regulate LT forma-
tion in a mouse model of allergic asthma leading to attenuated airway
inflammation [45].
The in vivo anti-inflammatory properties of TQ were extensively
studied in two main inflammatory diseases, ulcerative colitis and EAE.
Ulcerative colitis is an inflammatory disease characterized by symptoms
of acute inflammation, ulceration and bleeding of colonic mucosa [46].
Inflammatory factors such as eicosanoids and ROS play a role in the pro-
gressionof this disease [46]. It hasbeen established that substanceswith
anti-inflammatory and/or anti-oxidant properties tend to significantly
improve these symptoms [47,48]. Intra-colonic injection of rats with
3% acetic acid causes severe colitis in rats. Mahgoub demonstrated
that pretreatment of rats with 5 mg/kg TQ for 3 days provided partial
protection against acetic acid-induced colitis compared to the control-
treated rats [32]. Indeed, a dose of 10 mg/kg of TQ led to complete pro-
tection of rats against acetic acid-induced colitis [32].Interestingly,
10 mg/kg TQ led to a greater protection against acetic acid-induced co-
litis in rats compared to 500 mg/kg sulfasalazine, an anti-colitis drug
[32]. In a more recent study performed by Duncker and colleagues, the
anti-inflammatory role of TQ, extracted from N. sativa seeds, was inves-
tigated in ovalbumin-sensitized BALB/c mice against allergic diarrhea
[34]. Pre-treatment with TQ (13 μg/kg) caused a reduction in intestinal
mast cell numbers and MMCP-1 expression compared to the control
sample, thereby reducing the overall clinical scores of ovalbumin-
induced allergic diarrhea. TQ treatment had no significant effects on
total plasma IgE or ovalbumin-specific IgE levels, and the intra-gastric
treatment of mice with TQ did not affect IL-4, IL-5, IL-10 or IFNγsecre-
tion by mesenteric lymphocytes after ex vivo re-stimulation with oval-
bumin [34].Recently,TQwasdemonstratedtosignificantly reduce NO
production in LPS-challenged primary glial cells isolated from Wistar
rats [49].
TQ was also found to be effective against EAE, a T cell-mediated au-
toimmune disease of the central nervous system. Studies suggest that
oxidative stress is one of the major causes underlying the progression
of EAE, which can be of increased severity upon astrocyte proliferation
and infiltration of inflammatory cells [50]. Experimental evidence indi-
cates that a daily dose of 1 mg/kg TQ in a rat model of EAE ameliorated
the clinical sign of EAE [31]. TQ treatment led to a significant inhibition
of perivascular puffing and infiltration of mononuclear cells in the brain
and spinal cord, and a significant increase in glutathione levels in red
blood cells [31]. Glutathione is a tripeptide commonly used as a detox-
ifying agent due to its preventive effect against damage caused by oxi-
dative stress mediated by ROS [51]. Amelioration of EAE symptoms by
TQ administration has also been shown in Lewis rats with induced
EAE [52]. The ability of TQ to induce glutathione levels has also been
confirmed in NO-deficient hypertensive rats, leading to attenuated hy-
pertension and renal damage [53]. Similar effects were reported in the
kidneys of Wistar albino rats with induced acute renal toxicity [54],
the liver tissue of Wistar albino rats with induced-hepatocarcinoma
[55], and the kidney and liver tissues of streptozotocin diabetic rats
[56].Hence,thebeneficial effects of TQ against EAE are due to its ability
to act as a glutathione inducer, allowing TQ to serve as an anti-oxidant
and anti-inflammatory agent. This suggests that TQ could be a promis-
ing therapeutic agent in the treatment of EAE and similar disorders
such as multiple sclerosis. Moreover, a recent study demonstrated that
a single 3 mg/kg dose of TQseems to exert significant protective effects
in rat adjuvant-induced model of RA [57] and a guinea pig model of
asthma [58]. Very recently, feeding Sprague Dawley rats a diet supple-
mented with N. sativa fixed oil (4%) and essential oil (0.3%) for
56 days significantly enhanced the activity of several anti-oxidant en-
zymes including glutathione S-transferase (GST), glutathione reductase
(GR), glutathione peroxidase (GPx), catalase (CAT), and superoxide dis-
mutase (SOD) [59].
Intraperitoneal injection of TQ (0.5, 1, and 5 mg/kg) in rats was asso-
ciated with a significant dose-dependent reduction in paw edema by
38.9%, 56.6%, and 104.9%, respectively [26]. In addition, granuloma for-
mation was also significantly inhibited in rats that were intraperitoneal-
ly injected with TQ (3 and 5 mg/kg) by 13.0% and 48.1%, respectively
[26]. These effects were comparable to those caused by indomethacin
(3 mg/kg), which inhibited edema by 46.9% and granuloma formation
by 34.4% [26]. It was suggested that diminished levels of eicosanoids
and lipid peroxidation may be behind the anti-inflammatory activity
of TQ [26]. Hajhashemi and colleagues suggested that TQ may exert
very similar effects in mice [27].
3. Effects on cellular immunity
3.1. Effects of N. sativa on cellular immunity
Aside from its established anti-inflammatory, anti-oxidant, and anti-
tumor activities, research initiatives are growing extensively to assess
the potential of N. sativa to modulate adaptive immunity. Haq and col-
leagues examined the effects of whole and soluble extracts of N. sativa
seeds on human peripheral blood mononuclear cells (PBMC) response
to different mitogens in vitro [60].N. sativa extracts exhibited potent
stimulatory effects on PBMC response to pooled allogeneic cells, but
not to phytohemagglutinin (PHA) or concanavalin A (ConA), two
T cell mitogens [60].N. sativa extracts increased the secretion of IL-3
from PBMCs cultured in presence or absence of pooled allogeneic cells,
but no effects on IL-2 secretion from mitogen-stimulated PBMCs were
observed [60]. Later on, and using mixed lymphocyte cultures, the
same group demonstrated that whole and purified protein extracts of
N. sativa seeds exerted a stimulatory and suppressive roles on un-
stimulated lymphocytes and pokeweed mitogen (PWM)-activated lym-
phocytes, respectively [61].N. sativa extracts had no effect on the secre-
tion of IL-4 from lymphocytes, both in presence and absence of PWM
[61]. IL-8 secretion was suppressed by N. sativa extracts when the lym-
phocytes were left un-stimulated, but it was enhanced in PWM-
activated lymphocytes [61].Thesefindings indicate that N. sativa exerts
profound stimulatory effects on cellular immunity. In vivo,N. sativa oil
has also been reported to stimulate CD4
+
(helper) T lymphocytes in a
murine cytomegalovirus (CMV) model using BALB/c mice [62]. More-
over, oral administration of N. sativa fixed oil (2 ml/kg) for a period of
298 A.F. Majdalawieh, M.W. Fayyad / International Immunopharmacology 28 (2015) 295–304
12 weeks was demonstrated to cause a significant decrease in leukocyte
and platelet counts in Wistar-Kyoto rats [63].Inanotherin vivo study, it
was demonstrated that oral administration of N. sativa oil significantly
improved lymphocyte count (i.e. lymphocyte proliferation) in the pe-
ripheral blood of streptozotocin (STZ)-induced diabetic hamsters [64].
The possible immunomodulatory effects of the volatile oil of N. sativa
seeds were evaluated in Long–Evans rats that were challenged with a
specific antigen (typhoid TH) [65]. Oral administration of N. sativa oil
in antigen-challenged rats significantly decreased splenocyte and neu-
trophil counts while increasing peripheral lymphocyte and monocyte
counts [65]. Ebaid and colleagues evaluated the immunomodulatory
potential of N. sativa oil by assessing its ability to ameliorate the cellular
immunological changes that accompany the treatment with chloram-
phenicol, an antibiotic [66]. Oral administration of chloramphenicol in
albino rats led to a significant increase in total leukocyte count, a de-
crease in neutrophil and lymphocyte count, and a decrease in the values
of both rosette and plaque formingcells [66]. Intriguingly, oral adminis-
tration of N. sativa oil (90 mg/kg/day) for 30–60 days almost completely
restored the indicated immunological parameters back to normal levels
in a time-dependent manner [66], indicating that N. sativa oil can poten-
tially enhance cellular immune responses in vivo.
Onifade and colleagues presented a case study performed on a
46-year old human immunodeficiency virus (HIV)-infected man, who
displayed complete recovery and sero-reversion of HIV infection after
treatment with N. sativa concoction (60% N. sativa seeds and 40%
honey) for a period of 6 months [67]. The study revealed that a daily
consumption of 20 ml N. sativa concoction led to the disappearance of
fever, diarrhea, and multiple pruritic lesions as early as day 5, day 7,
and day 20 post administration of the N. sativa concoction [67].CD4
+
cell count significantly dropped from 250 cells/mm
3
to 160 cells/mm
3
despite significant reduction in HIV viral load (~27,000 copies/ml
to ~1000 copies/ml) after 30 days of the N. sativa concoction regime
[67]. By the end of the study (i.e. after 6 months), HIV screening was
sero-negative, and the CD4
+
cell count significantly increased to 650
cells/mm
3
with undetectable viral (HIV-RNA) load, parameters that
persisted at least 2 years after the completion of the N. sativa concoction
therapy [67]. This case study uncovers the therapeutic efficacy of
N. sativa against HIV infection. However, such studies must be per-
formed using N. sativa seeds without any additives to conclusively con-
firm a therapeutic effect of N. sativa in the treatment of HIV infection.
3.2. Effects of TQ on cellular immunity
Despite the fact that numerous studies investigated the anti-
inflammatory, and anti-oxidant, and anti-tumor activities of TQ, rela-
tively little research has been conducted to examine its role in modulat-
ing specific cellular and humoral responses. El Gazzar investigated the
possible modulatory effect of TQ on the production of Th2 cytokines,
IL-5, IL-10, and IL-13, from LPS-activated RBL-2H3 cells, a rat mast cell
line [70].TQ(10μM) significantly suppressed the expression of IL-5
and IL-13, but had no effect on IL-10 expression [68]. The expression
of IL-5 and IL-13 is regulated by several transcription factors including
globin transcription factor (GATA), activator protein 1 (AP-1), and nu-
clear factor of activated T cells (NF-AT). Further analysis revealed that
TQ suppresses IL-5 and IL-13 expression by blocking GATA, but not
AP-1 or NF-AT, promoter binding and transcriptional activity via
inhibited expression of GATA-1 and GATA-2 [68].
A study by Xuan and colleagues investigated whether TQ has any ef-
fect on LPS-induced dendritic cell (DC) maturation, survival, and cyto-
kine release using mouse bone marrow-derived DCs; chief regulators
of innate and adaptive immune responses [69].LPSisknowntotrigger
DC maturation and cytokine production. It was demonstrated that LPS-
activated DCs expressed significantly higher levels of CD11c and major
histocompatibility complex II (MHC-II) (surface markers), CD40 and
CD86 (co-stimulatory molecules), and CD54 (adhesion molecule), fac-
tors that mediate DC-T cell clustering and antigen presentation [69].
TQ (1–20 μM) co-treatment abrogated, in a dose-dependent manner,
LPS stimulatory effects with regard to surface marker expression and
DC-T cell clustering [69]. Furthermore, TQ co-treatment significantly
hampered LPS-induced release of IL-10, IL-12, and TNFαfrom DCs as
well as LPS-induced phosphorylation of pro-survival factors protein ki-
nase B (AKT) and extracellular signal-regulated kinase 1/2 (ERK1/2)
[69]. Therefore, TQ plays an inhibitory role towards LPS-induced DC
maturation, survival, and cytokine release.
In an in vivo study, the ability of TQ to improve gestational diabetes-
associated complications and T cell immune responses in rat offspring
was examined by Badr and colleagues [70]. Gestational diabetes was
triggered in female Swiss albino rats by administration of STZ, which
was associated with several postpartum complications including
reduced number of neonates, increased serum levels of the pro-
inflammatory cytokines (IL-1β,IL-6,andTNFα), decreased IL-2 serum
level, suppressed proliferation of superantigen (staphylococcal entero-
toxin B)-stimulated T lymphocytes, and reduced number of circulating
and thymus-homing CD4
+
(helper) and CD8
+
(cytotoxic) T lympho-
cytes, due to apoptosis, in the neonates [70]. Oral administration of TQ
(20 mg/kg/day) in diabetic mothers during pregnancy and lactation pe-
riods significantly increased the number and mean body weight of neo-
nates, and it restored serum levels of the indicated pro-inflammatory
cytokines, IL-2 serum level, the proliferative capacity of T lymphocytes,
as well as the number of circulating and thymus-homing CD4
+
and
CD8
+
T lymphocytes in the offspring at birth and 6 weeks after birth
[70]. Furthermore, TQ treatment restored T cell receptor (TCR)/CD28-
mediated F-actin polymerization; an event that is crucial for T cell acti-
vation and immunologic synapse formation, which was hampered in
the offspring of rats with STZ-induced gestational diabetes [70].
Mohany and colleagues investigated the immunomodulatory
effect of TQ on pesticide-induced immunotoxicity in male albino rats
[71]. Immunotoxicity was induced by a daily oral administration
(for 28 days) of imidacloprid (IC); an insecticide that caused a signifi-
cant increase in the catalytic activity of hepatic enzymes (alanine ami-
notransferase (ALT), aspartate aminotransferase (AST), and alkaline
phosphatase (ALP)), and malondialdehyde (MDA) serum level [71].IC
treatment was also associated with a significant decline in total leuko-
cyte counts, phagocytic activity, chemokine expression, and chemotax-
is, as well as the appearance of severe histopathological lesions in the
liver, spleen, and thymus tissues of the challenged animals [71].Intra-
peritoneal injection of TQ (1 mg/kg, once every 7 days) reversed the
IC-induced immunological, biochemical, and histopathological effects,
leading to augmented totalleukocyte count, phagocytic activity, chemo-
kine expression, and chemotaxis, as well as decreased activity of hepatic
enzymes and serum MDA levels [71]. These findings underscore the po-
tential of TQ to regulate various aspects of cellular immune responses.
4. Effects on humoral immunity
4.1. Effects of N. sativa on humoral immunity
In an in vitro study using splenic mixed lymphocyte cultures, an
aqueous extract and an ethyl acetate column chromatographic fraction
of N. sativa seeds significantly enhanced the proliferation of lymphocytes
cultured in presence of ConA, but not LPS [72].Thesefindings indicate
that N. sativa exerts profound suppressive effects on humoral immune
responses. A study performed by Islam and colleagues investigated po-
tential immunomodulatory effects of the volatile oil of N. sativa seeds
in Long-Evans rats that were challenged with a specific antigen (typhoid
TH) [65]. Results showed that oral administration of N. sativa oil in
antigen-challenged rats significantly reduced serum antibody titer [65].
Recently, Ebaid and colleagues evaluated the immunomodulatory po-
tential of N. sativa oil by assessing its ability to ameliorate humoral-
mediated immunological changes that accompany the treatment with
chloramphenicol, an antibiotic [66]. Similarly, oral administration of
chloramphenicol in albino rats led to a very low hemagglutination titer
299A.F. Majdalawieh, M.W. Fayyad / International Immunopharmacology 28 (2015) 295–304
[66]. Oral administration of N. sativa oil (90 mg/kg/day) for 30–60 days
almost completely restored the indicated immunological parameter
back to normal levels in a time-dependent manner [66], indicating that
N. sativa oil can potentially enhance humoral immune responses in
vivo. Sapmaz and colleagues have recently reported that administration
of N. sativa oil in rats led to a significant decrease in serum IgA, IgM, and
complement component 3 (C3) levels, which were induced by formalde-
hyde inhalation [73]. These findings suggest that N. sativa can
significantly decrease acute antibody responses and C3 levels in
formaldehyde-challenged rats, exerting an immunoregulatory role in
humoral immunity.
4.2. Effects of TQ on humoral immunity
The only findings suggesting a possible immunomodulatory role
of TQ in regulating humoral immune responses were reported by
Mohany and colleagues who investigated TQ effects on pesticide-
induced immunotoxicity in male albino rats [71]. Among several
biochemical and histopathological changes, IC treatment caused a sig-
nificant decline in total Ig levels (especially IgGs) and a significant inhi-
bition of antibody hemagglutination [71]. Intraperitoneal injection of TQ
(1 mg/kg, once every 7 days) reversed the IC-induced immunological
effects, leading to significantly increased total Ig levels and antibody
hemagglutination [71]. The findings of this study provided hints that
TQ may potentially modulate the outcome of humoral immune
responses.
5. Effects on Th1/Th2 paradigm
5.1. Effects of N. sativa on Th1/Th2 paradigm
Once activated, CD4
+
T helper cells further differentiate into Th1 or
Th2 cells, which specialize in thesecretion of Th1 cytokines (IL-2, IL-12,
IFNγ,andTNFα) and Th2cytokines (IL-4, IL-5, IL-10, and IL-13) [74].Ul-
timately, the decision to differentiate into Th1 or Th2 cells tips the bal-
ance either towards cellular or humoral immune response, and hence,
agents that can influence the Th1/Th2 balance can potentially alter the
outcome of the adaptive immune response in various diseases and med-
ical conditions [74].
A crude extract of N. sativa seeds was shown to have no significant
effects on IL-2 and IL-4 secretion from resting and activated human
PBMCs [60].N. sativa extracts had no effect on the secretion of IL-4
from lymphocytes, both in presence and absence of PWM [61].Howev-
er, whole and purified protein extracts of N. sativa seeds were shown to
significantly enhance the production of TNFαfrom un-stimulated and
PWM-activated lymphocytes [61]. In an attempt to explain the anti-
viral activity of N. sativa oil against CMV infection in BALB/c mice,
Salem and Hossain demonstrated that intraperitoneal injection of
N. sativa oil (100 μg/100 μl/mouse) elevated serum IFNγlevels in
CMV-infected mice, but not in un-infected mice, 3 days post treatment
[62]. Büyüköztürk and colleagues evaluated the effects of N. sativa oil
on cytokine production of splenic mononuclear cells (MNCs) in
ovalbumin-sensitized BALB/c mice [75]. It was demonstrated that oral
administration of N. sativa oil (0.3 ml/mouse/day) for 1 month had no
significant effects on the production of IL-4, IL-10, or IFNγfrom splenic
MNCs in ovalbumin-sensitized BALB/c mice, suggesting that N. sativa
does not possess immunomodulatory properties with regard to Th1
and Th2 cell responsiveness to allergen stimulation [75]. Using an aque-
ous extract of N. sativa, we assessed the potential of the extract to mod-
ulate lymphocyte proliferation and alterTh1 and/or Th2 cytokine profile
in vitro [39].N. sativa aqueous extract (1–100 μg/ml) significantly en-
hanced the proliferation of BALB/c splenocytes in a time- and dose-
dependent manner [39]. Our study further revealed that the aqueous
extract of N. sativa favored Th2 cytokine secretion and inhibited Th1 cy-
tokine secretion in a dose-dependent manner [39]. The secretion of IL-4
and IL-10 was significantly enhanced when splenocytes were treated
with the aqueous extract of N. sativa (50–100 μg/ml) in absence of
ConA. The stimulatory effect of the aqueous extract of N. sativa on IL-4
and IL-10 secretion was potent enough to significantly enhance the
secretion of IL-4 and IL-10 from splenocytes co-treated with ConA
[39]. Conversely, IFNγsecretion from splenocytes was significantly
inhibited by the aqueous extract of N. sativa even in the presence of
ConA [39]. In a recent in vivo study, oral administration of an ethanolic
extract of N. sativa (200 mg/kg/day) led to a significant increase in
serum IL-10, but not IL-4 or IFNγ, levels in male Wistar rats after 24 h
of treatment [76].Thesefindings indicate that N. sativa may play a crit-
ical role in modulating the balance of Th1/Th2 lymphocytes, leading to
altered Th1/Th2 cytokine profiles. However, the reported effects of
N. sativa on the Th1/Th2 paradigm are inconsistent, most likely due dif-
ferent experimental conditions including cell type, species, dose, mode
of administration, and method of detection. Future studies are required
to shed more light on the modulatory effects of N. sativa on Th1/Th2 cy-
tokine balance using different in vitro and in vivo model systems. Such
investigation is crucial given that altering the Th1/Th2 cytokine balance
may lead to various medical conditions. For instance, Th2cytokines pro-
duced by stimulated mast cells play a critical role in regulating allergic
inflammatory responses, and indeed, immunomodulation leading to
enhanced Th1 response and suppressed Th2 response has been pro-
posed as an immunotherapeutic approach to prevent and treat various
allergic reactions [77].
5.2. Effects of TQ on Th1/Th2 paradigm
Noteworthy, while a number of studies have evaluated a possible
role of N. sativa extracts in Th1/Th2 cell polarization [39,75,76],
compelling experimental evidence suggesting that TQ may influence
the Th1/Th2 balanceis largely lacking. The only study that may suggest
a role of TQ in regulating Th1/Th2 differentiation revealed that TQ
(10 μM) can reduce the production of cytokines that induce Th2 differ-
entiation (e.g. IL-5 and IL-13), but not IL-10, from LPS-activated RBL-
2H3 rat mast cells by down-regulating the transcriptional activity of
GATA-1 and GATA-2, but not AP-1 or NF-AT [68].Futurein vitro and in
vivo studies should examine the likely possibility that TQ may regulate
Th1/Th2 differentiation.
6. Effects on NK cytotoxic activity
6.1. Effects of N. sativa on NK cytotoxic activity
A line research has identified the enhancement of the cytotoxic ac-
tivity of NK cells as a mechanism underlying the anti-cancer effects ex-
hibited by N. sativa [39,78–82]. El-Kadi and colleagues performed an in
vivo study onhealthy volunteers to evaluate theimmunomodulatory ef-
fects of N. sativa seeds oil with regard to the ratio of helper to suppressor
T cells and the cytotoxic activity of NK cells [78,79]. The study revealed
that the ratio of helper to suppressor T cells and NK cytotoxic activity
were significantly enhanced in participants who ingested N. sativa oil
for 4 weeks [78,79]. Along the same lines, another in vivo study done
on mice revealed that 1-week oral consumption of an aqueous extract
of N. sativa resulted in a significant increase in splenic NK cell count
and a significant enhancement of splenic NK cytotoxic activity against
YAC-1 mouse tumor cells [81].Thesein vivo findings are in agreement
with those of several in vitro studies. Abuharfeil and colleagues demon-
strated that a fresh aqueous extract of N. sativa (50 and 100 μg/ml) re-
sulted in a significant increase in the cytotoxic activity of splenic NK
cells against YAC-1 cells (% cytotoxicity 43.7 ± 3.6 and 62.7 ± 5.6 at
200:1 effector:target (E:T) ratio, 45.7 ± 5.7 and 44.6 ± 6.2 at 100:1
E:T ratio, and 13.6 ± 2.7 and 18.3 ± 3.1 at 50:1 E:T ratio, respectively)
[80]. In fact, when compared to the old dried aqueous extract and the
ethanolic extract of N. sativa, the fresh aqueous extract of N. sativa ap-
peared to exhibit a greater potency in inducing NK cytotoxic
activity [80].In vitro experimental evidence from our laboratory
300 A.F. Majdalawieh, M.W. Fayyad / International Immunopharmacology 28 (2015) 295–304
provided further confirmation that an aqueous extract of N. sativa sig-
nificantly enhances killing of YAC-1 cells due to an increased NK cyto-
toxic activity, leading to 14% (3 folds) and 23% (4.5 folds) cytotoxicity
at 50 μg/ml and 100 μg/ml concentrations, respectively, at 200:1 E:T
ratio [39]. Importantly, the lack of a significant, direct cytotoxic effect
of N. sativa extract against YAC-1 cells in absence of NK cells indicated
that the enhanced killing of YAC-1 cells is due to the ability of N. sativa
extract to improve NK cytotoxic activity rather than exerting immedi-
ate, directcytotoxic effect by the extract [39].Wehavereportedsimilar
observations in which aqueous extracts (100 μg/ml) of black pepper
(P. nigrum) and cardamom (E. cardamomum) caused a significant in-
crease (35% and 45% cytotoxicity, respectively) in the NK cytotoxic ac-
tivity against YAC-1 cells at 200:1 E:T ratio [3]. Collectively, these in
vitro and in vivo findings strongly suggest that N. sativa significantly
augments the cytotoxic potential of NK cells against tumor cells,
which seemsto be at least one mechanismof action that explainsthe re-
ported anti-tumor effects of N. sativa and perhaps other herbs. Notewor-
thy, the effects of an aqueous extract of N. sativa on NK cytotoxic activity
was assessed using splenocytes obtained from C57/BL6 mice in our
study [39], whereas Abuharfeil and colleagues evaluated the cytotoxic
activity of NK cells in the presence of an aqueous extract of N. sativa
using splenocytes obtained from BALB/c mice [80], leading to similar
findings. This indicates that the enhancement of NK cytotoxic activity
caused by N. sativa seems to be common to different strains of mice
and perhaps to different species. However, further research is certainly
required to confirm this possibility using splenocytes from a wide range
of mice strains and different animal models. In another in vitro study,
Shabsoug and colleagues used an aqueous extract of N. sativa (10–500
μg/ml) and reported a significantly enhanced cytotoxic activity (26.6–
67.7% cytotoxicity) of NK cells previously isolated from human blood
against K562 tumor cells [82]. The improved cytotoxic potential of NK
cells was primarily due to the ability of N. sativa extract to significantly
up-regulate the expression of IFNγand TNFα; cytokines that induce po-
tent tumoricidal effects against tumor cells [82]. Moreover, the same
study revealed that treatment of NK cells with an aqueous extract of
N. sativa (10–500 μg/ml) led to a significant increase in the activity of
granzyme A (GZMA) and N-acetyl-β-D-glucosaminidase (NAGase);
key proteolytic enzymes involved in the killing event by NK cells against
target cells [82]. Collectively, these findings point to the augmentation
of NK cytotoxic activity against tumor cells as a plausible, effective
mechanism that may provide at least partial explanation of the reported
potent anti-tumor effects displayed by different extracts of N. sativa
seeds. Contrary to these findings, an early in vivo study showed that in-
traperitoneal injection of N. sativa oil (100 μg/100 ml/mouse) for 7 days
led to a significant decrease in the splenic NK cell count in non-infected
and CMV-infected BALB/c mice [62]. Interestingly, however, N. sativa oil
treatment caused a significant suppression of NK cytotoxic activity in
CMV-infected, but not in non-infected, mice [62]. The same study re-
vealed that in vitro treatment of splenic NK cells isolated form non-
infected mice with N. sativa oil (100 μg/ml) significantly decreased
their cytotoxic activity against YAC-1 cells [62]. The discrepancy be-
tween these different in vitro and in vivo studies with regard to the
immunopotentiating effects of N. sativa extracts on NK cytotoxic activity
needs to be resolved. Despite the fact that some of the signaling mole-
cules that seem to be involved in N. sativa-induced stimulation of NK
cytotoxic activity have been identified, our understanding of the exact
signal transduction pathways and cellular factors employed in
these pathways is far from lucid. Hence, future well-designed in vitro
and in vivo studies should be directed at identifying the targeted recep-
tors, the cytosolic proteins, and the transcription factors that are in-
volved in the reported N. sativa-induced immunomodulation of NK
cytotoxic activity. Additionally, we believe that the immunopotentiating
effects of N. sativa on NK cytotoxic activity should be validated both in
vitro and in vivo using a wide range of primary and transformed NK
cells, numerous primary tumors and cancer cell lines, and different ani-
mal models.
6.2. Effects of TQ on NK cytotoxic activity
Although enhancement of NK cytotoxic function has been demon-
strated by a number of studies as a plausible mechanism that, at least
partially, mediates the anti-tumor activity of N. sativa [39,78–82],com-
pelling experimental evidence suggesting that TQ particularly utilizes
this mechanism of action to manifest its anti-tumor activity is largely
lacking. In a very recent study, Salim and colleagues reported in vitro
and in vivo anti-leukemic effects of TQ on murine leukemia WEHI-3
cells [83]. In that study, the researchers concluded that TQ promoted
NK cytotoxic activity [83]. However, no clear experimental evidence
was provided to support this conclusion. This likely possibility should
be examined in the future to evaluate whether or not TQ does exert
its anti-tumor effects, at least partially, by provoking NK cytotoxic activ-
ity against tumor cells.
7. Signaling pathways underlying the immunomodulatory and
anti-inflammatory effects of TQ
Several studies have been conducted to unfold the molecular mech-
anisms and signal transduction pathways employed by TQ to manifest
their immunomodulatory and ant-inflammatory effects. While increas-
ing levels of glutathione seems to be a mechanism underlying the ben-
eficial effects of TQ against some inflammatory disorders, other
mechanisms may also be in play. Nuclear factor κB(NF-κB) comprises
a family of ubiquitously expressed, eukaryotic transcription factors
that are critically involved in various biological processes including in-
flammation [21]. Mohamed and colleagues demonstrated that TQ treat-
ment inhibited NF-κB signaling in the brain and spinal cord of rats with
induced EAE [31], suggesting that NF-κB could potentially be a molecu-
lar target for TQ. Later studies supported such findings. An in vitro study
using human proximal tubular epithelial cells that werestimulated with
advanced glycation end products (AGEs) revealed that TQ led to a
significant suppression of AGE-induced NF-κB activation and IL-6 ex-
pression [84]. El Gazzar demonstrated that TQ treatment led to a signif-
icant suppression of LPS-induced TNFαproduction in the rat basophil
cell line (RBL-2H3) [68]. Further investigation revealed that such an ef-
fect is due to the ability of TQ to inhibit LPS-induced NF-κB signaling by
preventing the translocation of p65 to the nucleus [85].Noticeably,TQ
did not cause a change in the cytosolic activation or nuclear expression
of NF-κB. Rather, TQ increased the nuclearlevels of the repressive NF-κB
p50 homodimer while decreasing the nuclear levels of the trans-
activating NF-κB p65:p50 heterodimer [85]. Similarly, TQ was demon-
strated to exhibit a dose-dependent inhibitory role against angiotensin
II-triggered NF-κB activation and IL-6 expression in human proximal tu-
bular epithelial cells with maximum inhibitory effect at a concentration
of 500 nM [86]. Sethi and colleagues investigated in detail the effect of
TQ on the NF-κB signaling pathway in human chronic myeloid leukemia
cells (KBM-5) [87]. It was demonstrated that TQ inhibited TNFα-
induced NF-κB activation in a time- and dose-dependent manner via
suppressing inhibitor of NF-κB kinase (IKK) activity, leading to sup-
pressed phosphorylation, and hence, degradation, of inhibitor of NF-
κBα(IκBα)[87]. Consistently, Chehl and colleagues demonstrated
that TQ (25–75 μM) significantly reduces the expression of IL-1β,
TNFα, monocyte chemoattractant protein 1 (MCP-1), and COX-2 in
pancreatic ductal adenocarcinoma (PDA) cells, HS766T cells, AsPC-1
cells, and MIA-PaCa cells in a time- and dose-dependent manner [88].
The TQ-mediated inhibited expression of these proinflammatory medi-
ators was due to blocked nuclear translocation of p65 leading to sup-
pressed NF-κB activation [88]. Using isolated human RA fibroblast-like
synoviocytes, TQ was shown to block LPS-induced activation of p38
mitogen-activated protein kinase (MAPK), ERK1/2, and NF-κB, leading
to suppressed expression of proinflammatory mediators IL-1β, TNFα,
matrix metalloproteinase 13 (MMP-13), COX-2, and prostaglandin E2
(PGE2) [57]. Thus, it is likely that TQ exerts its anti-inflammatory effects
by modulating a number of factors that are critically involved several
301A.F. Majdalawieh, M.W. Fayyad / International Immunopharmacology 28 (2015) 295–304
signaling pathways including the NF-κB pathway, p38 MAPK pathway,
and ERK1/2 pathway. These studies, however, do not rule out the possi-
bility that other signaling pathways may also be implicated in the TQ-
mediated anti-inflammatory effects.
Peroxisome proliferator-activated receptor γ(PPARγ) is a nuclear
receptor that serves as a transcription factor to turn on the expression
of various gene products in various cell types [89]. Active PPARγhas
been shown byseveral studies to exert anti-inflammatory roles by sup-
pressing the expression of a wide range of pro-inflammatory genes in-
cluding IL-1β, IL-6, TNFα, inducible nitric oxide synthase (iNOS), and
MMP-9 [89]. Indeed, numerous reports indicated that PPARγactivation
reduces inflammation due to suppressed NF-κB activity by several mo-
lecular mechanisms including decreased IκBαphosphorylation, cova-
lent modifications of NF-κB subunits leading to abrogated NF-κB-DNA
interaction, and induced NF-κB nuclear export [89]. Using MCF-7
human breast cancer cells, Woo and colleagues established a novel
relationship between TQ and PPARγsignaling [90]. In their study,
TQ (20–80 μM) specifically enhanced the transcriptional activity
of PPARγ, but not PPARαor PPARβ/δ, in MCF-7 cells in a dose-
dependent manner [90]. TQ-induced activation of PPARγwas abolished
when cells were co-treated with GW9662, a specific inhibitor of PPARγ,
and when a dominant negative mutant form of PPARγwas over-
expressed in cells [90]. Docking studies revealed that TQ up-regulates
PPARγactivity via physical interaction with 7 polar and 6 nonpolar res-
idues thought to be critical for PPARγactivity [90].Thus, these findings
highlight a novel molecular mechanism involving positive regulation
imposed by TQ towards PPARγactivity, culminating in the reported
anti-inflammatory effects.
Table 1
A brief summary comparing the immunomodulatory and anti-inflammatory activities of N. sativa and TQ.
Activity N. sativa TQ
Inflammation •Down-regulation of COX, 5-LO, and 5-HETE due to inhibited formation
of TXB2 and LTB4 metabolites [24,25]
•Reduction of carrageenan-induced hind paw edema and cotton seed
pellet granuloma formation [26–29]
•Amelioration of RA, EAE, ulcerative colitis, allergic rhinitis, allergic
diarrhea, eczema, allergic asthma, COPD, and DTH [7,30–38,41]
•Suppression of IL-6, TNFα, and NO production [38–40]
•Elevation of IL-10 level [38]
•Reduction of plasma glutathione peroxidase, catalase, and erythrocyte
superoxide dismutase activity [41]
•Inhibition of ROS formation and lipid peroxidation [41,42]
•Prevention of oxidative stress in blood, liver, spleen, intestines, and
brain [38,43]
•Down-regulation of COX, 5-LO, 5-HETE, and LT due to inhibited
formation of TXB2, LTB4, and LTC4 metabolites [24,25,44]
•Reduction of carrageenan-induced hind paw edema and cotton
seed pellet granuloma formation [26,27]
•Amelioration of EAE, ulcerative colitis, allergic diarrhea, allergic asthma,
and RA [31–34,45,52,57,58]
•Inhibition of eicosanoids and ROS formation and lipid peroxidation
[26,27,51]
•Suppression of NO production [49]
•No effect on the production of IL-4, IL-5, IL-10, and IFNγfrom
lymphocytes [34]
•Prevention of oxidative stress by increasing glutathione levels in blood,
kidneys, and liver [53–56]
•Elevation of the activity of several anti-oxidant enzymes
(GST,GR, GPx, CAT, and SOD) [59]
•No effect on IL-10 production from mast cells [68]
•Reduction of serum levels of pro-inflammatory cytokines
(IL-1β, IL-6, and TNFα)[70]
•Suppression of release of hepatic enzymes ALT, AST, and ALP post
liver damage [71]
Cellular immunity •Enhancement of the proliferative capacity of splenocytes and
T lymphocytes [39,72]
•Stimulation of PBMC response to pooled allogeneic cells [60]
•Elevation of IL-3 secretion from PBMCs [60]
•No effect on IL-2 and IL-4 secretion from PBMCs and lymphocytes,
respectively [60,61]
•Suppression and elevation of IL-8 secretion from un-stimulated and
PWM-activated lymphocytes, respectively [61]
•Stimulation of CD4
+
T lymphocytes [62,67]
•Reduction in leukocyte, splenocyte, neutrophil, and platelet counts
[63,65,66]
•Elevation in peripheral lymphocyte and monocyte counts [64,65]
•Therapeutic role against HIV infection due to enhanced CD4
+
T cell count [67]
•Suppression of IL-5 and IL-13 secretion by mast cells via inhibiting
GATA-1 and GATA-2 transcription factors [68]
•Inhibition of DC-T cell clustering [69]
•Inhibition of DC maturation, survival, and cytokine release
(IL-10, IL-12, and TNFα)[69]
•Elevation of IL-2 serum level [70]
•Enhancement of the proliferative capacity of T lymphocytes,
number of circulating and thymus-homing CD4
+
and CD8
+
T lymphocytes [70]
•Restoration of TCR/CD28-mediated F-actin polymerization [70]
•Elevation of total leukocyte count, phagocytic activity,
chemokine expression, and chemotaxis [71]
Humoral immunity •Reduction of serum antibody titer [65]
•Suppression of B lymphocyte proliferation [72]
•Reduction of serum IgA, IgM, and C3 levels [73]
•Elevation of hemagglutination titer [66]
•Elevation of total Ig levels (especially IgGs) and antibody
hemagglutination [71]
Th1/Th2 paradigm •Elevation of IL-4 and IL-10 secretion from lymphocytes [39]
•Suppression of IFNγsecretion from splenocytes [39]
•No effect on IL-2 and IL-4 secretion from PBMCs [60]
•Elevation of TNFαproduction from lymphocytes [61]
•Elevation of serum IFNγlevels in CMV-infected mice, but not in
un-infected mice [62]
•No effect on the secretion of IL-4, IL-10, or IFNγfrom splenic
MNCs [75]
•Elevation of serum IL-10, but not IL-4 or IFNγ, level [76]
•No effect on the production of IL-4, IL-5, IL-10, and IFNγfrom
lymphocytes [34]
•No effect on IL-10 secretion from mast cells [68]
•Suppression of IL-5 and IL-13 secretion by mast cells via inhibiting
GATA-1 and GATA-2 transcription factors [68]
NK cytotoxicity •Enhancement of mouse splenic NK cytotoxic activity against
YAC-1 tumor cells [39,80,81]
•Enhancement of human peripheral NK cytotoxic activity against
K562 tumor cells [78,79,82]
•Enhancement of mouse splenic NK cell count [81]
•Enhancement of NK cytotoxic activity is due to increased
expression of IFNγ, TNFα, GZMA, and NAGase [82]
•Suppression of NK cytotoxic activity in CMV-infected, but
not in non-infected, mice against YAC-1 cells [62]
•No reported findings
302 A.F. Majdalawieh, M.W. Fayyad / International Immunopharmacology 28 (2015) 295–304
Badr and colleagues investigated the immunomodulatory effects
and protective effects of TQ against diabetes-associated complications
in the offspring of Swiss albino mice with STZ-induced gestational dia-
betes, suggesting a regulatory role of TQ on the phosphatidylinositol-
3-kinase (PI3K)/AKT signaling pathway underlying its immuno-
protective effects [91].Thesefindings substantiate the immunomodula-
tory role that TQ can exert to instigate immuno-protective responses
and improve cellular immunity.
8. Conclusion
The experimental evidence pointing to the immunomodulatory ac-
tivity of N. sativa and its major active ingredient, TQ, cannot be
undermined. Besides their well-documented anti-tumor and anti-
microbial properties, the anti-inflammatory effects of N. sativa and TQ,
both in vitro and in vivo, are evident. Although scarce, experimental
findings suggest that N. sativa can alter cellular and humoral immune
responses. The bulk of these findings denote that N. sativa oil and
extracts can potentially suppress humoral immune responses while en-
hancing cellular immune responses. Yet, more carefully-designed in
vitro and in vivo studies are needed to further elucidate their immune-
potentiating properties towards various aspects of cellular and humoral
immune responses. In particular, the likely immunomodulatory effects
of TQ on humoral immunity, Th1/Th2 differentiation, and NK cytotoxic
activity need to be further assessed and validated. While a few signaling
pathways (e.g. NF-κB, PI3K/AKT, and MAPK) have been proposed to be
targeted by N. sativa and TQ, our understanding of the exact molecular
and cellular mechanisms underlying the immunomodulatory effects of
N. sativa and TQ remains largely dubious. Hence, future efforts should
focus on dissecting the signaling pathways and the mechanisms of ac-
tion by which N. sativa and TQ manifest their immunomodulatory and
anti-inflammatory activities. Table 1 provides a brief summary compar-
ing the immunomodulatory and anti-inflammatory activities of N. sativa
and TQ.
Noteworthy, N. sativa extracts and oil contain chemical constituents
other than TQ including thymohydroquinone (THQ), dithymoquinone
(DTQ), thymol (THY), carvacrol, nigellimine-N-oxide, nigellicine, and
nigellidine. The chemical structures of these compounds are shown in
Fig. 2. Such constituents may possess immunomodulatory and anti-
inflammatory potential, but future studies are needed to examine this
possibility. Collectively, the available data with regard to the immuno-
modulatory and anti-inflammatory effects of N. sativa and TQ strongly
suggest that different extracts of N. sativa and TQ may serve as potent
therapeutic agents towards the regulation of immune reactions impli-
cated in a wide range of infectious and non-infectiousdiseases including
cancer. We speculate that future studies will substantiate the immuno-
regulatory functions of N. sativa and TQ, and hence, confirm their possi-
ble therapeutic efficacy against various diseases and medical conditions.
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