Health benefits of black cumin are largely vested on its antioxidant property. Here, a summary of recent studies focused on their antioxidant properties in cell-based in vitro models and in vivo models, covering the last five years, is presented. Being a potential source of natural antioxidants, black cumin lowered the reactive oxygen species (ROS) level while upregulating antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT), and molecules, such as glutathione (GSH), as evident in several studies [26,27]. El-Gindy et al. reported a significant rise in blood TAC and a reduction in malondialdehyde (MDA) in rabbits supplemented with 600 mg/kg of black cumin seeds [28]. In Wister rats given with NSO (1 mL/kg), there was a significant reduction in ROS and nitrous oxide production in amygdala, thereby attenuating chlorpyrifos-induced oxidative stress [29]. TQ treatment resulted in the reduction of intracellular ROS and protection against hydrogen peroxide-induced neurotoxicity in human SH-SY5Y cells by a mechanism that involves upregulation of antioxidant related genes (SOD and CAT), as well as signaling genes, such as c-Jun N-terminal kinase (JNK), extracellular signal-regulated protein kinase (ERK)1/2, p53, protein kinase B (Akt) 1, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) [30]. In adult male rats exposed to contaminated drinking water with lead acetate (2000 ppm) for five weeks, TQ (5 mg/kg/day) ameliorated toxic effects by inducing activities of CAT, glutathione reductase (GR), glutathione peroxidase (GPx), and SOD, and by increasing GSH level in liver tissues [31]. TQ treatment has also been shown to reduce oxidative stress markers (superoxide, hydrogen peroxide, and nitric oxide) and attenuate oxidative stress in lipopolysaccharide (LPS)/interferon-gamma (IFNγ) or H₂O₂-activated BV-2 microglia by promoting antioxidant enzymes (SOD and CAT), and GSH level, downregulating pro-oxidant genes and upregulating antioxidant genes [32]. A meta-analysis of five studies using 293 human subjects suggests that black cumin supplementation may have a beneficial role as an antioxidant by improving SOD levels without affecting MDA level and total antioxidant capacity [33]. With these recent data, it can be concluded that black cumin (in the form of NSO) and its main ingredient TQ have potential antioxidant values that underlie their protective actions against oxidative stress-induced cellular pathology. Further clinical trials are needed to determine the protective functions of black cumin and its compounds against oxidative stress-induced cellular pathology occurred in different diseases condition.

Anti-inflammatory activities are important pharmacological properties of black cumin and TQ [34]. Here, in addition to the antioxidant properties, recent developments on the anti-inflammatory potentials of black cumin seeds, covering the last five years, are focused on. In low-grade inflammation in human pre-adipocytes, freshly extracted NSO reduced the interleukin-6 (IL-6) level, while stored NSO reduced IL-1β level [35]. Following NSO treatment (400 mg/kg) in rats with carrageenan-induced paw edema, there was a significant improvement in the pro-inflammatory cytokines IL-6, IL-12, and tumor necrosis factor (TNF)-α in paw exudates and sera [36]. Moreover, topical application of balm stick containing 10% NSO in rats with paw edema substantially mitigated acute and sub-acute inflammation with a marked edema inhibition (60.64%), and a reduced leucocytes count (43.55% lower than control), and TNF-α level (50% lower than control) on the inflammation area [34].

As a major bioactive, TQ is the key compound responsible for the anti-inflammatory property of black cumin. Hossen et al. reported that TQ inhibited pro-inflammatory factors, including nitric oxide (NO), nitric oxide synthase (iNOS), TNF-α, IL-6, IL-1β, and cyclooxygenase (COX) 2 in LPS-stimulated murine macrophage-like RAW264.7 cells, involving a mechanism that includes the inhibition of IRAK-linked AP-1/ NF-κB pathways [37]. TQ also promoted the autophosphorylation of TANK-binding kinase 1 (TBK1), reduced the mRNA expression of interferons (IFN-α and IFN-β), and downregulated the IRF-3 signaling pathways in LPS-stimulated murine macrophage-like RAW264.7 cells [38]. Current evidence of anti-inflammatory potentials of black cumin and TQ are promising, however, most of the studies so far have been conducted in animal models. Future studies should focus on determining the anti-inflammatory potential in ameliorating human disease conditions.

3.3 Protection against Neurological Disorders

Black cumin and TQ have shown their therapeutic promises against a range of neurological conditions, including neurodegenerative disorders (Alzheimer’s disease (AD), and Parkinson’s disease (PD)), ischemic stroke and acute brain injury, anxiety and depression, epilepsy, and schizophrenia (Table 1). Moreover, black cumin and TQ were shown to protect against various chemical-induced neuronal injury in experimental conditions (Table 1). The neuroprotective potentials of black cumin and TQ mostly stem from antioxidative and anti-inflammatory properties [47] (Figure 3).

3.4 Anti-Cancer Effects

Black cumin and its compounds are widely known for their potent anticancer actions. Accumulating evidence suggests that chemical constituents of black cumin seeds are chemopreventive and potent in inhibiting cell proliferation and provoking apoptosis (Table 2).

4. Molecular Mechanisms Underlying the Pharmacological Effects across Health and Disease Conditions

Black cumin and TQ were shown to exert diverse pharmacological and health effects through modulating multiple cellular signaling systems. The notable molecular pathways targeted by black cumin and TQ are Nrf2, NF-κB, TLR, SIRT1, AMPK-SIRT1-PGC-1α, PPAR, and PI3K/Akt signaling, which are shared across health/disease conditions (Figure 6).

Antioxidant activity of black cumin and TQ is amongst the pharmacological effects that underlie many of its health benefits and has been manifested by their capacity to enhance expression of enzymatic (such as SOD, GPx, CAT, and HO-1) and non-enzymatic (such as GSH) antioxidants, lowering various oxidative markers (such as ROS, MDA, LPO, and 4-HNE). The genetic expression of these antioxidants molecules is under the transcriptional regulation of Nrf2.

Activation of Nrf2 by either cellular redox status or pharmacological intervention leads to the up-regulation of over 250 genes encoding proteins that are involved in antioxidant defense systems, redox homeostasis, and xenobiotic detoxification [81]. Increased expression of antioxidant molecules and subsequent decline in oxidative markers by black cumin and TQ in various pharmacological effects indicate the involvement of Nrf2 activation [51,55,58,69,132,136].

In addition to its potentials on activating cellular antioxidant defense system, black cumin can directly scavenge free radicals, as demonstrated in several in vitro chemical assays like DPPH assay [35,292–294]. However, by virtue, TQ has a relatively poor capacity to quench free radicals because of its oxidized form [295]. This observation strengthens the idea that TQ can exert its antioxidant capacity by activating the Nrf2-dependent antioxidant defense system. However, thymohydroquinone, the reduced form of TQ, possesses a high radical-scavenging capacity [295]. It has been speculated that the conversion of TQ to thymohydroquinone can occur in cells and that the electron transport chain may have an important role in the antioxidant action of TQ.

While toll-like receptors (TLRs) signaling ensures protective immune response by recognizing invading pathogens and tissue-derived endogenous molecules, its overactivation perturbs the immune homeostasis by sustained release of pro-inflammatory mediators and subsequently underlies the development of many inflammatory diseases [296]. TQ may improve inflammatory response in Alzheimer’s disease model by downregulating the expression of TLRs signaling components as well as their downstream effectors NF-κB and IRF-3 [54].

Modulatory role of TQ in autophagy-an evolutionarily conserved cellular process that recycles defective and unwanted cell components and invading pathogens to retain cellular homeostasis has also been documented [297]. Protection against neuroinflammation by TQ in LPS-activated BV-2 microglia involved autophagy induction through activation and nuclear accumulation of SIRT1 [50]. Mitophagy, a form of autophagy that clears defective mitochondria, is regulated by parkin and Drp1 expression. An alteration of parkin and Drp1 expression may lead to impairment of mitophagy triggering apoptosis and neurodegeneration in the brain. Rotenone hindered parkin-mediated autophagy by upregulating Drp1 expression, which was ameliorated by TQ treatment [59].

The anticancer potentials of black cumin and TQ are vested on their capacity to regulate various cellular pathways that are implicated in proliferation, apoptosis, cell cycle regulation, carcinogenesis, angiogenesis, and metastasis [121]. Most of the anticancer actions of black cumin and TQ are reported to mediate by regulating cellular redox systems [117] through which both TQ and black cumin can inhibit cell proliferation, migration/invasion, and tumor growth by directly acting on growth factor signaling systems, such as EGFR/ERK1/2, Akt/mTOR/S6, Wnt, β-catenin, and VEGF signaling [121,298,299]. TQ can prevent cancer development by its antioxidant function and can hinder cancer progression through its pro-oxidant function [121]. Besides, TQ enhanced chemosensitivity to chemotherapeutics and chemopreventive molecules by downregulating inflammatory signaling pathways and enhancing tumor-suppressing genes [116,118,120,300].

As a master upstream kinase, LKB1 phosphorylates and activates AMPK and many other kinases that play a fundamental role in the regulation of cell growth and metabolism [301]. The LKB1–AMPK pathway acts as a cell metabolic checkpoint, arresting cell growth under low intracellular ATP conditions, such as in nutrient-deficient states [301]. Energy overload may suppress LKB1–AMPK signaling, leading to increased cancer risk in patients with obesity or diabetes. Whereas, activation of LKB1–AMPK signaling might contribute to the suppression of cancer risk and, thus, pharmacological modulators, such as TQ, which was shown to activate LKB1–AMPK signaling [106], could have therapeutic promise in cancer prevention.

Apart from the aforementioned mechanism, there still remain other (albeit not less significant) signaling systems that are targeted by black cumin and TQ, such as unfolded protein response (UPR). Triggering of endoplasmic reticulum (ER) stress is a common phenomenon in several pathological conditions such as hypoxia/reoxygenation and oxidative stress. ER homeostasis is crucial for proteostasis and its disruption results in the buildup of unfolded and misfolded proteins in the ER lumen. Consequently, UPR is activated to resolve this protein-folding defect and thus to restore ER homeostasis. In the case of an insufficient UPR, pharmacological activation can play a therapeutic role in mitigating ER stress. Attenuation of ER stress by TQ suggests its protective role in maintaining proteostasis. Moreover, black cumin nanoemulsion promoted Aβ clearance, thus maintained protein homeostasis in the brain, by upregulating LRP1 [52], a type I transmembrane glycoprotein expressed abundantly in neurons that facilitate trafficking and degradation of Aβ [302].