See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/274402033

Thymoquinone and its therapeutic potentials

Article in Pharmacological Research · March 2015

DOI: 10.1016/j.phrs.2015.03.011 · Source: PubMed

CITATIONS READS

473 2,434

4 authors, including:

Sara Darakhshan Abasalt Hosseinzadeh Colagar

Razi University University of Mazandaran
11 PUBLICATIONS 799 CITATIONS 120 PUBLICATIONS 2,831 CITATIONS

SEE PROFILE SEE PROFILE

Sajjad Sisakhtnezhad
Razi University
41 PUBLICATIONS 881 CITATIONS

SEE PROFILE

All content following this page was uploaded by Sara Darakhshan on 24 March 2020.

The user has requested enhancement of the downloaded file.

 Pharmacological Research 95–96 (2015) 138–158

Contents lists available at ScienceDirect

Pharmacological Research
journal homepage: www.elsevier.com/locate/yphrs

Review

Thymoquinone and its therapeutic potentials

Sara Darakhshan a,b,∗ , Ali Bidmeshki Pour a,∗∗ , Abasalt Hosseinzadeh Colagar c ,
Sajjad Sisakhtnezhad a
a
Department of Biology, Faculty of Science, Razi University, Kermanshah, Iran
b
Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, Kermanshah University of Medical Sciences, Kermanshah, Iran
c
Department of Molecular and Cell Biology, Faculty of Basic Sciences, University of Mazandaran, Babolsar, Iran

a r t i c l e i n f o a b s t r a c t

Article history: Herbal medicine has attracted great attention in the recent years and is increasingly used as alternatives
Received 8 February 2015 to chemical drugs. Several lines of evidence support the positive impact of medicinal plants in the pre-
Received in revised form 16 March 2015 vention and cure of a wide range of diseases. Thymoquinone (TQ) is the most abundant constituent of
Accepted 16 March 2015
the volatile oil of Nigella sativa seeds and most properties of N sativa are mainly attributed to TQ. A num-
Available online 28 March 2015
ber of pharmacological actions of TQ have been investigated including anti-oxidant, anti-inflammatory,
immunomodulatory, anti-histaminic, anti-microbial and anti-tumor effects. It has also gastroprotective,
Chemical compounds studied in this article:
hepatoprotective, nephroprotective and neuroprotective activities. In addition, positive effects of TQ in
1,2-Dimethyl-hydrazine (PubChem CID:
1322)
cardiovascular disorders, diabetes, reproductive disorders and respiratory ailments, as well as in the
Acetaminophen (PubChem CID: 1983) treatment of bone complications as well as fibrosis have been shown. In addition, a large body of data
Benzo(a)pyrene (PubChem CID: 2336) shows that TQ has very low adverse effects and no serious toxicity.
Bleomycin (PubChem CID: 5360373) More recently, a great deal of attention has been given to this dietary phytochemical with an increasing
Bortezomib (PubChem CID: 387447) interest to investigate it in pre-clinical and clinical researches for assessing its health benefits. Here we
Butylated hydroxytoluene (PubChem CID:) report on and analyze numerous properties of the active ingredient of N. sativa seeds, TQ, in the context of
Cisplatin (PubChem CID: 84691)
Cyclophosphamide (PubChem CID: 2907)
Gentamicin (PubChem CID: 3467)
Indomethacin (PubChem CID: 3715)
Mercuric chloride (PubChem CID:)
Methotrexate (PubChem CID: 126941)
Omeprazole (PubChem CID: 4594)
Paracetamol (PubChem CID:)
Silybin (PubChem CID: 5213)
Streptozocin (PubChem CID: 5300)
Sulfasalazine (PubChem CID: 5353980)
Tamoxifen (PubChem CID: 2733526)
Thioacetamide (PubChem CID: 2723949)
Vancomycin (PubChem CID: 14969)

Abbreviations: AGP, ␣1-acid glycoprotein; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ATP, adenosine triphosphate;
BMP, bone morphogenetic protein; BSA, bovine serum albumin; CAT, catalase; CDK, cyclin-dependent kinase; COX, cyclooxygenase; EAE, experimental allergic encephali-
tis; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; GSH, glutathione; GSH-Px, glutathione peroxidase;
GST, glutathione-S-transferase; HDL, high-density lipoprotein; JAK, janus kinase; JNK, Jun-N-terminal kinase; LDH, lactate dehydrogenase; LDL, low-density lipoprotein;
LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; Mcl-1, myeloid cell leukemia sequence 1; MDA, malondialdehyde; MMP, matrix metalloproteinase;
MPO, myeloperoxidase; NADH, nicotinamide adenine dineucleotide; NADPH, nicotinamide adenine dineucleotide phosphate; NDEA, N-nitrosodiethylamine; NF-␬B, nuclear
factor-kappaB; NLRP3, nucleotide binding domain and leucine rich repeat containing family pyrin domain containing 3; NO, nitric oxide; Nrf2, nuclear factor erythroid
derived 2-related factor 2; OVA, ovalbumin; PAF, platelet-activating factor; PARP, poly ADP-ribose polymerase; PGD2, prostaglandin D2; PGE2, prostaglandin E2; PI3K, phos-
phatidylinositol 3-kinase; PPAR, peroxisome proliferator-activated receptor; PTEN, phosphatase and tensin homolog; SOD, superoxide dismutase; STAT3, signal transducer
and activator of transcription 3; TBARS, thiobarbituric acid reactive substances; TGF-␤, transforming growth factor-beta; Th-1 and Th-2, helper T-cells; TIMP-1, tissue inhibitor
of metalloproteinase-1; TLR4, toll-like receptor 4; TNF-␣, tumor necrosis factor-alpha; TQ, thymoquinone; VEGF, vascular endothelial growth factor; ␣-SMA, alpha-smooth
muscle actin.
∗ Corresponding author at: Department of Biology, Faculty of Science, Razi University, Kermanshah, Iran. Tel.: +98 8334274545; fax: +98 8334274545.
∗ ∗ Corresponding author.
E-mail address: [email protected] (S. Darakhshan).

http://dx.doi.org/10.1016/j.phrs.2015.03.011
1043-6618/© 2015 Elsevier Ltd. All rights reserved.
 S. Darakhshan et al. / Pharmacological Research 95–96 (2015) 138–158 139

Keywords: its therapeutic potentials for a wide range of illnesses. We also summarize the drug’s possible mechanisms
Thymoquinone of action. The evidence reported sugests that TQ should be developed as a novel drug in clinical trials.
Black cumin © 2015 Elsevier Ltd. All rights reserved.
Nigella sativa
Anti-oxidants
Anti-cancer
Review

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
1.1. Pharmacological properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
1.2. Anti-oxidative, anti-inflammatory and immunomodulatory activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
1.3. Anti-cancer effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
1.4. Anti-microbial activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
1.5. Hepatoprotective effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
1.6. Hypoglycemic and anti-diabetic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
1.7. Gastroprotective effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
1.8. Neuroprotective effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
1.9. TQ and cardiovascular problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
1.10. TQ against respiratory diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
1.11. Nephroprotective effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
1.12. Effects of TQ on bone and joint complications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
1.13. Effects of TQ on reproductive system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
1.14. Hypolipidemic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
1.15. Anti-histaminic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
1.16. TQ and fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
1.17. Molecular mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
1.17.1. Cell cycle and proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
1.17.2. Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
1.17.3. Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
1.17.4. Migration, invasion and metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
1.17.5. Inflammation and Oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
1.18. Safety and adverse effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
2. Discussion and future viewpoints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

1. Introduction ethnomedicine to treat ailments and symptoms including, asthma,

bronchitis, inflammation, eczema, fever, influenza, hypertension,
Plants are invaluable sources of new drugs and interest in cough, headache, dizziness, diabetes, kidney and liver dysfunctions,
medicinal plants has increased due to increased efficiency of new nervous disorders, rheumatism, cancer and related inflammatory
plant-derived drugs. In addition, because of the concerns about the diseases, gastrointestinal problems, and overall for general well-
side effects of conventional medicine, the use of natural products as being [1,4,5].
a replacement for the use of synthetic drugs has risen considerably N. sativa seeds (NS) contain diverse but well-characterized
in the last decades. The wide utilization of herbal drugs has encour- chemical components; they include both fixed and essential
aged scientists to investigate their impressive effects on health, and (volatile) oil, proteins and amino acids, carbohydrates, alkaloids,
a large number of medicinal plants and their active extracted ingre- organic acids, saponins, crude fibers, vitamins, and minerals
dients are extensively studied for their potentials to protect cells [6].
from injuries. Thymoquinone (2-isopropyl-5-methylbenzo-1, 4-quinone)
Among the promising medicinal plants is Nigella sativa (also (TQ), the most abundant constituent of the volatile oil also present
called black cumin, black seed). N. sativa Linn as a member of the in the fixed oil, is the biologically active compound of NS seeds
botanical family of Ranunculaceae is an annual herbaceous [1]. It [1]. TQ as a bioactive component is found in many medicinal
has illustrious and religious background and its seeds and oil have plants. Besides Ranunculaceae, the presence of this compound has
been commonly used as a traditional remedy to treat a variety of been confirmed in several genera of the Lamiaceae family such
health conditions for more than 2000 years [2]. Black seed is known as Monarda, and the Cupressaceae family such as Juniperus [7].
as the curative black cumin in the Holy Bible and is described as the Although, TQ holds a great potential as an anti-oxidant and an
Melanthion of Hippocrates and Discroides and as the Gith of Pliny anti-inflammatory agent, it also has a vast array of other benefits.
[3]. Furthermore, the Prophet Muhammad (PBUH) advised: “Hold TQ, as a naturally derived agent, has lately received particular
onto use the black cumin, because it can heal every disease except consideration and has been extensively studied for its therapeutic
death” [2]. properties.
The seeds, which are the rich source of the active ingredients of In this review, we focus on the therapeutic potential of TQ in
plant, have long been used in the Middle and Far East as a traditional disease conditions. The evidence we report suggests that TQ should
medicine for a wide range of pathological conditions [4]. It is used in be investigated further in clinical trials.
140 S. Darakhshan et al. / Pharmacological Research 95–96 (2015) 138–158

Fig. 1. The Nigella sativa plant (a), its flower (b) and seeds (c); and the chemical structure of bioactive component of seeds, thymoquinone (TQ) (d).

Table 1 analogs of TQ and their effects on disease models are presented in

The significant properties of thymoquinone (TQ).
Table 2.
Systematic/IUPAC name 2-Isopropyl-5-methylbenzo-1,4-quinone There are different routes for administration of TQ including
Molecular formula C10 H12 O2 intravenous (i.v.) [18,19], intraperitoneal (i.p.) [20–22], and oral
Molar mass 164.20 g mol−1
subacute and subchronic administration [20,23–26]. However, oral
Appearance Crystalline and dark yellow
CAS number 490-91-5
administration could lead to biotransformation due to the metabo-
PubChem CID 10281 lizing activity of the liver enzymes such as DT-diaphorase (a quinine
reductase) that catalyzes reduction of TQ into a hydroquinone [27].
El-Dakhakhany [28] reported an LD50 of 10 mg/kg for TQ when
1.1. Pharmacological properties injected intraperitoneally in rats. Another study reported that doses
of 4, 8, 12.5, 25 and 50 mg/kg i.p. in mice did not alter the bio-
The chemical composition of NS seeds is rich, and diverse active chemical parameters, including serum alanine transaminase (ALT)
chemical components have been isolated from them. The seeds and lactate dehydrogenase (LDH) [29]. In the same study, doses of
were shown to contain a fixed oil (>30%, wt/wt) and a volatile oil TQ higher than 50 mg/kg body weight by intraperitoneal injection
(0.40–0.45%) [8]. The active principles include thymoquinone, thy- were lethal to mice and the LD50 was 90.3 mg/kg [29]. In many other
mohydroquinone, dithymoquinone (nigellone), thymol, carvacrol, studies conducted to determine anti-inflammatory, anti-cancer,
nigellicine, nigellidine and ␣-hedrin [9]. Nevertheless, most of the anti-oxidant and cytoprotective effects of TQ the investigators have
known actions have been attributed to TQ. used doses between 5 and 12.5 mg/kg, injected intraperitoneally in
TQ is the most bioactive component of seeds also of the volatile mice and rats without important toxicity [24,29–31]. A number of
oil (18.4–24%) [1], it was first extracted by El-Dakhakhny [10] using studies administered oral TQ dose in the range of 10–100 mg/kg
thin layer chromatography on silica gel. Illustration of NS plant, body weight without any reported toxic or lethal effects [32–36].
seeds of plant and the chemical structure of TQ are presented in The maximum tolerated dose for intraperitoneal injection was
Fig. 1 and the most relevant properties of TQ are summarized in 22.5 mg/kg in male and 15 mg/kg in female rats, whereas for oral
Table 1. ingestion it was 250 mg/kg, in both male and female rats [37].
TQ exists in tautomeric forms including the enol form, the keto The calculated clearance of TQ following intravenous admin-
form and mixtures. The keto form being the major fraction (∼90%), istration was 7.19 ml/kg/min, and the estimated volume of
and is responsible for the pharmacological properties of this com- distribution at steady state (Vss) was 700.90 ml/kg. At vari-
pound [4]. TQ is a hydrophobic molecule, thus its solubility is a ance, after oral administration the apparent clearance value was
challenge that could affect its bioavailability and cause limitations 12.30 ml/min/kg and Vss was 5109.46 ml/kg. The calculated absorp-
in drug formulation. In addition, it’s solubility dependent on the tion half-life (T1/2 ) of TQ was about 217 min, and it was rapidly
time as its solubility ranges from 549 to 669 ␮g/ml in aqueous removed from plasma [38]. The UV–vis spectra of TQ had one sharp
solutions at 24 h, increasing at 72 h to 665–740 ␮g/ml [11]. There- peak (max ) at 254–257 nm [11]. TQ was highly sensitive to light,
fore, recently attempts have been made to synthesize novel analogs even after a short period of exposure. A short period of light expo-
of TQ with enhanced bioavailability and activity. Some synthetic sure led to severe degradation, independent of the solution pH and

Table 2
Some analogs have been developed for enhanced bioavailability and activity of TQ.

Types Disease models Refs

Molecular micelle modified poly (d,l lactide-co-glycolide) (PLGA) MDA-MB-231 breast cancer cells [12]
nanoparticles
Solid lipid nanoparticles (SLNs) Paracetamol-induced liver fibrosis and/or cirrhosis [13]
TQ-encapsulated chitosan nanoparticles Brain diseases [14]
TQ-loaded liposomes (TQ-LP) MCF-7 and T47D breast cancer cell lines [15]
Caryophyllyl and germacryl conjugates as well as fatty acid conjugates HL-60 human leukemia, multidrug-resistant KB-V1/Vbl cervix, 518A2 melanoma [16]
and MCF-7/Topo breast cancer cells
TQ-loaded nanostructured lipid carriers (TQ-NLCs) Gastroprotective effects and inhibition of the formation of ethanol-induced ulcers [17]
 S. Darakhshan et al. / Pharmacological Research 95–96 (2015) 138–158 141

solvent type. In addition, it was unstable in aqueous solutions, par- CYP-b5 reductase or NADH-ubiquinone oxidoreductase, or a one-
ticularly at an alkaline pH. TQ stability decreased with rising pH; step two-electron reduction by NADPH-quinone oxidoreductase to
at alkaline pH, it suffered the highest degradation rate with the produce thymohydroquinone [44] (Fig. 2). When these metabo-
minimal degradation being at acidic pH [11]. lites are formed they can remove free radicals, an effect that can
Protein–drug interactions are an important factor on the replace the endogenous anti-oxidant defense molecules, viz. GSH
pharmacokinetics and pharmacological properties of drugs. The and superoxide dismutase (SOD), and prevent lipid peroxidation.
estimated percentages of TQ-protein binding in rabbit and human These forms were highly reactive toward different redox states
plasma were 99.19 and 98.99, respectively [38]. Therefore, TQ rep- of hemoglobin and myoglobin that led to recovery of these from
resented a compound with quick elimination and relatively slower oxidative stress [45].
absorption after oral administration. Anti-oxidant enzymes, such as GSH, SOD, catalase (CAT),
Lupidi et al. evaluated the interactions between TQ and human glutathione-S-transferase (GST), and glutathione peroxidase (GSH-
serum albumin (HSA). They showed that the association between Px) constitute the main pool of the anti-oxidant system of most
TQ and HSA does not affect the secondary structure of HSA. The cells. It is well known that anti-oxidant enzymes are responsible
thermodynamic analysis of the HSA/TQ complex formation showed for neutralizing the free radical-induced oxidative damage [46]. TQ
that the binding process was spontaneous and the hydrophobic induced the expression and/or activity of GST [47–49], GSH-Px [49],
interactions were main intermolecular forces stabilizing the com- SOD [32,42,48], and glutathione reductase [49]. TQ improved the
plex [39]. In addition, El-Najjar et al. [40] studied the effect of TQ plasma and liver anti-oxidant capacity and expression of liver anti-
binding with bovine serum albumin (BSA) and ␣1-acid glycoprotein oxidant genes. In models with induced hypercholesterolemia it can
(AGP) on its anti-cancer activity. The results suggested that cova- up-regulate the genes coding for GST, GSH-Px and CAT leading to
lent binding of TQ to BSA led to losing the TQ anti-cancer activity elevation of hepatic levels of these enzyme to overcome oxidative
against tested cancer cells, but the TQ anti-cancer effects was not stress induced during diethylnitrosamine metabolism [27,50].
affected when it is bound to AGP. The generation of free oxygen radicals is one of the possi-
The lack of bioavailability and pharmacokinetic parameters, and ble mechanisms by which several drugs exert their toxicity. TQ
formulation problems delayed the usage of TQ in clinical phase. had protective effects on several organs against oxidative dam-
Hence, more investigation is needed for a better understanding of age induced by a variety of free radical-generating agents and it
TQ pharmacological properties meant for future clinical develop- had also cytoprotective properties. It protected kidney from cis-
ment. platin [30], doxorubicin [51], gentamicin [52], vancomycin [53] and
mercuric chloride [54]-induced nephrotoxicity; and liver from car-
1.2. Anti-oxidative, anti-inflammatory and immunomodulatory bon tetrachloride [24,29], cyclophosphamide [55], acetaminophen
activities [56] and aflatoxin B1 (AFB1) [57]-induced hepatotoxicity. More-
over, it showed that TQ protected heart from doxorubicin [26,41]
Currently, the use of natural anti-oxidant compounds and phy- and cyclophosphamide [58]-induced toxicity. It also alleviated
tochemicals as a therapy in diseases related to oxidative stress has lung injuries induced by cyclophosphamide [59], toluene [36] and
gained massive interest for their abilities to quench free radicals bleomycin [60]. TQ has been shown to enhance detoxification and
and the protection of body against oxidative stress-induced patho- inhibit benzo(a)pyrene-induced fore-stomach tumors [61]. It had
genesis. Furthermore, natural immunomodulators help the body also protective role in the prevention of gentamicin ototoxicity [62].
by stimulate and strengthen the immune system. In this regard, TQ pre-treatment (5 mg/kg) also restored completely 1,2-
TQ as a natural compound has become an imperative agent in dimethyl-hydrazine (DMH)-induced oxidative stress, which occurs
pharmacological toxicity studies due its potent anti-oxidant, anti- in initiation stage of colon tumor. TQ corrected the oxidative status
inflammatory, and immune enhancement capacity [4]. and malondialdehyde (MDA), CAT, SOD and GST-Px level at promo-
Anti-oxidant enzymes are essential part of the cellular defense tion, with a reduction in tumor incidence and dysplasia degree [63].
against reactive oxygen species (ROS). Reactive oxygen species such TQ has been shown to have anti-oxidative and anti-inflammatory
as superoxide anion radical (O2 −• ), hydroxyl radical (• OH) and per- efficacy in hippocampal neurodegeneration model after chronic
oxyl radical (ROO• ) are continually generated in cells by aerobic toluene exposure [33]. TQ ameliorated most of the toxic effects
metabolism also by exogenous sources such as UV radiation and related to streptozotocin (STZ) and preserved beta-cell integrity by
environmental pollution. Generation of ROS induces oxidization decreasing oxidative stress [64]. NS oil and TQ have anti-oxidant
of biomolecules including membrane lipids, proteins and nucleic properties also in radiation-injured brain tissue, especially against
acids. nitrosative stress in the brain tissue of the irradiated rats [65].
TQ is a potent phytochemical anti-oxidant due to the scavenging TQ exhibited anti-oxidant/anti-inflammatory activities in
activity against several ROS including superoxide anion, hydroxyl experimental models, thus may be a clinically viable agent against
radical and singlet molecular oxygen [32,41], and thus it can antag- a variety of inflammatory conditions. Inflammation as a part of the
onize the adverse effects resulting from elevated ROS levels in complex biological response to harmful stimuli is mainly mediated
various disorders. by two enzymes, cyclooxygenase and lipoxygenase, which gener-
It has been shown that TQ and tert-butylhydroquinone ate prostaglandins and leukotrienes, respectively [41]. Houghton
(TBHQ), a synthetic structurally-related, powerfully inhibited iron- et al. [31] showed that the anti-inflammatory action of TQ resulted
dependent microsomal lipid peroxidation [42]. The anti-oxidative from prevention of eicosanoids generation, such as thromboxane
potential of TQ may be related to the redox properties of the B2 and leukotriene (LT) B4, by inhibiting both cyclooxygenase
quinone structure of molecule and unrestricted ability of TQ to cross and 5-lipoxygenase, and in part via non-enzymatic peroxida-
from physiological barriers and easy access to subcellular compart- tion of membrane lipids. TQ induced a significant inhibition on
ments, all of which help the radical scavenging effects [42,43]. LTC4 synthase activity [66]. Furthermore, in vitro treatment of
Under physiological conditions, TQ reacts with glutathione calcium- or ionophore-stimulated neutrophils with TQ inhibited
(GSH), NADH and NADPH through a spontaneous reaction to form 5-lipoxygenase products and 5-hydroxy-eicosa-tetra-enoic acid
reduced species, glutathionyl-dihydro-TQ, after rapid reaction with production [67].
GSH, and dihydrothymoquinone, after slow reaction with NADH It was a potent anti-inflammatory agent in an asthmatic murine
and NADPH [44]. TQ may undergo a two-step process of one- model [68]. It was a strong inhibitor of inflammatory cell aggrega-
electron reduction by microsomal NADPH CYP reductase, NADH tion in bronchoalveolar lavage (BAL) fluid and lung tissues, and of
142 S. Darakhshan et al. / Pharmacological Research 95–96 (2015) 138–158

Fig. 2. Mechanism of oxido-reduction cycling of TQ. In enzymatic reaction by a two-step one-electron reduction, or by a one-step two-electron reduction converts TQ
into thymohydroquinone. In one electron reduction of TQ, microsomal NADPH cytochrome P450 reductase, microsomal NADH cytochrome-b5 reductase, and mitochondrial
NADH ubiquinone oxidoreductase, catalyze the conversion of TQ to semiquinone. Subsequently, semiquinone is converted into thymohydroquinone. Alternatively, a one-step
reaction causing direct formation of thymohydroquinone may take place. TQ may also be reduced in a non-enzymatic reaction through interaction with GSH to generate
glutathionyl-dihydro-TQ. The reduced products of TQ, glutathionyl-dihydro-TQ and thymohydroquinone, is known for its anti-oxidant activity. A semiquinone produced
by one-electron reduction can contribute to the pro-oxidant effect of TQ in the tumor environment. Superoxide anion generated through oxidation of reduced TQ can
be detoxified by SOD and CAT. In the absence of detoxificating enzymes, which is common in numerous cancers, the accumulation of superoxide may contribute to the
pro-oxidant effect of TQ.

the expression of TGF-␤1 and inducible nitric oxide (NO) synthase activation of polymorphonuclear cells, and maintained the homeo-
(iNOS) mRNA, all converging in explaining its potency in antago- stasis in the cytokine imbalance. Tekeoglu et al. [74] reported that
nizing airway inflammation [68]. El Gazzar et al. [21] showed the administration of TQ had an action similar to methotrexate in
anti-inflammatory effect of TQ in a mouse model of allergic lung adjuvant-induced arthritis in rats. Furthermore, Vaillancourt et al.
inflammation. TQ attenuated pulmonary inflammation in allergic reported that TQ (5 mg/kg/day) significantly reduced the serum lev-
asthma by decreasing lung eosinophilia, and Th2 cytokines and els of 4-hydroxynonenal, IL-1␤ and TNF-␣, as well as of a number of
inflammatory cell infiltration in the lung. It also decreased the ele- factors known to be involved in rheumatoid arthritis pathogenesis,
vated levels of ovalbumin (OVA)-specific IgE and IgG1 in serum. It such as alkaline phosphatase (ALP) and tartrate-resistant acid phos-
inhibited significantly allergen-induced lung eosinophilic inflam- phatase. The protective effects of TQ against rheumatoid arthritis
mation and represented an inhibitory influence on IL-4, IL-5 and were also evident from the decrease in arthritis scoring and bone
IL-13, with some effect in inducing IFN-␥ production in the BAL fluid histology [75]. Increase in the levels of pro-inflammatory media-
[21]. Administration of TQ also down-regulated 5-lipoxygenase tors including IL-1␤, IL-6, TNF-␣, IFN-␥ and PGE2, and decreased
expression, and LTB4 and LTC4 production by lung cells in airway level of IL-10 was reported in arthritic rats, which were corrected
inflammation [69]. In addition, TQ inhibited COX-2 expression in with TQ [69].
a mouse model of OVA-induced allergic airway inflammation [70]. Experimental allergic encephalitis (EAE) as a T-cell medi-
Administration of TQ significantly reduced the ocular symptoms in ated autoimmune disease is a widely accepted animal model
allergic conjunctivitis by attenuating the recruitment of eosinophils for the human multiple sclerosis (MS). The EAE rats treated
and reducing the levels of IgE, histamine and cytokines. Further- with TQ showed higher GSH level, no perivascular inflammation
more, it blunted in mice immunized and exposed to OVA the mRNA and no disease symptoms compared with EAE untreated ani-
expression and serum level of interleukins including IL-4, -5 and mals. Furthermore, TQ treatment inhibited NF-␬B activation in
-13, as well TGF-␤ [71]. encephalomyelitis in rats [76]. By these data, TQ was shown to have
In arthritis-induced animal models, the administration of TQ an anti-oxidative stress effect leading to therapeutic effects in EAE
was effective against rheumatoid arthritis [72] and autoimmune animals [73].
encephalomyelitis [73]. Umar et al. [72] have demonstrated the TQ attenuated the pro-inflammatory and oxidative responses
anti-arthritic effect of TQ in collagen induced arthritis. They mainly by modulating NF-␬B and TNF-␣ production [77]. In addi-
found that TQ suppressed the increase of NO and myeloper- tion to significant down-regulation of TNF-␣, IL-1␤ and COX-2, TQ
oxidase (MPO), along with enhanced the activity of GSH, also inhibited the constitutive TNF-␣-mediated activation of NF-␬B
CAT and SOD. Moreover, TQ abolished the accumulation and and reduced the transport of NF-␬B from the cytosol to the nucleus
 S. Darakhshan et al. / Pharmacological Research 95–96 (2015) 138–158 143

[78]. Furthermore, it inhibited lipopolysaccharide (LPS)-induced anti-neoplastic agents, as well was a chemosensitizer when used
TNF-␣ production in the rat basophil cell line, RBL-2H3 by a mech- in combination with chemotherapeutic agents, while it was mini-
anism involving NF-␬B. Experimental studies have reported that mally toxic to normal cells.
TQ reduced TNF-␣, interleuken-6 and IL-1␤ in blood and tissues, TQ was shown to have a dual role, and depending on the cel-
and protected tissues by reducing inflammation [74,75]. TQ inhib- lular microenvironment, it may act as an anti-oxidant or as a
ited IL-6 signaling [79]; and it is possible that the role of TQ in EAE pro-oxidant. It exerted its biological functions by ROS generation in
[76], rheumatoid arthritis [72], allergic asthma [69], allergic lung tumor cells where it acted as pro-oxidant. TQ reacted with amino or
inflammation [21], experimental colitis [80], and immunomodula- thiol groups of amino acids, undergone a series of oxido-reduction
tory effects [4] is due to suppression of IL-6 signaling. Furthermore, reactions, and then metabolized to semiquinone or thymohydro-
TQ suppressed NO overproduction and iNOS expression induced by quinone, ultimately leading to the production of ROS [94].
different agents in various animal tissues [20,48,81–83]. It dimin- TQ inhibited the growth of the breast cancer cell lines MCF-
ished iNOS levels in LPS-activated rat peritoneal macrophages and 7, MDA-MB-231 and BT-474, exerted strong anti-proliferative
thus suppressed the production of NO by macrophages [22,84], an activity, and increased the cytotoxicity of doxorubicin (DOX) and 5-
effect that may be useful in ameliorating the inflammatory and fluorouracil (5-FU), when combined with them. It showed that the
autoimmune conditions. TQ also acted in vitro as inhibitor of COX-1 anti-cancer effects of TQ resulted from the ability of the compound
and -2 [85], also inhibiting p38 in murine macrophages [22]. to increase PPAR-␥ activity and to down-regulate the expression of
The immunomodulatory effect of TQ was observed also in mixed pro-apoptotic genes including Bcl-2, Bcl-xL and survivin [95]. TQ
lymphocyte cultures, where it caused decreased secretion in the also induced cell cycle arrest by suppression of cyclin D1, cyclin E,
levels of the cytokines IL-1␤ and -8 [86]. It enhanced the survival and the cyclin dependent kinase (CDK) inhibitor p27 expression in
and activity of antigen-specific CD8T cells thus protecting against T-47D and MDA-MB-468 breast cancer cells. TQ induced cell cycle
tumor growth [87]. TQ might regulate immunity also by influenc- arrest in G1 phase during initial time incubation, while extended
ing dendritic cell functions such as maturation, cell pH (by affecting exposure to TQ resulted in loss of mitochondrial membrane poten-
Na+ /H+ activity), oxidative burst, migration and cytokine release tial thus inducing apoptosis through release of cytochrome c and
and survival. Dendritic cell volume may also be affected by TQ [88]. interfering Akt activation [96].
TQ may enhance the activation of Nrf2, thereby raising the expres- It also inhibited DOX-resistant MCF-7/DOX cell proliferation. TQ
sion of heme oxygenase-1 (HO-1). TQ induced HO-1 expression in arrested MCF-7/DOX cells at G2/M phase and increased cellular
HaCaT cells by activating Nrf2 through ROS mediated phosphoryla- levels of p53 and p21 proteins. TQ-induced apoptosis was associ-
tion of Akt and adenosine monophosphate-activated protein kinase ated with changes in mitochondrial membrane potential, activation
(AMPK) as upstream targets [89]. of caspases and PARP cleavage in MCF-7/DOX cells. In addition,
In addition to TQ, components from other herbs are effective TQ treatment increased Bax/Bcl2 ratio via up-regulating Bax and
on HO-1 and Nrf2. A chemically synthesized kavalactone deriva- down-regulating Bcl2 proteins [97]. In addition, it was douns to
tive, which isolated from Piper methysticum, is capable of inhibiting be a radiosensitizer, such that using TQ in combination with ion-
LPS-stimulated iNOS induction and NO production via activation izing radiation such as ␥-radiation (2.5 Gy) was found to exert a
of Nrf2 signaling and HO-1 induction in microglial cells [90]. Some synergistic cytotoxicity against breast cancer cells [98].
small activators of Nrf2/HO-1 such as carnosol, a phenolic diterpene TQ was cytotoxic toward SiHa cells, human cervical squamous
in Rosmarinus officinalis and supercurcumin are effective modu- carcinoma, with even higher efficacy when compared to cisplatin in
lators of inflammation, and up-regulate HO-1 and down-regulate a pathway involving elevation of the p53 activity and the Bax/Bcl-2
the inflammatory response TNF-␣, PGE2 and nitrite [91]. Conse- ratio [99]. TQ has also shown cytotoxicity against BG-1 and human
quently, activation of Nrf2 and HO-1 moderate oxidative stress and ovarian adenocarcinoma cells [93]. The cytotoxicity of TQ has been
inflammation. Thus, anti-inflammatory and anti-carcinogenesis reported in PC3 prostate cancer cell xenograft tumor growing in
properties of TQ, in part, can be the result of induction of Nrf2 and SCID mice. TQ (6 mg/kg daily for 15 days) inhibited tumor angio-
HO-1 expression. genesis and tumor growth by suppressing Akt and extracellular
Reinforcement of body’s anti-oxidant store is believed to inhibit signal-regulated signaling pathways. It also significantly potenti-
oxidative stress-induced tissue damage and neoplastic transfor- ated the apoptotic effects of thalidomide and bortezomib [100]. TQ
mation of cells, and have therapeutic relevance; hence TQ in (at the dose of 20 mg/kg) repressed hormone-refractory prostate
therapeutic perspective as a cytoprotective agent against oxida- cancer, by suppressing the expression of androgen receptor and
tive damage, autoimmune and other inflammatory complications the transcription factor E2F-1 [101].
in many tissues may be promising option. Intragastric administration of TQ induced growth inhibition and
apoptosis in the human osteosarcoma cell line SaOS-2, and blocked
1.3. Anti-cancer effects human umbilical vein endothelial cell (HUVEC) tube formation. TQ
significantly down-regulated NF-␬B DNA-binding activity, as well
Among the novel anti-neoplastic drugs that are currently under as the expression of XIAP, survivin and vascular endothelial growth
investigation, bioactive natural products have gained consider- factor (VEGF) in SaOS-2 cells, while up-regulating the expression of
able attention. Natural compounds and phytochemicals may be cleaved caspase-3 and Smac. Overall, TQ effectively inhibited tumor
exceptional resource for anti-cancer agents, and may be valu- growth and angiogenesis by inhibition of NF-␬B and downstream
able alternatives to synthetic drugs in cancer therapy, or used to effector molecules in osteosarcoma [102].
enhance their effect and reduce their dosing and limiting their The effect of TQ on M059K and M059J human glioblastoma cells
toxicity [92]. An ideal cancer therapeutic agent is one that exerts was reported. Cytotoxicity of TQ resulted from induction of telom-
its anti-cancer effect with partial cytotoxicity over normal tis- ere shortening, DNA damage and apoptosis in these cells [103].
sues. One of the advantages of TQ is that its anti-cancer effects It was also demonstrated that TQ modulates potently proteasome
has been shown to be activated more specifically against can- complex activity in U87 MG and T98G malignant glioma cells. Inhi-
cer cells than normal cells [93]. As detailed below, among a bition of this complex led to intracellular accumulation of p53
wide spectrum of phytochemicals TQ is a compound exhibiting and Bax in malignant cells, and may be linked to accumulation
promising anti-carcinogenic, anti-neoplastic, anti-proliferative and of ubiquitin conjugates at the onset of apoptotic events [104]. In
anti-mutagenic activities against miscellaneous tumor models. It addition, TQ may have anti-metastatic activities since it inhibited
also is chemopreventive, and reduces the toxic effects of standard in vitro migration, adhesion and invasion of human glioblastoma
144 S. Darakhshan et al. / Pharmacological Research 95–96 (2015) 138–158

U87 and CCF-STTG1 cells. At the molecular level, this was medi- Topical application of a TQ-rich fraction obtained from NS extract
ated by a drastic down-regulation of Focal Adhesion Kinase (FAK), inhibited chemically induced mouse skin papillomagenesis [117].
associated with a reduction of extracellular signal-regulated kinase TQ suppressed also 20-methylcholanthrene-induced fibrosarcoma
(ERK) phosphorylation as well matrix metalloproteinase (MMP)-2 tumorigenesis through inhibition of tumor incidence and of tumor
and -9 secretion [105]. TQ also was found to be cytotoxic in the burden [47].
mouse neuroblastoma cell line Neuro-2a, where it triggered apo- TQ has been reported to prevent the proliferation and induce
ptosis as evidenced by elevation of Bax/Bcl2 ratio, cytochrome c apoptosis in the human chronic myeloid leukemia KBM-5 cells by
release from mitochondria, activation of caspases-3 and -9, and suppressing TNF-induced NF-␬B expression [118]. It also degraded
down-regulation of XIAP [106]. ␣ and ␤ tubulin of human astrocytoma cells in p53 deficient Jurkat
Studies demonstrated the anti-cancer effect of TQ in colorec- cells [119]. TQ induced growth inhibition and apoptosis in some
tal carcinoma [107], a multistep process, in which oxygen radicals primary effusion lymphoma (PEL) cell lines. TQ treatment led to
play a critical role during the initiation, promotion, and progres- down-regulation of constitutive activation of AKT via generation
sion phases. This agent can abrogate the stress response pathway of ROS, conformational changes in Bax protein, loss of mitochon-
sensor CHEK1 and contribute to apoptosis in colorectal cancer cells drial membrane potential and release of cytochrome c into the
[108]. Furthermore, in a HCT116 colorectal cancer cells xenograft cytoplasm, and caspase-dependent apoptosis. TQ-induced signal-
model, a three-time weekly intraperitoneal injection of 20 mg/kg ing caused caspase-9/3 activation and PARP cleavage and sensitized
TQ reduced the tumor size. This occurred through TQ-induced apo- TRAIL-mediated apoptosis in PEL cell lines [120]. It also inhibited
ptosis involving an up-regulation of both p53 and p21 expression the proliferation of CD138+ cells isolated from multiple myeloma
[108]. It inhibited also the proliferation of human colon cancer (MM) patient samples. Furthermore, in a xenograft mouse model
cells Caco-2, HCT-116, LoVo, DLD-1 and HT-29 by increasing the with MM cell lines, it potentiated the apoptotic effects of borte-
phosphorylation states of the mitogen-activated protein kinases zomib through modulation of various markers for survival and
(MAPK), JNK and ERK [109]. TQ was efficacious in protecting and angiogenesis, such as Ki-67, VEGF, Bcl-2 and p65 expression [121].
curing DMH-induced initiation phase of colon cancer, while exert- TQ inhibited IL-6-induced Akt phosphorylation and IL-6-induced
ing a protective role at promotion. These effects may be related to STAT3 expression. The effects of TQ on STAT3 phosphorylation cor-
its capacity in prevention of DMH-induced oxidative stress [63]. TQ related with the suppression of upstream protein tyrosine kinases
(orally and daily 375 mg/kg body weight for 12 weeks) decreased JAK2 and c-Src. It also suppressed both inducible and constitutive
the number of large polyps in the small intestine and reduced polyp STAT3 activation, which makes it a potentially effective suppressor
growth through selective induction of apoptosis. Moreover, it inter- of tumor cell survival, proliferation and angiogenesis [79].
fered with polyp progression in ApcMin mice, which best resembles TQ induced G2/M blockade in A549 human non-small cancer
the FAP (familial adenomatous polyposis, an autosomal dominantly lung cells (NSCLC). Microtubule depolymerization induced by TQ
inherited disease) phenotype, through modulating Wnt signaling. was followed by apoptosis. In addition, it distorted spindle orga-
TQ reduced cell proliferation in the villi, down-regulated c-myc nization and suppressed tubulin polymerization by direct tubulin
expression, and ␤-catenin translocation to the membrane in the binding [122]. Thus, like other tubulin assembly inhibitors TQ can
polyps of ApcMin mice [110]. be classified as a microtubule-depolymerizing agent against can-
TQ was found also to prevent fore-stomach carcinogenesis [61]. cers. TQ was able to induce apoptosis in both NSCLC and SCLC lung
Moreover, TQ had chemosensitisation effect; it enhanced the 5-FU- cancer cell lines, NCI-H460 and NCI-H146. It down-regulated NF-␬B
induced killing of gastric cancer cells by modulating Bcl-2 and Bax expression, and may be useful in overcoming cisplatin resistance
expression and increasing the release of cytochrome c from the resulted from overexpression of this factor. The combination of TQ
mitochondria. In addition the combined treatment of TQ with 5-FU and cisplatin was shown to be an active therapeutic combination
represented a more effective anti-tumor manager than either agent against lung cancer both in vitro and in vivo [123].
alone in a xenograft tumor mouse model [111]. Additionally, TQ exhibited chemosensitisation effects and sen-
TQ had cytotoxic effect against pancreatic cancer and decreased sitized pancreatic cancer cells to the chemotherapeutic drugs
viability in pancreatic cancer cells [112]. TQ inhibited the expres- gemcitabine and oxaliplatin. TQ augmented anti-tumor activity of
sion of Bcl-2, Bcl-xL, Mcl-1, survivin, and XIAP, and induced that of gemcitabine and oxaliplatin in pancreatic cancer (HPAC or BxPC3
Bax. Furthermore, it suppressed COX-2 expression, PGE2 accumu- cells) [112]. TQ (3 mg/mouse intragastric) alone or combined with
lation and NF-␬B activation [112]. either gemcitabine or oxaliplatin caused a substantial decrease in
Moreover, a potential role has been reported for TQ as a chemo- tumor weight [112]. TQ was effectively synergizing also with cis-
preventive agent at the early stage of skin tumourigenesis [113]. It platin also in lung cancer NCI-460 and NCI-146 cells [123].
induced cell-cycle arrest and apoptosis in neoplastic keratinocytes TQ acted as an enhancer for the anti-cancer effect of DOX in
and squamous carcinoma cell lines A431, Hep2 and RPMI 2650 in HL-60 leukemia and 518A2 melanoma cell lines. It significantly
both dose- and time-dependent manner, by inhibition of Akt and increased the growth inhibition by DOX in HL-60 and multidrug-
JNK phosphorylation [114]. Pretreatment of HR-1 hairless mouse resistant MCF-7/Topo cells when TQ was added [124]. In mice, TQ
skin with TQ attenuated 12-O-tetradecanoylphorbol-13-acetate (10 mg/kg per day) administered in drinking water improved the
(TPA)-induced expression of COX-2, and suppressed NF-␬B signal- anti-tumor effect of ifosfamide and attenuated ifosfamide-induced
ing and the phosphorylation of Akt, JNK and the p38 MAP kinase. Fanconi syndrome [125].
Besides, topical application of TQ induced the expression of HO-1 TQ also showed a powerful chemopreventive agent against
and GST in mouse skin. It also had inhibitory effects on the phospho- fore-stomach tumor induced by B(a)P (a well known clastogen)
rylation and degradation of I␬Ba, along with nuclear translocation and enhanced detoxification in mice [126]. Administration of
and the DNA binding of NF-␬B [115]. TQ inhibited migration and TQ in drinking water one week before, during and after B(a)P
metastasis of both human and mouse melanoma cells, also in vivo treatment resulted in suppression of B(a)P-induced tumourigen-
in the B16F10 mouse melanoma model. It decreased the num- esis. In this regard, anti-oxidant and anti-inflammatory activities
ber of tumor nodules in lungs; this effect was accompanied by a and enhancement of detoxification processes were considered
decrease in expression of NLRP3 inflammasome resulting in reduc- as the possible modes of TQ action against tumourigenesis [61].
tion of proteolytic cleavage of caspase-1. Inactivation of caspase-1 TQ had anti-mutagenic and anti-clastogenic (anti-chromosomal
by TQ resulted in inhibition of IL-1␤ and -18. Furthermore, treat- damaging) activities [42,126] and was effective in inhibiting B(a)P-
ment of melanoma cells with TQ blocked NF-␬B activity [116]. induced clastogenicity in mice. Daily intake of TQ (supplemented
 S. Darakhshan et al. / Pharmacological Research 95–96 (2015) 138–158 145

0.01% in drinking water for 28 days) after and before or during tissue, and could regress following TQ administration. The levels of
exposure to B(a)P significantly decreased the frequencies of chro- MDA and NO were found to be meaningfully lower in the groups
mosomal aberrations in bone marrow cells [126]. given TQ [136]. TQ also was a protective and anti-proliferative
In spite of many types cytotoxic drugs having been developed against tissue injury resulted from prostatitis-induced by E. coli.
for clinical applications, cancer chemotherapy is always accompa- Increases in MDA levels and histological damage caused by E. coli
nied with adverse effects, which may be serious in some cases. were improved considerably after TQ treatment, which particularly
In addition, the cure rate of chemotherapy is limited through the increased the activity of GSH-Px and reduced the activities of CAT
development of resistance. As such, there is increasing interest and SOD [137]. Therefore, TQ could be a clinically valuable agent in
in natural products to complement conventional medicine. The the prevention of prostatitis caused by bacteria.
information reported above, alongside that reported elsewhere TQ may also be used as a potential therapeutic agent to nor-
[127,128] indicates TQ as one of the most promising candidate malize the dysregulated insulin production observed in highly
drugs for the purpose of enhancing the anti-tumor potentials active anti-retroviral therapy treated HIV-1 positive patients [138].
and/or reducing toxicity of chemotherapy. TQ possessed anti-tuberculosis activity against clinical isolates of
Mycobacterium tuberculosis [139]. In addition, TQ exhibited anti-
1.4. Anti-microbial activities fungal activity against Candida albicans, Candida tropicalis, and
Candida krusei [140] as well as the pathogenic dermatophyte strains
In recent decades, the field of ethnobotanical research as a Trichophyton mentagrophytes, Microsporum canis and Microsporum
source for natural anti-microbial drugs and compounds has been gypseum [141]. The ether extract of TQ (and to a lesser extent NS)
expanding considerably. The usage of antibiotics and antibacterial markedly inhibited the growth of eight species of dermatophytes:
chemotherapeutics is becoming less effective, because of resistance four species of Trichophyton rubrum and one each of Trichophyton
to them and side effects. interdigitale, T. mentagrophytes, Epidermophyton floccosum and M.
The anti-bacterial activity of TQ was reported against some canis [142].
bacterial strains, including Escherichia coli, Streptococcus faecalis, Treatment of TQ had a protective effect on schistosomiasis.
Staphylococcus aureus, Peudomonas aeruginosa, and Bacillus sub- Treatment of Schistosoma mansoni-infected mice with NS oil or
tilis [129]. TQ exhibited a significant bactericidal activity especially TQ induced a protective effect on the infection-induced genotoxi-
against Gram-positive cocci, and prevented cell adhesion to glass city. This effect was evidenced by reduction in the percentage of
slides surface and avoided biofilm formation in human pathogenic chromosomal aberrations such as the numbers of chromosome
strains. TQ supplementation was effective on the strong biofilm deletions and tetraploidy induced by schistosomiasis [143]. More-
formers S. aureus, Streptococcus salivarius and Streptococcus oralis; over, a virtuous anti-bacterial action against Paenibacillus larvae
the biofilm former of Enterococcus faecalis, Gemella haemolysans, was observed by TQ (MIC values ranging from 8 to 16 mg/ml) [144].
Pseudomonas aeruginosa and Streptococcus mitis decreased but was These recommendations have made TQ a one of the preferred
not completely suppressed [130]. It has also been reported that TQ natural anti-microbial agent against many infections. However,
exhibits potent growth inhibitory against Gram-positive bacteria at since the exact mechanisms of the TQ anti-microbial activities are
low concentrations. In addition to reducing the number of adherent not clear, further works are required to investigate the main mech-
bacteria, TQ had an effect on the metabolic activity of cells embed- anisms by which TQ affect these organisms.
ded in biofilm. It efficiently killed staphylococci in suspension and
prevented biofilms formation [131]. 1.5. Hepatoprotective effects
TQ possesses a selective anti-bacterial and resistance modifying
activity against oral bacteria. The oral strains S. aureus, Streptococcus In recent years, attention have been drawn to numerous plants
mutans and S. salivarius were sensitive to TQ with MIC values rang- and plant-derived compounds for the treatment of liver ailments.
ing from 8 to 64 ␮g/ml. It has been reported that TQ had synergistic The protective effect of TQ on liver injures have been demon-
effects in combination with anti-bacterial agents. By synergic effect, strated by some studies. TQ could neutralize the negative influences
TQ reduced at least a 4-fold the tetracycline MICs value. In the case of various damaging agents on liver tissue. Oral administration
of benzalkonium chloride, an 8-fold reduction in MICs values was of TQ (100 mg/kg) resulted in substantial protection against the
observed particularly for S. aureus and Vibrio paraheamolyticus. In hepatotoxicity of carbon tetrachloride (CCl4). TQ inhibited the
addition, TQ inhibited the DAPI efflux activity, and the rate of DAPI non-enzymatic lipid peroxidation in liver homogenates and this
accumulation in clinical isolates was enhanced with TQ [132]. suggests that the protective action of TQ against CCl4-induced tox-
The broth microdilution (BMD) method is widely used for deter- icity may be mediated through the anti-oxidant properties of TQ.
mination of minimum inhibitory concentrations of anti-microbial The anti-oxidative nature of TQ as an inhibitor of lipid peroxi-
agents. TQ vapors (32–512 ␮g/mL) caused the complete inhibition dation in liver compares with that of butylated hydroxytoluene
of staphylococcal growth in adjoining wells within a microtiter (BHT) as a standard anti-oxidant [24]. TQ protected isolated rat
plate and this can significantly influence results of the standard hepatocytes in suspension culture against tert-butylhydroperoxide
BMD assay [133]. (TBHP)-induced toxicity. It prevented depletion of intracellular
The epithelial cells pre-treated also the cells post-treated with glutathione, and maintained the integrity of cell membranes as
TQ decreased the number of Streptococcus pyogens that were able evidenced by decreasing leakage of ALT and aspartate aminotrans-
to attach to the kidney epithelial cells in vitro in a dose depen- ferase (AST), as well trypan blue uptake. This protective role of TQ
dent manner [134]. TQ treatment reduced sepsis-related morbidity is comparable with that of silybin, a known hepatoprotective agent
and mortality in mice following LPS and live E. coli, and improved possessing anti-oxidant properties [43]. TQ oral injection efficiently
both renal and hepatic biomarker profiles. Moreover, mice treated reduced acetaminophen-induced hepatotoxicity, as evidenced by
with TQ exhibited lower levels of TNF-␣ and IL-2 [135]. This could decreased serum ALT activities. This positive effect is possibly
be a strong candidate to protect against and/or treat this devas- through increase resistance to oxidative and nitrosative stress and
tating condition for which no drug is suitably effective to date. its ability to enhance GST as well the mitochondrial energy (ATP)
Prostatitis plays a major role in mortality related to prostate dis- production. TQ produced less nitrate/nitrite than acetaminophen-
eases. TQ because of its anti-oxidant and anti-inflammatory effects treated mice and it inhibited nitric oxide production [56]. On the
improved acute bacterial prostatitis (ABP) induced by P. aeruginosa. other side, TQ supplementation induced quinone reductase and
ABP induced by P. aeruginosa had its oxidative effect on prostate glutathione transferase in liver; thus, it may increase resistance
146 S. Darakhshan et al. / Pharmacological Research 95–96 (2015) 138–158

to acetaminophen-induced toxicity through induction of phase-2 evaluate the potentially positive activities of TQ on the induction of
enzymes [27]. Lipid peroxide accumulation and reduction of GSH chromosomal aberrations [143]. Oral TQ treatment greatly reduced
content, and GST and DT-diaphorase activities were observed in the liver injury and tumor markers expression, prevented hepatic nod-
liver of B(a)P-treated tumor-bearing mice. Mice treated with TQ ule formation and reduced tumor multiplicity in NDEA-induced
along with B(a)P showed normal hepatic lipid peroxides, and nor- hepatic cancer bearing rats. It decreased the damaging alterations
mal enzyme content and activities compared to the control group by abrogating cell proliferation, by strongly inducing G1/S arrest in
[61]. cell cycle, and thus exhibited an advantageous role in the treatment
TQ showed positive effects against cadmium-induced hepato- of hepatocellular carcinoma [150].
toxicity in mice. Pre-exposure with TQ prior to cadmium exposure TQ caused also concentration dependent genotoxicity in hep-
decreased oxidative protein damage; it increased the expression atocyte primary cultures, and thus it induced chromosomal
of SOD, and prevented the reduction of CAT when exposed to aberrations and micronucleated cells [151].
cadmium. There was also an elevation in GSH and non-protein In general, these studies showed that TQ may be a potential
thiol (NP-SH) levels after TQ exposure [145]. TQ also ameliorated candidate for the therapy of hepatic fibrosis, carcinogenesis and
liver dysfunction induced by cyclophosphamide in rats through inflammation, although its use is potentially associated with health
up-regulation of anti-oxidant mechanisms [55]. TQ considerably risk [151]; thus further researches are needed to determine the
inhibited tamoxifen-induced hepatic GSH depletion. Consistently, effects of TQ on the liver enzymes and histology alternations and
it normalized the activity of SOD, inhibited the rise in TNF-␣ and its risk to benefit ratio.
ameliorated the histopathological changes; and in conclusion, it
protected against tamoxifen-induced hepatotoxicity in female rats 1.6. Hypoglycemic and anti-diabetic effects
[146].
Treatment with TQ was protective against cypermethrin- Diabetes mellitus is usually associated with many problems.
induced hepatotoxicity in mice, as proved by a decrease in serum Diabetes in humans induces chronic complications such as car-
AST and ALT activity. It also exhibited protective action against the diovascular damage, nephropathy and neuropathy. A scientific
cypermethrin-induced necrosis of hepatic cells, with degeneration, investigation of traditional plant remedies for diabetes may lead
dilatation of sinusoids and dissociated remark cordons in livers of for the development of alternative drugs and therapeutic strategies.
mice. Moreover, it induced cell proliferation leading to enhanced More than 1200 species of plants reported to have been used to treat
regeneration after tissue damage [147]. TQ is a promising agent diabetes and/or investigated for anti-diabetic activities, including
in maintenance of normal hepatic function during treatment with N. sativa [152].
anti-tuberculosis drugs and had therapeutic effect against anti- TQ has anti-diabetic activities apart from possessing many other
tuberculosis drugs-induced liver damage. TQ administration for 8 valuable pharmacological actions. The most common model of
weeks (3 days/week) attenuated the increases in the levels of hep- human diabetes is the streptozotocin (STZ)-induced diabetes in
atic AST, ALT and ALP enzymes, and caused a consequent recovery animal models. TQ exerted anti-hyperglycemic effects and ame-
toward normalization indicating stabilization of plasma membrane liorated blood glucose levels in gestational diabetes mellitus. Daily
as well as repair of hepatic tissues [148]. TQ was protective against gastric administration of TQ (50 mg/kg for 30 days) to STZ-induced
aflatoxin B1 (AFB1)-induced hepatotoxicity. The AST, ALT, ALP and diabetic hamsters efficiently reduced both fasting blood glucose
MDA levels were significantly lower in groups that received TQ and glycated hemoglobin levels, and reduced the gluconeogen-
compared with AFB1, and liver sections of AFB1 intoxicated mice esis observed under the diabetic condition. Glucose production
showed necrosis and degradation while treatment with TQ helped was considerably lower in hamsters treated with TQ, since the
to normalize liver architecture [57]. Solid lipid nanoparticles (SLNs) glucose-lowering effect of TQ is not related directly to insulin
of TQ acted against paracetamol-induced liver cirrhosis and fibrosis action; also, it decreased gluconeogenesis by suppressing the syn-
[13]. TQ remarkably reduced thioacetamide-induced liver fibrosis thesis of gluconeogenic enzymes [153]. TQ improved the glycemic
and inflammation. This effect accompanied by reduced ␣-smooth status in STZ-nicotinamide-induced diabetic rats. Oral administra-
muscle actin (␣-SMA) protein and mRNA expression, collagen- tion of TQ for 6 weeks resulted in a major decrease in plasma
and tissue inhibitor of metalloproteinase-1 (TIMP-1). Moreover, TQ glucose and an increase in insulin levels. TQ normalized the per-
down-regulated the expression of toll-like receptor 4 (TLR4) and turbed carbohydrate metabolism by enhancing glucose utilization
decreased pro-inflammatory cytokine levels. It also inhibited PI3K and by decreasing hepatic glucose production. TQ at 80 mg/kg dose
phosphorylation, enhanced the phosphorylation AMPK and liver decreased the activities of the gluconeogenic enzymes glucose-6-
kinase B (LKB)-1 [149]. phosphtse and fructose-1,6-bisphosphatase [25]. Treatment of rats
TQ has been shown to possess anti-oxidant and anti- with TQ decreased the diabetes-induced increases in tissue MDA
inflammatory effects both in vitro and in vivo, and these roles and serum glucose, and increased serum insulin and tissue SOD.
are directly connected to hepatoprotection. It may reduce oxida- TQ improved most of the toxic effects of STZ, including DNA dam-
tive stress through direct anti-oxidant effect as well as induction age, mitochondrial vacuolization and fragmentation, and preserved
of endogenous anti-oxidant enzymes leading to the prevention beta-cell integrity by decreasing oxidative stress. Thus, the anti-
of liver toxicity. TQ improved liver anti-oxidant capacity and diabetic action of TQ could be due, in part, to amelioration of the
enhanced the expression of liver anti-oxidant genes in hyper- cellular and subcellular structures of beta-cells [64]. Treatment of
cholesterolemic rats. It increased the expression of SOD1, CAT, STZ-diabetic rats with TQ blocked the expression of COX-2 enzyme,
GSH-Px genes and elevated hepatic SOD and GSH-Px levels [50]. lipid peroxidation and MDA levels, along with increased the level
Administration of TQ was effective in increasing the activities of of SOD in the pancreatic tissue [154].
quinone reductase [27,47] and glutathione transferase [27], which Nutritional supplementation of gestational diabetes mothers
makes it a promising manager against chemical carcinogenesis and with TQ during pregnancy and lactation periods improved diabetic
toxicity in liver. It was also reported that the oral administration of complications and maintained an efficient T cell immune response
TQ in bile duct ligated rats, reduced liver oxidative damage and in their offspring, providing a protection in later life. The restora-
ductular proliferation [82]. tive effect of TQ on IL-2 level and T-cell proliferation, and the
TQ had protective role against chromosomal aberrations following rescue of both circulating and thymus homing T cells in
induced by schistosomiasis infection in mice. Spleen and bone mar- the offspring of diabetic mothers enhanced the immune response
row cells in both in vitro and in vivo experiments were used to [155]. Similarly, the data indicated a decrease in the percentage of
 S. Darakhshan et al. / Pharmacological Research 95–96 (2015) 138–158 147

abortions, an increase in the number of successful pregnancies and effect than sulfasalazine, an anti-colitis drug [80]. Likewise, TQ pro-
an improvement of mortality among new pups born to diabetic tected against the ulcerating effect of alcohol and diminished most
mothers. TQ improved aberrant hydroperoxide and ROS levels in of the biochemical adverse effects induced by alcohol in gastric
pups and these results may be mediated by increase in the levels mucosa. It affected CAT activity in gastric tissue [48]. It attenuated
of GST, GSH and CAT, and a decrease in DNA damage [156]. acetic acid-induced colitis as evidenced by the blinted release of
Accumulation of advanced glycation end-products in tissues and histamine, to which the prevention of GSH depletion and lipid per-
serum plays important role in diabetes-associated complications. oxidation may have also helped [80]. In addition, an anti-histaminic
TQ had anti-glycating effect and reduced diabetic problems due to effect is an important defensive mechanism against gastric injury
protein glycation [157]. It regulated the plasma concentrations of [166] and TQ has a potent anti-histaminic effect [80].
cholesterol and triglycerides; plasma triglyceride and cholesterol TQ had a helpful role in indomethacin-induced gastric injury
levels diminished significantly in the diabetic rats treated with TQ model and was able to decrease acidity in indomethacin-induced
[158]. Orally administration of TQ (10 mg/rat/day) affected experi- gastric ulcer models [167]. TQ treatment was also able to reduce
mental diabetic neuropathy. TQ has been associated with recovery the number of macrophages and the gravity of gastric mucosal
of the histopathologic changes in sciatic nerves, and myelin break- lesions, and it was effective in controlling bacterial translocation
down decreased meaningfully after treatment with TQ [159]. and improving intestinal barrier function in rats [168].
Oxidative stress and inflammation play a crucial role in the Altogether, these studies confirm the gastroprotective effects of
progression of diabetic problems. Inhibition of adenosine kinase TQ in animal models and this makes it possible to use TQ as a natural
by ABT702, an adenosine kinase inhibitor, decreased extracellular drug against gastrointestinal defects in human.
adenosine levels to moderate renal injury in STZ-induced diabetes.
In fact, the nephroprotective effects of ABT702 could be attributed 1.8. Neuroprotective effects
to the reduction in renal inflammation and oxidative stress in dia-
betic mice [161]. STZ-induced diabetes caused nephropathy, and TQ TQ has demonstrated several beneficial neuropharmacological
therapy resulted in renal morphologic and functional improvement properties. Administration of TQ powerfully induced protec-
[160]. The anti-oxidant activity of TQ may relieve damage to beta- tion in cultured rat primary hippocampal and human induced
cells caused by STZ. TQ was effective for beta-cell protection against pluripotent stem cell (hiPSC)-derived neurons cells against ␣-
damage, through the down-regulation of inflammatory activity synuclein-induced synaptic toxicity [169]. TQ protected against
mediated by NO pathway [22]. TQ administered intraperitoneally MPP+- and rotenone-induced cell death in primary dopaminergic
in diabetic rats at 3 mg/kg normalized the elevated levels of the neurons in cell cultures obtained from patients with Parkinson’s
pro-inflammatory cytokines IL-1␤ and TNF-␣ [22]. STZ-diabetes disease. It sheltered cultured dopaminergic TH immunoreactive
induce an increase in heart and brain NO and MDA concentrations, cells from degeneration induced by MPP+ and rotenone toxicities;
changes that were mitigated by post-treatment of rats with TQ. and the anti-oxidant properties of TQ appeared to play an impor-
STZ-diabetes induced a decrease in GST, GSH and CAT, and these tant role in this respect [170]. TQ also inhibited oxidative stress and
lowered levels were improved by TQ administration. Serum car- neuropathy in STZ-induced diabetic rats. It reduced norepinephrine
diac creatine kinase muscle and brain types (CK-MB) was decreased and dopamine and enhanced serotonin levels [162]. TQ suppressed
in the diabetic rats, which recovered with TQ administration. Dur- morphine-induced brain oxidative stress, and development of mor-
ing diabetes, there was a marked increase in norepinephrine and phine tolerance and dependence in mice. The elevation in brain
dopamine and a marked decrease in serotonin level; these were glutamate level and the increase in the expression of brain iNOS
partly reversed by TQ orally [162]. and NO overproduction by morphine can be reduced by TQ [171].
These findings provide scientific bases to the widespread use of TQ has helpful properties against neurotoxic effects of lead (Pb)
NS seeds as an anti-diabetic remedy in Middle East folk medicine (a ubiquitous heavy metal) in rats. Co-treatment of lead-exposed
[163]; still the information on TQ action is still insufficient espe- rats with TQ distinctly reduced the incidence of lead-induced brain
cially as far as efficacy on diabetic complications is concerned. lesions [172]. TQ by anti-oxidant properties protected brain tissue
of irradiated rats from radiation-induced nitrosative damage [65].
1.7. Gastroprotective effects TQ had a protective role against ethanol-induced neuronal apopto-
sis in primary rat cortical neurons by decreasing ethanol-mediated
Ample evidence exists on the protective effect of TQ on gastroin- mitochondria-dependent apoptosis [173]. It caused morphologic
testinal tract. TQ possessed protecting effect against gastric lesions, improvement and prevented neurodegeneration in the hippocam-
which may be related to the protection of the gastric mucosal redox pus after chronic toluene exposure, and the distorted nerve cells
state. Anti-oxidant property of TQ corrected ischemia/reperfusion were mostly absent in the TQ-treated rats [33].
(I/R)-induced gastric dysfunction and stomach ulcer in rats. It Beta-amyloid (A␤) peptides are considered to play a main role in
increased GSH level and SOD activity, and reduced MDA content the pathogenesis of Alzheimer’s disease (AD) and compounds that
and MPO activity [164]. Gastric I/R-mediated alteration in acid can prevent pathways of A␤-induced neurotoxicity may be poten-
concentration, acid output and pepsin was markedly hindered by tial therapeutic agents for treatment of AD. TQ protected against
pre-treatment with TQ. The gastroprotective effects of TQ resulted A␤-induced toxicity and the impairment of synaptic function, as
from inhibition of the proton pump (H+ /K+ -ATPase), acid secretion well as decreased ␣␤ aggregation in primary hippocampal and
and neutrophil infiltration, though improving mucin secretion and cortical neurons. Treatment with TQ inhibited ␤-amyloid peptide
content, and NO production. By reducing gastric oxidative injury, 1–42 sequence A␤(1–42)-induced neurotoxicity, and efficiently
TQ combated the I/R-induced lipid peroxidation, and prevented attenuated A␤(1–42)-induced mitochondrial membrane poten-
depletion of GSH and SOD. All these mechanisms maintained nor- tial collapse in cultured hippocampal neurons, also suppressing
mal gastric mucosal barrier integrity. In addition, TQ corrected the ROS generation caused by A␤(1–42) [174]. TQ also abridged A␤-
altered parameters in a comparable manner to that of the refer- induced toxicity through inhibition of mitochondrial dysfunction
ence drug used, omeprazole and combination of it with omeprazole and oxidative stress in PC12 cells (derived from a pheochromo-
was synergic [165]. The administration of TQ on the acetic acid- cytoma of a rat adrenal medulla); moreover it protected rats
induced colitis by intracolonic injection of 3% acetic acid showed against transient forebrain ischemia-induced damage in the hip-
that pretreatment of animals for 3 days with 10 mg/kg TQ led to pocampus [34,173]. A significant increase in thiobarbituric acid
complete protection against colitis, with a similar or even higher reactive substances (TBARS) content, NO level and activity of
148 S. Darakhshan et al. / Pharmacological Research 95–96 (2015) 138–158

acetylcholine esterase was observed in A␤ exposure, which was 1.9. TQ and cardiovascular problems
normalized by TQ pre-treatment. Furthermore, TQ could reduce
neuronal damage and loss through counteracting oxidative stress, Cardiovascular diseases remain one of the leading causes of
via ameliorating glutathione and its dependent enzymes (GSH-Px, death worldwide. Several lines of evidence suggest that TQ is a
glutathione reductase) which are depleted by A␤(25–35) [175]. therapeutic option in cardiovascular complications. Doxorubicin
TQ has the potential to reduce A␤(1–40)-induced neuronal cell (DOX) has a wide spectrum of anti-tumor activity along with
death in primary cultured cerebellar granule neurons (CGNs). Pre- cardiotoxicity as a major side effect. Pretreatment with TQ pro-
treatment of CGNs with TQ and subsequent exposure to A␤(1–40) tected against DOX-induced cardiotoxicity without compromising
protected CGNs against the neurotoxic effects of the latter. In its anti-tumor activity [26,41]. TQ (8 mg/kg/day) administered with
addition, CGNs were better preserved with intact cell bodies, exten- drinking water starting 5 days before a single injection of DOX
sive neurite networks, a loss of condensed chromatin and less and continued during the experimental period improved the DOX-
free radical generation than those exposed to A␤(1–40) alone induced heart toxicity in mice. This finding was supported by
[176]. significant declines in serum LDH and creatine kinase elevated lev-
I/R injury resulting from stroke leads to metabolic distress, els, and by histopathological improvement of cardiac tissue [26]. TQ
oxidative stress and neuroinflammation, making it likely that acted as an inhibitor of lipid peroxidation and superoxide radical
therapeutic intervention strategy needed which affect oxida- scavenger in DOX-induced cardiotoxicity in rats [41]. Cyclophos-
tive stress and inflammation [177]. TQ was effective against phamide increases serum creatine kinase, creatinine, urea, LDH,
transient forebrain ischemia-induced damage in the rat hip- cholesterol, triglycerides and TNF-␣. In heart tissue, it also increases
pocampus. TQ administration (5 mg/kg/day orally) 5 days before TBARS and total nitrate/nitrite, and reduces GSH-Px, CAT and ATP
ischemia continued during the reperfusion time attenuated fore- levels. Supplementation by TQ resulted in a complete reversal
brain ischemia-induced neuronal damage, as revealed by reduction of all these changes to their control values [58]. Moreover, TQ
in the number of dead hippocampal neuronal cells. Pretreatment exhibited protection against the cypermethrin-induced necrosis,
of ischemic rats with TQ decreased the elevated levels of MDA, and degeneration, and loss of striation in heart; it resulted in reversal
improved GSH content, CAT and SOD activities to normal levels of cypermethrin-induced oxidative stress and lipid peroxidation
[83]. [147].
In traditional medicine, NS was known as an anti-convulsant and The state of hyperhomocysteinemia (HHcy) appears to be
some studies also have shown anti-convulsant effects of NS and TQ. accompanied by high risk of coronary, cerebral and peripheral vas-
Hosseinzadeh and Parvardeh [23] investigated the anti-convulsant cular disease; and the pathogenesis of HHcy is known to be linked
activity of TQ, using a single dose of pentylenetetrazol (90 mg/kg) as with free radical formation. Pretreatment of rats with a dose of
petit mal epilepsy model in mice. This anti-convulsant activity was 100 mg/kg TQ orally, for one week almost entirely protected against
through an opioid receptor-mediated improvement in GABAergic methionine-induced HHcy. Consequently, utilizing TQ against the
tone. The anti-nociceptive effect of morphine was increased in cardionegative impacts of HHcy may be valued [35]. Intravenous
TQ-pretreated mice. TQ produced anti-nociceptive effects mostly administration of TQ to rats decreased the arterial blood pressure
through the supraspinal opioid systems, mainly ␮- and ␬-opioid and the heart rate in a dose-dependent manner [21]. It was effec-
receptor subtypes [20]. TQ was beneficial for lessening neuropathic tive in protection of rats against N-nitro-l-arginine methyl ester
pain following spinal cord injury (SCI) and it had analgesic/anti- (l-NAME)-induced hypertension, perhaps via anti-oxidant action
nociceptive effects on central pain following SCI. Also total oxidant [181]. Treatment with TQ decreased the elevated creatinine, and
status, NO, MDA, IL-1␤, and TNF-␣ levels were lower in the TQ increased GSH levels compared to normal levels, and inhibited the
groups than in the control group [178]. TQ showed anti-anxiety production of superoxide radicals in enzymatic and non-enzymatic
activity through GABAergic and nitriergic modulation. TQ (10 and systems. It reduced the increase in systolic blood pressure induced
20 mg/kg) produced considerable anti-anxiety effects in unstressed by l-NAME in a dose-dependent manner [181].
mice without altering nitrite levels, but only the higher dose Pretreatment with TQ markedly prevented diesel exhaust
(20 mg/kg) of TQ augmented the GABA content in unstressed mice. particles-induced pulmonary and cardiovascular changes. Pretreat-
In stressed mice, TQ (20 mg/kg) showed anxiolytic effects, with ment of mice with TQ prohibited diesel exhaust particles-induced
a decrease in plasma nitrite and reversal of the reduced brain decrease of systolic blood pressure, increased SOD activity and
GABA content [81]. Additionally, TQ demonstrated anti-depressant decreased IL-6 level. It also stopped the decline in platelet numbers
effects. It further demonstrated anti-oxidant effects by reducing [182].
TBARS and increasing reduced GSH levels. It may be suggested
that TQ is a potential candidate for the management of depression 1.10. TQ against respiratory diseases
[179]. A double-blinded placebo controlled randomized trial was
done where TQ (dose of 1 mg/kg) was administered as an adjunc- The therapeutic effects of the NS seeds on respiratory problems
tive therapy to 22 children with refractory epilepsy, and its effects including asthma and dyspnea have been described in Iranian
on frequency of seizures were compared with those of a placebo. ancient medical books [183]. TQ can neutralize the negative results
The patients were assigned in two groups and received either TQ of some injuring agents. TQ showed anti-apoptotic effect and
or placebo for a period of four weeks, and then, during the two attenuated lung injury induced by chronic toluene exposure in rats
weeks of wash out period, they received only their preexisting anti- [36]. It also alleviated the progression of pulmonary fibrosis that
epileptic drugs; after cross-overing, they received TQ or placebo for induced by bleomycin in rats. TQ counteracted emphysema in air
a period of four weeks again. From this study, it concluded that TQ alveoli, inflammatory cell infiltration, lymphoid hyperplastic cells
had anti-epileptic effects [180]. activation surrounding the bronchioles, and the overexpression
TQ showed a very pleotropic favorable effect on experimen- of activated form of NF-␬B in lung tissue induced by bleomycin. It
tal allergic EAE [76] and suppressed NF-␬B activation in brain and also restored anti-oxidant enzyme activity of SOD and GST toward
spinal cord of EAE [76]. normal values [60]. TQ was protective against cyclophosphamide-
These results indicate TQ as a promising tool, if properly used, in induced pulmonary damage. Cyclophosphamide increased the
the prevention and treatment of a variety of nerves disorders such level of serum biomarkers total protein, LDH and TNF-␣. Treatment
as ischemia-reperfusion injury, depression, seizure, Alzheimer and of rats with TQ 7 days before and after cyclophosphamide injection
Parkinson’s disease. reduced the alterations in lung and serum biomarkers associated
 S. Darakhshan et al. / Pharmacological Research 95–96 (2015) 138–158 149

with inflammatory reactions, with less lipid peroxidation and protective against vancomycin-induced kidney injuries. The levels
restoration of anti-oxidants. It also improved cyclophosphamide- of serum blood urea nitrogen, creatinine and MDA were increased
induced histopathological changes in lung tissue [59]. In addition, in the vancomycin group, and activities of SOD and GSH-Px in kid-
TQ relaxed pulmonary arterial rings and caused a concentration- ney tissue were reduced. TQ administration ameliorated noticeably
dependent decrease in the tension of the pulmonary arterial rings these changes [53]. Administration of TQ caused renal morphologic
precontracted by phenylephrine. This relaxant effect may be due and functional improvement after STZ-induced diabetes in rats
to activation of ATP-sensitive potassium channels and probably [160]. TQ in the drinking water (5 mg/kg per day) before and during
by non-competitive blocking of serotonin, alpha1 and endothelin ifosfamide treatment improved the severity of ifosfamide-induced
receptors [184]. renal damage. It significantly amended ifosfamide-induced phos-
TQ had potential anti-inflammatory role during the allergic phaturia, glucosuria, elevated serum creatinine and urea, and
response in the lung. Intraperitoneal injection of TQ before air- normalized creatinine clearance rate. Moreover, it prevented renal
way challenge of OVA-sensitized mice attenuated allergic airway GSH depletion and lipid peroxide accumulation via anti-oxidant
inflammation by preventing Th2 cytokines and eosinophil infiltra- mechanisms [125]. Furthermore, TQ had positive effects on DOX-
tion and goblet cell hyperplasia in the airways, both in vivo and induced hyperlipidemic nephropathy in rats and decreased serum
in vitro. TQ showed a significant effect in inhibition of IL-4, -5 and triglycerides and cholesterol. Treatment with TQ (10 mg/kg/day)
-13, and in induction of IFN-␥ production in the BAL fluid [21]. TQ repressed DOX-induced proteinuria and albuminuria, also lowered
had an anti-inflammatory action during the allergic response in total triglycerides, total cholesterol and lipid peroxidation in the
the lung through the inhibition of Th2-driven immune response. kidneys. Moreover, non-protein sulfhydryl (NPSH) content and CAT
Injection of TQ intraperitoneally for 5 days before the first OVA activity in the kidneys of TQ-treated DOX group were markedly
challenge diminished airway inflammation, and this attenuation elevated compared with DOX alone [51]. DOX-induced nephrosis
of inflammation was associated to the inhibition of COX-2 protein involved a redox imbalance in renal tissue; this was reversed by
expression and PGD2 production [70]. TQ. Animals treated with TQ showed a reduced renal damage with
Chronic airway inflammation is a key feature of bronchial normalization of the elevated levels of serum urea, creatinine and
asthma and leukotrienes are potent inflammatory mediators that urinary albumin excretion, as equivalents of MDA, accompanied by
play a role in the pathophysiology of asthma. Administration of substantial increases in the activities of SOD and GST, along with
TQ before OVA challenge inhibited expression by lung cells of 5- reduction of the renal oxidase NOX-4 level [187].
lipoxygenase, the main enzyme in leukotriene biosynthesis, and TQ also was effective in hepatorenal dysfunction induced by
moderated the levels of LTB4 and LTC4. This was accompanied by a renal I/R. Renal I/R resulted in an increase in MDA level and reduc-
decrease in Th2 cytokines, BAL fluid and lung tissue eosinophilia tion in GST and SOD activity in liver and kidney tissues, and
[69]. It also caused a concentration-dependent decrease in the TQ treatment caused the reversal of these changes. It reduced
tension of the tracheal smooth muscle precontracted by carba- spermidine/spermine N-1-acetyl-transferase (SSAT), a catabolic
chol in the guinea pig. TQ induced relaxation of guinea pig’s enzyme that participates in polyamine metabolism, and CYP3A1
isolated trachea, mediated by inhibition of lipoxygenase products gene expression in liver and noticeably in kidney of rats [188].
of arachidonic acid metabolism and by non-selective blocking of TQ administration protected the kidneys from oxidative damage
the histamine and serotonin receptors [185]. caused by pyelonephritis. In the pyelonephritis, SOD and CAT activ-
Furthermore, TQ has been shown to be useful for treatment of ity, and MDA levels were significantly abnormal; and levels of
acute respiratory distress syndrome in a rat model [186]. Intra- these factors repaired in the pyelonephritis treated by TQ [189].
venous administration of TQ induced rises in the intratracheal Supplementation of TQ exhibited protective action against the
pressure, without any effect in the respiratory rate in urethane- cypermethrin-induced-sloughing off epithelial cell, shrinkage of
anaesthetized guinea pig [19]. These results from TQ demonstrate glomeruli, and necrosis of renal tubules in kidneys of mice [147].
its value in respiratory disorders, and further support the tradi- These data suggest that TQ can be applicable as a protective agent
tional use of black seeds to treat respiratory complaints such as for nephropathies.
bronchial asthma.
1.12. Effects of TQ on bone and joint complications
1.11. Nephroprotective effects
The effects of TQ on bone metabolism, bone formation and bone
Reports have shown protective effects of TQ on kidneys in healing as well disorders related to bone was reported in some
various pathogenic conditions. TQ provided an improvement in studies. TQ may be advantageous in osteogenesis. Kirui et al. [190]
renal lesions resulted from various toxic agents because it showed examined the physiological responses of TQ in the femoral defect
a defensive effect, in a part by attenuating oxidative stress and animal model and showed sustained delivery of drug was effec-
inflammation. TQ supplementation prevented gentamicin-induced tive in bone healing. It has shown that the alveolar bone loss due
acute renal failure in rats by improving mitochondrial function and to periodontitis was reduced by gastric TQ given to rats. TQ also
augmenting ATP production. It reduced the nephrotoxicity indexes reduced the number of osteoclasts and raised osteoblastic activ-
and degenerative changes induced by gentamicin. It also reversed ity [191]. TQ had anabolic effects and induced the proliferation
the gentamicin-induced increase in blood urea nitrogen, creatinine, and the mineralization of MC3T3-E1 osteoblast cells. It induced
and TBARS, which is accompanied with the rise in the total anti- the expression of differentiation related genes including ALP (an
oxidant status in renal cortex, including GSH level, GSH-Px and early marker for mature osteoblasts), osteocalcin and osteopontin
CAT activities [52]. TQ protected against mercuric chloride (HgCl2 )- (phenotypic markers for the later stage of osteoblast differentia-
induced renal damage in rats. The decline of anti-oxidant enzymes, tion); it also increased the expression of BMP-2, and up-regulated
increase of serum creatinine, proliferative response and histolog- the phosphorylation of ERK signaling pathway and activated MAPK
ical damage caused by HgCl2 are ameliorated by TQ usage [54]. pathway. TQ’s effect on cell maturation was accompanied by an
TQ, when administered in drinking water (50 mg/l), improved the increase in BMP-2 expression and activation of the ERK pathway.
cisplatin-induced acute kidney damage, as well developed cisplatin The ability of TQ to enhance the expression levels of early, interme-
therapeutic effects on rodent models. This was marked by signifi- diate and late differentiation markers indicates that it can be used
cant reduction in serum urea and creatinine, and improvement in for interference at various levels from early to terminal stage of
polyuria, kidney weight, and creatinine clearance [30]. It was also differentiation process [192]. In addition, systemic use of TQ in rats
150 S. Darakhshan et al. / Pharmacological Research 95–96 (2015) 138–158

resulted in measurable acceleration of bone formation. Systemic 1.14. Hypolipidemic effects

administration of 10 mg/kg of TQ can promote bone formation and
may be beneficial in prevention of relapse following the rapid max- TQ was capable of lowering plasma cholesterol level in animals,
illary expansion (RME) procedure. In light of potent anti-oxidant in part, because of its anti-oxidant activity. It attenuated hepatic
properties, TQ may prove to have an imperative role in accelerat- oxidative stress induced by high cholesterol diet in rabbits [196].
ing bone formation and in the shortening of the retention period It also had protective effect on development of atherosclerosis
involved in RME. TQ reduced the ROS production and the level of in cholesterol-fed rabbits. Administration of TQ with cholesterol-
pro-inflammatory cytokines IL-1␣ and -6 and TNF-␣, cytokines that enriched diet reduced total cholesterol, low-density lipoprotein
can lead to differentiation of osteoclast precursors and osteoclast (LDL)-cholesterol, triglycerides and TBARS concentrations, while
activity to cause bone resorption [193]. increased high-density lipoprotein (HDL)-cholesterol as well as
A link between inflammation and bone homeostasis has been glutathione content compared to control group [200].
attributed to the effects of IL-1␤, IL-6, TNF-␣, IFN-␥ and PGE2 that TQ supplementation along with ethanol and high fat diet
are abundantly expressed in patients with RA and in the arthritic markedly decreased the levels of serum lipase, amylase and
joints of rat with collagen-induced arthritis. Blockade of these caspase-1. A dose of 100 mg/kg body weight was found to provide
resulted in a reduction of disease severity and bone resorption optimal effect on pancreas against high fat diet-induced pathologi-
[194]. Treatment with TQ shifted the balance of cytokines toward cal changes [201]. In addition, TQ generated a hypocholesterolemic
a bone protecting pattern that acted to both decrease levels of effect in diabetic rats through regulation of cholesterol in two main
TNF-␣, IL-1␤, IFN-␥ and IL-6, and raise the levels of IL-10 [72] a mechanisms, firstly by increasing uptake of LDL-cholesterol via
potent anti-inflammatory cytokine that limits cartilage and bone up-regulation of hepatic LDL receptor gene and, secondly, by sup-
pathology in RA [195]. From these results, TQ appears to have car- pressing the 3-hydroxy-3-methylglutaryl-coenzyme A reductase
tilage/bone protective effects mediated through the inhibition of (HMG-CoAR) gene [158]. It also prevented cardiovascular disease
pro-inflammatory and induction of anti-inflammatory mediators. risks and parameters via a reduction in HMG-CoAR, along with
TQ prominently abolished also LPS-induced IL-1␤, TNF-␣, MMP-13, an increase in arylesterase activity. TQ may thus be used in the
COX-2, and PGE2 induction in isolated RA fibroblast-like syn- protection of hyperlipidemia and atherosclerotic problems [202].
oviocytes. Furthermore, LPS-induced phosphorylation of p38 MAP It had positive effects on DOX-induced hyperlipidemic nephropa-
kinase and NF-␬B-p65 were also blunted by TQ. The oral adminis- thy in rats and could decrease serum triglycerides and cholesterol,
tration of 5 mg/kg/day TQ in a rat adjuvant-induced arthritis model as shown by the reduction in total triglycerides, total cholesterol
of RA significantly decreased the serum levels of H2O2-induced 4- and lipid peroxidation in the kidneys after treatment with TQ
hydroxynonenal, and increased bone turnover markers, such as ALP (10 mg/kg/day) [51]. The results, taken together support a role for
and tartrate-resistant acid phosphatase. The protective effects of TQ TQ in the treatment of hyperlipidemia/dyslipidemia and associated
against articular diseases were also manifest from the reduction in complications.
arthritis scoring and bone resorption [75].
1.15. Anti-histaminic effects

1.13. Effects of TQ on reproductive system Histamine is released by basophils and mast cells, producing
allergic reactions. Some of anti-inflammatory effects of TQ have
Acute bacterial prostatitis (ABP) induced by P. aeruginosa, which been related to its role in inhibition of histamine production and/or
has an oxidative effect on prostate tissue, regressed following TQ release. It reduced the ocular symptoms in allergic conjunctivitis by
administration as shown by biochemical and histological results. attenuating histamine in mice exposed to OVA [71]. TQ induced
In the ABP groups given TQ, the levels of MDA, SOD and NO, and relaxation of guinea pig isolated trachea, which is mediated by
the GSH-Px activity were found to be normal compared to control non-selective blockade of the histamine receptors. It abolished the
group, indicating an antioxidant activity of TQ [136]. It also had a effects of histamine and serotonin on the tracheal and ileum smooth
protective effect against tissue injury in ABP-induced by E. coli. TQ muscles [185]. Moreover, TQ protected against acetic acid-induced
reduced MDA, and improved the activities of GSH-Px, CAT and SOD colitis in rats by its ability to inhibit the release of the mediators
[137]. TQ, through its anti-oxidant and anti-inflammatory activi- platelet-activating factor (PAF) and histamine [80], and it is proven
ties, protected the testes against the injurious effect of cadmium that a main mechanism by which TQ acts against gastric injury is
exposure. It decreased the cadmium-induced reductions in serum an anti-histaminic action [166].
testosterone, elevated testicular glutathione and SOD activity, also
decreased the elevations of testicular MDA, NO and cadmium ion 1.16. TQ and fibrosis
levels resulting from cadmium chloride administration. In addition,
the cadmium-induced expression of iNOS, TNF-␣, COX-2 and NF-␬B TQ has been investigated for its anti-oxidant and anti-
was diminished by TQ in testicular tissue [196]. inflammatory activities in some models since its isolation in 1960s;
TQ had favorable effect against lead (Pb)-induced inhibition of however, its possible anti-fibrotic effect is yet not very clear.
rat testicular functions. It protected against Pb-induced impair- TQ attenuated liver fibrosis through hindering PI3K and down-
ment of testicular steroidogenic and spermatogenic functions. regulating TLR4 signaling pathways in activated rat hepatic stellate
When co-administrated with lead, TQ significantly improved the cell line, T-HSC/Cl-6. It induced apoptosis, reduced the expression
low plasma testosterone level and the decreased epididymal sperm of CD14 and TLR4, and suppressed the expression of collagen-I and
count caused by lead [197]. TQ treatment declined interstitial space PI3K, and Akt phosphorylation. Furthermore, the levels of serum
dilatation in methotrexate testicular injuries in mouse model; ALT and AST decreased by TQ [203]. TQ substantially attenuated
it reversed histological alterations, augmented total anti-oxidant thioacetamide-induced liver fibrosis and inflammation. It down-
capacity and blocked the increase in the MPO activity, which regulated the expression of TLR4, inhibited PI3K phosphorylation,
occurred in methotrexate-treated group [198]. TQ was cytotoxic activated LKB1-AMPK signaling pathway, and decreased extracel-
toward breast cancer [95,96], ovarian adenocarcinoma [93], cervi- lular matrix accumulation [149]. Solid lipid nanoparticles (SLNs)
cal squamous carcinoma [99], and prostate cancer [100,101]. formulation of TQ inhibited paracetamol-induced liver cirrhosis
In conclusion, considering these data, the use of TQ could be a and fibrosis [13]. Pretreatment with TQ blocked the LPS-induced
viable option for reproductive system diseases. pro-inflammatory response in LX2, an immortalized human
 S. Darakhshan et al. / Pharmacological Research 95–96 (2015) 138–158 151

hepatic stellate cell line, as demonstrated by a reduced IL- migration, invasion, and tube formation [100]. It blocked angio-
6 and MCP-1 mRNA expression [203]. It also blocked the genesis in vitro and in vivo, so preventing tumor growth [102,118].
bleomycin-induced pulmonary fibrosis in rats by attenuation of
bleomycin-induced oxidative stress and through NF-␬B inhibition 1.17.4. Migration, invasion and metastasis
[60]. The most lethal feature of cancer is its ability to spread or metas-
These results suggested that TQ can reduce fibrosis and inflam- tasize to distant sites. TQ was reported to control invasion and
mation, and may be a potential candidate for fibrosis therapy. metastasis of cancer [105,116,118,121].
However, the exact effects of TQ on fibrosis in organs including
liver, lung, skeletal muscle, heart, and kidney are not yet clear; and 1.17.5. Inflammation and Oxidative stress
efforts in this direction are still needed. The alleviation of oxidative stress and inflammation is a ratio-
nal strategy to prevent various chronic diseases. Cells are equipped
1.17. Molecular mechanisms with a wide array of cytoprotective factors, which upon activa-
tion protect cells from oxidative and inflammatory insults. TQ
The TQ mechanisms of action are complex because various have been shown to induce the expression and/or activities of
actions have been observed. TQ has been shown to target multi- cytoprotective proteins involved in cellular anti-oxidant defense
ple factors in many pathophysiological conditions. These pathways and/or the inactivation of electrophilic carcinogens, thereby pre-
include cell cycle progression, proliferation, apoptosis, angiogene- venting oxidative stress [145,146,164,165]. It also acted on the
sis, migration, invasion and metastasis of tumor in cancer models. immune system by modulating the levels of immune mediators
Moreover, it inhibits oxidative damage of cellular components [68,70].
and inflammatory responses. TQ also modulates proteins, which TQ down-regulates the expression of pro-inflammatory medi-
involved in metabolism processes. The molecular targets modified ators such as COX-2 [73,196], iNOS [84,196], 5-lipoxygenase
by TQ are briefly discussed here below and summarized in Table 3. [31,67,70], TNF-␣ [22,72] and inhibit the activation of transcription
factor NF-␬B [77,118], Akt and ERK signaling pathways [100].
1.17.1. Cell cycle and proliferation Overall, it is likely that TQ modifies a number of molecular tar-
Uncontrolled growth and proliferation of cancer cells are impor- gets; however, the molecular mechanisms underlying the effects of
tant features in carcinogenesis, which causes increase in size of the drug remain not fully understood and further work is needed
tumor and become problematic to cure. Expression and/or activity also in this respect.
of cell cycle progression and proliferation regulators is affected by
TQ, leading to cell cycle detention, DNA damage and apoptosis. Evi- 1.18. Safety and adverse effects
dence indicates TQ has the ability to arrest cancer cells at different
phases of the cell cycle; G0/G1 [93,113,205], G1/S [101] and G2/M The growing interest in phytomedicine; brings along the issue
[97,113]. of their safety, and the legal requirements to meet health stan-
dards. It has been shown that the seeds and oil of NS plant are
1.17.2. Apoptosis characterized by a very low degree of toxicity [1]. Many studies
P53 is related to cell cycle regulation and apoptosis induc- were carried out to assess the toxicological properties of TQ in vitro
tion, and TQ induced both p53-dependent [97,108] and p53- and in vivo [37,151,203], and only a limited number of reports on
independent [119,205] mechanisms of apoptosis. TQ plays a major the potentially toxic effects of TQ exist.
role in repression of cancer via induction of pro-apoptotic fac- In the pathological conditions where TQ has been shown to be a
tors and/or down-regulation of anti-apoptotic proteins. It moreover promising prophylactic it also has been shown to be endowed with
regulates the caspase pathways [79,108,112,120,205]. a relatively low toxicity [26,29,30,145].
Apoptosis may occur also as a consequence of ROS production. TQ is a very well-tolerated drug in mice. Gali-Muhtasib et al.
TQ is an anti-oxidant or pro-oxidant depending on its concentra- [107] showed that TQ administered for 20 consecutive days did
tion. TQ is anti-oxidant at lower concentrations and most of the not induce death in Balb/c mice or affect their mean body weight,
studies elucidating the mechanism have addressed on the anti- which is a very sensitive parameter for toxicity in rodents. TQ was
oxidant effects. However, TQ has been shown at least in one report not associated with any clinically important changes in neurologi-
to be pro-oxidant at high concentrations. TQ is known to induce cal function, laboratory variables or vital signs. TQ administered at
apoptotic cell death via, in part, direct involvement of oxidants 1 mg/kg/day was mostly well tolerated [180]. Badary et al. [208]
[206]. indicated that addition of TQ in the drinking water of mice at
The anti-oxidant/pro-oxidant role of TQ depends also on the concentrations of up to 0.03% for 3 months led to no sign of toxic-
environment where it is present [109]. TQ with its respective ity, except for a decrease in fasting plasma glucose concentration.
semiquinone radicals may induce apoptosis in cancer cells by gen- Sustained delivery of TQ for 30 days using Tri-Calcium Phosphate
erating ROS [123]. TQ generates ROS and causes low expression Lysine (TCPL) capsule loaded with 0.02 grams of TQ to adult male
of pro-survival genes, conformational changes in pro-apoptotic rats have shown little or no side effects on the major vital and
proteins, loss of mitochondrial membrane potential leading to acti- reproductive organs [190].
vation of caspase-9, -3, and PARP cleavage and caspase-dependent It is notable that the effective dose of TQ was found to be safe
apoptosis [106,111,120]. These results place TQ in a class of plant- and no toxicity was reported in subchronic administration of
derived anti-oxidants, which also exhibit pro-oxidant properties. TQ in rats with doses of 90 mg/kg/day [208]. The same authors
reported hypoactivity and difficulty in respiration as signs of
1.17.3. Angiogenesis toxicity at high doses, 24 h after TQ administration at 2–3 g/kg,
Of relevance to tumor growth, angiogenesis is essential for sup- with a decrease in tissue (liver, kidneys, and heart) GSH content,
plying oxygen and nutrients. A major finding of TQ is that it has causing liver and kidney toxicity. This toxicity evidenced by rise
potent anti-angiogenic effect. TQ is a selective blocker of VEGF, a in plasma metabolites and enzymes; plasma urea and creatinine
key pro-angiogenic molecule, and abolished proliferation and tubu- concentrations, and the enzyme activities of ALT, LDH, and creatine
logenesis of endothelial colony forming cells [207]. Endothelial cell phosphokinase were increased [37]. Moreover, it has shown TQ
migration shows a critical step in the angiogenesis, and TQ effec- increased the rate of necrotic cells at concentrations between
tively inhibited human umbilical vein endothelial cell (HUVEC) 2.5 and 20 ␮M. Furthermore, it caused concentration dependent
152 S. Darakhshan et al. / Pharmacological Research 95–96 (2015) 138–158

Table 3
Molecular mechanisms underlying activities of TQ (↓: reduction; ↑: increase).

Processes Molecular targets/pathways Experimental models/Cell types Refs

↓ PCNA, Ki67, cyclin D1, cyclin E, Cdk4; ↑ p21 → G1/S transition arrest NDEA-induced hepatocellular carcinoma [150]
↑ p53 and p21; ↓ cyclin D1 → induction of G1 phase cell cycle arrest HCT116 colorectal cancer cells [108]
Cell cycle and cell ↑ p21 and p27; ↓ E2F-1 and androgen receptor; ↓ Cdk-4, Cdk-2 and cyclin A → LNCaP prostate cancer cells [101]
proliferation blockade of G1/S phase
↑ Expression of the CDK-inhibitor p16; and ↓ cyclin D1 expression → G0/G1 Papilloma (SP-1) cells [113]
phase arrest
↓ IL-6-induced Akt phosphorylation and ↓ IL-6-induced STAT3 expression; ↓ U266 multiple myeloma cells [79]
c-Src and JAK-2 activation; caspase-3 activation and PARP cleavage → sub-G1
accumulation
↑ PTEN; ↓ PI3K/Akt pathway; ↑ p53 and p21 protein expression; ↓ cyclin B1 MCF-7/DOX breast cancer cells [97]
and cdc25 levels → G2/M cell cycle arrest

↑ PPAR-␥ and PPAR-␤/␦; activation of caspase-7, -8 and -9; ↓ expression of MCF-7 breast cancer cells [95]
Bcl-2, Bcl-xl and surviving
↓ c-myc expression; ↓ ␤-catenin translocation; ↓ phosphorylation of Akt and Apc Min
mice colorectal cancer [110]
GSK3␤; ↓ MEK1/2 pathway
↓ CHEK1 (checkpoint kinase 1 homolog); ↑ caspase-3 activity → DNA damage p53−/− HCT116 colorectal carcinoma cells [108]
and apoptosis
↑ Bax and caspase-3; ↓ Bcl-2 and Bcl-xL; ↓ Mcl-1, survivin and XIAP HPAC human pancreatic cells [112]
↑ Bax/Bcl2 ratio, ↑ release of cytochrome c from mitochondria; activation of Neuro-2a neuroblastoma cell line [106]
caspase-3 and -9; ↓ expression of XIAP
Apoptosis ↓ Cyclin D1, Bcl-2, Bcl-xL, survivin, Mcl-1 U266 multiple myeloma cells [101]
↓ bcl-2; ↑ bax; activation of caspase-3 and -9; ↑ release of cytochrome c from Xenograft mouse model of gastric cancer [111]
the mitochondria
↓ Expression of Bcl-2, Bcl-xL, survivin and Mcl-1 proteins, PARP cleavage U266 multiple myeloma [79]
↑ Levels of p53 and Bax U87 MG and T98G malignant glioblastoma cells [104]
↑ Expression of cytochrome c; ↑ Bax/Bcl-2 ratio; ↓ phosphorylation of Akt and A431 and Hep2 squamous cell carcinoma cell [114]
JNK lines
↑ Activation of caspases and PARP cleavage; ↑ Bax/Bcl2 ratio MCF-7/DOX cells [97]
↑ Bax and cytoplasmic cytochrome c; activation of caspase-3; ↓ Bcl-2 and T47D and MDA-MB-468 breast cancer cells [96]
survivin; ↓ phosphorylation of Akt and GSK3␤; ↓ EGF-induced
phosphorylation of Akt, PTEN, PDK1, and Bad
↓ Phosphorylation of JNK and ERK; ↑ activation of caspase-3, and -7 DLD-1 human colon cancer cells [109]
↓ I␬B␣ degradation and phosphorylation; ↓ p65 phosphorylation and nuclear Human chronic myeloid leukemia KBM5 cells [118]
translocation; ↓ TNF-␣-induced NF-␬B-regulated gene products, including
IAP1, IAP2, XIAP, Bcl-2, Bcl-xL, survivin, COX-2, cyclin D1, c-Myc, MMP-9 →
cell death and ↓ tumor growth
↓ Constitutive activation of AKT; activation of caspase-9, -3; and sensitized Primary effusion lymphoma cells [120]
TRAIL-mediated apoptosis
↓ Bcl-2, Bcl-xL and survivin; ↑ PPAR-␥ MDA-MB-231 breast cancer cells [95]
↑ ␣ and ␤ tubulin degradation; ↑ p73 expression → cell apoptosis Human astrocytoma cells and in Jurkat cells [119]
↓ NF-␬B, Ki67, XIAP and survivin; ↑ expression of cleaved caspase-3 and Smac; Osteosarcoma (SaOS-2) cells xenograft tumors [102]
↓ expression of CD34 and VEGF in nude mice
↓ Telomerase activity; induction of DNA damage M059J and M059K human glioblastoma cells [103]

↓ VEGF expression and ↓ VEGF-induced AKT/ERK activation Xenograft human prostate cancer (PC3) model [100]
Angiogenesis in mouse
↓ TNF-␣-induced expression of VEGF Human chronic myeloid leukemia KBM5 cells [118]
↓ NF-␬B pathway, ↓ AKT and ERK signaling pathways, ↓ constitutive and U266 multiple myeloma cells [79]
IL-6-induced STAT3 phosphorylation

↓ Chemokine receptor-4 (CXCR4), COX-2 MMP-9 and p65 expression U266 multiple myeloma cells [121]
Cell migration,
↓ Expression of inflammosome marker NLRP3; ↓ secretion of IL-1␤ and IL-18; B16F10 melanoma cells in C57/BL6 mice [116]
invasion and
↓ NF-␬B activity
metastasis
↓ MMP-2 and -9; ↓ ERK phosphorylation; ↓ FAK U87 and CCF-STTG1 glioblastoma cells [105]
↓ TNF-␣-induced expression of MMP-9 KBM5 myeloid leukemia cells [118]

↓ IL-4, -5 and -13; ↓ LTB4 and LTC4 levels; ↑ IFN-␥ Mouse model of allergic airway inflammation [69]
↓ IL-1␤, IL-6, TNF-␣, IFN-␥ and PGE2; ↑ IL-10 Collagen-induced arthritic rats [72]
↓ TGF-␤1, iNOS, COX-2 and prostaglandin expression Allergic airway inflammation [68,70]
↓ TNF-␣ and IL-2 levels LPS and live E. coli-induced sepsis [135]
↓ NO, MDA, IL-1␤, and TNF-␣ levels Spinal cord injury (SCI) [178]
↓ IL-6 and NF-␬B activation Encephalomyelitis model [73,76]
Inflammatory ↓ COX-2 expression; ↓ PGE2 accumulation and ↓ activation of NF-␬B HPAC human pancreatic cancer cells [112]
responses ↓ COX-2; ↓ NF-␬B signaling (phosphorylation of Akt, JNK and p38 MAP kinase) HR-1 hairless mouse skin [115]
↓ lipid peroxidation; ↓ expression of COX-2 and MDA; ↑ SOD level Pancreatic tissue of STZ-diabetic rats [154]
↓ LPS-induced IL-1␤, TNF-␣, MMP-13, COX-2; ↓ PGE2, NF-␬B, p65, p38 and Rheumatoid arthritis [75]
ERK1/2 phosphorylation
↓ PKC, PAF, histamine release and iNOS expression; ↓ GSH depletion and lipid Peritoneal mast cells [80]
peroxidation
↓ expression of PI3K, CD14 and TLR4, collagen-I and Akt phosphorylation Activated rat hepatic stellate cell line, [203]
T-HSC/Cl-6
↓ expression of brain iNOS and NO Morphine-induced oxidative stress [171]
 S. Darakhshan et al. / Pharmacological Research 95–96 (2015) 138–158 153

Table 3 (Continued)

Processes Molecular targets/pathways Experimental models/Cell types Refs

↓ NO and iNOS expression and/or production Supernatants of LPS-stimulated macrophages [84]

↓ MDA and NO level; ↑ activities of CAT, GSH-Px and SOD Acute bacterial prostatitis induced by P. [136,137]
aeruginosa or E. coli
↓ Level of MDA; ↑ GSH content, CAT and SOD activities; ↓ lipid peroxidation I/R injury in rat hippocampus [34]
↑ GST, GSH-Px, SOD and CAT Hypercholesterolemia [27,50]
↓ NO and MPO; ↑ GSH, CAT and SOD Collagen-induced arthritis [72]
↑ HO-1 and GST HR-1 hairless mouse skin [115]
↑ Content of GSH and SOD activity; ↓ MDA content and MPO activity; ↓ NO I/R-induced gastric dysfunction and ulcer in [164,165]
production rats
↓ NO and MDA; ↑ GSH and CAT; also ↓ norepinephrine and dopamine STZ-diabetes model [162]
Oxidative stress
↓ lipid peroxidation; ↓ AST and ALT activity Cypermethrin-induced hepatotoxicity [147]
↓ AST, ALT, ALP and MDA levels Aflatoxin B1-induced hepatotoxicity [57]
↑ GSH content, and GST and DT-diaphorase activities; ↓ lipid peroxide Liver of B(a)P-treated tumor-bearing mice [61]
accumulation
↑ SOD, CAT, GSH and non-protein thiol (NP-SH) levels Cadmium-induced hepatotoxicity [145]
↓ AST, ALT and ALP levels Anti-tuberculosis drugs-induced liver damage [148]
↓ MDA; ↑CAT, SOD and GST-Px 1,2-Dimethyl-hydrazine (DMH)-induced colon [63]
tumor
↓ MDA, ↑ activities of SOD and GST; along with ↓ NOX-4 level DOX-induced nephrotoxicity [187]
↓ TBARS; ↑ GSH level, GSH-Px and CAT activities Gentamicin-induced acute renal failure in rats [52]
↓ Hepatic GSH depletion; ↑ activity of SOD, ↓ TNF-␣ Tamoxifen-induced hepatotoxicity in female [146]
rats
↓ Kidney tissue MDA; ↑ activities of SOD and GSH-Px in kidney tissue Vancomycin-induced kidney injuries [53]
↓ ALT activity; ↑ GST as well ATP production Acetaminophen-induced hepatotoxicity [56]

↑ Hepatic LDL receptor gene; ↓ 3-hydroxy-3-methylglutaryl-coenzyme A Diabetic rats [158]

reductase (HMG-CoAR) gene → hypocholesterolemic effect
↓ Serum lipase, amylase, MPO, and oxidative stress index Ethanol and high fat diet [201]
Components
↓ Total cholesterol, LDL-cholesterol, triglycerides and TBARS; ↑ Cholesterol-fed rabbits [199]
involved in
HDL-cholesterol concentration
metabolism
↓ Activities of glucose-6-phosphtse and fructose-1,6-bisphosphatase → ↓ STZ-nicotinamide-induced diabetic rats [25]
gluconeogenesis
↑ Alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase Adjuvant-induced arthritis rat model [75]
↑ ALP, osteocalcin, osteopontin and BMP-2; ↑ phosphorylation of ERK signaling MC3T3-E1 osteoblast cells [192]
pathway and activated MAPK pathway → osteogenesis
↓ MDA level; ↑ GST and SOD activity in liver and kidney tissues; ↓ Hepatorenal dysfunction induced by renal I/R [188]
spermidine/spermine N-1-acetyl-transferase (SSAT) and ↓ CYP3A1 gene
expression in liver and kidney
↓ Creatine kinase, LDH, cholesterol, and TNF-␣; ↓ TBARS; and ↑ ATP levels Cyclophosphamide-induced cardiotoxicity [58]

genotoxic effect in hepatocyte primary cultures, i.e. an increase oral ingestion, TQ was biotransformed in gastrointestinal tract or
of the frequency of chromosomal aberrations and micronucleated metabolized in the liver [37].
cells [151]. The lack of preclinical studies with TQ reporting the maximum
Importantly however, TQ had selective cytotoxic effects and also tolerated dose (MTD), which is defined as the highest dose is safe to
these, as shown above, have potential therapeutic application. In administer in the absence of intolerable side effects, is regarded as a
this respect, it appears that TQ kills tumor cells efficiently with- limitation in using TQ in clinical settings [37]. Further research both
out cytotoxicity to normal cells. The selective cytotoxicity of TQ for at the clinical and preclinical level is thus needed to determine the
malignant cells compared to normal osteoblasts [205], mouse nor- therapeutic effective dose of TQ in various diseases. In particular,
mal kidney cells [93], normal human lung fibroblasts [103] and Vero the effect of TQ on ionic channels, especially calcium channels, is
cells [99] has been described. In addition, normal cell lines such still unclear and needs to be more investigated.
as primary mouse keratinocytes and Madin-Darby canine kidney
(MDCK) cells are reported to be resistant to the cytotoxic effects of
TQ (IC50 = 101 ␮M) [16,113]. 2. Discussion and future viewpoints
TQ, while depolymerized the microtubule network and disrupt-
ing the mitotic spindle organization of A549 cells, did not affect Nowadays extensive research is focusing on herbal products
the microtubule network of the more normal HUVEC cells and at as an alternative medicine and traditional medicinal plants have
below the IC50 concentration [122]. TQ degraded ␣ and ␤ tubulin received much attention due to several factors such as cheap cost,
of human astrocytoma and Jurkat cells, without affecting normal easy availability, safety, and efficacy. Moreover, many plants and
human fibroblast cells [119]. their products are used based on religious and cultural traditions.
Not all is, however, straight forwards, as mentioned above TQ N. sativa seeds, from which TQ is extracted, have been used by
might be metabolized to reactive species and increase oxidative diverse human cultures around the world for centuries to treat
stress, which contributes to the depletion of anti-oxidant enzymes many problems [4].
and damage to DNA in hepatocytes treated with high TQ concen- TQ, as an example of phytochemicals, has attracted noteworthy
trations [151]. scientific attention in recent years for its high biological activity
The route of administration could have an influence on TQ toxic- and low systemic toxicity that can make it a promising alternative
ity outcome. Rats that received intraperitoneal TQ showed toxicity to conventional therapeutic drugs. Plant-based anti-oxidants have
signs, which were related to acute pancreatitis. Meanwhile, rats, recently gained popularity due to their role as dietary supple-
which received oral ingestion, showed transient toxicity. This can ments with minimal side effects. Additionally, the use of naturally
be explained by the fact that intraperitoneal injection resulted in occurring agents to prevent the development or recurrence of
whole absorption of TQ into systemic circulation; whereas with cancers has become widely accepted as a realistic option for
154 S. Darakhshan et al. / Pharmacological Research 95–96 (2015) 138–158

fighting diseases. The need for new treatments for cancer, which for nose-to-brain targeting: a pharmacoscintigraphic study, Int. J. Nanomed.
have fewer adverse effects, has generated interest in exploiting the 7 (2012) 5705–5718, PMID: 23180965.
[15] F. Odeh, S.I. Ismail, R. Abu-Dahab, I.S. Mahmoud, A. Al Bawab, Thymoquinone
anti-cancer properties of dietary phytochemicals and other natural in liposomes: a study of loading efficiency and biological activity towards
products. A potential anti-cancer drug should exhibit selective breast cancer, Drug Deliv. 19 (2012) 371–377, PMID: 23043626.
and specific cytotoxicity against cancerous cell leaving the nor- [16] K. Effenberger, S. Breyer, R. Schobert, Terpene conjugates of the Nigella sativa
seed-oil constituent thymoquinone with enhanced efficacy in cancer cells,
mal cells unharmed. Extensive results show that TQ has these Chem. Biodivers. 7 (2010) 129–139, PMID: 20087986.
properties. [17] S.I. Abdelwahab, B.Y. Sheikh, M.M. Taha, C.W. How, R. Abdullah, U. Yagoub,
There is no doubt that in vitro and in vivo research has demon- et al., Thymoquinone-loaded nanostructured lipid carriers: preparation,
gastroprotection, in vitro toxicity, and pharmacokinetic properties after
strated the therapeutic potential of TQ against cell cultures and
extravascular administration, Int. J. Nanomed. 8 (2013) 2163–2172, PMID:
animal models with the emphasis on the mechanism of action. 23818776.
However, there are no clinical data upon which to base dosing [18] K.E. El Tahir, M.M. Ashour, M.M. Al-Harbi, The cardiovascular actions of
the volatile oil of the black seed (Nigella sativa) in rats: elucidation of
recommendations in humans and current understanding about its
the mechanism of action, Gen. Pharmacol. 24 (1993) 1123–1131, PMID:
action in the body is not satisfactory. There is a paucity of clinical 8270171.
studies testing TQ in human patients; We believe that it is now [19] K.E. El Tahir, M.M. Ashour, M.M. Al-Harbi, The respiratory effects of the volatile
appropriate that TQ should move from testing on the bench to clin- oil of the black seed (Nigella sativa) in guinea-pigs: elucidation of the mech-
anism(s) of action, Gen. Pharmacol. 24 (1993) 1115–1122, PMID: 8270170.
ical experimentation, in which explore effects of TQ on immune [20] A.M. Abdel-Fattah, K. Matsumoto, H. Watanabe, Antinociceptive effects of
system, liver as well as upon other vital tissues, areas in which its Nigella sativa oil and its major component, thymoquinone, in mice, Eur. J.
potential toxicity has emerged. Pharmacol. 400 (2000) 89–97, PMID: 10913589.
[21] M. El Gazzar, R. El Mezayen, J.C. Marecki, M.R. Nicolls, A. Canastar, S.C. Dre-
Nevertheless, considerable amount of information about TQ skin, Anti-inflammatory effect of thymoquinone in a mouse model of allergic
regarding its molecular activity, drug toxicity, and novel drug deliv- lung inflammation, Int. Immunopharmacol. 6 (2006) 1135–1142, PMID:
ery approaches are now available for researchers. Results emerging 16714217.
[22] A. El-Mahmoudy, Y. Shimizu, T. Shiina, H. Matsuyama, M. El-Sayed, T. Take-
from such studies will substantially improve the therapeutic appli- waki, Successful abrogation by thymoquinone against induction of diabetes
cation of TQ in clinical settings for a wide range of illnesses once mellitus with streptozotocin via nitric oxide inhibitory mechanism, Int.
we have clear information on the conditions for its safe use. The Immunopharmacol. 5 (2005) 195–207, PMID: 15589481.
[23] H. Hosseinzadeh, S. Parvardeh, Anticonvulsant effects of thymoquinone, the
efficacy of TQ should be measured based on the nature of the dis-
major constituent of Nigella sativa seeds, in mice, Phytomedicine 11 (2004)
eases. As detailed above TQ may synergize with more conventional 56–64, PMID: 14971722.
chemotherapeutic drugs. Its use in combination therapies may be at [24] M.N. Nagi, K. Alam, O.A. Badary, O.A. Al-Shabanah, H.A. Al-Sawaf, A.M. Al-
Bekairi, Thymoquinone protects against carbon tetrachloride hepatotoxicity
lower dosage, reduce the dosage of the concomitant drug optimiz-
in mice via an antioxidant mechanism, Biochem. Mol. Biol. Int. 47 (1999)
ing efficacy vs toxicity; it might also overcome to drug resistance 153–159.
problem [112,123]. [25] L. Pari, C. Sankaranarayanan, Beneficial effects of thymoquinone on hepatic
key enzymes in streptozotocin-nicotinamide induced diabetic rats, Life Sci.
85 (2009) 830–834, PMID: 19903489.
Conflict of interest statement [26] O.A. Al-Shabanah, O.A. Badary, M.N. Nagi, N.M. Al-Gharably, A.C. Al-Rikabi,
A.M. Al-Bekairi, Thymoquinone protects against doxorubicin-induced car-
diotoxicity without compromising its antitumor activity, J. Exp. Clin. Cancer
None declared. Res. 17 (1998) 193–198, PMID 9700580.
[27] M.N. Nagi, H.A. Almakki, Thymoquinone supplementation induces quinone
reductase and glutathione transferase in mice liver: possible role in protec-
References tion against chemical carcinogenesis and toxicity, Phytother. Res. 23 (2009)
1295–1298.
[1] B.H. Ali, G. Blunden, Pharmacological and toxicological properties of Nigella [28] M. El-Dakhakhny, Studies on the Egyptian Nigella sativa L. Some pharmaco-
sativa, Phytother. Res. 17 (2003) 299–305, PMID: 12722128. logical properties of the seeds’ active principle in comparison to its dihydro
[2] W.G. Goreja, Black Seed: Nature’s Miracle Remedy, NY7 Amazing Herbs Press, compound and its polymer, Arzneimittelforschung 15 (1965) 1227–1229,
New York, 2003. PMID: 4380349.
[3] M. Junemann, Three Great Healing Herbs, Twin Laked WI7 Lotus Light Publi- [29] M.A. Mansour, O.T. Ginawi, T. El-Hadiyah, A.S. El-Khatib, O.A. Al-Shabanah,
cations, 1998. H.A. Al-Sawaf, Effects of volatile oil constituents of Nigella sativa on car-
[4] M.L. Salem, Immunomodulatory and therapeutic properties of the Nigella bon tetrachloride-induced hepatotoxicity in mice: evidence for antioxidant
sativa L. seed, Int. Immunopharmacol. 5 (2005) 1749–1770, PMID: 16275613. effects of thymoquinone, Res. Commun. Mol. Pathol. Pharmacol. 110 (2001)
[5] M.A. Khan, Chemical composition and medicinal properties of Nigella sativa 239–251, PMID: 12760491.
Linn, Inflammopharmacology 7 (1999) 15–35, PMID: 17657444. [30] O.A. Badary, M.N. Nagi, O.A. Al-Shabanah, H.A. Al-Sawaf, M.O. Al-Sohaibani,
[6] H. Gali-Muhtasib, N. El-Najjar, R. Schneider-Stock, The medicinal potential A.M. Al-Bekairi, Thymoquinone ameliorates the nephrotoxicity induced by
of black seed (Nigella sativa) and its components, Adv. Phytomed. 2 (2006) cisplatin in rodents and potentiates its antitumor activity, Can. J. Physiol.
133–153. Pharmacol. 75 (1997) 1356–1361, PMID 9534946.
[7] J. Taborsky, M. Kunt, P. Kloucek, J. Lachman, V. Zeleny, L. Kokoska, Iden- [31] P.J. Houghton, R. Zarka, B. De las Heras, J.R. Hoult, Fixed oil of Nigella
tification of potential sources of thymoquinone and related compounds in sativa and derived thymoquinone inhibit eicosanoid generation in leuko-
Asteraceae, Cupressaceae, Lamiaceae, and Ranunculaceae families, Cent. Eur. cytes and membrane lipid peroxidation, Planta Med. 61 (1995) 33–36, PMID:
J. Chem. 10 (2012) 1899–1906. 7700988.
[8] H.M. El-Fatatry, Isolation and structure assignment of an antimicrobial prin- [32] M.A. Mansour, M.N. Nagi, A.S. El-Khatib, A.M. Al-Bekairi, Effects of thy-
ciple from the volatile oil of Nigella sativa L. seeds, Pharmazie 30 (1975) moquinone on antioxidant enzyme activities, lipid peroxidation and
109–111, PMID: 238225. DT-diaphorase in different tissues of mice: a possible mechanism of action,
[9] M.A. Randhawa, M.S. Al-Ghamdi, A review of the pharmaco-therapeutic Cell Biochem. Funct. 20 (2002) 143–151, PMID: 11979510.
effects of Nigella sativa, Pak. J. Med. Res. 41 (2002) 77–83. [33] M. Kanter, Nigella sativa and derived thymoquinone prevents hippocampal
[10] M. El-Dakhakhny, Studies on the chemical constitution of Egyptian N. sativa neurodegeneration after chronic toluene exposure in rats, Neurochem. Res.
L. seeds, Planta Med. 11 (1963) 465–470. 33 (2008) 579–588, PMID: 17929168.
[11] J.M. Salmani, S. Asghar, H. Lv, J. Zhou, Aqueous solubility and degradation [34] A.A. Al-Majed, F.A. Al-Omar, M.N. Nagi, Neuroprotective effects of thymo-
kinetics of the phytochemical anticancer thymoquinone; probing the effects quinone against transient forebrain ischemia in the rat hippocampus, Eur. J.
of solvents, pH and light, Molecules 19 (2014) 5925–5939, PMID: 24815311. Pharmacol. 543 (2006) 40–47, PMID: 16828080.
[12] G.M. Ganea, S.O. Fakayode, J.N. Losso, C.F. van Nostrum, C.M. Sabliov, I.M. [35] S.C. El-Saleh, O.A. Al-Sagair, M.I. Al-Khalaf, Thymoquinone and Nigella sativa
Warner, Delivery of phytochemical thymoquinone using molecular micelle oil protection against methionine-induced hyperhomocysteinemia in rats,
modified poly(d,l lactide-co-glycolide) (PLGA) nanoparticles, Nanotechnol- Int. J. Cardiol. 93 (2004) 19–23, PMID: 14729430.
ogy 21 (2010) 285104, PMID: 20585163. [36] M. Kanter, Thymoquinone attenuates lung injury induced by chronic toluene
[13] A. Singh, I. Ahmad, S. Akhter, G.K. Jain, Z. Iqbal, S. Talegaonkar, et al., Nanocar- exposure in rats, Toxicol. Ind. Health 27 (2011) 387–395, PMID: 21088054.
rier based formulation of Thymoquinone improves oral delivery: stability [37] M.M. Abukhader, The effect of route of administration in thymoquinone tox-
assessment, in vitro and in vivo studies, Colloids Surf. B: Biointerfaces 102 icity in male and female rats, Indian J. Pharm. Sci. 74 (2012) 195–200, PMID:
(2013) 822–832, PMID: 23104039. 23440704.
[14] S. Alam, Z.I. Khan, G. Mustafa, M. Kumar, F. Islam, A. Bhatnagar, et al., Develop- [38] K.M. Alkharfy, A. Ahmad, R.M. Khan, W.M. Al-Shagha, Pharmacokinetic
ment and evaluation of thymoquinone-encapsulated chitosan nanoparticles plasma behaviors of intravenous and oral bioavailability of thymoquinone in
 S. Darakhshan et al. / Pharmacological Research 95–96 (2015) 138–158 155

a rabbit model, Eur. J. Drug Metab. Pharmacokinet. (2014), PMID: 24924310 pancreatic ␤-cells of streptozotocin-induced diabetic rats, J. Diabetes 2 (2010)
[Epub ahead of print]. 256–266, PMID: 20923501.
[39] G. Lupidi, A. Scire, E. Camaioni, K.H. Khalife, G. De Sanctis, F. Tanfani, et al., [65] A. Ahlatci, A. Kuzhan, S. Taysi, O.C. Demirtas, H.E. Alkis, M. Tarakcioglu,
Thymoquinone, a potential therapeutic agent of Nigella sativa, binds to site et al., Radiation-modifying abilities of Nigella sativa and thymoquinone on
I of human serum albumin, Phytomedicine (2010) 17714–17720, PMID: radiation-induced nitrosative stress in the brain tissue, Phytomedicine 21
20171066. (2014) 740–744, PMID: 24268807.
[40] N. El-Najjar, R.A. Ketola, T. Nissilä, T. Mauriala, M. Antopolsky, J. Jänis, et al., [66] M. Mansour, S. Tornhamre, Inhibition of 5-lipoxygenase and leukotriene C4
Impact of protein binding on the analytical detectability and anticancer activ- synthase in human blood cells by thymoquinone, J. Enzyme Inhib. Med. Chem.
ity of thymoquinone, J. Chem. Biol. 4 (2011) 97–107, PMID: 22229047. 19 (2004) 431–436, PMID: 15648658.
[41] M.N. Nagi, M.A. Mansour, Protective effect of thymoquinone against [67] M. El-Dakhakhny, N.J. Madi, N. Lembert, H.P. Ammon, Nigella sativa oil, nigel-
doxorubicin-induced cardiotoxicity in rats: a possible mechanism of protec- lone and derived thymoquinone inhibit synthesis of 5-lipoxygenase products
tion, Pharmacol. Res. 41 (2000) 283–289, PMID: 10675279. in polymorphonuclear leukocytes from rats, J. Ethnopharmacol. 81 (2002)
[42] O.A. Badary, R.A. Taha, A.M. Gamal El-Din, M.H. Abdel-Wahab, Thymoquinone 161–164, PMID: 12065147.
is a potent superoxide anion scavenger, Drug Chem. Toxicol. 26 (2003) 87–98, [68] E.S.M. Ammar, N.M. Gameil, N.M. Shawky, M.A. Nader, Comparative evalua-
PMID: 12816394. tion of anti-inflammatory properties of thymoquinone and curcumin using
[43] M.H. Daba, M.S. Abdel-Rahman, Hepatoprotective activity of thymoquinone an asthmatic murine model, Int. Immunopharmacol. 11 (2011) 2232–2236,
in isolated rat hepatocytes, Toxicol. Lett. 95 (1998) 23–29, PMID: 9650643. PMID: 22051975.
[44] K.H. Khalife, G. Lupidi, Non enzymatic reduction of thymoquinone in physio- [69] M. El Gazzar, R. El Mezayen, M.R. Nicolls, J.C. Marecki, S.C. Dreskin, Down-
logical conditions, Free Radic. Res. 41 (2007) 153–161, PMID: 17364941. regulation of leukotriene biosynthesis by thymoquinone attenuates airway
[45] K.H. Khalife, G. Lupidi, Reduction of hypervalent states of myoglobin and inflammation in a mouse model of allergic asthma, Biochim. Biophys. Acta
hemoglobin to their ferrous forms by thymoquinone: the role of GSH, NADH 1760 (2006) 1088–1095, PMID: 16624488.
and NADPH, Biochim. Biophys. Acta 1780 (2008) 627–637, PMID: 18206117. [70] R. El Mezayen, M. El Gazzar, M.R. Nicolls, J.C. Marecki, S.C. Dreskin, H.
[46] H.J. Harzallah, A. Neffati, I. Skandrani, E. Maaloul, L. Chekir-Ghedira, et al., Nomiyama, Effect of thymoquinone on cyclooxygenase expression and
Antioxidant and antigenotoxic activities of Globularia alypum leaves extracts, prostaglandin production in a mouse model of allergic airway inflammation,
J. Med. Plant Res. 4 (2010) 2048–2053. Immunol. Lett. 106 (2006) 72–81, PMID: 16762422.
[47] O.A. Badary, A.M. Gamal El-Din, Inhibitory effects of thymoquinone against [71] K. Hayat, M.B. Asim, M. Nawaz, M. Li, L. Zhang, N. Sun, Ameliorative effect of
20-methylcholanthrene-induced fibrosarcoma tumorigenesis, Cancer Detect. thymoquinone on ovalbumin-induced allergic conjunctivitis in Balb/c mice,
Prev. 25 (2001) 362–368, PMID: 11531013. Curr. Eye Res. 36 (2011) 591–598, PMID: 21604966.
[48] M. Kanter, H. Demir, C. Karakaya, H. Ozbek, Gastroprotective activity of Nigella [72] S. Umar, J. Zargan, K. Umar, S. Ahmad, C.K. Katiyar, H.A. Khan, Modulation of
sativa L oil and its constituent, thymoquinone against acute alcohol-induced the oxidative stress and inflammatory cytokine response by thymoquinone in
gastric mucosal injury in rats, World J. Gastroenterol. 11 (2005) 6662–6666, the collagen induced arthritis in Wistar rats, Chem. Biol. Interact. 197 (2012)
PMID: 16425361. 40–46, PMID: 22450443.
[49] F. Elbarbry, A. Ragheb, T. Marfleet, A. Shoker, Modulation of hepatic [73] A. Mohamed, A. Shoker, F. Bendjelloul, A. Mare, M. Alzrigh, H. Benghuzzi,
drug metabolizing enzymes by dietary doses of thymoquinone in female et al., Improvement of experimental allergic encephalomyelitis (EAE) by thy-
New Zealand White rabbits, Phytother. Res. 26 (2012) 1726–1730, PMID: moquinone; an oxidative stress inhibitor, Biomed. Sci. Instrum. 39 (2003)
22422469. 440–445, PMID: 12724933.
[50] M. Ismail, G. Al-Naqeep, K.W. Chan, Nigella sativa thymoquinone-rich fraction [74] I. Tekeoglu, A. Dogan, L. Demiralp, Effects of thymoquinone (volatile oil of
greatly improves plasma antioxidant capacity and expression of antioxidant black cumin) on rheumatoid arthritis in rat models, Phytother. Res. 20 (2006)
genes in hypercholesterolemic rats, Free Radic. Biol. Med. 48 (2010) 664–672, 869–871, PMID: 16835876.
PMID: 20005291. [75] F. Vaillancourt, P. Silva, Q. Shi, H. Fahmi, J.C. Fernandes, M. Benderdour,
[51] O.A. Badary, A.B. Abdel-Naim, M.H. Abdel-Wahab, F.M. Hamada, The influ- Elucidation of molecular mechanisms underlying the protective effects of
ence of thymoquinone on doxorubicin-induced hyperlipidemic nephropathy thymoquinone against rheumatoid arthritis, J. Cell. Biochem. 112 (2011)
in rats, Toxicology 143 (2000) 219–226, PMID: 10755708. 107–117, PMID: 20872780.
[52] M.M. Sayed-Ahmed, M.N. Nagi, Thymoquinone supplementation prevents the [76] A. Mohamed, D.M. Afridi, O. Garani, M. Tucci, Thymoquinone inhibits the
development of gentamicin-induced acute renal toxicity in rats, Clin. Exp. activation of NF-kappaB in the brain and spinal cord of experimental autoim-
Pharmacol. Physiol. 34 (2007) 399–405, PMID: 17439407. mune encephalomyelitis, Biomed. Sci. Instrum. 41 (2005) 388–393, PMID:
[53] F. Basarslan, N. Yilmaz, S. Ates, T. Ozgur, M. Tutanc, V.K. Motor, et al., Protec- 15850137.
tive effects of thymoquinone on vancomycin-induced nephrotoxicity in rats, [77] A.A. Sayed, M. Morcos, Thymoquinone decreases AGE-induced NF-kappaB
Hum. Exp. Toxicol. 31 (2012) 726–733, PMID: 22318306. activation in proximal tubular epithelial cells, Phytother. Res. 21 (2007)
[54] A.M. Fouda, M.H. Daba, G.M. Dahab, O.A. Sharaf El-Din, Thymoquinone 898–899, PMID: 17582594.
ameliorates renal oxidative damage and proliferative response induced by [78] N. Chehl, G. Chipitsyna, Q. Gong, C.J. Yeo, H.A. Arafat, Anti-inflammatory
mercuric chloride in rats, Basic Clin. Pharmacol. Toxicol. 103 (2008) 109–118, effects of the Nigella sativa seed extract, thymoquinone, in pancreatic cancer
PMID: 18816292. cells, HPB (Oxf.) 11 (2009) 373–381, PMID: 19768141.
[55] F.Q. Alenzi, S. El-Bolkiny Yel, M.L. Salem, Protective effects of Nigella sativa oil [79] F. Li, P. Rajendran, G. Sethi, Thymoquinone inhibits proliferation, induces
and thymoquinone against toxicity induced by the anticancer drug cyclophos- apoptosis and chemosensitizes human multiple myeloma cells through sup-
phamide, Br. J. Biomed. Sci. 67 (2010) 20–28, PMID: 20373678. pression of signal transducer and activator of transcription 3 activation
[56] M.N. Nagi, H.A. Almakki, M.M. Sayed-Ahmed, A.M. Al-Bekairi, Thymoquinone pathway, Br. J. Pharmacol. 161 (2010) 541–554, PMID: 20880395.
supplementation reverses acetaminophen-induced oxidative stress, nitric [80] A.A. Mahgoub, Thymoquinone protects against experimental colitis in rats,
oxide production and energy decline in mice liver, Food Chem. Toxicol. 48 Toxicol. Lett. 143 (2003) 133–143, PMID: 12749817.
(2010) 2361–2365, PMID: 20561950. [81] N. Gilhotra, D. Dhingra, Thymoquinone produced antianxiety-like effects in
[57] A. Nili-Ahmadabadi, F. Tavakoli, G.R. Hasanzadeh, H.R. Rahimi, O. Sabze- mice through modulation of GABA and NO levels, Pharmacol. Rep. 63 (2011)
vari, Protective effect of pretreatment with thymoquinone against Aflatoxin 660–669, PMID: 21857076.
B1 induced liver toxicity in mice, DARU 19 (2011) 282–287, PMID: [82] S. Oguz, M. Kanter, M. Erboga, C. Erenoglu, Protective effects of thymoquinone
22615670. against cholestatic oxidative stress and hepatic damage after biliary obstruc-
[58] M.N. Nagi, O.A. Al-Shabanah, M.M. Hafez, M.M. Sayed-Ahmed, Thymoquinone tion in rats, J. Mol. Histol. 43 (2012) 151–159, PMID: 22270828.
supplementation attenuates cyclophosphamide-induced cardiotoxicity in [83] H. Hosseinzadeh, S. Parvardeh, M.N. Asl, H.R. Sadeghnia, T. Ziaee, Effect
rats, J. Biochem. Mol. Toxicol. 25 (2011) 135–142, PMID: 20957680. of thymoquinone and Nigella sativa seeds oil on lipid peroxidation level
[59] G.M. Suddek, N.A. Ashry, N.M. Gameil, Thymoquinone attenuates during global cerebral ischemia-reperfusion injury in rat hippocampus, Phy-
cyclophosphamide-induced pulmonary injury in rats, Inflammophar- tomedicine 14 (2007) 621–627, PMID: 17291733.
macology 21 (2013) 427–435, PMID: 23196752. [84] A. El-Mahmoudy, H. Matsuyama, M.A. Borgan, Y. Shimizu, M.G. El-Sayed,
[60] D. El-Khouly, W.M. El-Bakly, A.S. Awad, H.O. El-Mesallamy, E. El-Demerdash, N. Minamoto, et al., Thymoquinone suppresses expression of inducible
Thymoquinone blocks lung injury and fibrosis by attenuating bleomycin- nitric oxide synthase in rat macrophages, Int. Immunopharmacol. 2 (2002)
induced oxidative stress and activation of nuclear factor Kappa-B in rats, 1603–1611, PMID: 12433061.
Toxicology 302 (2012) 106–113, PMID: 22982510. [85] P. Marsik, L. Kokoska, P. Landa, A. Nepovim, P. Soudek, T. Vanek, In vitro
[61] O.A. Badary, O.A. Al-Shabanah, M.N. Nagi, A.C. Al-Rikabi, M.M. Elmazar, Inhi- inhibitory effects of thymol and quinones of Nigella sativa seeds on
bition of benzo(a)pyrene-induced forestomach carcinogenesis in mice by cyclooxygenase-1- and -2-catalyzed prostaglandin E2 biosyntheses, Planta
thymoquinone, Eur. J. Cancer Prev. 8 (1999) 435–440, PMID: 10548399. Med. 71 (2005) 739–742, PMID: 16142638.
[62] M. Sagit, F. Korkmaz, S.G. Gürgen, M. Kaya, A. Akcadag, I. Ozcan, The protec- [86] A. Haq, P.I. Lobo, M. Al-Tufail, N.R. Rama, S.T. Al-Sedairy, Immunomodulatory
tive role of thymoquinone in the prevention of gentamicin ototoxicity, Am. J. effect of Nigella sativa proteins fractionated by ion exchange chromatography,
Otolaryngol. 35 (2014) 603–609, PMID: 25087465. Int. J. Immunopharmacol. 21 (1999) 283–295, PMID: 10408636.
[63] H. Jrah-Harzallah, S. Ben-Hadj-Khalifa, W.Y. Almawi, A. Maaloul, Z. Houas, T. [87] N.T. Xuan, E. Shumilina, S.M. Qadri, F. Götz, F. Lang, Effect of thymoquinone
Mahjoub, Effect of thymoquinone on 1,2-dimethyl-hydrazine-induced oxida- on mouse dendritic cells, Cell. Physiol. Biochem. 25 (2010) 307–314, PMID:
tive stress during initiation and promotion of colon carcinogenesis, Eur. J. 20110691.
Cancer 49 (2013) 1127–1135, PMID: 23131834. [88] W. Yang, M. Bhandaru, V. Pasham, D. Bobbala, C. Zelenak, K. Jilani, et al., Effect
[64] N.E. Abdelmeguid, R. Fakhoury, S.M. Kamal, R.J. Al Wafai, Effects of Nigella of thymoquinone on cytosolic pH and Na+/H+ exchanger activity in mouse
sativa and thymoquinone on biochemical and subcellular changes in dendritic cells, Cell. Physiol. Biochem. 29 (2012) 21–30, PMID: 22415071.
156 S. Darakhshan et al. / Pharmacological Research 95–96 (2015) 138–158

[89] J. Kundu, D.H. Kim, J.K. Kundu, K.S. Chun, Thymoquinone induces heme [113] H.U. Gali-Muhtasib, W.G. Abou Kheir, L.A. Kheir, N. Darwiche, P.A. Crooks,
oxygenase-1 expression in HaCaT cells via Nrf2/ARE activation: Akt and Molecular pathway for thymoquinone-induced cell cycle arrest and apopto-
AMPK␣ as upstream targets, Food Chem. Toxicol. 65 (2014) 18–26, PMID: sis in neoplastic keratinocytes, Anticancer Drugs 15 (2004) 389–399, PMID:
24355171. 15057144.
[90] R. Terazawa, N. Akimoto, T. Kato, T. Itoh, Y. Fujita, N. Hamada, et al., A kavalac- [114] S. Das, K.K. Dey, G. Dey, I. Pal, A. Majumder, S. MaitiChoudhury, et al., Anti-
tone derivative inhibits lipopolysaccharide-stimulated iNOS induction and neoplastic and apoptotic potential of traditional medicines thymoquinone
NO production through activation of Nrf2 signaling in BV2 microglial cells, and diosgenin in squamous cell carcinoma, PLoS ONE 7 (2012) e46641, PMID:
Pharmacol. Res. 71 (2013) 34–43, PMID: 23419834. 23077516.
[91] R. Foresti, S.K. Bains, T.S. Pitchumony, L.E. de Castro Brás, F. Drago, J.L. Dubois- [115] J.K. Kundu, L. Liu, J.W. Shin, Y.J. Surh, Thymoquinone inhibits phorbol ester-
Randé, et al., Small molecule activators of the Nrf2-HO-1 antioxidant axis induced activation of NF-␬B and expression of COX-2, and induces expression
modulate heme metabolism and inflammation in BV2 microglia cells, Phar- of cytoprotective enzymes in mouse skin in vivo, Biochem. Biophys. Res.
macol. Res. 76 (2013) 132–148, PMID: 23942037. Commun. 438 (2013) 721–727, PMID: 23911786.
[92] L. Reddy, B. Odhav, K.D. Bhoola, Natural products for cancer prevention: a [116] I. Ahmad, K.M. Muneer, I.A. Tamimi, M.E. Chang, M.O. Ata, N. Yusuf,
global perspective, Pharmacol. Ther. 99 (2003) 1–13, PMID: 12804695. Thymoquinone suppresses metastasis of melanoma cells by inhibition of
[93] A.M. Shoieb, M. Elgayyar, P.S. Dudrick, J.L. Bell, P.K. Tithof, In vitro inhibition NLRP3 inflammasome, Toxicol. Appl. Pharmacol. 270 (2013) 70–76, PMID:
of growth and induction of apoptosis in cancer cell lines by thymoquinone, 23583630.
Int. J. Oncol. 22 (2003) 107–113, PMID: 12469192. [117] M.J. Salomi, S.C. Nair, K.R. Panikkar, Inhibitory effects of Nigella sativa and
[94] M. Khader, P.M. Eckl, N. Bresgen, Effects of aqueous extracts of medicinal saffron (Crocus sativus) on chemical carcinogenesis in mice, Nutr. Cancer 16
plants on MNNG-treated rat hepatocytes in primary cultures, J. Ethnophar- (1991) 67–72, PMID: 1923908.
macol. 112 (2007) 199–202, PMID: 17324542. [118] G. Sethi, K.S. Ahn, B.B. Aggarwal, Targeting nuclear factor-kappa B activation
[95] C.C. Woo, S.Y. Loo, V. Gee, C.W. Yap, G. Sethi, A.P. Kumar, et al., Anticancer pathway by thymoquinone: role in suppression of antiapoptotic gene prod-
activity of thymoquinone in breast cancer cells: possible involvement of ucts and enhancement of apoptosis, Mol. Cancer Res. 6 (2008) 1059–1070,
PPAR-␥ pathway, Biochem. Pharmacol. 82 (2011) 464–475, PMID: 21679698. PMID: 18567808.
[96] S. Rajput, B.N. Kumar, K.K. Dey, I. Pal, A. Parekh, M. Mandal, Molecular tar- [119] M. Alhosin, A. Ibrahim, A. Boukhari, T. Sharif, J.P. Gies, C. Auger, et al.,
geting of Akt by thymoquinone promotes G(1) arrest through translation Anti-neoplastic agent thymoquinone induces degradation of ␣ and ␤ tubu-
inhibition of cyclin D1 and induces apoptosis in breast cancer cells, Life Sci. lin proteins in human cancer cells without affecting their level in normal
93 (2013) 783–790, PMID: 24044882. human fibroblasts, Investig. New Drugs 30 (2012) 1813–1819, PMID:
[97] E.S.A. Arafa, Q. Zhu, Z.I. Shah, G. Wani, B.M. Barakat, I. Racoma, et al., 21881916.
Thymoquinone up-regulates PTEN expression and induces apoptosis in [120] A.R. Hussain, M. Ahmed, S. Ahmed, P. Manogaran, L.C. Platanias, S.N. Alvi, et al.,
doxorubicin-resistant human breast cancer cells, Mutat. Res. 706 (2011) Thymoquinone suppresses growth and induces apoptosis via generation of
28–35, PMID: 21040738. reactive oxygen species in primary effusion lymphoma, Free Radic. Biol. Med.
[98] R. Velho-Pereira, A. Kumar, B.N. Pandey, A.G. Jagtap, K.P. Mishra, Radiosensi- 50 (2011) 978–987, PMID: 21215312.
tization in human breast carcinoma cells by thymoquinone: role of cell cycle [121] K.S. Siveen, N. Mustafa, F. Li, R. Kannaiyan, K.S. Ahn, A.P. Kumar, et al., Thy-
and apoptosis, Cell Biol. Int. 35 (2011) 1025–1029, PMID: 21557727. moquinone overcomes chemoresistance and enhances the anticancer effects
[99] W.K. Ng, L.S. Yazan, M. Ismail, Thymoquinone from Nigella sativa was more of bortezomib through abrogation of NF-␬B regulated gene products in mul-
potent than cisplatin in eliminating of SiHa cells via apoptosis with down- tiple myeloma xenograft mouse model, Oncotarget 5 (2014) 634–648, PMID:
regulation of Bcl-2 protein, Toxicol. In Vitro 25 (2011) 1392–1398, PMID: 24504138.
21609759. [122] B.R. Acharya, A. Chatterjee, A. Ganguli, S. Bhattacharya, G. Chakrabarti, Thymo-
[100] T. Yi, S.G. Cho, Z. Yi, X. Pang, M. Rodriguez, Y. Wang, et al., Thymoquinone quinone inhibits microtubule polymerization by tubulin binding and causes
inhibits tumor angiogenesis and tumor growth through suppressing AKT and mitotic arrest following apoptosis in A549 cells, Biochimie 97 (2014) 78–91,
extracellular signal-regulated kinase signaling pathways, Mol. Cancer Ther. 7 PMID: 24113316.
(2008) 1789–1796, PMID: 18644991. [123] S.H. Jafri, J. Glass, R. Shi, S. Zhang, M. Prince, H. Kleiner-Hancock, Thymo-
[101] A.O. Kaseb, K. Chinnakannu, D. Chen, A. Sivanandam, S. Tejwani, M. Menon, quinone and cisplatin as a therapeutic combination in lung cancer: in vitro
et al., Androgen receptor-and E2F-1-targeted thymoquinone therapy for hor- and in vivo, J. Exp. Clin. Cancer Res. 29 (2010) 87, PMID: 20594324.
mone refractory prostate cancer, Cancer Res. 67 (2007) 7782–7788, PMID: [124] K. Effenberger-Neidnicht, R. Schobert, Combinatorial effects of thymoquinone
17699783. on the anti-cancer activity of doxorubicin, Cancer Chemother. Pharmacol. 67
[102] L. Peng, A. Liu, Y. Shen, H.Z. Xu, S.Z. Yang, X.Z. Ying, et al., Antitumor and anti- (2011) 867–874, PMID: 20582416.
angiogenesis effects of thymoquinone on osteosarcoma through the NF-␬B [125] O.A. Badary, Thymoquinone attenuates ifosfamide-induced Fanconi syn-
pathway, Oncol. Rep. 29 (2013) 571–578, PMID: 23232982. drome in rats and enhances its antitumor activity in mice, J. Ethnopharmacol.
[103] R.L. Gurung, S.N. Lim, A.K. Khaw, J.F. Soon, K. Shenoy, S. Mohamed Ali, et al., 67 (1999) 135–142, PMID: 10619376.
Thymoquinone induces telomere shortening, DNA damage and apoptosis in [126] O.A. Badary, M.F. Abd-Ellah, M.A. El-Mahdy, S.A. Salama, F.M. Hamada, Anti-
human glioblastoma cells, PLoS ONE 5 (2010) e12124, PMID: 20711342. clastogenic activity of thymoquinone against benzo(a)pyrene in mice, Food
[104] V. Cecarini, L. Quassinti, A. Di Blasio, L. Bonfili, M. Bramucci, G. Lupidi, et al., Chem. Toxicol. 45 (2007) 88–92, PMID: 17011106.
Effects of thymoquinone on isolated and cellular proteasomes, FEBS J. 277 [127] C.C. Woo, A.P. Kumar, G. Sethi, K.H. Tan, Thymoquinone: potential cure for
(2010) 2128–2141, PMID: 20412058. inflammatory disorders and cancer, Biochem. Pharmacol. 83 (2012) 443–451,
[105] K. Kolli-Bouhafs, A. Boukhari, A. Abusnina, E. Velot, J.P. Gies, C. Lugnier, et al., PMID: 22005518.
Thymoquinone reduces migration and invasion of human glioblastoma cells [128] R. Schneider-Stock, I.H. Fakhoury, A.M. Zaki, C.O. El-Baba, H.U. Gali-Muhtasib,
associated with FAK, MMP-2 and MMP-9 down-regulation, Investig. New Thymoquinone: fifty years of success in the battle against cancer models, Drug
Drugs 30 (2012) 2121–2131, PMID: 22170088. Discov. Today 19 (2014) 18–30, PMID: 24001594.
[106] A. Paramasivam, S. Sambantham, J. Shabnam, S. Raghunandhakumar, B. [129] M.A. Khan, M.K. Ashfaq, H.S. Zuberi, M.S. Mahmood, A.H. Gilani, The in vivo
Anandan, R. Rajiv, et al., Anti-cancer effects of thymoquinone in mouse antifungal activity of the aqueous extract from Nigella sativa seeds, Phytother.
neuroblastoma (Neuro-2a) cells through caspase-3 activation with down- Res. 17 (2003) 183–186, PMID: 12601685.
regulation of XIAP, Toxicol. Lett. 213 (2012) 151–159, PMID: 22732633. [130] K. Chaieb, B. Kouidhi, H. Jrah, K. Mahdouani, A. Bakhrouf, Antibacterial activ-
[107] H. Gali-Muhtasib, M. Ocker, D. Kuester, S. Krueger, Z. El-Hajj, A. Diestel, et al., ity of Thymoquinone, an active principle of Nigella sativa and its potency to
Thymoquinone reduces mouse colon tumor cell invasion and inhibits tumor prevent bacterial biofilm formation, BMC Complement. Altern. Med. 13 (11)
growth in murine colon cancer models, J. Cell. Mol. Med. 12 (2008) 330–342, (2011) 29, PMID: 21489272.
PMID: 18366456. [131] L. Kokoska, J. Havlik, I. Valterova, H. Sovova, M. Sajfrtova, I. Jankovska, Com-
[108] H. Gali-Muhtasib, D. Kuester, C. Mawrin, K. Bajbouj, A. Diestel, M. Ocker, et al., parison of chemical composition and antibacterial activity of Nigella sativa
Thymoquinone triggers inactivation of the stress response pathway sensor seed essential oils obtained by different extraction methods, J. Food Prot. 71
CHEK1 and contributes to apoptosis in colorectal cancer cells, Cancer Res. 68 (2008) 2475–2480, PMID: 19244901.
(2008) 5609–5618, PMID: 18632613. [132] B. Kouidhi, T. Zmantar, H. Jrah, Y. Souiden, K. Chaieb, K. Mahdouani, et al.,
[109] N. El-Najjar, M. Chatila, H. Moukadem, H. Vuorela, M. Ocker, M. Gandesiri, Antibacterial and resistance-modifying activities of thymoquinone against
et al., Reactive oxygen species mediate thymoquinone-induced apoptosis oral pathogens, Ann. Clin. Microbiol. Antimicrob. 10 (2011) 29, PMID:
and activate ERK and JNK signaling, Apoptosis 15 (2010) 183–195, PMID: 21707998.
19882352. [133] P. Novy, P. Kloucek, J. Rondevaldova, J. Havlik, L. Kourimska, L. Kokoska, Thy-
[110] M. Lang, M. Borgmann, G. Oberhuber, R. Evstatiev, K. Jimenez, K.W. Dammann, moquinone vapor significantly affects the results of Staphylococcus aureus
Thymoquinone attenuates tumor growth in ApcMin mice by interference sensitivity tests using the standard broth microdilution method, Fitoterapia
with Wnt-signaling, Mol. Cancer 12 (2013) 41, PMID: 23668310. 94 (2014) 102–107, PMID: 24508861.
[111] X. Lei, X. Lv, M. Liu, Z. Yang, M. Ji, X. Guo, et al., Thymoquinone inhibits growth [134] S. Hull Vance, H. Benghuzzi, M. Tucci, Inhibition of bacterial attachment to kid-
and augments 5-fluorouracil-induced apoptosis in gastric cancer cells both ney epithelial cells using thymoquinone – biomed 2010, Biomed. Sci. Instrum.
in vitro and in vivo, Biochem. Biophys. Res. Commun. 417 (2012) 864–868, 46 (2010) 69–74, PMID: 20467074.
PMID: 22206670. [135] K.M. Alkharfy, N.M. Al-Daghri, O.S. Al-Attas, M.S. Alokail, The protective effect
[112] S. Banerjee, A.O. Kaseb, Z. Wang, D. Kong, M. Mohammad, S. Padhye, et al., of thymoquinone against sepsis syndrome morbidity and mortality in mice,
Antitumor activity of gemcitabine and oxaliplatin is augmented by thy- Int. Immunopharmacol. 11 (2011) 250–254, PMID: 21145996.
moquinone in pancreatic cancer, Cancer Res. 69 (2009) 5575–5583, PMID: [136] M.M. Rifaioglu, A. Nacar, R. Yuksel, Z. Yonden, M. Karcioglu, O.U. Zorba,
19549912. et al., Antioxidative and anti-inflammatory effect of thymoquinone in an
 S. Darakhshan et al. / Pharmacological Research 95–96 (2015) 138–158 157

acute Pseudomonas prostatitis rat model, Urol. Int. 91 (2013) 474–481, PMID: streptozotocin-induced diabetes through anti-inflammatory and anti-
24008979. oxidant mechanisms, Pharmacol. Res. 85 (2014) 45–54, PMID: 24841126.
[137] M. Inci, M. Davarci, M. Inci, S. Motor, F.R. Yalcinkaya, E. Nacar, et al., Anti- [162] N.M. Hamdy, R.A. Taha, Effects of Nigella sativa oil and thymoquinone on
inflammatory and antioxidant activity of thymoquinone in a rat model of oxidative stress and neuropathy in streptozotocin-induced diabetic rats,
acute bacterial prostatitis, Hum. Exp. Toxicol. 32 (2013) 354–361, PMID: Pharmacology 84 (2009) 127–134, PMID: 19684436.
22872606. [163] A. Al-Hader, M. Aqel, Z. Hasan, Hypoglycemic effect of the volatile oil of Nigella
[138] S. Chandra, D. Mondal, K.C. Agrawal, HIV-1 protease inhibitor induced oxida- sativa seeds, Int. J. Pharm. 31 (1993) 96–100.
tive stress suppresses glucose stimulated insulin release: protection with [164] H.S. El-Abhar, D.M. Abdallah, S. Saleh, Gastroprotective activity of Nigella
thymoquinone, Exp. Biol. Med. (Maywood) 234 (2009) 442–453, PMID: sativa oil and its constituent, thymoquinone against gastric mucosal injury
19234050. induced by ischaemia/reperfusion in rats, J. Ethnopharmacol. 84 (2003)
[139] M.A. Randhawa, In vitro antituberculous activity of thymoquinone, an active 251–258, PMID: 12648823.
principle of Nigella sativa, J. Ayub Med. Coll. Abbottabad 23 (2011) 78–81, [165] M.A. Magdy, E.A. Hanan, E.M. Nabila, Thymoquinone: novel gastroprotective
PMID: 24800349. mechanisms, Eur. J. Pharmacol. 697 (2012) 126–131, PMID: 23051678.
[140] A. Piras, A. Rosa, B. Marongiua, S. Porcedda, D. Falconieri, M.A. Dessi, et al., [166] A. Kahraman, N. Erkasap, T. Köken, M. Serteser, F. Aktepe, S. Erkasap, The
Chemical composition and in vitro bioactivity of the volatile and fixed oils of antioxidative and antihistaminic properties of quercetin in ethanol-induced
Nigella sativa L. extracted by supercritical carbon dioxide, Ind. Crop Prod. 46 gastric lesions, Toxicology 183 (2003) 133–142, PMID: 12504347.
(2013) 317–323. [167] R. Zaman, M.S. Akhtar, M.S. Khan, Gastroprotective and anti-secretory effect
[141] H. Mahmoudvand, A. Sepahvand, S. Jahanbakhsh, B. Ezatpour, S.A. Ayatollahi of Nigella sativa seed and its extracts in indomethacin-treated rats, Pak. J. Biol.
Mousavi, Evaluation of antifungal activities of the essential oil and various Sci. 7 (2004) 995–1000.
extracts of Nigella sativa and its main component, thymoquinone against [168] M. Kapan, R. Tekin, A. Onder, U. Firat, O. Evliyaoglu, F. Taskesen, et al., Thy-
pathogenic dermatophyte strains, J. Mycol. Med. 24 (2014) e155–e161, PMID: moquinone ameliorates bacterial translocation and inflammatory response
25442918. in rats with intestinal obstruction, Int. J. Surg. 10 (2012) 484–488, PMID:
[142] S.H. Aljabre, M.A. Randhawa, N. Akhtar, O.M. Alakloby, A.M. Alqurashi, A. 22750428.
Aldossary, Antidermatophyte activity of ether extract of Nigella sativa and [169] A.H. Alhebshi, A. Odawara, M. Gotoh, I. Suzuki, Thymoquinone protects
its active principle, thymoquinone, J. Ethnopharmacol. 101 (2005) 116–119, cultured hippocampal and human induced pluripotent stem cells-derived
PMID: 15908151. neurons against ␣-synuclein-induced synapse damage, Neurosci. Lett. 570
[143] E.I. Aboul-Ela, Cytogenetic studies on Nigella sativa seeds extract and thy- (2014) 126–131, PMID: 24080376.
moquinone on mouse cells infected with schistosomiasis using karyotyping, [170] K. Radad, R. Moldzio, M. Taha, W.D. Rausch, Thymoquinone protects dopamin-
Mutat. Res. 516 (2002) 11–17, PMID: 11943605. ergic neurons against MPP+ and rotenone, Phytother. Res. 23 (2009) 696–700,
[144] J. Flesar, J. Havlik, P. Kloucek, V. Rada, D. Titera, M. Bednar, et al., In vitro PMID: 19089849.
growth-inhibitory effect of plant-derived extracts and compounds against [171] A.O. Abdel-Zaher, M.G. Mostafa, H.M. Farghly, M.M. Hamdy, G.A. Omran, N.K.
Paenibacillus larvae and their acute oral toxicity to adult honey bees, Vet. Al-Shaibani, Inhibition of brain oxidative stress and inducible nitric oxide
Microbiol. 145 (2010) 129–133, PMID: 20409652. synthase expression by thymoquinone attenuates the development of mor-
[145] M.F. Zafeer, M. Waseem, S. Chaudhary, S. Parvez, Cadmium-induced hepa- phine tolerance and dependence in mice, Eur. J. Pharmacol. 702 (2013) 62–70,
totoxicity and its abrogation by thymoquinone, J. Biochem. Mol. Toxicol. 26 PMID: 23376567.
(2012) 199–205, PMID: 22539463. [172] K. Radad, K. Hassanein, M. Al-Shraim, R. Moldzio, W.D. Rausch, Thymoquinone
[146] G.M. Suddek, Protective role of thymoquinone against liver damage induced ameliorates lead-induced brain damage in Sprague Dawley rats, Exp. Toxicol.
by tamoxifen in female rats, Can. J. Physiol. Pharmacol. 92 (2014) 640–644, Pathol. 66 (2014) 13–17, PMID: 23910425.
PMID: 24941454. [173] I. Ullah, N. Ullah, M.I. Naseer, H.Y. Lee, M.O. Kim, Neuroprotection with
[147] S. Ince, I. Kucukkurt, H. Huseyin Demirel, R. Turkmen, E. Sever, Thymoquinone metformin and thymoquinone against ethanol-induced apoptotic neurode-
attenuates cypermethrin induced oxidative stress in Swiss albino mice, Pestic. generation in prenatal rat cortical neurons, BMC Neurosci. 13 (2012) 11,
Biochem. Phys. 104 (2012) 229–235. PMID: 22260211.
[148] A. Jaswal, N. Sinha, M. Bhadauria, S. Shrivastava, S. Shukla, Therapeutic poten- [174] A.H. Alhebshi, M. Gotoh, I. Suzuki, Thymoquinone protects cultured rat pri-
tial of thymoquinone against anti-tuberculosis drugs induced liver damage, mary neurons against amyloid ␤-induced neurotoxicity, Biochem. Biophys.
Environ. Toxicol. Pharmacol. 36 (2013) 779–786, PMID: 23958970. Res. Commun. 433 (2013) 362–367, PMID: 23537659.
[149] T. Bai, Y. Yang, Y.L. Wu, S. Jiang, J.J. Lee, L.H. Lian, et al., Thymoquinone allevi- [175] A. Khan, K. Vaibhav, H. Javed, M.M. Khan, R. Tabassum, M.E. Ahmed, et al.,
ates thioacetamide-induced hepatic fibrosis and inflammation by activating Attenuation of A␤-induced neurotoxicity by thymoquinone via inhibition
LKB1-AMPK signaling pathway in mice, Int. Immunopharmacol. 19 (2014) of mitochondrial dysfunction and oxidative stress, Mol. Cell. Biochem. 369
351–357, PMID: 24560906. (2012) 55–65, PMID: 22752387.
[150] S. Raghunandhakumar, A. Paramasivam, S. Senthilraja, C. Naveenku- [176] N. Ismail, M. Ismail, M. Mazlan, L.A. Latiff, M.U. Imam, S. Iqbal, et al.,
mar, S. Asokkumar, J. Binuclara, et al., Thymoquinone inhibits cell Thymoquinone prevents ␤-amyloid neurotoxicity in primary cultured cere-
proliferation through regulation of G1/S phase cell cycle transition in N- bellar granule neurons, Cell. Mol. Neurobiol. 33 (2013) 1159–1169, PMID:
nitrosodiethylamine-induced experimental rat hepatocellular carcinoma, 24101432.
Toxicol. Lett. 223 (2013) 60–72, PMID: 24012840. [177] E. Esposito, M. Cordaro, S. Cuzzocrea, Roles of fatty acid ethanolamides (FAE)
[151] M. Khader, N. Bresgen, P.M. Eckl, In vitro toxicological properties of thymo- in traumatic and ischemic brain injury, Pharmacol. Res. 86 (2014) 26–31,
quinone, Food Chem. Toxicol. 47 (2009) 129–133, PMID: 19010375. PMID: 24874648.
[152] R.J. Marles, N.R. Farnsworth, Antidiabetic plants and their active constituents, [178] F. Celik, C. Göçmez, H. Karaman, K. Kamaşak, I. Kaplan, E. Akıl, et al., Thera-
Phytomedicine 2 (1995) 137–189, PMID: 23196156. peutic effects of thymoquinone in a model of neuropathic pain, Curr. Ther.
[153] K.M. Fararh, Y. Shimizu, T. Shiina, H. Nikami, M.M. Ghanem, T. Takewaki, Thy- Res. Clin. Exp. 76 (2014) 11–16, PMID: 25031663.
moquinone reduces hepatic glucose production in diabetic hamsters, Res. Vet. [179] M. Aquib, A.K. Najmi, M. Akhtar, Antidepressant effect of thymoquinone in
Sci. 79 (2005) 219–223, PMID: 16054891. animal models of depression, Drug Res. (Stuttg.) (2014), PMID: 25207705
[154] R.J. Al Wafai, Nigella sativa and thymoquinone suppress cyclooxygenase-2 and [Epub ahead of print].
oxidative stress in pancreatic tissue of streptozotocin-induced diabetic rats, [180] J. Akhondian, H. Kianifar, M. Raoofziaee, A. Moayedpour, M.B. Toosi, M. Kha-
Pancreas 42 (2013) 841–849, PMID: 23429494. jedaluee, The effect of thymoquinone on intractable pediatric seizures (pilot
[155] G. Badr, S. Alwasel, H. Ebaid, M. Mohany, I. Alhazza, Perinatal supple- study), Epilepsy Res. 93 (2011) 39–43, PMID: 21112742.
mentation with thymoquinone improves diabetic complications and T cell [181] M.M. Khattab, M.N. Nagi, Thymoquinone supplementation attenuates hyper-
immune responses in rat offspring, Cell. Immunol. 267 (2011) 133–140, PMID: tension and renal damage in nitric oxide deficient hypertensive rats,
21295287. Phytother. Res. 21 (2007) 410–414, PMID: 17236176.
[156] S. Kapoor, Emerging clinical and therapeutic applications of Nigella sativa [182] A. Nemmar, S. Al-Salam, S. Zia, F. Marzouqi, A. Al-Dhaheri, D. Subramaniyan,
in gastroenterology, World J. Gastroenterol. 15 (2009) 2170–2171, PMID: et al., Contrasting actions of diesel exhaust particles on the pulmonary and
19418593. cardiovascular systems and the effects of thymoquinone, Br. J. Pharmacol. 164
[157] S. Anwar, M.A. Khan, A. Sadaf, H. Younus, A structural study on the protection (2011) 1871–1882, PMID: 21501145.
of glycation of superoxide dismutase by thymoquinone, Int. J. Biol. Macromol. [183] A. Sharafkhandy, Ave-Sina. Law in Medicine. Interpreter, Ministry of Guidance
69 (2014) 476–481, PMID: 24933520. Publication, Teheran, 1990, pp. 314.
[158] G. Al-Naqeep, M. Ismail, Z. Allaudin, Regulation of low-density lipopro- [184] G.M. Suddek, Thymoquinone-induced relaxation of isolated rat pulmonary
tein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase gene artery, J. Ethnopharmacol. 127 (2010) 210–214, PMID: 19961917.
expression by thymoquinone-rich fraction and thymoquinone in HepG2 cells, [185] A.A. Al-Majed, M.H. Daba, Y.A. Asiri, O.A. Al-Shabanah, A.A. Mostafa, H.A.
J. Nutrigenet. Nutrigenomics 2 (2009) 163–172, PMID: 19887822. El-Kashef, Thymoquinone-induced relaxation of guinea-pig isolated tra-
[159] M. Kanter, Effects of Nigella sativa and its major constituent, thymoquinone chea, Res. Commun. Mol. Pathol. Pharmacol. 110 (2001) 333–345, PMID:
on sciatic nerves in experimental diabetic neuropathy, Neurochem. Res. 33 12889525.
(2008) 87–96, PMID: 17713854. [186] A.F. Isik, I. Kati, I. Bayram, H. Ozbek, A new agent for treatment of acute res-
[160] M. Kanter, Protective effects of thymoquinone on streptozotocin- piratory distress syndrome: thymoquinone. An experimental study in a rat
induced diabetic nephropathy, J. Mol. Histol. 40 (2009) 107–115, PMID: model, Eur. J. Cardiothorac. Surg. 28 (2005) 301–305, PMID: 15949945.
19484499. [187] N.M. Elsherbiny, M. El-Sherbiny, Thymoquinone attenuates doxorubicin-
[161] C. Pye, N.M. Elsherbiny, A.S. Ibrahim, G.I. Liou, A. Chadli, M. Al- induced nephrotoxicity in rats: role of Nrf2 and NOX4, Chem. Biol. Interact.
Shabrawey, et al., Adenosine kinase inhibition protects the kidney against 223C (2014) 102–108, PMID: 25268985.
158 S. Darakhshan et al. / Pharmacological Research 95–96 (2015) 138–158

[188] A.S. Awad, R. Kamel, M.A. Sherief, Effect of thymoquinone on hepatorenal [198] A. Gökçe, S. Oktar, A. Koc, Z. Yonden, Protective effects of thymoquinone
dysfunction and alteration of CYP3A1 and spermidine/spermine N-1-acetyl- against methotrexate-induced testicular injury, Hum. Exp. Toxicol. 30 (2011)
transferase gene expression induced by renal ischaemia-reperfusion in rats, 897–903, PMID: 20813795.
J. Pharm. Pharmacol. 63 (2011) 1037–1042, PMID: 21718287. [199] A. Attia, A. Ragheb, T. Sylwestrowicz, A. Shoker, Attenuation of high
[189] O. Evirgen, A. Gökçe, O.H. Ozturk, E. Nacar, Y. Onlen, B. Ozer, et al., Effect of cholesterol-induced oxidative stress in rabbit liver by thymoquinone, Eur.
thymoquinone on oxidative stress in Escherichia coli-induced pyelonephritis J. Gastroenterol. Hepatol. 22 (2010) 826–834, PMID: 20173644.
in rats, Curr. Ther. Res. Clin. Exp. 72 (2011) 204–215, PMID: 24653507. [200] M.A. Nader, D.S. El-Agamy, G.M. Suddek, Protective effects of propolis and
[190] P.K. Kirui, J. Cameron, H.A. Benghuzzi, M. Tucci, R. Patel, F. Adah, et al., Effects thymoquinone on development of atherosclerosis in cholesterol-fed rabbits,
of sustained delivery of thymoqiunone on bone healing of male rats, Biomed. Arch. Pharm. Res. 33 (2010) 637–643, PMID: 20422375.
Sci. Instrum. 40 (2004) 111–116, PMID: 15133944. [201] P. Suguna, A. Geetha, R. Aruna, G.V. Siva, Effect of thymoquinone on ethanol
[191] H. Ozdemir, M.I. Kara, K. Erciyas, H. Ozer, S. Ay, Preventive effects and high fat diet induced chronic pancreatitis – a dose response study in rats,
of thymoquinone in a rat periodontitis model: a morphometric and Indian J. Exp. Biol. 51 (2013) 292–302.
histopathological study, J. Periodontal Res. 47 (2012) 74–80, PMID: [202] S. Ahmad, Z.H. Beg, Hypolipidemic and antioxidant activities of thymo-
21992581. quinone and limonene in atherogenic suspension fed rats, Food Chem. 138
[192] A. Wirries, A.K. Schubert, R. Zimmermann, S. Jabari, S. Ruchholtz, N. El-Najjar, (2013) 1116–1124, PMID: 23411222.
Thymoquinone accelerates osteoblast differentiation and activates bone mor- [203] T. Bai, L.H. Lian, Y.L. Wu, Y. Wan, J.X. Nan, Thymoquinone attenuates liver
phogenetic protein-2 and ERK pathway, Int. Immunopharmacol. 15 (2013) fibrosis via PI3K and TLR4 signaling pathways in activated hepatic stellate
381–386, PMID: 23333454. cells, Int. Immunopharmacol. 15 (2013) 275–281, PMID: 23318601.
[193] M.I. Kara, K. Erciyas, A.B. Altan, M. Ozkut, S. Ay, S. Inan, Thymoquinone accel- [205] M. Roepke, A. Diestel, K. Bajbouj, D. Walluscheck, P. Schonfeld, A. Roessner,
erates new bone formation in the rapid maxillary expansion procedure, Arch. et al., Lack of p53 augments thymoquinone-induced apoptosis and caspase
Oral Biol. 57 (2012) 357–363, PMID: 22036504. activation in human osteosarcoma cells, Cancer Biol. Ther. 6 (2007) 160–169,
[194] G. Schett, C. Stach, J. Zwerina, R. Voll, B. Manger, How antirheumatic drugs pro- PMID: 17218778.
tect joints from damage in rheumatoid arthritis, Arthritis Rheum. 58 (2008) [206] H. Zubair, H.Y. Khan, A. Sohail, S. Azim, M.F. Ullah, A. Ahmad, et al., Redox
2936–2948, PMID: 18821703. cycling of endogenous copper by thymoquinone leads to ROS-mediated
[195] Y. Juarranz, C. Abad, C. Martinez, A. Arranz, I. Gutierrez-Cañas, F. Rosignoli, DNA breakage and consequent cell death: putative anticancer mechanism
et al., Protective effect of vasoactive intestinal peptide on bone destruction of antioxidants, Cell Death Dis. 4 (2013) e660, PMID: 23744360.
in the collagen-induced arthritis model of rheumatoid arthritis, Arthritis Res. [207] S. Dragoni, U. Laforenza, E. Bonetti, F. Lodola, C. Bottino, R. Berra-Romani,
Ther. 7 (2005) R1034–R1045, PMID: 16207319. et al., Vascular endothelial growth factor stimulates endothelial colony
[196] A.A. Fouad, I. Jresat, Thymoquinone therapy abrogates toxic effect of cadmium forming cells proliferation and tubulogenesis by inducing oscillations in
on rat testes, Andrologia (2014), http://dx.doi.org/10.1111/and.12281, PMID: intracellular Ca2+ concentration, Stem Cells 29 (2011) 1898–1907, PMID:
24735446. 21905169.
[197] A. Mabrouk, H.B. Cheikh, Thymoquinone supplementation ameliorates lead- [208] O.A. Badary, O.A. Al-Shabanah, M.N. Nagi, A.M. Al-Bekairi, M.M.A. Elmazar,
induced testis function impairment in adult rats, Toxicol. Ind. Health (2014), Acute and subchronic toxicity of thymoquinone in mice, Drug Dev. Res. 44
pii: 0748233714548474, PMID: 25216800. (1998) 56–61.

View publication stats