In vitro potential antiviral SARS-CoV-19- activity of natural product thymohydroquinone and dithymoquinone from Nigella sativa.

Inflammation, oxidation, and compromised immunity all increase the dangers of COVID-19, whereas many pharmaceutical protocols may lead to increased immunity such as ingesting from sources containing vitamin E and zinc. A global search for natural remedies to fight COVID-19 has emerged, to assist in the treatment of this infamous coronavirus. Nigella satvia is a world-renowned plant, an esteemed herbal remedy, which can be used as a liquid medicine to increase immunity while decreasing the dangers of acute respiratory distress syndrome. Thymoqinone (TQ), dithymoqinone (DTQ) and thymohydroquinone (THQ), are major compounds of the essential oil contained in N.sativa. A current study aims to discover the antiviral activity of two compounds, Thymohydroquinone and Dithymoquinone, which are synthesized through simple chemical procedures, deriving from thymoquinone, which happens to be a major compound of Nigella sativa. A half-maximal cytotoxic concentration, "CC50", was calculated by MTT assay for each individual drug, The sample showed anti-SARS-CoV-2 activity at non-cytotoxic nanomolar concentrations in vitro with a low selectivity index (CC50/IC50 = 31.74/23.15 = 1.4), whereby Dimthymoquinone shows high cytotoxicity.

Introduction

The severe acute respiratory syndrome, coronavirus-2 (SARS-CoV-2), known internationally as the cause of COVID-19, also known as the coronavirus, was declared a pandemic in March of 2020. The main goal, world-wide, has been to discovera drug withenough potential antiviral activity to face this pandemic and overcome it; a drug having limited side effects, unlike synthetic drugs which are known to have a variety of aftereffects. With that specific and lofty ambition, our team set outfor collective information regarding the potential of natural plants aspossible antivirals, and publishedsome papers in regards to the activity of medicinal plants which were previously used to treat SARS –CoV-19, due to their similarity and host receptor. Regrettably, SARS-CoV-19 has infected thousands of people whereby every single country has suffered huge losses. Moreover, the death rate with COVID-19 is higher than that of SAES-CoV and MERS-CoV-19 combined. Therefore, our ambitious task has proven quite difficult.

Seeds of Nigella sativa L. (also commonly known as black cumin or black seed) are widely used in traditional Islamic medicine and for culinary purposes worldwide. Nigella seed oil is becoming popular within the Islamic world and beyond. Composition of Nigella seed oil is known to be location-dependent. We investigated the composition of Nigella seed oil prepared by solvent- or by the cold press-extraction ofNigella seeds grown in Morocco. Oil extraction yield was 37% and 27% when solvent or cold press extraction methods were used, respectively. In terms of its oil major components, the composition of Nigella seed oil from Morocco is similar to that ofother Mediterranean countries, who are commonlyknown for their Nigella seed-oil quality [1].

The antibacterial activity of the essential oil of N.sativawas determined against a panel of strains of bacteria. The GC–MS analysis showed that the major constituents of the oil were monoterpene hydrocarbons and phenolic monoterpenes, whereby theresults of antibacterial activity confirmed the possibility of using Nigella sativaessential oils or some of their components in biological and pharmaceutical preparations [2].

To fight against coronavirus by using the constituents of Nigella sativa L, one important question is posed; Which partis the most important, the essential oil or the actual seed?

Many reviews were conducted on plants and their secondary metabolites, which have shown activity against SARS-CoV. Numerous scientific reports on the potential of plants and secondary metabolites against SARS-CoV infection, whereby many of the compounds had been studied through computational studies [4], [5], [6]. confirmed that the primary host receptor for SARS-CoV-2 is the human angiotensin-converting enzyme 2 (ACE2).

Ye et al. overviewed the existing knowledge about 7 human coronaviruses(HCoVs), with a focus on the history of their discoveriesas well as their zoonotic origins and interspecies transmissions. They also compared and contrasted the different HCoVs from a perspective of virus evolution and genome recombination [7].

Lo et al. reported their experience on the evaluation of SARS-CoV-2 RNA shedding in clinical specimens and clinical features of all 10 patients in Macau, and recommended the assessment of both fecal and respiratory specimens for enhancing diagnostic sensitivity becausethere were notany specific antiviral drugs available for the treatment of this sudden and lethal disease.Many drugs have been used in thistherapy. Yang et al. [8] reported that greater than 85% of COVID-19 patients in China have been receiving Traditional Chinese Medicine (TCM) treatment,and presented strong clinical evidence showing the beneficial effect of TCM in the treatment of thesepatients [8].

Zhou and Zhaopointed out the great importance of using therapeutic neutralizing antibodies (NAbs) to control the spread and re-emergence of SARS-CoV-2 and assert that the development of NAbsshould therefore be a high priority in future considerations [9].

The two main components of black seed essential oil, Thymoquinone (TQ) and Thymohydroquinone (THQ), were investigated for their antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Shigella flexneri, Salmonella typhimurium, Salmonella enteritidis and Staphylococcus aureus [10], [11].

Thymohydroquinone (2), also referred to as Dihydrothymoquinone or Hydrothymoquinone, is the phenolic hydroquinone derivative of Thymoquinon, Thymohydroquinone is the first reductive product of Thymoquinone, which is a significant co-product of the essential oil in Nigella sativa.

Several studies onthebiological activity of Thymoquinone have been discussed in literature; oxidative stress, hepatoprotection, the anti-tumor proliferation, the anti-inflammatory, the hypertension, the anti-microbial, neuropathy pain, gastroenterological, the kidney, renal, and the heart [12], [13], [14]. In contrast to Thymoquinone, there is no concern over Dithymoquinone or hydrothymoqunon.

N. sativa seeds contain 36 –38% fixed oil and low concentrations of some unusual unsaturated fatty acids. Different components were characterized from the oil: the major ones were TQ (28–57%) and para-cymene (7– 15%), whereaslow concentrations of the dimeric form of TQ (Dithymoquinone), and only a minimal quantity of Dihydrothymoquinone (DHTQ) were detected in the oil [15]. DHTQ could be formed in the body after TQ ingestion, following the action of reductases, as reported for DT-diaphorase, which catalyzes reduction of TQ to DHTQ in different organs [16].

Thymoquinone (TQ), the main, active ingredient of black seed oil, possesses antioxidant, anti-inflammatory, antiviral, antimicrobial, immunomodulatory and anticoagulant activities[17].

Using POM Theory, arelatively new computational method for virtually screening many compounds from natural source, we screened20 compounds derived from Nigella sativa, Artemisia herba alba and thymus. The choice of these plants, as previously discussed, was made basedon their role in traditional medicine for curing a variety of diseases, with special focus on influenza, fever, and colds. Unfortunately, only a few of these plants have been screened against SARS-CoV, with verylimited references reporting these plants as a possible antiviral source.From amongst these selected plants, we choose only 5 compounds, Thymole, Thymoquinone, Dithydrothymoqinon (DHQ) and Dithmoquinon (DTQ) and Artemisinin (ARTM) all show promising results. So, we beganthe work protocol by studying in vitro, antiviral activity of these compounds against COVID-19.

The aim of thiscurrent study is to understand and emphasize the potential antiviral activity of Thymohydroquinon (THQ), and dithymoquinon(DTQ), whereby themany papers study the effect of Nigella sativa as antiviral [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18].However, there is not a single study of antiviral compounds derived from this plant, documenting antiviral activity against SARS-Cov19.

Material and methods

Mterial and equipment

Thymoquinone, solvent, acetone, ethanol, glacial acetic acid and TLC , HPLC-grade methanol were purchased from Sigma–Aldrich.

FT IR Spectra (Brüker Tensor 27with ATR configuration), H1NMR (BrükerAvance 400 NMR spectrometer), UV–Visable spectra (Varian Cary® 50 Scan spectrometer in a 1.0 cm square cuvette).

Synthesis of compounds (1, 3)

Thymoquinone was used for synthesis two compound HTQ and DTQ.

Synthesis hydrothymoquinone HTQ (2)

Thymoquinone (0.50 g, 3.1 mmol) was dissolved in glacial acetic acid (10.0 mL). Zinc (0.75 g) powder was added, and the reaction mixture was stirred at room temperature for 12 h. The progress of the reaction was monitored by TLC. After completion of the reaction, acetic acid was concentrated under vacuum and neutralized by Na2CO3 (2 M solution, 50 mL). Then, dichloromethane (50 mL) was added, and the organic materials were partitioned between the two phases. The organic layer was separated, dried over anhydrous MgSO4, and evaporated under reduced pressure,the seprated compound was then purified by column chromatography using silica gel as the stationary phase and hexane:ethyl acetate (9:1) mobile phase. White solid, 288 mg, yield 60%. was optained [16].

Characterization of compound 2 HTQ

The obtained FTIR spectra of the bending alkane vibration band, the bending δ(C—H) vibration of the resultingabsorption band is displayed here [19]. The strong band appeared at 1231 cm−1 due to stretching vibrations of ν(C—O) of aromatic ether. The two medium absorption bands at 1124 cm−1 and 900 cm−1 attributed to bending vibration of δ (CC) of α,β-unsaturated alkene., broad band appears at 3300 cm−1 corresponding stretching vibrations of νOH . 1H NMR (CDCl3, 500 MHz, δ in ppm): 1.04–1.06 (d, j = 1.6, 6H), 1.98 (s, CH3), 3.3 (m, 1H,CH), 6.42 (d, CH, j = 2.4), 8.35 (d, CH, j = 2.4). MS detection show apeak at m/z 165 [M−H] corresponding to the formation of HTQ.Also the absence of peak at m/z 163 [M−H] relayed to TQ confirmed the complete reaction which also confirmed by comparison the compound by authentic sample on TLC System ethyl acetate:hexane (1:9). All spectral data confirmed the presence of pure HTQ compound, and its purity was confirmed by HPLC.

Synthesis of Dithymoquinone DTQ (3)

In a 500 mL glass beaker, compound  1  (0.50 g) was dissolved in 5.0 mLacetone. The bright yellow solution was gently rotated along the inner surfaces of the beaker until complete evaporation occurred, into a thin, crystalline layer. The resulting thin layer (solid state) of  1  was exposed to UV lamp (345 ƛ max) in a fume hood at room temperature. The reaction was found to be greater than 99% complete after 8 h. The photodimerization reaction was monitored by TLC. Crude product was dissolved in asmall amount of DCM, loaded on silica gel, and then purified by column chromatography using silica gel as the stationary phase and hexane:ethyl acetate (9:1) mobile phase. Compound (DTQ) was dissolved in a minimal volume of ethyl acetate, transferred to a smaller Erlenmeyer flask, and then evaporated to dryness over gentle heat. Crystallization of  compound  was performed using ethanol to give fine, pale yellow needle-like crystals,ultra-pure water and cold 2-propanol, re-centrifuged and lyophilized overnight to dryness. The method was follow as the method decriped by Elsharqawy et al., [20].

Characterization of compound 3 DTQ

Compound 3, 110 mg, 22% yield, m.p. 200.5 °C,UVmax  250 nm and UVmin  380 nm. IR (solid state): 3060 cm−1(vinylic CC-H stretch), 2969–2872 cm−1(C—H stretch of aliphatic groups), H-NMR (600 MHz), δ 6.70 (s,2H), 3. (s, 2H), 3.12–3.06 (septet, j = 6.6,2H), 1.22 (s, 6H), 1.16–1.13 (2d, j = 7.2, 6.6, 12H). ESIMS: 329 [M + 1].

Cytotoxicity assay

To assess the half maximal cytotoxic concentration (CC50), stock solutions of the test compounds were prepared in 10% DMSO in ddH2O and diluted further to the working solutions with DMEM. The cytotoxic activity of the extracts was tested in VERO-E6 cells by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method with minor modifications. Briefly, the cells were seeded in 96 well-plates (100 µl/well at a density of 3 × 105 cells/ml) and incubated for 24 h at 37 °C in 5% CO2. After 24 h, cells were treated with various concentrations of the tested compounds in triplicates. 24 h later, the supernatant was discarded, and cell monolayers were washed with sterile 1x phosphate buffer saline (PBS) 3 times and MTT solution (20 µl of 5 mg/mL stock solution) was added to each well and incubated at 37 °C for 4 h followed by medium aspiration. In each well, the formed formazan crystals were dissolved with 200 µl of acidified isopropanol (0.04 M HCl in absolute isopropanol = 0.073 mL HCL in 50 mL isopropanol). Absorbance of formazan solutions was measured at λ max 540 nm with 620 nm as a reference wavelength using a multi-well plate reader. The percentage of cytotoxicity compared to the untreated cells was determined with the following equation.

The plot of % cytotoxicity versus sample concentration was used to calculate the concentration which exhibited 50% cytotoxicity (CC50) [21].

Inhibitory concentration 50 (IC50) determination

In 96-well tissue culture plates, 2.4 × 104 Vero-E6 cells were distributed in each well and incubated overnight at a humidified 37 °C incubator under 5% CO2 conditions. The cell monolayers were then washed once with 1x PBS and subjected to virus adsorption (hCoV-19/Egypt/NRC-03/2020 (Accession Number on GSAID: EPI_ISL_430820)) for 1 h at room temperature (RT). The cell monolayers were further overlaid with 100 μl of DMEM containing varying concentrations of the test compounds. Following incubation at 37 °C in a5% CO2 incubator for 72 h, the cells were fixed with 100 μl of 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet in distilled water for 15 min at RT. The crystal violet dye was then dissolved using 100 μl absolute methanol per well and the optical density of the color wasmeasured at 570 nm using Anthos Zenyth 200rt plate reader (Anthos Labtec Instruments, Heerhugowaard, Netherlands). The IC50 of the compound is that required to reduce the virus-induced cytopathic effect (CPE) by 50%, relative to the virus control.

Statistical analysis

Statistical test were carried out using GraphPad Prism 5.01 software. Data are presented as average of means . the IC 50 and CC50 curve represent the nonlinear fit of Normalize of Transform of the obtained data.

Result and discussion

Thymoquinone was used for synthesis hydrothymoquinone(HTQ) and dithymoquinone (DTQ) to study their antiviral activity.

Compounds HTQ and DTQ were synthesis by method descriped in material and methods part and the identification of copounds were follow by UV, IR, H1NMR and ESIMS, The purity of compounds was confirmed by HPLC. And the data were present in supplmantry material.

Antiviral assay

To identify the proper concentrations to define the antiviral activity of the selected drugs, half-maximal cytotoxic concentration “CC50” was calculated by MTT assay for each individual drug, The sample showed anti-SARS-CoV-2 activity at non-cytotoxic nanomolar concentrations in vitro with low aselectivity index (CC50/IC50, 31.74/23.15 = 1.4). Whereby dimthymoquinone shows high cytotoxicity as in Fig. 1 .

Fig. 1

Cytotoxic effect of compounds, Dimer (3) and DHT (2). Inhibitory concentration (IC50) values were calculated by nonlinear regression analysis of Graph pad prism software (version 5.01) by plotting log inhibitor versus normalize response (variable slop).

POM analyses of compounds 1–3

The three compounds 1–3 were also screened for the in-silico POM study to calculate various general properties along with the prediction of antiviral bioactivity. Data was analyzed and comparedwith astandard anti-malaria Artemisinin drug. Osiris and Molinspiration are two cheminformatic based software tools which help in calculation of toxicity risks, molecular properties as well as in the forecasting of bioactivity scoresof the screened compounds.

As our tested compounds 1–3 have a molecular weightof less than 500 g/mol, so they may be highly absorbed because most of the traded drugs, i.e., approximately 80% of them have molecular weights in this range (Table 1 ). In contrast to compounds 1 and 3 which have a negative drug-score, the dimer drug with amolecular weightof 328 g/mol also shows best drug likeness at92% with an exceptional drug-score at72%. The drug likeness of compounds 1 and 2 are −1.2 and −6.33 and their drug-scores are limited to 35% and 22%,respectively (Table 1).

Table 1

Structure of some compounds of Nigella sativa for antiviral screening against COVID-19.

Compound	Name, formula and molecular weight
Compound (1)	● Name: Thymoquinone ● Synonyms: 2-isopropyl-5-methylcyclohexa-2,5-diene-1,4-dione● Molecular formula: C10H12O2 ● Molecular weight: 164,20 g/mol
Compound (2)	● Name: 2,5-Dihydroxy-para-cymene ● Synonyms: 2-methyl-5-propan-2-ylbenzene-1,4-diol 2-isopropyl-5-methylbenzene-1,4-diol Thymoquinol;Thymohydroquinone;5-isopropyl-2-methylhydroquinone;2-Methyl-5-isopropylhydroquinone;2-Isopropyl-5-methyl-1,4-benzenediol;2-Methyl-5-isopropylbenzene-1,4-diol;2-isopropyl-5-methylbenzene-1,4-diol;2-methyl-5-propan-2-ylbenzene-1,4-diol;2-methyl-5-propan-2-yl-benzene-1,4-diol;2-Methyl-5-(1-methylethyl)1,4-benzenediol ● Molecular formula: C10H14 O2 ● Molecular weight: 166.22 g/mol ● Toxicity of compound: LD50 = 25 mg/kg [3]
Compound (3)	● Name: Dithymoquinone ● Synonyms: (4aR,8aS)-2,6-diisopropyl-4a,8a-dimethyl-4a,4b,8a,8b-tetrahydrobiphenylene-1,4,5,8-tetraone ● Molecular Formula: C20H24O4 ● Molecular weight: 328,17 g/mol

From Molinspiration data (Table 2 ) it was concluded that the series of tested compounds 1–3 satisfy the rule of Lipinski and behave as a drug and exclusively wherebyonly the dimer compound 3 has anuclear receptor ligand along with enzyme inhibition properties. The cLogP value of the compounds 1–3 falls in the standard range, (i.e., less than 5) therefore these compounds may be highly hydrophilic and thus, meet the criteria of market drugs (Table 3 ).

Table 2

Osiris analysis of compounds 1–3.

Compound	Toxicity Risks	Solubility	Drug-Score
Compound (1)
Compound (2)
Compound (3)

Table 3

Molinspiration analysis of compounds 1–3.

Compound	Molecular Properties	Bioactivity Scores
Compound (1)	cLogP 1.90TPSA 34.14natoms 12MW 164.20nON 2nOHNH 0nviolations 0nrotb 1	GPCR ligand -1.40Ion channel modulator -0.31Kinase inhibitor -1.27Nuclear receptor ligand -1.47Protease inhibitor -1.45Enzyme inhibitor -0.40
Compound (2)	cLogP 3.26TPSA 40.46natoms 12MW 166.22nON 2nOHNH 2nviolations 0nrotb 1volume 166.59	GPCR ligand -0.92Ion channel modulator -0.44Kinase inhibitor -1.06Nuclear receptor ligand -0.54Protease inhibitor -1.17Enzyme inhibitor -0.46
Compound (3)	cLogP 1.70TPSA 68.28natoms 24MW 328.41nON 4nOHNH 0nviolations 0nrotb 2volume 310.56	GPCR ligand -0.18Ion channel modulator -0.09Kinase inhibitor -0.48Nuclear receptor ligand 0.14Protease inhibitor -0.10Enzyme inhibitor 0.10

Identification of antiviral pharmacophore sitesof compounds 1–3

The invention of POM Theory leads us to identify each type of pharmacophore sites with real success, on the basis of semi-empirical data of about 7.000 antibacterial, antifungal, antitumor and antiviral commercial and new drugs. All details of therapeutic applications of POM Theory are given in the literature and the identification of different and various types of pharmacophore sites is well established [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50], [51], [52], [53], [54], [55], [56], [57], [58], [59], [60], [61], [62], [63], [64], [65], [66], [67], [68], [69], [70], [71], [72], [73], [74], [75], [76], [77], [78], [79], [80], [81], [82], [83], [84], [85], [86], [87], [88], [89], [90], [91], [92].

The Atomic charge calculation of compounds 1–3 (Table 4 ) show that all oxygen atoms are negatively charged (Fig. 2 ). The distance between any couple of two oxygen can be obtained after optimization of molecular structure by using the Petra program.

Table 4

Atomic charge of Oxygen of the antiviral (O,O’) pharmacophore sites of 1–3.

Compd.	O1	O2	O3	O4	Distance (Å)		Pharmacophore sites
1	−032	−0.32	---	---	O1-O2	5.99	One Medium Antiviral Site
2	−0.28	−0.28	---	---	O1-O2	5.99	One Medium Antiviral Site
3	−0.30	−0.30	−0.30	−0.30	O1-O2O3-O4O1-O3O2-O4	4.124.125.985.98	Two Medium AntiviralplusTwoStrong AntiviralSites

Fig. 2

Atomic charge of compounds 1–3.

Discussion

COVID-19 is considered a critical threat to public health, and what aggravated the situation is that there is no existing antiviral therapy that is clinically approved for the management of this disease. Searching for new drugs for this disease is a global duty for scientists, so the current study concern searching for new drugs from natural plants have a history in the treatment of many diseases, antiviral, antioxidant, antidiabetic and anticancer.

In this study, three proposed anti-COVID-19 supportive drugs/treatments; Thymoquinone (1), hydrothymoquinone (2), and Dithymoquinone (3) have been analysed, and their pharmacophore sites are characterized via bioinformatic POM analysis.  Osiris calculations of analysed compounds reveal that Thymoquinone and dihydrothymoquinone are relatively moresafe than hydroxychloroquine. The dithymoquinoneis the safest one. The most important antiviral activities of dithymoquinone and dihydrothymoquione on agree with the POM results obtained and the tested Thymoquinone derivatives via a synergic mechanism.

In vitro study the results revaled amoung two investigated compound, HTQ has anti-SARS-CoV-2 activity at non-cytotoxic nanomolar concentrations in vitro with low aselectivity index (CC50/IC50, 31.74/23.15 = 1.4). Whereby DTQ shows high cytotoxicity, So the compounds isolated from N.sativia and nigela sativa plant may be used in treatment of CoV-19 after further clinical studies.

Mode of action still has not been identified, however, Molecular docking studies on the TQ showed a notable antiviral activity against a SARS-CoV-19 strain isolated from Egypt, whereby astudy revaled that TQ haspotent antiviral activity through binding to the receptor. Its binding domain on the spike and envelopeproteins of SARSCoV-19, which may hinder virus entry in to the host cell and inhibit its ion chanal and pore-forming activity [92]. The authors of this current study suggest the same mechanism of HTQ inhibition, therefore this will be discussed in afuture study

Conclusion

The current study highlight in vitro antiviral studies on two compounds from three major compounds in Nigella sativa depending on bioinformatic analysis, which reveals the importance of three major compounds (TQ, DTQ, and HTQ), as antiviral agents. in vitro antiviral screening on two compounds (DTQ and HTQ) showed the tested drugs exhibited a promising in vitro activity against COVID-19, and have promising antiviral activities, further investigations in clinical trials to determine actual in vivo activity in the treatment of COVID-19 have recommended.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.