Nigella sativa L. and COVID-19: A Glance at The Anti-COVID-19 Chemical Constituents, Clinical Trials, Inventions, and Patent Literature

COVID-19 has had an impact on human quality of life and economics. Scientists have been identifying remedies for its prevention and treatment from all possible sources, including plants. Nigella sativa L. (NS) is an important medicinal plant of Islamic value. This review highlights the anti-COVID-19 potential, clinical trials, inventions, and patent literature related to NS and its major chemical constituents, like thymoquinone. The literature was collected from different databases, including Pubmed, Espacenet, and Patentscope. The literature supports the efficacy of NS, NS oil (NSO), and its chemical constituents against COVID-19. The clinical data imply that NS and NSO can prevent and treat COVID-19 patients with a faster recovery rate. Several inventions comprising NS and NSO have been claimed in patent applications to prevent/treat COVID-19. The patent literature cites NS as an immunomodulator, antioxidant, anti-inflammatory, a source of anti-SARS-CoV-2 compounds, and a plant having protective effects on the lungs. The available facts indicate that NS, NSO, and its various compositions have all the attributes to be used as a promising remedy to prevent, manage, and treat COVID-19 among high-risk people as well as for the therapy of COVID-19 patients of all age groups as a monotherapy or a combination therapy. Many compositions of NS in combination with countless medicinal herbs and medicines are still unexplored. Accordingly, the authors foresee a bright scope in developing NS-based anti-COVID-19 composition for clinical use in the future.


Introduction
COVID-19, a pandemic transmissible sickness, is triggered by SARS-CoV-2. As of 1 April 2022, this disease has infected >488 million individuals, led to about 6.16 million mortalities, and affected the quality of life and worldwide economy since its inception in 2019 [1]. COVID-19 cases have been documented around the globe, wherein the United States, India, Brazil, France, and Germany have reported the majority of cases [1]. The clinical features of COVID-19 are similar to normal flu (cough, sore throat, high fever, headache, sneezing, myalgia, breathlessness, etc.), and it mainly targets the respiratory system, leading to acute respiratory distress syndrome. If untreated on time, COVID-19 may be fatal, and the patient may die due to pneumonia, respiratory failure, and damage to other vital organs due to cytokine storm [2]. Some vaccines (Janssen, Moderna, and Pfizer-BioNTech), new drugs (remdesivir, molnupiravir, favipiravir, and a combination of nirmatrelvir with ritonavir), and supportive therapy (a combination of paracetamol, vitamin C, and zinc) have been established to prevent/treat COVID19 [2][3][4]. The drugs used to prevent/treat COVID-19 act on different enzymes/proteins of SARS-CoV-2, including RNA-dependent-RNA-polymerase, helicase, main protease, non-structural proteins (Nsp9 and Nsp15), and RNA replicase [2][3][4]. Many new drugs and their combinations are also in clinical trials [3,4]. Despite the development of many vaccines and some drugs, the battle against COVID-19 is far from over. The identification and the spread of new contagious SARS-CoV-2 strains (α, β, γ, δ, Omicron, etc.) also pose challenges to the existing vaccines and treatment of COVID-19 [5]. Accordingly, the development of alternative and supporting therapies has been perceived as a promising way to combat COVID-19 [6][7][8][9][10]. Many studies have demonstrated the effectiveness of health supplements (zinc, vitamin C, vitamin D, etc.) and herbal products (black seed, honey, ginger, garlic, etc.) to prevent/treat COVID-19 [11,12]. This review highlights the anti-COVID-19 potential of Nigella sativa L. (NS) and its major chemical constituents.

Materials and Methods
The non-patent literature search for the titled review was performed on the PubMed database and Sci-finder database, utilizing keywords COVID-19 or SARS-CoV-2 in combination with different keywords related to N. sativa (Black cumin, Black seed, and Kalonji). The relevant non-patent literature citing the prophylactic/therapeutic use of N. sativa against COVID-19 was reviewed and cited in this review. The patent searching was performed utilizing different patent databases (Sci-Finder, Espacenet, Patentscope, and USPTO). The patents/patent applications were segregated according to their patent family, and duplicate patent families were removed. The patent applications that exclusively provide clear information about the use of N. sativa or N. sativa-based inventions to treat COVID-19 have been included in this review. The patent searching results are provided as Supplementary Material (Table S1).

Introduction
Phytotherapy is one of the alternative approaches explored by the scientific community to discover and develop preventive and/or therapeutic regimens against the menace of SAR-CoV-2. Amongst thousands of ethnomedicinal plants which are known to be effective against infectious diseases and alleviate acute respiratory symptoms, N. sativa and its phytoconstituents are one of the extensively studied natural substances.
N. sativa belonging to the family Ranunculaceae is a nutrient-rich, annual flowering medicinal plant of significant historical and religious importance. It is usually recognized as black seed or black cumin in English, Habbatul barakah in Arabic, and Kalonji in Hindi and Urdu [13]. Its nutritional value and a wide array of therapeutic properties have been known to mankind since ancient times. Muslims consider black seed as a panacea, i.e., a remedy that can cure all diseases except death, while traditional medicine practitioners believed it to be 'the herb from heaven' [14]. Its beneficial uses are also mentioned by the 10th-century physician Avicenna in his book 'The Canon of medicine' [15]. Because of its health benefits and a broad spectrum of traditional medicinal uses, this nutraceutical herb has been described in the literature as a miraculous herb [16], prophetic medicine [17], habbat al barakah [18], a holy herb of the Middle East [19], a mine for multi cure [20], a novel beauty care ingredient [21], a complementary drug [22], a universal healer [23], a marvel herb [24], a black seed of miracle [25], blessed seed [26], and a wonder herb [27].
Although all parts of the black seed herbaceous plant are of biological and economic importance, NS seeds and their oil (NSO) are most commonly consumed for their health benefits and to treat diseases in various indigenous systems of medicines [13]. NS seeds, owing to their peculiar aroma, fragrance, and nutritional value, are also used as food additives. The nutritional value of black seed is due to its rich content of vegetable proteins and amino acids, fats, fibers, carbohydrates, minerals, and vitamins. In traditional and indigenous medicine, different dosage forms of black seeds have been employed to manage multiple acute and chronic sicknesses, including respiratory diseases (asthma, bronchitis), pain and inflammation, skin allergy, hypertension, diabetes, liver disorders, rheumatism, mental disorder, malaria, loss of hunger, insomnia, snake bite, indigestion, and dysmenorrhea [28,29]. Numerous preclinical and clinical studies have explored the health-promoting potential of this traditional medicinal plant and provided evidence in favor of its antioxidant, antidiabetic, cardioprotective, neuroprotective, analgesic and anti-inflammatory, antimicrobial, anthelmintic, anti-oxytocic, anti-obesity, anti-cancer, antiasthmatic, hepatoprotective, gastroprotective, anti-schistosomiasis, immunomodulatory, nephroprotective, and wound healing activities. Traditional uses, nutritional properties, and biological activities of NS have been thoroughly reviewed [13,15,[28][29][30][31].

Important Biologically Active Chemical Constituents of NS
The pharmacological activities of NS, in general, are credited to the existence of volatile components, especially TQ, but other secondary metabolites (alkaloids, pentacyclic triterpenes, phytosterols, flavonoids), including those present in NSO, have also been shown to exhibit therapeutic activities [34][35][36][37][38] (Figure 1). The chemical structure of selected important bioactive components of NS is presented in Figure 2. TQ (2-methyl-5-isopropyl-1, 4-benzoquinone; Figure 2) is a monoterpenoid and one of the key volatile components (30%-48%) of the NS volatile oil which exhibits an array of useful bioactivities [39]. It shows anti-inflammatory action by decreasing the appearance of pro-inflammatory cytokines (the COX-2, TNF-α, IL-6) and by upregulating the levels of IL-4 and IL-10, the anti-inflammatory cytokines [40]. Recently, Boskabady et al. demonstrated that TQ is effective in ameliorating lipopolysaccharide (LPS) generated lung inflammation in rats [41]. The immunomodulatory influences of TQ are mediated through activation of T cells, the appearance of CD62L and CD8+ T cells, and by helping in the formation of IFN-γ [42]. It is an effective chemotherapeutic agent against breast, bladder, cervical, colorectal, gastric, lung, prostate, pancreatic cancers, and leukemia in vitro and in vivo studies. It shows anticancer activity by producing apoptosis, autophagia, cell cycle arrest, controlling angiogenesis, and regulating signaling pathways [35,43,44]. Thymohydroquinone (THQ; Figure 2) exhibits stronger antioxidant activity and protects cell damage better than TQ [45]. Some other activities shown by TQ include neuroprotective, cardioprotective, antimicrobial, gastroprotective, antiarthritic, and antiasthmatic activities [39]. Nigellone has been reported to inhibit trachea contraction along with improved mucociliary clearance and thus is useful in the treatment of bronchitis and asthma [46]. Alpha-Hederin, a water-soluble pentacyclic triterpene of NS, exhibits anticancer activity by inducing apoptosis, anti-inflammatory, and bronchodilatory activity by raising the levels of cAMP that leads to elevated β2-adrenergic responsiveness [47,48,49].

Anti-COVID-19 Chemical Constituents of NS
Several research publications reviewed the therapeutic activities of NS and TQ viz., antiviral, anti-inflammatory, immunomodulatory, antioxidant, and broncho-dilatory activities and suggested their possible role in combating COVID-19 [50,51,52,53,54]. A TQ (2-methyl-5-isopropyl-1, 4-benzoquinone; Figure 2) is a monoterpenoid and one of the key volatile components (30%-48%) of the NS volatile oil which exhibits an array of useful bioactivities [39]. It shows anti-inflammatory action by decreasing the appearance of pro-inflammatory cytokines (the COX-2, TNF-α, IL-6) and by upregulating the levels of IL-4 and IL-10, the anti-inflammatory cytokines [40]. Recently, Boskabady et al. demonstrated that TQ is effective in ameliorating lipopolysaccharide (LPS) generated lung inflammation in rats [41]. The immunomodulatory influences of TQ are mediated through activation of T cells, the appearance of CD62L and CD8+ T cells, and by helping in the formation of IFNγ [42]. It is an effective chemotherapeutic agent against breast, bladder, cervical, colorectal, gastric, lung, prostate, pancreatic cancers, and leukemia in vitro and in vivo studies. It shows anticancer activity by producing apoptosis, autophagia, cell cycle arrest, controlling angiogenesis, and regulating signaling pathways [35,43,44]. Thymohydroquinone (THQ; Figure 2) exhibits stronger antioxidant activity and protects cell damage better than TQ [45]. Some other activities shown by TQ include neuroprotective, cardioprotective, antimicrobial, gastroprotective, antiarthritic, and anti-asthmatic activities [39]. Nigellone has been reported to inhibit trachea contraction along with improved mucociliary clearance and thus is useful in the treatment of bronchitis and asthma [46]. Alpha-Hederin, a watersoluble pentacyclic triterpene of NS, exhibits anticancer activity by inducing apoptosis, anti-inflammatory, and bronchodilatory activity by raising the levels of cAMP that leads to elevated β 2 -adrenergic responsiveness [47][48][49].

Anti-COVID-19 Chemical Constituents of NS
Several research publications reviewed the therapeutic activities of NS and TQ viz., antiviral, anti-inflammatory, immunomodulatory, antioxidant, and broncho-dilatory activities and suggested their possible role in combating COVID-19 [50][51][52][53][54]. A summary of the in silico, in vitro, and in vivo investigations done on bioactive phytoconstituents of NS since the break out of the COVID-19 pandemic is presented in Table 1. Data presented in Table 1 indicate that four in silico studies identified α-Hederin as the promising anti-COVID agent while DTQ in one in vitro and three in silico studies emerged as the lead anti-viral candidate. An in vivo study performed in experimental rats similarly exhibited the anti-SARS-CoV-2 efficacy of nigellidine [55]. Surprisingly, TQ was identified as a lead compound in only one in silico and one in vitro study. These phytochemicals have been shown to act at different molecular targets of SARS-CoV-2 (RdRp, 3CL pro , Nsp15, Nsp2, Nsp3, M pro , and ACE2) ( Figure 3).

Anti-COVID-19 Clinical Trials on NS and Its Chemical Constituents
A search for the clinical studies related to NS was conducted on the clinical trial database [67] utilizing the terms "N. sativa or its synonyms" and the name of its different important chemical constituents (TQ, THQ, DTQ or nigellone, nigellimine, nigellicine, nigellidine, nigellicimine, and nigellicimine N-oxide) on 20 February 2022. This search provided eight clinical studies belonging to NS and TQ to treat/prevent COVID-19. Two CTs have been conducted in Saudi Arabia, two in Pakistan, two in Tunisia, and one each in the United States, and Egypt. Two CTs were related to the prevention of COVID-19, whereas six CTs involved treatment of COVID-19 utilizing NS. Most of the CTs have been conducted employing the capsule (500 mg) containing an effective amount of NS and/or NSO. The CTs have also been done using a combination of NS and honey or Omega 3. The data of this search are summarized in Table 2.

Patent Summary
A patent search was carried out on 20 February 2022, using patent databases as mentioned in the literature [68][69][70][71]. The summary of the finalized patents/patent application is provided in Table 3.  . This patent application provides a protocol for the clinical study of the claimed composition but is silent about its data [77]. A synergistic composition to treat/prevent COVID-19 comprising metformin, Green Tea extract (epigallocatechin-3-gallate), blueberry extract (pterostilebene), NS extract (TQ), and broccoli extract (sulforaphane). The in vivo activity data demonstrated that the claimed composition enhanced type 2 monocytes, IL-10 (anti-inflammatory protein), and HGF-1 (regenerative protein). It also reduced lung injury, and IL-17 (inflammatory protein). However, no specific data have been provided against SARS-CoV-2 or COVID-19 treatment [78]. A method of treating inflammation associated with neurological damage due to COVID-19 utilizing a composition comprising green tea extract (epigallocatechin-3-gallate), blueberry extract (pterostilbene), NS extract (TQ), and broccoli extract (sulforaphane) [80]. A method of preventing or treating SARS-CoV-2 infection using a composition containing oregano oil (1), thyme oil (2), NSO (3), sumac oil (4), sesame oil (5), olibanum oil (6). Different combinations of these oils were made as combination A (1+2+3), combination B (1+2+3+4), combination C (1+2+3+4+5), and combination D (1+2+3+4+5+6). The in vitro analysis of these combinations demonstrated that these combinations digested the S-1 and S-2 subunits of the spike protein of SARS-CoV-2 and attenuated it. The clinical trial in a patient showed that the combination-A relieved sore throat and cough in 24 and 40 h, respectively [81]. A homogenized powdered composition consisting of hydroxypropyl methylcellulose particles, at least one signaling agent (menthol, strawberry, mint, spearmint, peppermint, eucalyptus, lavender, and citrus), and optionally one or more biologically active agents like NS. This document does not furnish any rationale for using NS in the description part [85].

Conclusions
The supplementation of the NS possesses a variety of advantageous effects on human well-being. Several compositions comprising NS and NSO have been tested clinically and have also been claimed in patent applications to prevent/treat COVID-19. The clinical studies and patent filings support the therapeutic efficacy of NS against COVID-19 owing to its immunomodulatory, antioxidant, antiviral, and anti-inflammatory effects. NS can prevent and treat COVID-19 patients with a faster recovery rate. Many compositions of NS in combination with many medicinal herbs and medicines are still unexplored. However, further clinical trials are recommended to establish the anti-COVID-19 potential of the unexplored NS compositions.

Discussion and Perspective
NS is one of the Quranic and prophetic medicinal plants with beneficial effects on human health [87]. The literature and clinical studies have demonstrated its efficacy in preventing and treating COVID-19. The summary of eight CTs on NS seeds and NSO is mentioned in Table 1. The data of six CTs have not been published. However, the data of two CTs (NCT04401202 and NCT04347382) involving the use of NS against COVID-19 have been published [52,88].
One of the published clinical studies (NCT04401202) concluded that NSO supplementation provides faster recovery of 62% of the mild COVID-19 patients on day 14 of the treatment. The normal revival time was also briefer than the control group. This study suggested that the reduction in the COVID-19 symptoms (anosmia, chills, runny nose, and loss of appetite) might be owing to the anti-inflammatory properties of NS [52]. The infection of SARS-CoV-2 causes the cytokine storm because of the liberation of leukocytes and pro-inflammatory cytokines [89,90]. The discussion part of the published CTs [52] stated that NS demonstrates immunostimulant and anti-inflammatory effects by restricting the release of pro-inflammatory cytokines [54]. In addition, NS also displays antioxidant and antiviral effects [54]. Accordingly, drugs like NS can stop the cytokine storm and are useful to prevent/treat COVID-19.
The published results of another CT (NCT04347382) affirmed that the combination of honey and NSO improved the COVID-19 symptoms, viral clearance, and mortality among COVID-19 patients. This report also advocated the use of the combination of honey and NS along with other treatments of COVID-19 [88]. According to this study, the combination of honey and NS provided its anti-COVID-19 activity due to their antioxidative/antiviral/immunostimulant chemical constituents (phenolic compounds, flavonoids, and zinc) that attack the multiple sites (lowering the expression of ACE-2 receptor, RdRp, Mpro protease, etc.,) of SARS-CoV-2.
Many in silico, in vitro, and in vivo studies have demonstrated potential anti-COVID-19 activity of the main chemical constituents of NS due to their diverse mechanisms of action against SARS-CoV-2 (Table 1) (Figure 3). TQ is one of the main chemical constituents of NSO possessing antioxidant, anti-inflammatory, and antiviral activities [31,92]. It is disclosed that TQ may bind the hydrophobic component of SARS-CoV-2 because of its lipophilicity. This causes activation of the virus. TQ also reduces the generation of cytokine storm in COVID-19 patients because of its anti-inflammatory potential [93]. TQ has also demonstrated SARS-CoV-2 inhibitory activity and can be an efficacious adjuvant for the therapy of COVID-19 [94].
Many findings have also established the potential of NS seeds and TQ in reducing thrombus formation [95]. It is a well-known fact that thrombus formation causes multiple organ collapse and fatality among COVID-19 patients. Therefore, NS may be used as a therapeutic formulation, including its nano-formulations [96] to treat COVID-19, and may also be used as a supportive therapy with anti-COVID-19 medicines [4,6,97,98].
NS and its oil possess antiviral activity, cardioprotective activity, gastroprotective activity, neuroprotective activity, anticancer activity, antidiabetic activity, antioxidant activity, anti-obesity activity, immunomodulatory activity, antihistaminic activity, and antiinflammatory activity [99,100] (Figure 1). NS and NSO inhibit the activity of SARS-CoV-2 through the diverse mechanism of action ( Figure 3). All these facts, in combination with the data of the CTs based on NS, indicate the potential of NS for the management of COVID-19 in normal COVID-19 patients as well as among the high-risk population.
A patent application is filed for a novel and inventive research of industrial application [101]. Our search revealed several patent applications connected to the practice of different compositions of NS to prevent/treat COVID-19 ( Table 2). Most of these patents were published in 2021. Like non-patent literature, the patent literature also cites NS as an immunomodulator (activates natural killer cells, interferon, and dendritic cells), suppresses virus multiplication by its chemical constituents like TQ, protects lungs from pathology by blocking the activities of several inflammatory mediators that perform a major function in the COVID-19 pathology, several chemical constituents of NS, including TQ, nigellidine, and alpha-heridin have displayed anti-COVID-19 activity, the extracts of NS showed BCG like activity, and many NS compositions are in clinical trial against COVID-19 [73,75,77].
NS also has antihistaminic, antioxidant, antiallergic, and anti-asthmatic effects [74]. NS is also a source of zinc [77], which has also demonstrated activity against SARS-CoV-2 [102]. Many publications have also stated the usefulness of NS against chronic diseases like cancer, cardiovascular diseases, and diabetes, as mentioned above [78]. It is acceptably recognized that immunocompromised patients with little levels of natural killer cells, interferon, and dendritic cells are vulnerable to SARS-CoV-2 infection. COVID-19 patients suffer from cytokine storm owing to increased activity of the pro-inflammatory mediators in the body, and the malfunction of the lung among COVID-19 patients lead to severe consequences [73,75,77]. All these facts indicate that NS and its various compositions have all the attributes to be used as a promising remedy for the management of COVID-19 among high-risk individuals as well as to treat normal COVID-19 patients as a monotherapy or a combination therapy.