Thymoquinone is Knocking at the Door of Clinical Trial

This review aimed at summarizing the therapeutic potentials in a mechanistic context of a vastly studied quinone, naturally derived product from the seed oil of Nigella sativa called thymoquinone (TQ). The proposed underlyng mechanism may be attributed to its lowdose-mediated antioxidative effect while pro-oxidative at high doses. The TQ is able to form unstable and stable redox compounds in the presence of a variety of internal or external factors such as light, pH, catalyst. Antioxidant-mediated cytoprotectivity while pro-oxidative effect mediated cytotoxicity are the features of TQ with the possibility of redox balancing capacity. In addition, induced autophagy with the redox balancing potential may be a novel consideration in the cancer chemotherapy. Up to date investigated therapeutic potentials of TQ areantioxidant, anti-inflammatory, antimicrobial, anticancer, immunomodulatory, neuro-, gastro-, cardio-, hepatoand nephroprotective. It also imparts beneficial effects in oral hygene, metabolic disorders, diabetes (especially type 2), reproductive and respiratory tract disorders, fibrosis, bone formation and decay. TQ needs clinical trial reports, despite of having a massive animal model data, combinatorial effects with some currently used chemotherapeutic agents as well as action against resistant antibiotics making TQ interesting. TQ should be taken into pharmaceutical account as a novel drug to be developed for clinical trials. Thymoquinone is Knocking at the Door of Clinical Trial REVIEW


Introduction
The use of medicinal plants and their recipes are off the mark because it's bearing information from the past.This deeply rooted portion in the human civilization has been incorporated into the traditional medicine of virtually all human cultures.Plants are the deport of the-

Thymoquinone (TQ) Thymoquinone
(2-isopropyl-5-methylbenzo-1, 4-quinone) (TQ), isolated in 1960s and documen-ted in 1970s is abundantly present in volatile oil as well as fixed oil in N. sativa seeds [1].It is also found together with its few derivatives such as dithymoquinone, thymohydroquinone and thymol (Figure 1) [2,3].However, TQ is evident to have in some species of the family Cupressaceae, Lamiaceae and Monarda.TQ exists in keto (~90%) and enol (~10%) forms.The former one is responsible for its pharmacological properties.TQ is a fat soluble molecule.Solubility in aqueous medium at 24-72 h ranges from 0.549-0.740mg/mL.However, it is unstable in aqueous solution, especially at an alkaline pH.Intravenous (i.v.) administration of TQ followed plasma clearance of 7.19 mL/kg/ min and volume of distribution (steady state) was 700.90 mL/kg; for oral (p.o.) administration that was 12.30 mL/kg/min and 5109.46 mL/kg, respectively [4].The protein binding nature of TQ in human is 98.99% with a plasma half-life of 217 min.TQ is highly light sensitive and provides UV-vis maximum absorbance at 254-257 nm [5].

Antioxidant potential of TQ
The antioxidant potential of TQ is thought to be linked to its conversion of keto form to enol from.The thymohydroquinone/di-hydrothymoquinone is the ultimate molecule which may be obtained by the step-wise reduction of keto-TQ in mammalian microsome by the help of NADPH CYP reductase, NADH CYP-b5 reductase, NADH-ubiquinone oxidoreductase or NADH-quinone oxidoreductase.TQ has potential scavenging power of reactive oxygen species (ROS), superoxide radical (O 2 •-), hydroxyl radical ( • OH) and singlet oxygen molecules.The latter one is more common in plants.It is also a potential redundant of nitric oxide (NO), lipid peroxidation (LP), malonylaldehyde (MDA), myeloperoxidase (MPO) and thiobarbituric acid reactive substances (TBARS) formations.In addition, TQ stimulates to augment the molecular bio-antioxidant systems such as reduced glutathione (GSH), glutathione peroxidase (GPx), glutathione-S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT) [3].

Antimicrobial activities of TQ
The TQ activity against a number bacteria, fungi, protozoa and viruses is thought to be linked to its antioxidant-induced pro-oxidative effects of high concentration, prevention in cell adhesion and biofilm formation.The pathogenic Gram positive such as Bacillus subtilis, Enterococcus faecalis, Gemella haemolysans, Staphylococcus aureus, Streptococcus faecalis, S. mitis, S. oralis, S. pyogens and S. salivarius; and Gram negative species such as Escherichia coli, Pseudomonas aeruginosa and Vibrio paraheamolyticus as well as a Gram positive and Gram negative setting bacteria called Mycobacterium tuberculosis were effectively inhibited within the minimum inhibitory concentration (MIC) values between 8 and 512 µg/mL [3,6,7].In addition TQ is found as an antibacterial resistance modifier [8], while synergistic to particular antibiotic agents [3,6,7].According to Shi et al.TQ significantly reduced the growth of Cronobacter sakazakii with the higher concentration (30 µM) and temperature (55 °C) applied [9].The fungal species Candida albicans, C. krusei, C. tropicalis, Epidermophyton floccosum, Microsporum canis, M. gypseum, Trichophyton interdigitale, T. mentagrophytes and T. rubrum were found to be susceptible to TQ [10,11].Moreover, TQ is also effective against Schistosoma mansoni and Paenibacillus larvae (MIC: 8 to 16 mg/mL) [6,3].TQ at dosesup to 25 µg/mL showed strong anti-Leishmania activities with the half-minimal inhibitory concentration (IC 50 ) values between 1.16 and 2.6 μg/mL toL eishmania tropica and L. infantum [12].
A recent study conducted on avian influenza virus (H9N2) infected birds (n=15) suggests TQ with curcumin having activity against H9N2, where an increased cytokine gene expression was observed [13], which may connect to an inflammation-mediated cytotoxic effect in the viral cell.
A number of tests performed in animal models suggesting that TQ is an effective inhibitor of TNF-α, IL-1β, IL-2, IL-3, IL-5, IL-12, IL-13 other than IL-10, phosphorylation of pro-survival factors, protein kinase B (Akt), ERK1/2, antigen-stimulated T lymphocytes, serum levels of MDA, ALP, ALT, AST and down-regulator of the expression of TGF-β, heme oxygenase-1 (HO-1), GATA-1 and GATA-2 (transcriptor family having ability to bind to DNA sequence), while up-regulator of leukocyte count, phagocytic activity, nuclear factor erythroid derived 2-related factor (Nrf2), chemokinase expression and chemotaxis.TQ may influence T helper cell ration, Th-1/ Th-2 balance and can trigger the cell maturation also.Otherwise, TQ is evident to produce an antileukemic effect on murine leukemia WEHI-3 cells via natural killer (NK) cytotoxic activity pathway.In addition, TQ on pesticide-induced immunotoxicity in male albino rats causing a noteworthy declining in total Ig levels (especially IgGs) with a significant inhibition of hemagglutination [3,25].It has been suggested that, an increased level of glutathione may be related to the TQ activity against inflammatory disorders, despite of NF-κB up-regulation in the brain and spinal cord of rats.The latter one is associated with the inhibition of translocation of p65 other than p50 to the nucleus.Otherwise, TQ has suppressive effects on the production of pro-inflammatory/inflammatory cytokines, TNF-α, NF-κB and its kinase (IKK), monocyte chemoattractant protein 1 (MCP-1), COX-1 and -2, PG-E2, iNOS, and MMP-9.Additionally, TQ up-regulates PPAR-γ, which has an important role in inflammation reduction as well as control over the phosphatidylinositol-3-kinase (PI3K)/Akt balance [25].

TQ in cancer
TQ in various cancer models exhibited promising anti-carcinogenic, anti-neoplastic, anti-proliferative and anti-mutagenic activities [3].It also has chemosensitizing, chemopreventive as well as toxicity reducing capabilities with some standard chemotherapeutic agents, including cyclophosphamide (CP), doxorubicin (DOX) and 5-fluorouracil (5-FU).Otherwise, low concentration-mediated antioxidant and high concentration-mediated pro-oxidant capabilities, giving it's a sword with a dual role, where the former one is responsible for its cytoprotective and the latter one with cytotoxic behavior.TQ is an up-regulator of PPAR-γ, Bax/Bcl-2 ration, Caspase-9/3, smooth muscle actin (Smac), HO-1, p53 and p21; while down-regulator of the pro-apoptotic genes including Bcl-2, Bcl-xL and cell surviving.The TQ-mediated cell cycle arresting is thought to be due to its suppression effects of cyclin dependent kinase (CDK), cyclin D1, cyclin E, androgen receptor, transcription factor E2F-1, NF-κB DNA-binding activity, expression of XIAP, vascular endothelial growth factor (VEGF), Ki-67, focal adhesion kinase (FAK), Mcl-1, c-Myc expression, β-catenin translocation, phosphorylation of extracellular signal-regulated kinase (ERK), and MMP-2 and -9, secretion in different tumor cell lines [26].In a number of human colon cancer cell lines, TQ reduced the phosphorylation states of the MAPK, Jun-N-terminal kinase (JNK) and ERK, tumor nodules and proteolytic cleavage of Caspase-1 (resulting inhibition of IL-1β and IL-18) [3].
TQ may be acting as a microtubule-depolymerizing agent as it distorted spindle organization and suppressed tubulin polymerization by direct tubulin binding in human lung cancer cells [27].However, along with pro-oxidant capacity, TQ may cause loss of mitochondrial membrane potential, inducing apoptosis through the release of cytochrome C and interfering with Akt activation.It is noteworthy that, an ideal anti-cancer agent must have to exert cytotoxicity towards cancerous cell other than the normal cells besides it.To be noted that most of the anti-cancerous chemotherapeutic drugs act by inducing a chronic ROS-mediated detrimental effects towards the cancer cells.In this context, chronic oxidation-mediated genotoxic as well as mutagenic effects are also a major consideration.Although, TQ exhibited a concentration dependent genotoxicity with chromosomal aberrations and micronucleated cells in hepatocyte primary cultures, but there is no evidence for its mutagenic effects [3].Otherwise, TQ (5 mg/mL, p.o.) decreased T cell exhaustion and apoptosis by modulating the expression of Bcl-2, PD-1, Bax, and Caspase-3 on the radiation-exposed rats [28].In addition, TQ inhibited TWIST1 promoter activity and decreased its expression, leading to the inhibition of cancer cell migration, invasion and metastasis [29].Apoptosis and autophagy processes were suspected in human oral cancer cells with TQ treatment.It has been found that, in SASVO3 oral cancer cells TQ arrested the cell cycle at G1 phase where Bcl-2, Bid and Caspase-9 were down-regulated, while LC311, Beclim 1, class III PI3K complex, Rubicon and Arg family proteins were up-regulated [30].In addition, TQ in a mouse cell line induced apoptosis through the down-regulation of Caspase-3 and blocking of p38β MAPK with an increased expression of pro-apoptotic proteins Bid and Bad, causing activation of p53.TQ was also found to prevent oral neoplasm and expression of cytokeratin on 7.12 dimethyl benz (a) anthracene (DMBA)-induced in hamster buccal cancerous cells [31].

TQ on cell cycle and other related events
A study performed on different animal models including cancer cell lines derived from human, saying that TQ can affect cell cycle progression and proliferation, leading to arrest at G0/G1/S/ G2/M phases of the cell cycle (depending on the TQ-treatment, type of cancer cell and mode of activity), causing DNA damage and apoptosis.However, apoptosis may also occur due to its ROS production in anti-oxidant-mediated pro-oxidant depending pathway.In this occasion, overproduction of ROS may cause down-regulation of pro-survival genes, conformational changes in pro-apoptotic proteins, disturbance or even loss of mitochondrial membrane potential leading to activation of Caspase-3, and -9, and cleavage of poly ADP-ribose polymerase (PARP) and Caspase-dependent apoptotic cell death [32].In addition, angiogenesis is a well-known method of supplying O 2 and nutrient demanding of the cells.TQ found as a selective blocker of VEGF (a key pro-angiogenic molecule), thus abolishing proliferation and tubulogenesis of the cancerous cells [33].However, TQ is also evident to inhibit migration, invasion, tubular formation as well as metastasis of cancer cells [33][34][35][36].

Mitochondria targeting TQ and its cationic derivatives
The cationic derivatives of TQ namely SkQT1 (decyltriphenylphosphonium) and SkQ1 (dodecyltriphenylphosphonium) (Figure 1) were suggested as rechargeable antioxidants.However, SkQT1 was found to be a prominent inhibitor of the production of MDA in rat heart and hydrogen peroxide (H 2 O 2 )-induced apoptosis in human fibroblasts.At high concentration they can decrease the mitochondrial membrane potential.As the interior region of mitochondria is more negatively charged than the exterior, thus these positively charged quinine derivatives may readily penetrate through the biomembrane.The ationic derivatives are considered asbetter MDA pump in cancerous cells [37,38], although, in normal cells, the MDA pumps are negligible by these compounds [39].However, TQ at 0.03-100 μg/mL when tested in H 2 O 2 )-induced oxidative stress in human neuronal cells was found to preserve the mitochondrial metabolic enzymes, reducing intracellular ROS levels, preserving morphological architecture, and modulating the expression of genes related to SOD1, SOD2, and CAT and signaling genes-p53, Akt1, ERK1/2, p38 MAPK, JNK, and NF-κβ [40].

TQ in dental caries
The Gram positive bacteria mainly-S.mutans and S. sobrinus iscapable to cause fermentation of carbohydrates resulting in acid production, and the subsequent destruction of the hard tissues of the tooth; thus the initiation and progression of dental caries.TQ is already found to have a strong inhibitory effect to these pathogenic species.Otherwise, rats challenged with S. mutans when treated with TQ at a dose of 10 mg/kg in drinking water significantly reduced the caries score and plaque index in comparison to the control groups [41].

TQ in periodontal and gingival diseases
These inflammatory phenomena are generally related to the Gram negative bacteria, occurring in connective tissue, causing destruction to the gingiva around the teeth.However, it is evident that these may associated with several systemic conditions, such as diabetes mellitus (DM), cardiovascular (CVS) and pulmonary (PMD) diseases, low birth weight, and related other conditions.Otherwise, improper oral hygiene and microbiome control are the few other important causes for periodontal diseases.A research conducted on rats (TQ in drinking water) suggests a significant reduction of the periodontal indices and sub-gingival bacterial counts.It has also a preventive role in periodontal inflammation, irritation progression [7] andbone loss [3].

TQ on fibrosis
Apoptosis, reduced the expression of CD14, tolllike receptor 4 (TLR-4), collagen-1, PI3K, and Akt phosphorylation along with other anti-inflammatory phenomena is thought to be linked with the anti-fibrosis effect of TQ.A decreased level of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) phosphorylation are also consigned to it [42].Paracetamol and lipopolysaccharide (LPS)-induced hepatic fibrosis were found to alleviate by TQ-loaded solid lipid nanoparticles (SLNs) and TQ, respectively [43].In addition, its antihepatic fibrosis activity is regulated via inhibition of phosphorylation of AMPK, liver kinase B, proinflammatory cytokines as well as TLR-4 [14].
Moreover, TQ exhibited an antinociceptive effect through indirect activation of the supraspinal μ-1 and κ-opioid receptors in formalin-treated mice [48].Additionally, a significant reduction of the writhing and late phases in the formalin and acetic acid-induced mice and caragreenan-induced paw edema, and croton seed oil-induced ear edema in rats was also reported by TQ treatment [49,50].Otherwise, GABAergic and nitriergic modulatory anxiolytic and anticonvulsant effects are also evident [51].In epileptic and parkinsonism animal models, TQ augmented the activity of SOD with a descending level of NO and MDA [14].

TQ effects on respiratory system
The lung protective activity of TQ is also blotted with its antioxidant and anti-inflammatory-mediated cytoprotective effects.It was reported that, TQ results in down-regulation of NF-κB, total protein, lactase dehydroginase (LDH), TNF-α, endothelin receptors, Th-2 cytokines, eosinophil infiltration, goblet cell hyperplasia and arachidonic acid metabolism; while it up-regulates IFN-γ production, ATPsensitive potassium channels and augmentation of intratracheal pressure.Thus, the toluene-induced apoptotic effect, bleomycin-induced pulmonary fibrosis, cyclosporine induced pulmonary damage, CP-induced serum biomarker alterations, phenylephrine-induced pulmonary arterial contraction, OVA-induced inflammatory mediator release and carbachol-induced contraction in tracheal smooth muscle is diminished by TQ treatment in a number of animal models [3].

TQ on gastrointestinal tract (GIT)
Along with the down-toning of oxidative and inflammatory mediators, TQ inhibits proton pump, acid secretion, neutrophil infiltration, improvesthe volume and the secretion of mucin, reduces gastric macrophages and mucosal lesions, controls bacterial translocation and improves intestinal barrier.TQ is evident to show gastroprotectivity against ischemia/perfusion (I/R), acetic acid and indomethacininduced gastric injury in test animals [3].

TQ effects on cardiovascular system
In DOX-induced cardiotoxicity mice, TQ lowered the elevated serum LDH and creatine kinase (CK) levels [52].In addition, TQ also exhibited cardioprotective effect in cypermethrin [53] and CP [54] induced the cardiotoxicity in rodents.TQ, methionine-induced hyperhomocysteinemia with a decreased arterial blood pressure (BP) and heart rate, and N-nitro-L-arginine methyl ester (L-NAME)-induced elevated systolic blood pressure (SBP) [55].Moreover, in diesel exhaust particles-induced toxicity in pulmonary and cardiovascular system TQ exhibited to decline in platelet numbers [56].

TQ in metabolic syndrome
The effect on TQ on dyslipidemia is thought to be related to its antioxidant activity with a decline of hepatic HMG-CoA reductase activity, increased arylesterase activity, regulatory effects on genes influencing the cholesterol metabolism [90,91].In cholesterol-enriched dieted rabbits, TQ reduced total cholesterol (TC), low-density lipoprotein-C (LDL-C), triglycerides (TGs) andTBARS concentrations with an increasing level of high-density lipoprotein-C (HDL-C) and GSH contents in comparison to the control group [92].On the other hand, TQ has prominent regulatory effect on Apolipoprotein A-1 and -B100 genes that influence cholesterol metabolism in HepG2 cells [2].Hyperlipidemic rats (n=8) when treated with 10 and 20 mg/kg (p.o.) of TQ for 6 weeks significantly increased in insulin resistance, serum TC, TG, PPAR-γ gene overexpression with a significant decrease in HDL were observed.In addition, a reduction in MDA, IL-10 and Bcl proteins with an increased expression of TNF-α and BAX protein was also claimed [93].
The anti-obesity activity of TQ goes to link with its CVS protection, anticancer, insulin sensitivity and immunomodulatory effects.TQ with 0.5 and 1 mg/ kg/day (p.o.) reduced L-NAME-induced increased SBP via antioxidant effects through down-regulation of creatinine and O 2 •-levels withan up-regulation of the kidney GSH level.TQ is also evident to exhibit anti-diabetic effects against STZ [94], Cd and alloxan-induced hyperglycemia animals.In the former case TQ was found as a corrector of CK-MB and brain monoamines.Overall, anti-diabetic activity of TQ is thought to be related to its correction of dysregulated insulin production, augmentation in insulin secretion as well as pancreatic β-cell proliferative activities [2,3].A research done on diabetic rats, suggesting TQ to exhibit a synergistic effect with glibenclamide on glucose level via reducing CYP450 activity [95].

TQ effects on bone
In animal models TQ reduced the number of osteoclasts and raised osteoblastic activity with an increased in the proliferation and the mineralization events.TQ is also evident to induce the expression of differentiation related genes, osteocalcin and osteopontin, bone morphogenetic protein-2 (BMP-2), and augmented the phosphorylation of ERK and MAPK pathways [96].The i.v.administration of TQ promoted bone formation with a rapid maxillary expansion.Interestingly (i.g.), TQ in the presence of light produced rapid bone formation in RME [97].
In other respects, reduced levels of ROS and proinflammatory mediator production is a helpful phenomenon for the osteoclastic activity and differentiation of osteoclast precursors.

Safety and/or toxic profiles
Findings suggesting, TQ has relatively low toxicity as it is a well-tolerated drug in rodents.Mice treated with 0.03% TQ (in drinking water) for 3 months produced no toxic signs (except with a fasting hypoglycemic effect).Otherwise, rats treated with 90 mg/kg/day (subchronic) exhibited no toxicity.The LD 50 of TQ for mice calculated as 104.7 mg/kg and 870.9 mg/kg after oral and i.p. administration, respectively, whereas the LD 50 in rats was found to be 57.5 mg/kg and 794.3 mg/kg after oral and i.p. adminstration, respectively [14].In addition, TQ at high dose (2000-3000 mg/kg) is found to decrease the GSH content and causing liver and kidney toxicity with the enhanced plasma urea and creatinine levels withan increasing of ALT, LDH and creatinine phosphokinase activities [98].However, TQ is concentration dependently cell necrotic and genotoxic agent.According to Khalife et al.TQ at 10-80 µM with the same concentration of topotecan induced apoptosis through p53-independent mechanism with the S-phase cell cycle arrest and increased production of fragmented DNA [99].While TQ at 1.25 and 2.5 µM for a 43 weeks treatment reduced microsatellite instability independent of a functional mismatch repair system in Msh2 (loxP/loxP) Villin-Cre mice [100].TQ in CCl 4 -induced hepatic damaged mice exhibited a significant protective effect only at 12.5 mg/kg rather than 25 and 50 mg/kg.This may be due to its pro-oxidative effects at higher dose [101].In an adult patient with an advanced malignant cancer TQ was found at a maximum tolerance up to 2600 mg/day [102].To be noted that TQ evident to cause inhibition of phosphorylation of ERK and MAPK pathways in many cancer cells, which is also an essential fact for bone and teeth formation.Thus, during developmental phases, chemotherapy with TQ, it is a major concern.However, the LD 50 , phar-macokinetic behavior and a few other reports telling that TQ toxicity are not only dependent on its concentration, but also of its route administration

Discussion
Thymoquinone (TQ) the quinone, going after a cyclic oxido-reduction reaction produces semiquinone (by NADPH CYP and CYP-b5 reductase, NADH-ubiquinone oxidoreductase), thymohydroquinone (by NADPH-quinone oxidoreductase) and glutathionyldihydro-thymoquinone (by GSH) in in vivo.The last two products containing two active hydroxyl (-OH) group in their structure, are thought to be responsible for antioxidant capacity.According to Guin et al. [103] in an in vitro system, in neutral aprotic media, quinone undergoes a one proton (H + ) two electron or only two electron transfer reactions and produces semiquinone (reversible) and quinone dianion (quasireversible), respectively.In an acidic pH, stable dihydroquinone (irreversible) is the ultimate product under the process of two protons and two electron transfer.However, in basic medium (alkaline pH), quinone goes only a two step one electron transferring process and produces quinone dianion.The redox reaction of TQ in two systems has been shown in figure 2.
The potentiality of the quinone redox system depends on the stability of the reduced species, solvent polarity, supporting electrolytic nature, types of hydrogen bonding, presence of other anionic and cationic additives, ion pairing and proton donation and accepting behaviors.Cation from the supporting electrolyte forms an ion pair with semiquinone and quinone dianion.The hydroxyl quinones, by their OH functional group can alter the typical redox behavior owing to the formation of intra-molecular hydrogen bonds in the reduced species.The free radical generated via redox reaction is the pivotal role in quinine's biological activities.In this occasion anthracycline drugs are the best examples.Toxicities in different organs and chromosomal damaging properties are associated with their electron transfer processes involving the respiratory chain generating H 2 O 2 /O 2 •-/-OH.Through radical reactions, quinones initiate oxidative phosphorylation, complexation of phospholipids and peroxidations of several lipids.Thus, the redox property of quinones connects not only the therapeutic,but also the toxic effects.In this context, semiquinones may be the pioneer for toxic responses,as they can form O 2 •-via reoxidation process, leading to formation of H 2 O 2 and • OH, those are reported to degrade DNA molecule by abstracting hydrogen from deoxyribose residues [103].Compounds, like flavonoids and terpenic essential oils containing active OH group(s) in their structures are responsible antioxidant effect [104] and those having antioxidant capability, at high concentration may cause cytotoxicity by producing ROS itself (pro-oxidant effect).Thus, TQ through its redox reactions producing semiquinone and quinone dianion may impart its pro-oxidative cytotoxic effect.In the body system, by GSH; TQ reduced to glutathionyl-dihydro-TQ, is capable to show antioxidative effect.Otherwise, O 2 •-coming from the conversion of semiquinone to thymohydroquinone can be neutralized by SOD and CAT to H 2 O 2 and finally H 2 O. TQ has been found to induce cell death (apoptosis: via cell cycle arresting, pro-oxidant, expression of cellular proteins), anti-proliferation, anti-migration, anti-invasion, anti-metastasis and anti-angiogenesis effects on a number of animal-derived cancer cell lines.In addition, it exhibited as a chemosensitizer agent with DOX and 5-FU.The anthracycline, DOX is an inhibitor of the relaxer of supercoils in DNA, topoisomerase II essential for DNA transcription leading to stop the process of replication.DOX is also known for the production of quinone type free radicals, hence contributing to its cytotoxicity [105].Otherwise, 5-FU acts principally as a thymidylate synthase (TS) inhibitor.Thus inhibits DNA replication by blocking the synthesis of the neucleosides, pyrimidine thymidine [106].To be noted that not only PPAR-γ (mainly its γ2 and γ3 subunits) but also PPAR-α, -β/-δ are mainly responsible of adipocyte differentiation.Thus, the TQ anticancer activity on breast cancer cell lines may account for its up-regulation power of these classes of PPARs.
Moreover, TQ was found to stimulate the actions of CP, gemcitabine and oxaliplatin.CP (by N7) attaches the alkyl group to the guanine base results in cross-linking in between and within DNA strands (intra-strand and inter-strand), which eventually inhibits DNA replication.In addition the irreversibility of the linking results cell apoptosis [107].The gemcitabine is thought to replace the cytidine (a building block of nucleic acid), leading to stop DNA replication.In addition, it inhibits the synthesis of the enzyme ribonucleotide reductase required for production of deoxyribonucleotides in DNA replication and repair.It also causes cell apoptosis during DNA replication [108].Although, the mechanism of action of oxaliplatin is still poorly understood, its action pathway is thought to be similar that of the CP [109].Thus, the combine effects of TQ with the above anticancer agents may be due to its own mechanism as well as a multi-outlet-like weapon as it was observed that TQ is active against DOXresistant MCF-7 cell line [110].In addition, the acti-vity against antibiotic-resistant bacteria [8] may be accounted with this concept.
It is to be noted that the hydroquinonic form is more stable than the semiquinonic form, as the latter one is unstable and the process of formation is reversible.However, the semiquinone radicals are sometimes stabilized by forming a strong hydrogen bond between the anionic quinone oxygen and the phenolic -OH protons present at β-positions with respect to quinone oxygen [103].The ionizing radiation can generate damaging intermediates through the interaction with water, in which water is sequentially converted to • OH, H 2 O 2 , O 2 •-and ultimately molecular oxygen (O 2 ).The hydroxyl radical ( • OH) is extremely reactive and immediately removes electrons from any molecule in its path, turning that molecule into a free radical and thus propagating a chain reaction.While the H 2 O 2 causes damage to the DNA.TQ exhibited an antagonistic activity in radiation-exposed rats [28] may be due to its ROS neutralizing potentials.Otherwise, radiotherapy by using γ-rays induces ROS-mediated cell death and mitotic failure [111].TQ with this type of ray (2.5 Gy) was found to produce a synergistic cytotoxicity against breast cancer cells [112].However, TQ is a light sensitive quinone.There is an evidence suggesting TQ potentiating a rapid bone formation in the presence of light than the dark [97].Thus, in the presence of light TQ may oxidize in place of cellular components and causes an eventual redox balance by the above mentioned quinone stabilization pathway.In addition, after light exposure it can generate ROS (e.g.-semiquinone, O 2 •-), those are responsible for its pro-oxidant capacity, leading to an additive effect with γ-rays.Chemotherapeutic agents having self-redox balancing and induced autophagy properties may be a good target for cancer treatment.After induction of autophagy to the targeted cell, if it terminates the oxido-reduction reactions, letting out readily from the site of the normal cells belonging to the cancerous one, should not be capable to induce toxic effects on them.
TQ was found to induce autophagy in human oral cancer cell line [30] and redox balancing effect in renal tissue [70].Thus, there is a hope for this noble target.A general pathway of redox balancing of TQ is shown in Figure 3. TQ is already evident to have an anti-angiogenesis activity through the blocking of VEGF as well as arresting tubulogenesis [113].In case of tumor, immature cells undergoing swiftly proliferation, angiogenesis is essential for supplying required oxygen (O 2 ) and nutrients.Figure 2 says that TQ needs molecular O 2 in NADPH and NADH-dependent redox cycling process to form its short lived pro-oxidant semiquinone.This may deprive the cancer cells of O 2 .In addition, liberated O 2 •-may impart a proficient oxidative damage to this kind of rapidly proliferating cells.Thus, anti-angiogenesis potential of TQ may be a good target of cancer cell death.Responsible cytotoxic and cytoprotective conversions of TQ have been shown in Figure 4.
Generally, low levels of inflammation play protective response in host, which eliminates the initial causes of cell injury, necrotic cells and damaged tissues, and initiate tissue repair.However, chronic inflammation is detrimental to the body tissue and cellsand leads some diseases, including periodontitis, atherosclerosis, rheumatoid arthritis, hay fever and even cancer.TQ is evident to act against periodontitis and a number cancers in different animal models.In addition, in diesel exhaust particles-induced toxicity study, TQ reduced the platelet numbers [3],which may be linked to its anti-atherosclerosisactivity.Although TQ-mediated necrotic effect is still controversial, but it exhibited an anti-necrotic effect on renal tubules by cypermethrin-induced kidney damaged mice [53].Otherwise, in collagen induced arthritis in Wistar rats, TQ exhibited a significant anti-arthritic activity via reduction of oxidative stress with a down-regulation of the production of inflammatory cytokines [114].
The immuneprotective effectof TQ observed in lymphocyte cultures and HaCaT cells was thought to link with the modulatory effects of cell mediators as well as oxidative burst and cytokine release [3,25].Thus, antioxidant, pro-oxidant and anti-inflammatory, and immune modulatory effects of TQ are linked to each other.However, inflammation is a generic response whose mechanism is better to consider as an innate immunity, as it is specific for pathogens and events [115].
Beta-amyloid (Aβ) peptides are one of the wellknown contributors of Alzheimer's disease (AD).The collapse of the mitochondrial membrane potential and impairment of synaptic function is the main two functions of them.Ultimate result is the neuronal cell death.TQ was found an effective in-  hibitor of these peptides.TQ has GABAergic activities [3].Otherwise, strong control over oxidative stress and inflammatory mediators are leading to its potential neuroprotective role.Low levels of 5-HT are evident to cause sudden infant death syndrome (SIDS) as 5-HT controls heart rate and breath.Thus, up-regulation of 5-HT secretion by TQ may be helpful for intestinal movement, blood clotting and vasoconstriction, regulation of mood, appetite and sleep including improvement of cognitive functions such as learning and memory.Cardiac and respiratory protective effects of TQ also may be an account for it.Osteoblasts, the bone precursor cells secrete a collagen-proteoglycan matrix which binds calcium salts, leading to prebone (osteoid) matrix calcification.In this context ossification (process in which cartilage in transformed into bone) requires morphogenic proteins (such as BMP-1, BMP-2) and the activated transcription factor, CBFA1.Up-regulated expression of differentiation related genes, osteocalcin and osteopontin, BMP-2 with an augmenting phosphorylation of ERK and MAPK pathways [3] are the helpful events for the formation of bone.In addition, fighting against ROS and pro-inflammatory mediators-mediated detrimental effects may be the obliging dealings for osteoclastic activity, differentiation of osteocyte precursor as well as prevention of decay of bone.Phosphorylation is crucial for DNA transcription.Having difficulty in MAPK/ ERK (Ras-Raf-MEK-ERK) pathway leads uncontrolled growth of cells, which is a necessary step in cancer development.However, this route may be helpful for osteogenesis, and TQ found to up-regulate this pathway.
The high levels of LDL-C are linked to atherosclerosis, heart disease, stroke and other serious health conditions.LDL-C should be lower than 100 mg/dL.Otherwise, the too little HDL-c amount (< 40 mg/ dL) is also a major factor for coronary heart disease.However, LDL has anti-quorum sensing potential against S. aureus through binding with Apolipoprotein B. In human, MPO oxidatively damages HDL by oxidation of specific amino acid residues in Apolipoprotein A1, which is also a main cause of atherogenesis [116].An accumulated result, suggesting that, TQ is an anti-atherogenic agent.In addition, it caused augmentation of the lag time of LDL, which may initiate two events, firstly: available for LDL oxidation by the oxidized semiquinone moiety and secondly: inhibition of LDL receptor to pick up LDL molecules into the endosome.PPAR-γ, activated by PG-J2 and certain members of 5-HETE are responsible for shunting of insulin resistance as well as obesity, while PPAR-α (activated by LT-B4) for increasing insulin sensitivity.TQ is evident to upregulate these two receptors.Some recent study findings on TQ have been shown in Table 2. Table 2. Some important findings from 2015 to March, 2016 on thymoquinone.

Dose, route and period
Test system Activity Reference -In methicillin-resistant Staphylococcus aureus (strains=99) TQ exhibited MIC in the range of 8-16 μg/ mL and MIC 90 of 16 μg/mL against the tested strains. [8]

5-30 mM with heat
In Cronobactersakazakii Significantly reduced the population of C. sakazakii more rapidly with higher temperatures and increased concentrations of TQ.
TQ with curcumin increased cytokine gene expression in combination, leading to suppressed pathogenesis of H9N2 viruses.[12] InternatIonal archIves of MedIcIne In lambda-cyhalothrin (LCT) treated male Wistar rats (n=) Decreased MDA and NO levels with a down-regulation of NF-κB/p65 and pro-inflammatory genes expression and their levels.Increased GSH, immunoglobulins concentrations and antiinflammatory cytokine genes mRNA levels.[15] 0.4% (w/v) for 7 days In allergic rhinitis male rabbits (n=6).Significant decreased in intraepithelial and submucosal inflammation and goblet cell hypertrophy with maintaining normal cilial structure. [23] 50 mg/kg/day for 10 days In 5-Gy cobalt-60 gamma ray-induced cataracted Sprague-Dawley rats (n=9).

4-24 µM
In BT 549 cells.Inhibited TWIST1 promoter activity and decreased its expression, leading to the inhibition of cancer cell migration, invasion and metastasis.
Decreased SOD, MDA levels, active-Caspase 3 and Bax expression as well as apoptotic index in comparison to the torsion group. [44] 10 mg/kg/day (i.p.) for 7 days In ischemia rats (n=8).TQ decreased the levels of MDA, NO, TNF-α and IL-1 as well as motor neuron apoptosis, increased activities of SOD, GPx, CAT in the spinal cord. [45] 5 mg/kg/day (i.p.) for 7 days Traumatic brain injured rats (n=8).Increased the neuron densities in contralateral hippocampal regions other than SOD and GPx levels.TQ decreased the MDA levels.
TQ nanoparticles (94.8±6.61nm; drug content 99.86 ±0.35%) significantly improved locomotor and grip strength after i.n.rather than i.v.administration. [47] 5 mg/kg/day (p.o.) for 5 weeks Pb-induced liver damaged male rats TQ altered the Pb-induced Pb exposure increased in hepatic content, damaged hepatic histological structure, and changes in liver function by altering AST, ALT, ALP, γ-GT, and LDH.TQ also increased TAS level and decreased LP in the rat liver.
[57] 50 mg/kg/day (p.o.) for 30 days Cd-induced nephrotoxic male Wistar albino rats (n=8) Significantly decreased the over expression of NF-κB, number of apoptotic cells, LP with an increased levels of GSH, SOD, CAT, GPx in renal tissue. [71] 10 mg/kg/day (i.g.) for 15 days As-induced kidney damaged male Sprague-Dawley rats (n=8) TQ showed a protective role against arsenic-induced toxicity with impeding oxidative stress and apoptotic cell death in kidney.
[72] 50 mg/kg (p.o.) Testicular rats (n=8).Increased SOD, CAT, GPx, activity of PCNA and decreased the number of TUNEL-positive cells, MDA, LP in comparison to the control group. [82] InternatIonal archIves of MedIcIne In human MCF-7 breast cancer cells.TQ reactivated p21 and Maspin, induced pro-apoptotic gene Bax, down regulated the anti-apoptotic gene, Bcl-2 and arrested the cell cycle at G2/M phase.
[93] -Streptozotocin treated DM rats.Inhibited the increased endothelial NOS protein expression and suppressed the elevation of COX 2, TNF-α and IL 6 levels as well as reduced caspase 3 activity and the promotion of Akt levels.[94] With glibenclamide In diabetic rats.Exhibited a synergistic effect with glibenclamide on glucose level via reducing CYP450 activity.[95] 10-80 µM with topotecan With in human colorectal cancer cells.Both induced apoptosis through a p53-independent mechanism, while TQ by p21.S-phase cell cycle arrest was observed separately, while co-treatment increased the production of fragmented DNA.TQ increased the effectiveness of topotecan inhibiting proliferation and lowering toxicity through p53-and Bax/Bcl2-independent mechanisms. [99] 1.25 and 2.5 µM for 43 weeks In Msh2 (loxP/loxP) Villin-Cre mice Reduced microsatellite instability independent of a functional mismatch repair system.
[100] 0.125-128 μg/mL and 1, 2 and 5 mg/kg with liposomal preparation of TQ (Lip-TQ) (i.p.) for a 40 days On Candida albicans infected mice.Exhibited actvity against both fluconazole-susceptible and -resistant C. albicans.Lip-TQ was more effective than TQ. [117] InternatIonal archIves of MedIcIne In breast cancer cells.miR-34A up-regulation-mediated down-regulation of Rac1 expression followed by actin depolymerisation thereby disrupting the actin cytoskeleton leading to the reduction in the lamellipodia and filopodia formation on cell surfaces thus retarding cell migration.
[118] 20-100 µM In HepG2 cells.Exhibited G2/M phase cell cycle arrest, increased in the percentage of apoptotic cells and the ratio of Bax/Bcl-2, and decreased the expression of mRNA and protein level of vascular endothelial growth factor.
Synergistic effects with cisplatin.Significant reductions of cell proliferation with an increasing apoptosis, promoted cisplatin-induced pH2AX expression, while an inhibition to NF-κB.
A rapid elimination and relatively slower absorption profile in oral dose.
Prevented hyperglycaemia, hypertriglyceridemia, hypercholesterolaemia and elevated systolic blood pressure with impaired glucose tolerance and insulin resistance.Increased the levels of SOD, CAT and GSH.Furthermore, augmented the expression of mRNA of PPAR-α and PPAR-γ in HFD rats. [125] 25 and 50 mg/kg (p.o.) for 2 weeks In liver injured Sprague-Dawley rats (n=8) A reduction in HP and MDA, while increased in SOD and GPx levels.[126] 100 mg/kg/day (p.o.) for 60 days In NLRP3-mediated inflamed in the pancreas of albino Wistar rats (n=8).

Conclusion
Each treatment strategy is based on a particular action pathway of a substance, which is called the mechanism of action on that.It is noteworthy that by these days we have a number of novel drug delivery approaches.However, drugs with a clear cut action pathway(s) are still a concern to the drug scientists.
A known mode of action for any drug is not only a potential for its implementation to a particular disease, but also a concern of its stability, selection of routes and dosage.Metabolism and excretion profiles along with its nature inside the host are also other important concerns.It is doubtless that TQ is well-to-do with research evidence (in vitro and in vivo) in a number of animal models as well as human cell lines.However, only one phase-I clinical trial report is available.Data from the animal models show that TQ has a number of therapeutic potentials as low toxicity, potential antioxidant effects, and thus cytoprotection, with a combinatorial activity with currently used chemotherapeutic agents, activity against drug resistance (especially antibiotics) as well as antagonistic effects with well-known toxic substances making TQ to produce a trickle to the nose of clinical trial.This review suggests taking TQ into the account of clinical settings.

Figure 3 :
Figure 3: Possible general pathways of redox balancing capacity of thymoquinone.

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