Dietary phytochemicals against COVID‐19: A focus on thymoquinone

It has been three years since the outbreak of the Coronavirus disease 2019 (COVID-19) at the end of 2019 in Wuhan, China, which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). So far, over six million people around the world have died of COVID-19 (Murakami et al., 2023). With huge efforts from both academic and industrial organizations worldwide, the development of various types of vaccines and several new drugs has been made and the situation has been significantly improved to fight against the virus (Murakami et al., 2023). Regarding new medicines or treatments approved by Food and Drug Administration, USA, so far, they include anti-inflammatory agents (e.g., dexamethasone and Baricitinib), anti-SARS-CoV-2 (neutralizing) monoclonal antibody (mAbs), antivirals (Remdesivir, Paxlovid, and Molnupiravir), and antithrombotics. The mAbs can bind to the viral spike proteins, and block the virus attachment to host cell surface proteins, therefore reducing the viral load. Remdesivir is an inhibitor of RNA-dependent RNA-polymerase (RdRp), which is essential to virus genome replication. Paxlovid is a combination of Nirmatrelvir and Ritonavir, which acts as an oral SARS-CoV-2 Mpro (or 3CL) protease inhibitor to stop viral replication and a strong cytochrome P450 3A4 inhibitor as a booster of the pharmacokinetics of Nirmatrelvir, respectively (Murakami et al., 2023). However, new mutant variants of coronaviruses are emerging, which could cause the loss of the efficacy of the approved vaccines, mAbs, and small molecule antiviral drugs, exposing the elderly and those with chronic conditions, such as diabetes and cancers. Moreover, many drugs such as mAbs and Paxlovid are expensive and not easily available in developing countries. Therefore, novel vaccines, effective therapies, and affordable management, such as phytochemicals, are highly desirable. Plants, including food and traditional herbs, have benefited human beings over thousands of years. To fight coronavirus, herbal medicines have been explored to treat COVID patients across the world. Many nutrients in food play important roles by boosting the immune system and/or reducing viral respiratory tract infections (Omer et al., 2022). For example, Zinc as an antioxidant mineral (Dhawan et al., 2022), and vitamin D in food (Wimalawansa, 2022) can exhibit immunomodulatory and anti-inflammatory properties. Honey, rich in nutrients and bioactive compounds, is recommended by National Health Service, England to ease a cough in COVID-19 patients (www.nhs.uk). Numerous phytochemicals such as flavonoids including quercetin (e.g., apples, onions, parsley, and sage), kaempferol (spinach and kale), isoliquiritigenin (soybean and shallots), tangeretin (tangerine), and baicalein (Scutellaria baicalensis, Huang-qin) (Song et al., 2021) (Figure 1) are shown to be inhibitors of SARS-CoV-2 Mpro with a similar mechanism as Nirmatrelvir in Paxlovid (Mandal et al., 2021). Other phenolics such as epigallocatechin gallate (green tea) and hesperidin (citrus fruits) (Figure 1) could inhibit RdRp activity in a similar way as Remdesivir (Bahun et al., 2022; Omer et al., 2022). Thymoquinone (TQ, Figure 1) is the major bioactive compound in N. sativa, black cumin seed (Johnson-Ajinwo & Li, 2014). It shows antiviral, antimalarial, anticancer (Johnson-Ajinwo et al., 2018), anti-inflammatory, and immunomodulatory (Ahmad et al., 2020; Khazdair et al., 2021) activities. Various reviews relating to the roles of TQ and N. sativa in COVID-19 have been published (Abdelrahim et al., 2022; Ahmad et al., 2020; He et al., 2022; Islam et al., 2021; Khazdair et al., 2021; Kulyar et al., 2021; Taysi et al., 2022). Here, this update is focused on its direct applications in targeting the SARS-CoV-2 virus in silico, in vitro, in vivo, and in clinical trials. Docking studies suggested TQ can bind human angiotensin-converting enzyme 2 (ACE2) as the receptor of SARS-CoV-2 spike proteins (Jakhmola Mani et al., 2022; Srivastava et al., 2021; Xu et al., 2021) and a sheddase ADAM7 (a membrane-bound metalloprotease) associated with the modulation of ACE2 (He et al., 2022), thus blocking the virus entry to human cells. Further studies show that TQ binds to SARS-CoV-2 Mpro via interaction with the active site (His41 and Cys145) and the Glu166 (Abdallah et al., 2022; Khan et al., 2024; Ullah et al., 2022). TQ as an unsubstituted quinone has inherent reactivity to free cysteine via thiol addition reaction (Li et al., 2005; Lu et al., 2010). TQ can covalently bind to the free cysteines of human hemoglobin (unpublished). Thus, it is highly likely that TQ can also covalently bind to the free catalytic cysteine (Cys145) residue of the protease as Nirmatrelvir does. TQ was found to inhibit the replication of SARS-CoV-2 and SARS-CoV after infecting HEK293-ACE2 cells, with IC50 of 5.0 and 7.6 μM, respectively (Xu et al., 2021). TQ shows relatively high cytotoxicity in in vitro assessment of SARS-CoV-2 (hCoV-19/Egypt/NRC-03/2020) (Abdallah et al., 2022). TQ inhibits four further SARS-CoV-2 variants (Omicron, 614D, Delta, UK, Brazil) in infection of HEK293-ACE2 cells with an IC50 value range between 1 and 3 µM (Bencheqroun et al., 2022). Compared with IC50 of Nirmatrelvir at the nanomolar range (Rosales et al., 2022), these values seem to be much greater. Future structure modification of TQ (Johnson-Ajinwo et al., 2018) or its use in combination with other approved antiviral drugs could enhance its activity. TQ showed protective effects against acute kidney damage (AKI) in septic BALB/c mice caused by cecal ligation and puncture (Guo et al., 2020). However, there are no reported studies of the effect of TQ or N. sativa oils/extracts on the SARS-CoV-2 -infected animal models. A phase I clinical trial study showed that TQ-rich black cumin oil (containing 5% of TQ) was safe at 200 mg/adult/day (Thomas et al., 2022). A phase II clinical trial of oral N. sativa oil in adult patients with mild COVID-19 was carried out in Saudi Arabia (clinicaltrials.gov, Identifier: NCT04401202 on) (Koshak et al., 2021). A total of 173 patients, with an average age of 36 ± 11 years, were randomly divided into a treatment group (86) and a control group (87). The treatment group was treated with two N. sativa oil (NSO, 500 mg) capsules per day for 10 days, whilst the control group received standard treatment (Koshak et al., 2020). The percentages of recovered patients in the treatment and control groups were 62% (mean duration of recovery, 10.7 ± 3.2 days) and 36% (12.3 ± 2.8 days), respectively. These results indicated that NSO treatment could cause faster recovery of symptoms than usual care alone. A clinical trial was carried out using honey and N. sativa seeds (HNS) for the treatment of COVID-19 patients with moderate or severe conditions, from April to July 2020 in Pakistan (ClinicalTrials.gov; Identifier: NCT04347382). Among the moderate cases, 210 patients were randomly assigned to either receive a combination of honey (1 g/kg/day), N. sativa seeds (80 mg/kg/day), and standard care, or a placebo along with standard care. Among the severe cases, 50 were given HNS and 53 the placebo. HNS caused ~50% decrease in time taken to improve symptoms as compared with the placebo (moderate cases, 4 vs. 7 days; Hazard Ratio [HR], 6.1; p < 0.0001 and for severe cases, 6 vs. 13 days; HR, 4.0; p < 0.0001). HNS also cleared the virus earlier than the placebo in both moderate cases (6 vs. 10 days; HR, 5.5; p < 0.0001) and severe cases (8.5 vs. 12 days; HR, 4.3; p < 0.0001). In severe cases, the mortality rate was less than 25% in the HNS group than in the placebo group (4% vs. 18.9%; odds ratio (OR), 0.18; p = 0.03). HNS significantly improved symptoms, sped up viral load clearance, and reduced mortality in COVID-19 patients. A clinical trial was carried out to evaluate the efficacy and safety of a Persian herbal medicine formula (extract of the Glycyrrhiza glabra, Punica granatum, and Rheum palmatum) along with N. sativa powder among patients with COVID-19 from June to September 2020 in Iran (Registration: Iranian Registry of Clinical Trials [www.irct.ir]: IRCT20200330046899N1). In total, 79 cases (mean age, 59.1 ± 17.1 years) completed the trial. The combined treatment significantly improved the symptoms and decreased the mean hospitalization days by 3 ± 0.4 days (Setayesh et al., 2022). A clinical trial was carried out to study the effects of N. sativa and/or vitamin D3 on COVID-19 and the alleviation of symptoms (ClinicalTrials.gov, Identifier: NCT04981743). In total, 120 COVID-19 patients with mild to moderate symptoms were recruited and randomly assigned to four groups with 30 patients each. Groups 1–4 received an oral dose of 900 mg N. sativa twice daily for 14 days, 2000 IU of vitamin D3, 900 mg of N. sativa and 2000 IU of vitamin D3, and none of these two agents, respectively. All groups received standard therapy at the same time. The combination of N. sativa–vitamin D3 besides the standard therapy significantly alleviated most COVID-19 symptoms. Virus clearing test by quantitative polymerase chain reaction on the 7th and 14th day of treatment indicated that the N. sativa and vitamin D3 combination is the best among the four treatments, where the values of the OR on the 7th and 14th day were 0.13 and 0.09, respectively (Said et al., 2022). A randomized, double-blind, placebo-controlled phase II trial from May to September of 2021 in the USA (ClinicalTrials.gov, Identifier: NCT04914377) was conducted to assess the safety and efficacy of a patent thymoquinone formula (TQF) derived from N. sativa oil containing 1.7% of TQ. A total of 55 symptomatic SARS-CoV-2 adult patients (>18 years) were randomly assigned to receive oral TQF (29) or a placebo (26). The results showed that the median time-to-sustained-clinical-response (SCR) was 6 days in the TQF treatment versus 8 days in the placebo group (p = 0.77). However, while the SCR difference is not significant, the trend towards the benefit of using TQF is encouraging. There was no significant difference in the rate of adverse events (p = 0.16), indicating its safety, which is consistent with a previous Phase 1 study (Thomas et al., 2022). TQF significantly improved the total symptom burden faster (p < 0.001), and increased cytotoxic CD4+ (p = 0.04) and helper CD8+ (p = 0.04) central memory T lymphocytes (Bencheqroun et al., 2022). Four phase II clinical studies across the world using N. sativa alone or in combination with other herbs or honey or vitamin D3 have been conducted. All these studies indicated some benefits of treating COVID-19 patients with N. sativa. However, the quality of these studies varied and the numbers of patients recruited were generally small. The content of the bioactive compound-TQ might vary a lot across these studies, so the clinical performance of the N. sativa could be different as observed. Also, only single doses of these products were used. It would be good to carry out future clinical trials using standardized products by quantifying TQ content in the N. sativa extract or oils (Bencheqroun et al., 2022; Johnson-Ajinwo & Li, 2014) or even use the pure TQ formulation at different doses with a larger number of patients in a randomized, double-blind, and placebo-controlled manner (Bencheqroun et al., 2022). Considering the benefits of honey in managing COVID-19, the preparation of a combination of honey with N. sativa oil with a determined quantity of TQ is also recommended for future clinical investigation against COVID-19 or other diseases. Phytochemical constituents in foods, fruits, herbs, and spices may provide preventive and even adjunct therapeutic effects on COVID-19 patients via multiple mechanisms. The widely available and cheap N. sativa and its bioactive compound-TQ could be further explored for their application in fighting COVID-19.