Viral protein used in this experiment; https://zhanglab.ccmb.med.umich.edu/COVID-19/
Nigellidine binding to N terminus protease nsp3_QHD43415_3 (Fig 1)
This is known as Papain-like proteinase. It is
responsible for the cleavages located at the N-terminus of the replicase polyprotein. It participates together with nsp4 in the assembly of virally-induced cytoplasmic double-membrane vesicles necessary for viral replication. Nigellidine showed highest binding energy value of -7.61 and its ligand efficiency was also high (-0.35). According to the AutoDock result the position A having binding energy -7.61, forms some unstable bonds with amino acids THR943, LYS945, MET 1556, ALA1557, PRO1558, ILE1559 (Fig 1). Binding energy range of -7.59 to -4.56 (total posture 4) were found at position B. It showed an entry/exit pocket where nigellidine binds to TRP1632 through an N-O bond and other hydrogen bond with HIS1630, TRP1632, ALA1878 and THR1774. The ACE value was also good at this site (-284.28) and nigellidine affinity was also found. At site C binding energy (-6.18 & -5.05) was also high and the highest ACE value (-356.72) was observed. Here also nigellidine formed stable bond with GLY251 (C-C & O-C) and ASN 244 (N-O) and some hydrogen bond with other mentioned amino acids. From this observation it could be concluded that affinity of nigellidine at site was very high but then it may transferred to site B and C and may hamper the normal function of N-terminal protease of coronavirus.
Nigellidine binding to Proteinase 3CL-PRO or Mpro_QHD43415_5 (Fig 2).
This protein cleaves the C-terminus of replicase polyprotein at 11 sites. Recognizes substrates containing the core sequence [ILMVF]-Q-|-[SGACN]. Also able to bind an ADP-ribose-1''-phosphate (ADRP). It is noticed from both Patchdock and Autodock results nigellide binds with the high efficiency with the important amino acids in the active sites of the enzyme which could have altered its activity and viral metabolism significantly. The ACE value was found to be -265. 82. Active site biding energy was found to be -6.38 and -4.19 suggesting potential impairment due to drug binding effect. One previous report suggests Mpro protease structure in COVID-19 may bind nigellide molecule at it active site .
Nigellidine binding to Non-structural protein 2 nsp2_QHD43415_2; Fig 3
This protein plays a role in the modulation of host cell survival signaling pathway by interacting with host PHB and PHB2, which helps to maintain the functional integrity of the mitochondria and protecting cells from various stresses. According to Angeletti et.al., 2020 , NSP2 protein of COVID 19 has an entry pocket (Fig 3). Just at that position nigellidine bound with ACE value of -248.81 and -241.81 forming an H-bond with ALA 241. Same location with binding energy value of -6.28 (out of the pocket) and -5.86 (within the pocket) was observed in AutoDock result. The ligand efficiency was found –e site also.
Nigellidine binding to Nucleocapsid QHD43423–Fig 4
This is one of the important proteins that packages the positive strand viral genome RNA into a helical ribonucleocapsid (RNP) and plays a fundamental role during virion assembly by interacting viral genome and membrane protein M. It also plays an important role in enhancing the efficiency of subgenomic viral RNA transcription as well as viral replication. According to Luo et al., 2004,  nucleocapside of COVID – 19 has a conserved sequence of WPQIAQF which acts as the active site. Nigellidine was bound with the 6th Q residue stably in docking experiment. So, it could directly inhibit the activity of viral nucleocapsid. The highest binding energy value was -6.24 and the active site binding energy was -4.9. And the ligand efficiency values were -0.28 and -0.22.
Nigellidine binding to Spike Glycoprotein, data not shown
The spike glycoprotein interact with the angiotensin convertage enzyme 2 (ACE 2) receptor present at human cell surface and initiate the viral entry into the host cell. No satisfactory attachment of nigellidine was observed at the active site. The highest binding energy value was -6.11, whereas, presence of nigellidien at the central core was observed with -4.77 binding energy. The ligand efficiency ranged from -0.28 to -0.22. Though the highest ACE value was -340.50 and strong affinity was observed .
Human receptors, inflammatory signal molecules and other proteins
Nigellidine binding to IL1R, Fig 5
The highest binding energy for IL1R and nigellidine was -6.23 but that location was not at the active site. IL1R interact with its ligand through the amino acids TYR 127, VAL 16, ALA 109, GLU 11, ILE 110, LYS112, LEU 237, ASP 239, ALA 241, TYR261, ASP 260 and GLU 252. Among them nigellidine was found to interact with TYR127 (ACE value: -216.30) and TYR261 (ACE value: -136.97). Additionally it was found at the location LYS270, THR300 and HIS301 (ACE value: -64.99). This location is very close to ligand attachment site. It may also destabilize ligand interaction with receptor. At that location -5.67 and -5.23 binding energy value was also observed in Autodock result. And the ligand efficiency were -0.26 and -0.24.
Nigellidine binding to TNFR1, Fig 6
A strong affinity of nigellidine was found with TNFR1. According to Autodock, It showed a range of binding energy value -6.81 to -5.81. All the postures were found at a secondary attachment site of TNFR1. And their ligand efficiency value were also remarkable ranging from -0.31 to -0.26. Normally TNFR1 interact with its ligand involving the amino acids Met 11, SER 13, GLN 48, LYS 32, GLU 64, ASP 49, LYS 35, GLU 54, GLN 17, GLN 130, GLN 133 and THR 135. Among them, GLN 130, GLN 133 and THR 135 were present at the secondary attachment site which could be blocked by nigellidine. Similar results also analyzed in Patchdock result. Additionally some interaction was also observed at the primary binding sites according to ACE value where nigellidine was found to block GLN 17 and ASP 49. This indicated the strong TNFR1 inhibition affinity of nigellidine.
Nigellidine binding to TNFR2 Fig 7
In TNFR2 normal interaction with its ligand occurs through the amino acids ARG 113, CYS 74, SER 73, SER 59, CYS 71, TYR 103, ARE 13, GLN 109, GLN 63, TRP 67 and CYS 71. All the interactions were stabilized through hydrogen bond. Whereas, nigellidine formed H-bond with SER153, THR 151, ARG 158, ASP 175, ASN 149 and TYR61. In addition some stable bonds were also formed with GLN 63, GLN 109, PRO 144, ILE 162, GLY 145, LYS 120 and GLU 84. Most interestingly amino acids GLN 63, GLN 109 if occupied by nigellidine it will hamper the normal ligand interactions. Although the ACE value of TNFR2 – Nigellidine binding was moderate and the highest value was -258.71. But, the normal function could be hampered if nigellidine concentration will increase. The highest binding energy value for this interaction was -5.16 and the ligand efficiency value was ranged from -0.23 –0.19.
Results on rat experimental rat model
Present results suggest in the figure 8 that ALP, SGPT and SGOT and MDA remarkably lower in the NS treated tissues compared to that of their basal level. Serum urea and creatinine levels were also found to decline from that of vehicle treated group (data not shown). Nigellidine is the first indazole alkaloid which has been found with a sulfuryl group. The sulfated compound is possibly favorable for higher rate of solubility and increased bioavailability. So, this compound might have role in the induction of sulfotranseferase; SULTs that catalyzes sulfuryl group transfer. These enzymes may modulate phase II drug metabolism, drug-drug interactions. In the current study, we found no impact of nigellidine on Phenol catalyzing ASTIV or Steroid catalyzing EST expressions/activities. So this drug does not influence at least sulfation mediated adverse metabolic processes. This is demonstrated in the figure 8.
Our present finding has two parts. In the molecular docking strategies with both PatchDock and Autodock were run to verify the binding of nigellidine and its locations on different proteins of SARS CoV 2. It is noticed that the drug very effectively binds with the N terminus protease, nucleocapsid and Main protease which are absolutely important for viral maturations in proteins structures, RNA packaging and other functions. Another point is that during post infection period this virus has been reported to greatly impair the human immune system by the extravagant activities of different cytokines namely TNFα, IL1, IL6 and others. These signaling molecules perform after binding with their specific receptors like TNFR1/2, IL1R and IL6R respectively. So blocking of these cytokines or their receptors may help to break the cascade of cytokine signaling. This could be one of the steps to decrease the severity of the SARS CoV 2 infection. Other than nigellidine there are several compounds like thymoquinone groups of drugs, α-hederin and these compounds are reported to have significant therapeutic activities against different types of pathogen infections. The docking results revealed promising inhibitory potential of thymoquinone against Cag A and Vac A, H. pylori oncoproteins with comparison to the standard drug, metronidazole .
Complex glycan molecules are the frequent components on the viral and bacterial surfaces; sometimes lipopolysaccharides or glycated proteins generate significant and unwanted immunological reactions which instead potentiating, exhaust the host immune system. As for example, in the present case, SARS CoV 2 spike which is covered with a large number of NAG disfavors the proper presentation of the epitopic part at the time of MHC presentations. This glycan molecule also restricts proper drug targeting to the spike proteins. This has been demonstrated in our previous findings on some suitable epitope-screening from SARS CoV 2 spike . Our earlier study also demonstrated that Epigallocatechin gallate (EGCG) and Theaflavin gallate (TDG) are the potent binder to the CoV 2 spike channel . Increased cytokines and other hematopoetic parameters (oxidative stress markers) by LPS-administration have been shown to terminate by Nigella sativa (NS) extract . Cardio-respiratory and endothelial dysfunction is one of the major symptoms of SARS CoV 2 infection. And this dysfunction may be counteracted by NS and its component thymoquinone by restraining interleukin-1β, TNF-α and NF-κB signaling . Promising anxiolytic and anti-inflammatory activities of NS has been demonstrated . Hypertensive renin-angiotensin system is an obvious co-morbid condition in the elderly persons and that has been targeted in the SARS CoV 2 infection. Nevertheless, hypertension associated diabetic immune-suppressive status is the major target of this viral infection. So drug prohibition of these secondary disorders is of great importance in decreasing mortality rate. The NS component, nigellidine has been decisively shown to bind to the active sites of the IL1 and TNF-α receptors (fig 6 and 7). The immune response and pathogenicity of the H9N2 avian influenza virus has been restricted by the Nigella sativa . The use of N. sativa seed fixed oil can inhibit the inflammation of sinuses and respiratory airways, microbial infections, such as coryza, nasal congestion . Report revealed from some prospective study that, killed and re-assorted influenza virus supplemented with natural adjuvant like Nigella sativa has been more responsive than NS alone to generate IgG and IgM responses via augmented CD4+- and CD8+ signaling . A significant decline in the number of peripheral lymphocyte counts, mainly CD4 T and CD8 T cells in COVID-19 patients is shown to relate disease severity and further opportunistic infection. Several peripheral; tissues like spleen, lymph nodes, and lymphoid tissue and T lymphocytes have been shown to carry COVID-19 RNA. It is suggested that enhanced CD4+ mediated responses generated by NS in the decrease of HIV-RNA load in the patients. 
In the current study, we have decisively shown by molecular modeling that nigellidine can bind in the active sites of several important proteins of SARS CoV 2, several host receptors specific for SARS CoV 2 induced inflammatory markers IL1, IL6, TNF-α. Moreover, the extract from black cumin seed has been shown in experimental rat to be highly antioxidative, hepato- and reno-protective. Further studies are necessary to verify the potential effects of nigellidine in in vivo laboratory experimental animal model.