Therapeutic perspective of thymoquinone: A mechanistic treatise

Abstract The higher utilization of fruits and vegetables is well known to cure human maladies due to the presence of bioactive components. Among these compounds, thymoquinone, a monoterpene and significant constituent in the essential oil of Nigella sativa L., has attained attention by the researchers due to their pharmacologies perspectives such as prevention from cancer, antidiabetic and antiobesity, prevention from oxidative stress and cardioprotective disorder. Thymoquinone has been found to work as anticancer agent against different human and animal cancer stages including propagation, migration, and invasion. Thymoquinone as phytochemical also downregulated the Rac1 expression, mediated the miR‐34a upregulation, and increased the levels of miR‐34a through p53, as well as also regulated the pro‐ and antiapoptotic genes and decreased the phosphorylation of NF‐κB and IKKα/β. In addition, thymoquinone also lowered the metastasis and ERK1/2 and PI3K activities. The present review article has been piled by adapting narrative review method and highlights the diverse aspects of thymoquinone such as hepatoprotective, anti‐inflammatory, and antiaging through various pathways, and further utilization of this compound in diet has been proven effective against different types of cancers.


| INTRODUC TI ON
The higher utilization of fruits, vegetables, spices, and herbs has been used as practical strategy to prevent from the different types of human cancers and other maladies such as diabetes, cardiovascular disease, obesity due to the presence of bioactive compounds. Among these phytochemicals, thymoquinone from Nigella sativa has been found to be very effective against various human maladies such as cancer insurgence, diabetes prevalence, obesity, inflammation, cardiovascular disease, and oxidative stress, as well as also several infectious microbial diseases. It also prevents the cardiotoxicity induced by doxorubicin through suppression of the carcinogenesis activity, membrane lipid peroxidation, destruction of the Fe-NTA-induced oxidative stress, and inhibition of the eicosanoid production (Farah & Begum, 2003;Yimer et al., 2019). Nigella sativa has attracted healers in ancient civilizations and researchers in recent times.
Traditionally, it has been used in different forms to treat many diseases including asthma, hypertension, diabetes, inflammation, cough, bronchitis, headache, eczema, fever, dizziness, and influenza. Experimentally, it has been demonstrated that N. sativa extracts and the main constituent of their volatile oil, thymoquinone, possess antioxidant, anti-inflammatory, and hepatoprotective properties (Khader & Eckl, 2014). Being anticancer agent, it suppresses the proliferation, migration, and invasion stages and mediates the miR-34a upregulation through p53, and downregulates the Rac1 expression. Thymoquinone regulates pro-and antiapoptotic genes, and reduces the NF-κB and IKKα/β phosphorylation and ERK1/2 and PI3K activities (Imran et al., 2018).
Uncoupling protein-1 (UCP-1) is the index protein of the brown adipose tissue. In recent study conducted by group of researchers, they found that thymoquinone treatment to the experimental subjects, which significantly increased the serum total antioxidant capacity, caused reduction in waist circumference, body weight, and body mass index Namazi et al., 2018;Tüfek et al., 2015).
Glucagon-like peptide-1 (GLP-1) analogs have been found to improve the glycemic control, while administration of streptozotocin is used to induce the diabetes in experimental subjects. In diabetic rats, supplementation of thymoquinone by using oral route in dose-dependent manner exhibited elevation in plasma GLP-1 levels by lowering the hypergaphy (Harphoush et al., 2019;Lee, Kim, et al., 2019). In diet-induced obesity mouse model, supplemented thymoquinone at the rate of 20 mg/kg BW per day prevented diabetic phenotype via lowering the glucose concentration, fasting insulins, serum cholesterol, triglycerides, and inflammatory markers resistin and MCP-1, improving the glucose tolerance and insulin sensitivity, and enhancing the phosphorylated Akt level, whereas it phosphorylated SIRT-1 in skeletal muscle and phosphorylated SIRT-1 and AMPKα in liver (Shpetim et al., 2017).

| Cancer insurgence
Thymoquinone being potent anticancer agent significantly lowers the hypoxia-inducible factor-1α (HIF-1α) expression, inhibits interaction between HSP90 and HIF-1α, and boosts HIF-1α protein degradation in hypoxic cancer cells. It also suppresses the downstream genes of HIF-1α, exhibited alterations in concentrations of lactate, glucose, and ATP levels, which further linked with disturbance of anaerobic metabolic and induction of apoptosis (Lee, Kuo, et al., 2019;Lee, Kim, et al., 2019). Thymoquinone also suppresses the cancer cell stages and the activation of PI3K/Akt pathway in oral squamous cell carcinoma (Ren & Luo, 2019). Moreover, thymoquinone administration in AGS cell lines also lowers the cell propagation rate and causes the induction of apoptotic cell death, and downregulates the VEGF-A gene expression (Rashid et al., 2019). In recent silico docking study by Sumathi and their coworkers, apoptotic targets such as MDM2, Trail-R, Bak, Bax, Bcl-2, and DNA repair target PARP were effectively tumor cell spheres at subtoxic doses, (e) inhibition of cell adhesion, and (f) reduction of transcriptional activity of ß-catenin, respectively (Ndreshkjana et al., 2019). Thymoquinone also downregulates the expressions of containing plant homeodomain (PHD) and really interesting new gene (RING) finger domains 1 DNMT1,3A,3B, (UHRF1), HDAC1,4,9,G9A,KMT2A,B,C,D,E,and KDM1B genes in Jurkat cells and MDA-MB-468 cancer cell lines (Qadi et al., 2019). Multiple researchers and investigators determined the potent role of thymoquinone in combination with cyclophosphamide by applying different concentrations (0.5 mM-10 μM) against Her2 + breast cancer cells through inhibiting the proliferation through the accumulation of cells in sub-G1 (5.49%) and G1 (57.72%), whereas 12% cells were shifted from G2/M phase. On other side, combination of both compounds (0.5 mM-20 μM) showed 16.6% of arresting cells in sub-G1 and only 3.54% cells were remained in G2/M phase. Nonetheless, alleviation in PI3K/Akt signaling pathway via upregulating the PTEN and downregulating the Akt phosphorylation, and also reduction in expression of cyclin D1 were observed after both compounds' administration in Her-2 cells (Aumeeruddy & Mahomoodally, 2019;Bimonte et al., 2019;Khan et al., 2019). In different human cancer cell lines (T98 and LnCaP) and mouse embryonic fibroblast cell lines (3T3), thymoquinone dose-dependently lowered the cell numbers and induced apoptosis via activating the caspase-9 (Kus et al., 2018).
A study described by the Subburayan and their colleagues found that human glioma cells treated with thymoquinone showed inhibition in cell growth via inducing Par-4 expression, which triggers cellular senescence and prostate apoptosis response-4 (Par-4) tumor suppressor protein expressions. Enhancement in cellular size, G1 phase arrest, β-galactosidase staining, and expression of senescence markers (p21, p53, Rb), and reduction in cyclin E, lamin B1, and cyclin depended kinase-2 (CDK-2) were reported after thymoquinone treatment (Table 1; Figures 1 and 2).
Further, overexpression of Par-4 significantly increases the expression of p53 and its downstream target p21, and increases β-galactosidase-positive cells, while siRNA/shRNA-mediated knockdown of Par-4 reverses the thymoquinone-induced effects (Subburayan et al., 2018). In glioblastoma cells, thymoquinone also significantly suppresses the tumorigenic processes through proliferation, invasion, migration, counteract carcinogenesis, and angiogenesis (Chowdhury et al., 2018;Gruber et al., 2018;Johnson-Ajinwo et al., 2018). Anticancer effects are improved when it was combined with genistein bioactive compound against thyroid cancer cells. Both compounds momentously lowered the vascular endothelial growth factor-A, cell viability, messenger RNA expression levels of human telomerase reverse transcriptase, and nuclear factor kappa B genes, and also enhanced the tensin homolog and cyclin-dependent kinase inhibitor 1 (p21) mRNA expression levels and active CASP-3 protein level .
Hatiboglu and their colleagues explored the in vitro cytotoxic effect of thymoquinone dose-dependently on B16-F10 melanoma cell of mice, and intracerebral melanoma in vivo enhanced the cytotoxicity through inducing the apoptosis and DNA damage, enhancing the intracellular reactive oxygen species, suppressing the p-STAT3, and regulating the proapoptotic and antiapoptotic proteins (Hatiboglu et al., 2018). The significant and momentous enhancement in hippocampal neurons is linked with increment in the doublecortin expression on both gene and protein levels, and reduction in caspase-3 expression and the cleavage of poly-ADP ribose polymerase, which were observed after thymoquinone treatment. On other side, thymoquinone has not shown any affect on gene expression of synaptophysin, synapsin, AKT, NGF, NF-kB, p53, and Bax, and protein expression of nNOS and BDNF (Beker et al., 2018). A study reported by Diab-Assaf et al. (2018) supplemented thymoquinone in HTLV-1-negative (CEM and Jurkat) malignant T lymphocytes and HTLV-1-positive (C91-PL and HuT-102) malignant T lymphocytes, which prevented the proliferation, induced apoptosis, enhanced the DNA fragmentation, decreased Bcl-2α and TGFα expressions, and enhanced p53, TGF-β1, and p21 levels (Diab-Assaf et al., 2018).
A peer group of researchers and investigators investigated the effect of thymoquinone with different doses (2 and 4 mg/kg) dosedependently against MDA-MB-231 triple negative breast cancer in experimental mouse via inhibiting CXCR4 expression, tumor growth, and tumor vascularity along with suppressing the brain, lung, and bone metastases. Moreover, thymoquinone also suppresses the NF-κB binding to the CXCR4 promoter and downregulation of the nuclear factor kappa-light-chain-enhancer of activated B-cell (NF-κB) activation Shanmugam et al., 2018).
Breast cancer cells lines (MDA-MB-231 and MCF-7) treated with thymoquinone to experimental subjects exhibited cell cycle arrest at sub-G1 phase, and induced cell death (Kommineni et al., 2019). A peer group of researchers (Ekinci et al., 2018;Samarghandian et al., 2019) in another study evaluated the anticancer role of thymoquinone against human lung cancer cell line (A549 cells) via lowering the viability, inducing apoptotic cell death, enhancing the Bax/Bcl-2 ratio, upregulating the p53 expression, and activating the caspase-3 and caspase-9, respectively (Ekinci et al., 2018;Samarghandian et al., 2019). Similarly, thymoquinone significantly upregulated the p53, downregulated the Bcl2, and induced apoptosis in MCF-7 cells . Haron and their coworkers in another study investigated that thymoquinone suppressed the growth of Hep3B at IC50 < 16.7 μM for 72 hr, induced cell cycle arrest at the G1 checkpoint and non-phase-specific cell cycle arrest, and activated the caspases-3/7 (Haron et al., 2018). Temozolomide in combination with thymoquinone has synergistic cytotoxic effect on U87MG cells through lowering the cell invasion and matrix metalloproteinases 2, 9 (Pazhouhi et al., 2018). In addition, clinical data have been reported on in vitro studies to explore the role of thymoquinone as an anticancer agent. In vitro, thymoquinone in renal cell cancer lines (ACHN &786-O) of xenograft model markedly suppressed the metastatic capacity through AMPK/mTOR signaling pathway, inhibited the migration and invasion, induced autophagy, and suppressed the EMT dose-dependently .

| ANTID IAB E TI C ROLE
In recent study, thymoquinone has been found as potent antidiabetic agent in healthy male volunteers via decreasing the concentrations of the cholesterol and triglycerides, and enhancing the high-density lipoprotein, glucose-induced insulin secretion, and insulin sensitivity (Pelegrin et al., 2019). Daily administration of Nigella seed extracts with diverse doses, that is, 100, 200, and 400 mg/kg, by gavage to streptozotocin-induced diabetic rat caused improvements in atherogenic index and reductions in serum glucose and lipids. Improvement in vasorelaxant responses, reduction in VCAM-1 and LOX-1 expressions (vascular cells of aortic tissue), and enhancement in eNOS in mRNA expression level and function were reported after thymoquinone treatment (Abbasnezhad et al., 2019). Thymoquinone markedly decreased the fasting plasma glucose (FPG) and glycemic status in experimental volunteers (Askari et al., 2019). In another investigation, it has been reported that thymoquinone significantly prevented male Wistar experimental rats from streptozotocin-induced diabetes via lowering blood glucose level, and increasing insulin levels, and catalase and GSH activities. Furthermore, thymoquinone also enhanced the mean pancreatic islet diameter, and percentage of insulin immunoreactive parts, which improved the histopathological picture and hepatic glycogen contents (Abdelrazek et al., 2018). Safhi et al. (2019) investigated the effect of thymoquinone alone and combination of thymoquinone + fluoxetine in depressive type 2 TA B L E 1 Health-endorsing perspectives of thymoquinone

Disorders Mechanisms References
Anticancer Lowered the hypoxia-inducible factor-1α (HIF-1α) expression, suppressed the interaction between HSP90 and HIF-1α, and inhibited the downstream genes of HIF-1α Altered the lactate, glucose, and ATP levels Lee, Kuo, et al. (2019), Lee, Kim, et al. (2019) Suppressed the activation of PI3K/Akt pathway Ren and Luo (2019) Decreased the cell propagation rate and induced apoptotic cell death Downregulated the VEGF-A gene expression Rashid et al. (2019) Docked apoptotic targets such as Bak, Bax, MDM2, Bcl-2, Trail-R, and DNA repair target PARP Exhibited cell cycle arrest in the late apoptotic stage Downregulated the PARP gene expression endothelial growth factor. It also showed inhibition on C-reactive protein, E-selectin level, and interleukin-6 along with normalized plasma cardiac markers (creatine kinase and troponin), respectively (Atta et al., 2018;Ozer et al., 2018). Likewise, thymoquinone also prevented mature male Wistar rats from streptozotocin-induced diabetes via decreasing blood glucose concentrations and enhancing the insulin level (Abdelrazek et al., 2018).

| ANTI -INFL AMMATORY ROLE
Cerebral small vessel disease is covering a variety of abnormalityrelated small blood vessels that degrade the cognition, which further leads to stroke. Thymoquinone phytochemical is working as an antiinflammatory agent and prevented spontaneous hypertensive rats from the cerebral small vessel disease through various mechanisms such as reduction in systolic blood pressure, escape latency time, and the time spent. It also significantly increased along with momentous reduction in mRNA expression of IL-6, monocyte chemoattractant protein-1, IL-1β, and cyclooxygenase-2 in brain of spontaneous hy-  (Alemi et al., 2013;Rifaioglu et al., 2013).

| C ARD I OVA SCUL AR ROLE
In Langendorff-perfused rat hearts, thymoquinone has significant impact on myocardial ischemia/reperfusion (I/R) injury through lowering infarct size, creatine kinase-MB levels, and cardiac lactate dehydrogenase, promoting autophagy, improving cardiac function, and suppressing enedoxidative stress and apoptosis (Xiao et al., 2018).
Moreover, thymoquinone also prevents Wistar albino rats from the myocardial ischemia/reperfusion via enhancing the levels of p53 and Bax (Sezen et al., 2018). Adli and their coworkers investigated that supplementation of thymoquinone (40 mg kg −1 day −1 ) to adult male Wistar Albino rats prevented rats from the cisplatin (15 mg/ kg dose)-induced myocardial injury, which further caused reduction in edema, congestion, and pycnotic nuclei in myocardial fibers, and enhancement in antiapoptotic protein Bcl-2 level (Adalı et al., 2016). Thymoquinone also prevented Wistar rats induced by isoproterenol (125 mg/kg) by applying the different oral doses such as 12.5, 25, and 50 mg/kg through multiple mechanisms such as enhancement in SOD and myocardial ratio and reduction in lactate dehydrogenase and thiobarbituric acid levels from the myocardial injury (Ahmad & Beg, 2013;Randhawa et al., 2013;Tufail et al., 2020).
Cyclophosphamide is used to induce cardiotoxicity in experimental subjects by enhancing the concentrations of serum lactate dehydrogenase, urea, cholesterol, triglycerides, creatine kinase, creatinine, tumor necrosis factorα, total nitrate, and thiobarbituric acid reactive substances. It also reduced the glutathione, glutathione peroxidase, catalase, and adenosine triphosphate levels. On other side, thymoquinone at the rate of 50 mg/L in drinking water to experimental subjects reverted these changes (Nader et al., 2010;Nagi et al., 2011). Similarly, thymoquinone treated with rats with reperfusion injury showed momentous improvements in left ventricular function, attenuation in mitochondrial oxidative damage, reduction in myocardial infarct size and lactate dehydrogenase, enhancement in antioxidant enzymes, reduction in number of apoptotic cardiomyocytes, suppression of p53 acetylation, enhancement of mitochondrial function, upregulation of SIRT1 level, and reduction in production of H 2 O 2 , respectively (Lu et al., 2018). A study conducted by Gonca and Kurt showed that intraperitoneal supplementation of thymoquinone (10 mg/kg) to anesthetized rats prevented rats from the myocardial ischemia and ischemia-and reperfusion-induced ventricular arrhythmias via multiple pathways such as reduction in infarct size, arrhythmia scores, and incidence of ventricular tachycardia and ventricular fibrillation (Gonca & Kurt, 2015).
The supplementation of thymoquinone at the rate of 10 mg kg −1 day −1 in the drinking water normalized the levels of SK(Ca), eNOS, the components of the angiotensin system, and IK(Ca), and restoration of EDHF-mediated relaxations and NO− in the mesenteric artery of middle-aged rats (Idris-Khodja & Schini-Kerth, 2012). Thymoquinone phytochemical is also capable of lowering the aortic MDA and attenuating the atherogenesis in a rabbit model of atherosclerosis (Ragheb et al., 2011). Thymoquinone also decreases the oxidized low-density lipoprotein receptor-1 (LOX-1) gene, protein expression, macrophages and pro-inflammatory cytokine level in apolipoprotein E knockout (ApoE −/− ) male mice (Xu et al., 2018). In earlier study conducted by Lu and their colleagues, thymoquinone showed significant impact on rat hearts and neonatal rat (myocardial ischemia injury) caused momentous improvement in left ventricular function, reduction in myocardial infarct size, lactate dehydrogenase production, elevation of antioxidant enzymes, inhibition of p53 acetylation, enhancement of mitochondrial function, upregulation of SIRT1 expression, and reduction in number of apoptotic cardiomyocytes (Lu et al., 2018). Moreover, thymoquinone (0.2 ml/kg) in combination prevented experimental rats (Wistar albino) after myocardial ischemia from the lung injury and lowered the levels of p53 and Bax (Sezen et al., 2018). Likewise, thymoquinone formulated in liposome prevented experimental subjects from the cyclophosphamide-induced liver toxicity and higher serum bilirubin concentration (Laskar et al., 2016). In nonalcoholic steatohepatitis (NAFLD) liver of rats, thymoquinone (10, 20 mg/kg) lowered the MDA level, enhanced total antioxidant capacity, reduced hepatic TNFα, increased IL10, lowered BAX protein, and enhanced Bcl expression (Awad et al., 2016). Elevation in liver enzymes such as aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, total bilirubin, alkaline phosphatase, and gamma-glutamyltransferase was reported in female rats after inducing the hepatotoxicity by tamoxifen. It also used to prevent rats from the lipid peroxidation and leakage of antioxidant enzymes, and enhance the tumor necrosis factor-alpha in the liver, whereas thymoquinone supplementation (50 mg/kg/BW) reverted these changes (Suddek, 2014). Induction of carbon tetrachloride to experimental rats caused momentous elevation in alanine aminotransferase activity, reductions in glutathione concentrations, reductions in the NAD(P)H-quinone oxidoreductase activities and messenger RNA (mRNA) levels of glutathione S-transferase and microsomal epoxide hydrolase, and reduction in the glutathione and cysteine levels. On other side, thymoquinone application in corn oil (5 mg/kg) reverted these changes (El-Sayed, 2011;Erdemli et al., 2018). It also enhances the antioxidant enzyme levels (superoxide dismutase, glutathione level, catalase, glutathione peroxidase, and glutathione reductase) in liver tissues in experimental subjects (Mabrouk, 2017). In Wistar rats, thymoquinone application (500 mg kg −1 day −1 ) provides protection against intraperitoneally administrated cisplatin (12 mg/ kg/body weight)-induced hepatotoxicity via enhancing the antioxidant enzyme activities, that is, reduced glutathione contents, and decreasing MDA, TNFα, iNOS, and IL-1β (Al-Malki & Sayed, 2014).

| HEPATOPROTEC TIVE ROLE
Zafeer and their coworkers investigated the role of thymoquinone (10 μM) by providing the protection against the cadmium-induced oxidative stress in the liver of Swiss albino rats through attenuating the protein oxidation and rejuvenating the depleted antioxidants of cellular fraction (Zafeer et al., 2012). Similarly, aflatoxin in experimental subjects also known to induce hepatotoxicity via enhancing the concentrations of liver enzymes, that is, ALT, ALP, and AST along with malondialdehyde levels, and reduce the glutathione concentrations, whereas thymoquinone administration to mouse reverted these changes (Nili-Ahmadabadi et al., 2011). In addition, intraperitoneal supplementation of acetaminophen (500 mg/kg) significantly increased the concentrations of hepatic lipid peroxides, total nitrate/ nitrite, and serum ALT, and lowered the hepatic reduced glutathione and adenosine triphosphate in a time-dependent manner. On other side, different doses of thymoquinone (0.5, 1, and 2 mg kg day −1 ) to experimental mouse provide prevention against acetaminopheninduced hepatotoxicity dose-and time-dependently via lowering the serum alanine aminotransferase activities . and 10 mg/kg in rats exhibited neuromodulatory effect via inducing apoptotic cell death and Aβ formation resulting from glutamate administration (Cascella et al., 2018;Farkhondeh et al., 2018;Fouad et al., 2018). Thymoquinone also prevents rats from the progression of Parkinson's disease induced by rotenone. The supplementation of thymoquinone at the rate of 7.5 and 15 mg kg day −1 , po, in male Wistar rats prevented rotenone-induced motor defects and caused changes in the dynamin-related protein-1, parkin, dopamine, and TH levels in the striatum and substantia nigra of dopaminergic areas (Ebrahimi et al., 2017). Similarly, encapsulated thymoquinone in polylactic coglycolic acid chitosan nanoparticles considerably enhanced the locomotor activity and grip strength and lowered the ischemia infarct volume in the middle cerebral artery-occluded rats (Xiao et al., 2016). Ramachandran and Thangarajan (2016) investigated that effective role of solid lipid nanoparticles encapsulated thymoquinone (10 and 20 mg/kg) against 3-nitropropionic acid-induced huntington's disease animals via multiple mechanisms such as improving the muscle strength, movement, rigidity, and memory performances, attenuating the levels of NO, LPO, and protein carbonyls in 3-NP-induced animals, controlling the mitochondrial SDH inhibition, restoring the antioxidant defense system, and alleviating anticholinergic effect upon 3-NP induction. Moreover, thymoquinone also plays effective role against 3-NP toxicity by protecting the striatal structural microelements (Ramachandran & Thangarajan, 2016). In experimental rats, orally administrated thymoquinone (5 mg kg −1 day −1 ) enhanced the neuron density in contralateral hippocampal regions (CA1, CA2-3, and CA4) and lowered malondialdehyde level (Gülşen et al., 2016). With acrylamide-induced neurotoxicity in both in vitro and in vivo of male Wistar rats, thymoquinone dose-dependently (2.5, 5, and 10 mg/kg IP) significantly decreased abnormalities and lowered the level of MDA in cerebral cortex (Mehri et al., 2014).

| NEUROPROTEC TIVE ROLE
Thymoquinone also rescued dopaminergic neurons and decreased the release of lactate dehydrogenase, increased the mitochondrial membrane potential, enhanced lysosomal degradation, and inhibited mitochondria-mediated apoptotic cell death (Radad et al., 2015).
Thymoquinone (10 mg/kg) supplemented to male Wistar albino rats with spinal cord injury (SCI) lowered the histological features of spinal cord damage (Üstün et al., 2014). In animals, pentylenetetrazole (50 mg/kg) has been used to induce generalized seizures and mortality, prolong the onset of seizures, and lower the polyspike and epileptiform discharges and high-grade seizures. It also caused reduction in calmodulin-dependent protein kinase II (CaMKII), suppression in phosphorylation of cAMP response element-binding protein (CREB) in cortex and hippocampus, and decline in gamma-aminobutyric acid B1 receptor (GABAB1R) levels. It also enhanced the Bax, decreased Bcl-2 expression, and activated caspase-3. Thymoquinone in combination with vitamin C reversed these changes (Ullah et al., 2015).
In unilateral intrastriatal 6-hydroxydopamine (6-OHDA)-lesioned rats, diverse concentrations of thymoquinone (5 and 10 mg/kg BW) caused reduction in the number of neurons on the left side of the substantia nigra pars compacta, nitrite, and MDA level, and improved turning behavior (Sedaghat et al., 2014). In primary cultured cerebellar granule neurons, supplemented thymoquinone with different doses (0.1 and 1 μM) lessened the β-amyloid peptide 1-40 sequence by preventing neurotoxic effects and neural cell death (Ismail et al., 2013). Multiple evidences and findings are reported by different researchers and investigators and found preventive role | 1803 BUTT eT al.

| REPRODUC TIVE ROLE
Cadmium chloride is used to induce reproductive toxicity in male rats, while thymoquinone administration prevented the deleterious effects of cadmium chloride via activating testicular endocrine and antioxidant systems (Parhizkar et al., 2016;Sayed et al., 2014).
Similarly, orally administrated lead (20 mg body weight) has been known to cause toxicity in male albino rats and lowered the sperm count, testis and epididymal weights, motility and viability, and serum FSH, LH, testosterone, and estradiol levels. It also lowered the testicular antioxidant molecules, caused enhancement in sperm abnormalities, downregulated the aromatase gene expression, activated the caspase-3 apoptotic pathways, decreased the MDA and NO levels, and also exhibited the germinal epithelium sloughing, complete seminiferous tubules hyalinization, and hypocellularity.
Moreover, thymoquinone treated with experimental rats reverted these changes .
Moreover, morphine at the rate of 20 mg/kg applied to the experimental male mice caused momentous reductions in testis weight, germinal thickness, count, testosterone level, viability, morphology, and motility of sperm and enhancement in nitric oxide. On other side, different doses of thymoquinone (2, 10, and 20 mg/kg) and thymoquinone (2, 10, and 20 mg/kg) in combination with morphine (20 mg/ kg) lowered the nitric oxide level, enhanced the motility (total motility and progressive motility), germinal thickness, morphology, count, viability of sperm cells, and testosterone hormone (Miah et al., 2018;Salahshoor et al., 2018). It also improved the sperm fertility rate and prevented anomalies. Moreover, thymoquinone treatment momentously enhanced the spermatogenic cells, mean volumes of testis, leydig cells, and seminiferous tubules (Tüfek et al., 2015). From another model, diabetic Wistar male rats induced by intraperitoneal injection of STZ (65 mg/kg) enhanced the malondialdehyde and nitric oxide level, upregulated the NF-κB and nitric oxide, and lowered the antioxidant enzyme concentrations in testicles of diabetic rats, whereas oral-administrated thymoquinone reverted these changes in subjects.
Additionally, it also showed reductions in epididymal sperm count and improvement in low plasma testosterone level (Mabrouk & Ben Cheikh, 2016;Rathore et al., 2020).

| Antiarthritic action
In adjuvant-induced arthritis, thymoquinone treatment provides protection against rheumatoid arthritis via reducing the expressions of IL-1b and TNF-a in adjuvant-induced arthritis (Vaillancourt et al., 2013). Moreover, ovalbumin-induced asthma

| Effects of thymoquinone in respiratory diseases
Bronchial asthma is linked with airway inflammation and leukotrienes. Thymoquinone has been found to prevent from the deleterious effects induced by chemicals and environmental toxins. It also protects the lungs by exposing the toluene in rats (Kanter, 2011). Thymoquinone also prevents the deleterious effects of bleomycin on lung tissues of rats through lowering the pulmonary fibrosis development and activated NF-kb overexpression, as well as corrected emphysema in inflammatory cell infiltration, air alveoli, and lymphoid hyperplastic cell initiation (El-Khouly et al., 2012). It also works as an effective agent against cyclophosphamide-persuaded pulmonary impairment in rats (El-Khouly et al., 2012). Similarly, thymoquinone as bioactive compound has been found to reduce the levels of TNF-a, LDH, MDA, and total protein (Suddek, 2014). A study described by El Gazzar and colleagues investigated ameliorative role of thymoquinone against allergic airway inflammation via hampering Th-2 cytokine initiation, cell infiltration and hyperplasia, IL-4, IL-5, and IL-13, and also initiated the IFN-a production (El Gazzar et al., 2006).
It also attenuated the inflammation via lowering the COX-2 expression and PGD-2 production (El Mezayen et al., 2006).

| CON CLUS ION
Thymoquinone is a phytochemical compound found in the plant Nigella sativa. Evidently, thymoquinone is chemically known as 2-methyl-5-isopropyl-1, 4-benzoquinone, an active principal component of the volatile oil that exhibits wide spectrum of healthendorsing properties such as anti-inflammatory, hepatoprotective, antimicrobial, antitumor, antimutagenic, antiepileptic, neuroprotective, and nephroprotective, respectively. Health-associated perspectives of this bioactive compound led to medical applications.
In several anti-inflammatory and degenerative disorders such as cancer, thymoquinone also has been known to modify the multiple molecular and signaling pathways. Most important aspects of thymoquinone such as hepatoprotective, anti-inflammatory, and antiaging have been highlighted through various pathways, and further utilization of this compound in diet has been proven effective against different types of cancers.

ACK N OWLED G M ENTS
The authors are grateful to Higher Education Commission Islamabad, Pakistan, and Universities for providing facilities to carry out this activity.

CO N FLI C T O F I NTE R E S T
There is no conflict of interest among authors.

E TH I C A L A PPROVA L
Not Applicable.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data available on request from the authors.