Revisiting pharmacological potentials of Nigella sativa seed: A promising option for COVID‐19 prevention and cure

Nigella sativa seed and its active compounds have been historically recognized as an effective herbal panacea that can establish a balanced inflammatory response by suppressing chronic inflammation and promoting healthy immune response. The essential oil and other preparations of N. sativa seed have substantial therapeutic outcomes against immune disturbance, autophagy dysfunction, oxidative stress, ischemia, inflammation, in several COVID‐19 comorbidities such as diabetes, cardiovascular disorders, Kawasaki‐like diseases, and many bacterial and viral infections. Compelling evidence in the therapeutic efficiency of N. sativa along with the recent computational findings is strongly suggestive of combating emerged COVID‐19 pandemic. Also, being an available candidate in nutraceuticals, N. sativa seed oil could be immensely potential and feasible to prevent and cure COVID‐19. This review was aimed at revisiting the pharmacological benefits of N. sativa seed and its active metabolites that may constitute a potential basis for developing a novel preventive and therapeutic strategy against COVID‐19. Bioactive compounds of N. sativa seed, especially thymiquinone, α‐hederin, and nigellidine, could be alternative and promising herbal drugs to combat COVID‐19. Preclinical and clinical trials are required to delineate detailed mechanism of N. sativa's active components and to investigate their efficacy and potency under specific pathophysiological conditions of COVID‐19.


| INTRODUCTION
The recent outbreak of novel coronavirus is of serious global concern.
At the end of December 2019, novel coronavirus (2019-nCoV) or Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was identified in the Wuhan City of Hubei Province of China (Huang et al., 2020). Due to its highly contagious nature, within a very short time, this virus has spread over 210 countries and territories around the world and the coronavirus disease  has been stated as a pandemic by WHO on March 11, 2020(WHO, 2020b. As of August 4, 2020, 18,142,718 people had been infected by this virus and the number of deaths had totaled to 691,013 (WHO, 2020a). The majority of the COVID-19 patients in China were presented in the hospital with a severe infection of the lower respiratory tract in the form of pneumonia, which is similar to SARS-CoV and the middle east respiratory syndrome coronavirus (MERS-CoV) patients (Ralph et al., 2020).
It has also been noticed that younger patients who have strong immunity suffer from relatively less illness, while older patients who have several other health issues suffer a high illness. This virus triggers infection in the respiratory tract, nervous system, gastrointestinal tract, kidney, and liver of the patients (Ralph et al., 2020). Several neurologic signs including CAM-ICU (confusion assessment method for the intensive care unit) positive signs (acute change or a fluctuation in mental status, inattention, disorganized thinking, and altered level of consciousness), agitation, corticospinal tract signs, perfusion abnormalities, cerebral ischemic stroke (Helms et al., 2020), and gastrointestinal disorders (Gu, Han, & Wang, 2020) were reported in severe COVID-19 condition. In some cases, acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) may be developed which leads to a high mortality rate (X. Yang et al., 2020). Since the disease was identified, the highest efforts have continued to control this deadly outbreak. Despite it is far from the discovery of an effective vaccine against the COVID-19. Although a significant number of compounds have been proposed and the existing drugs have also been tested for repurposing, no specific therapy has been approved yet . In this reality, it is important to consider all kinds of possible options to fight against the COVID-19.
Recently, a molecular docking-based study identified nigellidine and α-hederin among the compounds of N. sativa as novel inhibitors of SARS-CoV-2 (Salim & Noureddine, 2020). Even, traditional practice that involves taking black cumin seed formula and its steam has been found to relieve the symptoms of the disease. All these evidence strongly suggests the therapeutic potentials of N. sativa seed and its active constituents against COVID-19. To gain further insight into the therapeutic benefits, we have revisited the pharmacological potentials of N. sativa seed and its bioactive constituents, and present a comprehensive outline on prospects of these natural products for the prevention and cure of COVID-19. The main goal of our current effort is to explore the possibility of any alternatives to prevent this deadly pandemic.
2 | COVID-19 PATHOPHYSIOLOGY AND POSSIBLE INTERVENTION BY N. SATIVA SEED Over the past two decades, substantial research progress has been made on the health-benefiting effects of N. sativa seed and its major active compound, thymoquinone on various physiological systems, including the immune system. However, the precise mechanism of how N. sativa may mediate protective effects against viral infection has not been clearly understood. The following sections summarize the current knowledge on N. sativa in mediating the protection against SARS-CoV-2-associated pathological conditions including immune dysfunction, autophagy dysfunction, oxidative stress, inflammation, and the co-morbidities such as diabetes, hyperglycemia, cardiovascular disorders, bacterial infection, and viral infection (Table 1). Ref.

| Autophagy
Autophagy is a natural, regulated, and catabolic mechanism that mediates the degradation of damaged-cellular components through the actions of the lysosomal system. Autophagy is involved in several physiological processes, namely, cell differentiation and development, starvation and degradation of cellular structures and thus maintaining homeostasis (Klionsky et al., 2016). The alteration of immune system is common in infections with highly pathogenic viruses which facilitates immune dysfunction. Therefore, to fight against viruses like SARS-CoV infections in the pulmonary system, a powerful immune response is needed (Li, Fan, et al., 2020;Li, Geng, et al., 2020). Being a part of the cell surveillance system, autophagy plays an important role in immune responses (Crotzer & Blum, 2010;Pan et al., 2016).
SARS-CoV-2 infection suppresses autophagy (Gassen et al., 2020;Hannan, Rahman, et al., 2020). While autophagy machinery is essential for their replication, some viruses evolved strategies to evade autophagy flux (Gassen et al., 2019). Pharmacological agents that induce autophagy may, therefore, have antiviral effects against SARS-CoV-2 (Dong & Levine, 2013). A study indicates that thymoquinone acts as a cardioprotective agent by promoting autophagy (Xiao et al., 2018). The conversion of LC3I to LC3II is an indicator of autophagic activity (Sohn et al., 2017). The study shows that pretreatment of thymoquinone augmented the expression of LC3II while expression of p62 was inhibited (Xiao et al., 2018), indicating the activation of autophagy. In addition, the effects of thymoquinone on autophagy in myocardium were partly eliminated when treated with chloroquine (autophagy inhibitor), supporting the probable contribution of autophagy in thymoquinone-facilitated cardioprotective properties.
Interestingly, some existing drugs having autophagy modulatory effects were found to be promising against SARS-CoV-2 infections (Shojaei, Koleini, et al., 2020;Shojaei, Suresh, Klionsky, Labouta, & Ghavami, 2020). Instead of antagonizing the viral effect, these drugs also suppress autophagy flux in a similar way the virus does, leading to increased accumulation of autophagosomes and subsequent activation of the apoptotic pathway that results in apoptotic death of SARS-CoV-2-infected cells, and thus hinder the virus replication (Shojaei, Suresh, et al., 2020). Based on this autophagy-dependent mechanism, it can be anticipated that suppressing the autophagy flux can also be a possible therapeutic option to fight against COVID-19. To the existing knowledge, it is not clear whether N. sativa or thymoquinone has similar autophagy modulatory effect, and therefore, further research is being proposed to underpin this possibility too.

| Inflammation and cytokine storm
Cytokine storm syndrome (CSS) might happen to patients with severe SARS-CoV-2 infection requiring intensive care (Mehta et al., 2020).
Excess production of immune cells (Channappanavar & Perlman, 2017) and mediators of pro-inflammatory and inflammatory cytokines cause cytokine storms (Huang et al., 2020;Li, Fan, et al., 2020;Li, Geng, et al., 2020;Mehta et al., 2020). Moreover, the characteristic inappropriate inflammatory response in SARS-CoV-2 infection is highly related to an attenuated function of type I and II IFNs along with an increased expression of IL-6 (Blanco-Melo et al., 2020) and C-X-C motif chemokine 10 (CXCL10), chemokine ligand 2 (CCL2), CCL3, and CCL4 (Huang et al., 2020). Cytokines, such as IL-1β, TNFα, and IL-17 are also responsible for Th17 type responses, causing vascular permeability and leakage developing a sign of severe SARS-CoV-2 infection (Wu & Yang, 2020). On the other hand, IL-37 is known to impede immune activities, inhibit MHC-II and TNF, IL-1, IL-6, IL-8 modulate inflammation during SARS-CoV-2 infection (Conti et al., 2020). NF-kB pathway positively regulates cytokines and chemokines (Conti et al., 2020). Upon recognition of the PAMPs of SARS-CoV-2 by pathogen recognition receptors (e.g., TLR) on the surface of infected-host cells or immune cells, NF-kB pathway is activated upregulating the synthesis and secretion of cytokines and chemokines (Conti et al., 2020). To mitigate SARS-CoV-2, it is necessary to inhibit Th17 type responses, cytokine secretion, and the master inflammatory regulator, NF-kB pathway. Several previous reports also corroborated that NF-kB suppression or regulation is positively related to the enhancement of IFN-mediated antiviral activity (Mahase, 2020).
Since phytochemicals have potential roles to modulate or suppress NF-kB activation and thereby inflammation, these could be alternatively used to fight against SARS-CoV-2 (Rahman, Biswas, & Kirkham, 2006). Phytochemicals also modulate MAP kinase, phosphatidylinositide kinase, JAK/STAT, TLR, and other pro-inflammatory signaling molecules (Yahfoufi, Alsadi, Jambi, & Matar, 2018). The volatile oil of N. sativa seed exhibited a remarkable pain-relieving effect in acetic acid-induced writhing, formalin, and tail-flick tests (Zakaria, Jais, & Ishak, 2018). There was a significant increase in antiinflammatory activity and decrease in level of histamine release with improved tracheal responsiveness in animal models experimented with various extracts, oil, and another bioactive compound, α-hederin of N. sativa (Boskabady et al., 2011;Ikhsan et al., 2018;Keyhanmanesh et al., 2013;Saadat et al., 2015). It has been reported that 5-lipoxygenase and leukotriene C4 synthase in human blood cells were inhibited by N. sativa oil and thymoquinone (Houghton, Zarka, de las Heras, & Hoult, 1995). Both enzymes can produce inflammatory mediators called leukotrienes and prostaglandins (Houghton et al., 1995;Mansour & Tornhamre, 2004). In addition, N. sativa seed fixed oil decreased the serum IgG1, IgG2a, IL-2, IL-12, IL-10, IFN-γ, and inflammatory cells in lung tissue of murine model of allergic asthma (Abbas et al., 2005). Considering the antiinflammatory actions of N. sativa seed and its different extracts, these might be potentially used for the prevention as well as cure of SARS-CoV-2 viral infection.

| Oxidative stress
Oxidative stress is considered as a biomarker for various disease conditions, such as neurological disorder, cancer, aging, and endocrine illness (Lupoli, Vannocci, Longo, Niccolai, & Pastore, 2018). In addition, many viral infections, such as human immunodeficiency virus (HIV), respiratory syncytial virus (RSV), influenza A, hepatitis viruses, Epstein-Barr virus (EBV), and other viruses induce oxidative stress (Ivanov, Bartosch, & Isaguliants, 2017). Oxidative stress in chronic viral hepatitis triggers liver fibrosis, cirrhosis, cancer, and metabolic dysfunction (Ivanov et al., 2017). In acute respiratory viral infections, oxidative stress increases lung tissue injury and epithelial barrier dysfunction and further it plays an important role in secondary infections (Ivanov et al., 2017). For example, in the presence of inflammatory signals, the influenza viruses damage lungs and airways. The virus activates monocytes and polymorphonuclear leukocytes to produce reactive oxygen species (ROS), a mediator of oxidative stress (Jacoby & Choi, 1994). The produced ROS then contributes to the pathogenesis of influenza virus infection (Peterhans, Grob, Burge, & Zanoni, 1987).
Many viral infections elicit "cytokine storm," which is responsible for activation of endothelial cells in lung capillary, infiltration of neutrophil, and increased oxidation. Some immune cells notably macrophages and neutrophils produce a lot of ROS (Loffredo et al., 2019;Perrone, Belser, Wadford, Katz, & Tumpey, 2013). Increased oxidative stress is responsible for pulmonary injuries, such as ALI and ARDS (Hecker, 2018;Yan et al., 2019). Among the pandemic viruses, coronaviruses and influenza viruses cause lethal lung injuries and death from ARDS which is a vital reason for fatality (Chen, Liu, et al., 2020;Chen, Zhou, et al., 2020;Fowler Iii et al., 2017;D. Wang et al., 2020).
Undoubtedly, elevated oxidative stress is an indication of ARDS that generates cellular injury, organ failure, and death. ARDS may lead to severe hypoxemia which cooperates with frequent uncontrolled inflammation, oxidative injury, and damage to the alveolar-capillary barrier (Meng, Zhao, & Zhang, 2019). According to recent clinical reports, the therapeutic time for COVID-19-infected patients is longer than 14 days. In addition, out of 29 patients with COVID-19 pneumonia, 27 (93%) showed increased CRP, a marker of inflammation and oxidative stress (Chen, Liu, et al., 2020;Chen, Zhou, et al., 2020). An early clinical study in Wuhan reported that 63 out of 73 patients (86.3%) with COVID-19 had a remarkable CRP level (Chen, Liu, et al., 2020;Chen, Zhou, et al., 2020). The elevated level of CRP was likely due to acute inflammatory response and subsequent cytokine storms during COVID-19 pathogenesis (Huang et al., 2020). hyperglycemia was reported in 51% of cases (Chen, Liu, et al., 2020;Chen, Zhou, et al., 2020). However, we should not overlook hyperglycemia, as it can lead to patient immune suppression and any further complications (Butler, Btaiche, & Alaniz, 2005).  induced platelet aggregation and blood coagulation (Enomoto et al., 2001). Besides, dichloromethane extract of N. sativa can partly contribute to antihypertensive activity by increasing discharge of chloride, sodium, potassium, and urea followed by diuresis (Zaoui et al., 2000). Black cumin seed oil resulted in lower levels of harmful LDL-cholesterol, and higher levels of beneficial HDL cholesterol in animal model indicating a protective role against atherosclerosis (Al-Naqeep, Al-Zubairi, Ismail, Amom, & Esa, 2011;Nader, El-Agamy, & Suddek, 2010). A reduction in serum cholesterol, triglyceride and glucose levels, and leucocyte and platelet counts, and a rise in Hb and PCV levels compared to control were observed after administrating N. sativa orally, which indicates that N. sativa oil has beneficial effects on hyperglycemia, hyperlipidemia, and certain types of anemia (Zaoui et al., 2002).
N. sativa might have beneficial roles to control the emerging fetal incidence of PIMS or Kawasaki-like disease associated with COVID-19 by modulating immune response. N. sativa extracts and/or its bioactive compounds such as thymoquinone, nigellone, and α-hederin exhibits antihistaminic, antieosinophilic, antileukotrienes, antiimmunoglobulin and reduced-proinflammatory cytokines (IL-2, IL-4, IL-5, IL-6, IL-12, and IL-13) in in vitro/in vivo models (A. Koshak, Koshak, & Heinrich, 2017). In addition, thymoquinone-rich extract produced potent favorable immunomodulation in asthma inflammation by suppressing IL-2, IL-6, and PGE2 in T-lymphocytes as well as IL-6 and PGE2 in monocytes (Koshak, Yousif, Fiebich, Koshak, & Heinrich, 2018). Though the exact mechanism of action in Kawasakilike disease is unclear, there has been a clear indication that an increase in circulating inflammatory molecules is seen during this incidence with an event of myocarditis (Belhadjer et al., 2020;Toubiana et al., 2020). Antihypertensive agents as well as antiinflammatory action of N. sativa along with its different extracts have proven beneficial effects for both prevention and cure of this condition (Abbas et al., 2005;Ojha et al., 2015). In addition, cytokine storm might affect patients with Kawasaki-like disease (Henderson et al., 2020;W. Wang, Gong, Zhu, Fu, & Zhang, 2015) and thymoquinone can prevent this incidence by its antiinflammatory and antioxidative activities (L.-P. Guo, Fan, et al., 2020;T. Guo, Liu, et al., 2020).

| Bacterial co-infection in COVID-19
Some studies have recently shown that many COVID-19 patients are developing secondary bacterial coinfections such as bacterial pneumonia and sepsis, which are considered a serious threat to severe  (Zhou, Yang, et al., 2020;Zhou, Yu, et al., 2020). Staphylococcus aureus is the main pathogen of secondary infections in influenza but Streptococcus pneumoniae and Haemophilus influenzae are also common (Low, 2008). Although, data are not available yet which pathogens are associated with secondary infections in COVID-19, the most commonly detected co-pathogens detected in a study of 30 COVID-19 patients in Qingdao, China were influenza A (60%) and influenza B (53%), followed by Mycoplasma pneumoniae (23%) and Legionella pneumophila (20%) (Xing et al., 2020). Another study found that about 50% of patients died because of viral infection, while the other 50% were caused by secondary infections (Ruan et al., 2020). In the case of A range of antibiotics can be used in secondary bacterial infection. Azithromycin has been found to prevent secondary bacterial complications in post-Zika and -Ebola virus infections (Madrid et al., 2015;Retallack et al., 2016). Moreover, in case of other respiratory viral infections, Azithromycin reveals to reduce the severity of bacterial co-infections (Bacharier et al., 2015). More than 20 years, bacteria-induced Whipple's diseases were treated with hydroxychloroquine (Lagier & Raoult, 2018). Some studies provided insightful information on the antiviral effects of chloroquine against SARS coronavirus (Keyaerts, Vijgen, Maes, Neyts, & Van Ranst, 2004;Rolain, Colson, & Raoult, 2007;Savarino, Di Trani, Donatelli, Cauda, & Cassone, 2006). The synergistic effect of hydroxychloroquine and azithromycin works against SARS-CoV-2 and prevent bacterial superinfections. In accordance with the report, COVID-19 patients treated with the combination of hydroxychloroquine and azithromycin were improved compared with patients treated with hydroxychloroquine only, and control group (Gautret et al., 2020). However, no effective drugs have been offered to treat COVID-19 patients yet.
A wide range of Gram-positive and Gram-negative bacteria was suppressed by thymoquinone, a molecule obtained from N. sativa seeds (Abdallah, 2017). N. sativa showed a significantly higher zone of inhibitions in various bacteria (Hasan, Nawahwi, & Malek, 2013;Maryam, Fatimah, Ebtesam, Abdulrahman, & Ineta, 2016). N. sativa essential oil and its bioactive compounds (thymoquinone, carvacrol, and pcymene) were found resistance modifiers against S. aureus and inhibited bacterial biofilm formation (Mouwakeh et al., 2018). A noticeable bactericidal activity of thymoquinone was observed in Gram-positive cocci associated with minimum inhibitory concentration (MIC) ranging from 8 to 32 μg/ml. In addition, the molecule revealed MIC at 22 μg/ml for S. aureus and 60 μg/ml for S. epidermidis (Chaieb et al., 2011). All of these can become a scientific basis for the investigation of potential uses of N. sativa seed as curative options for COVID-19 patients coinfected with bacteria.
Black seed oil was shown to increase the number and action of CD4+ T cells and augmented levels of IFN-γ thus overpowering on viral load in mice infected with cytomegalovirus (Forouzanfar, Bazzaz, & Hosseinzadeh, 2014). After administration of N. sativa oil, hepatitis C virus (HCV)-infected patients improved health conditions such as total protein, red blood cell, and platelet count, decreased fasting blood glucose, and postprandial glucose in both diabetic and non-diabetic HCV individuals (Barakat et al., 2013). Treatment with N. sativa for half a year was found to recover a 46-year-old HIV patient (Onifade et al., 2013). Furthermore, treating with a mixture of N. sativa and honey, a mid-aged woman with HIV positive got rid of HIV and the number of CD4+ T cells was good enough to declare that she is recovered from HIV infection (Onifade et al., 2013). Altogether, it has been strongly suggested that black cumin seed could be a potential natural product to treat several incurable infectious diseases such as HIV but the efficiency of black cumin is yet to be determined.
In HIV-infected patients, ROS may activate viral replication while oxidants are known to contribute to the loss of CD4 T cells by apoptosis. But antioxidants can suppress the apoptosis, which indicates a relation between antiviral and antioxidant actions (Peterhans, 1997).
In a study with hepatitis C virus (HCV)-infected individual surprisingly showed a decrease in viral load and an augmented total antioxidant activity, total protein and albumin, improved RBC and platelet counts when supplemented with black seed oil (Barakat et al., 2013). To investigate the antiviral effect of black seed oil, MCMV (murine cytomegalovirus) has been used as a model in a study where it was concluded that N. sativa oil can produce antiviral effect against MCMV infection by increasing level of serum of IFN-γ, number of CD4+ helper T-cells, suppressor function, and numbers of macrophages (Salem & Hossain, 2000b). The viral infection is known to be controlled by several components of immune system including natural killer cell (NK cells), and specific cells including CD4 and CD8 T-cells (Salem & Hossain, 2000a) and it is proved that N. sativa oil can induce antiviral cellular response by rising CD4 cells (Salem & Hossain, 2000b). The antiviral effects of thymoquinone against avian influenza virus (H9N2) and murine cytomegalovirus infection models were confirmed. N. sativa seed fixed oil had stimulatory effects on CD4+ Tlymphocytes in murine BALB/c cytomegalovirus model (Salem & Hossain, 2000b;Umar et al., 2016).
Coronavirus spike protein can utilize angiotensin converting enzyme 2 (ACE2) like a receptor for entering into cells (Kuhn, Li, Choe, & Farzan, 2004). The α-hederin gives better energy score when compared to chloroquine, hydroxychloroquine, and favipiravir. Preventive potentials of N. sativa seed oil against COVID is yet to be revealed. The molecular docking system discovered that N. sativa may inhibit COVID-19 through its main compound nigellidine and α-hederin. So further insights into in vivo and in vitro experiments with those active compounds are required (Salim & Noureddine, 2020).  Thymoquinone shows a set of therapeutic benefits with its antioxidant, antiinflammatory, antimicrobial, and anticonvulsant activities.
Another component, α-hederin also has come into view as novel therapeutic potential against viral diseases. Hopefully, inhibition of COVID-19 by these two compounds (α-hederin and nigellidine) of black cumin was reported in a molecular docking study, but further studies are warranted to evaluate the efficiency and explore the specific cellular and molecular mechanisms of their antiviral effects alone or in combination with other drugs.
F I G U R E 1 Mechanisms involved in pharmacological effects of N. sativa on COVID-19. Black cumin seed or its active compounds activate immune cells, initiate antigen presentation system, and make balance between Th1 and Th2 cytokines. The bioactive compounds of black cumin seed increase conversion of LC3II to LC3III, a marker of autophagy activation. Besides, black cumin decreases inflammation, oxidative stress (MDA) and increases anti-inflammation, ant-histamine responses, SOD, and GPx. Comorbodities increase the severity of SARS-CoV-2 infection. Black cumin may also be beneficial through improving comorbodity situation in SARS-CoV-2-infected patients. CVD, cardiovascular disease; KLD, Kawasaki-like diseases; PIMS, pediatric inflammatory multisystemic syndrome [Colour figure can be viewed at wileyonlinelibrary.com]

| CONCLUDING REMARKS
Where the whole world is hoping for an effective drug or vaccine to combat COVID-19, we have tried to draw our attention to the pharmacological potentials of black cumin seed and its bioactive compounds for further scientific concern to adopt an alternative strategic plan. Black cumin seed and most its bioactive components already have a proven history of boosting up immune systems and can be easily accessible for all classes of people along with self-awareness and -protection (Hannan, Islam, & Uddin, 2020) against COVID-19. This review can offer some valuable information for the bonafide public domain or organization, where firstly, N. sativa seed and oil can be considered as a first-aid kit as a preventive measure against COVID-19; and secondly, the bioactive compounds, thymoquinone, α-hederin, or nigellidine could be tested preclinically and clinically for drug development, efficacy, and potency under a specific pathophysiological condition in pursuit to control the deadliest pandemic. Md. Jamal Uddin https://orcid.org/0000-0003-2911-3255