HPLC analysis. The non-alkaloids of R. stricta were isolated using the HPLC method. Separation using the HPLC method confirmed the existence of nine non-alkaloid compounds (Table 1) and seven additional alkaloids as minor components.
Molecular Docking. Autodock vina was used to estimate the binding affinity and compounds possess into the binding site cavity of the RBD I.D. 6M0J (Fig. 2). Among all the compounds, compounds with a 0.8 kcal binding affinity were chosen. Lig 434 showed the least binding affinity, and its atoms involved in interaction with RBD protein by six hydrogen bonds, and the binding affinity was − 8.5 kcal/mol (Fig. 2A). Also, several electrostatic contacts involved. Lig 68 showed the same binding affinity − 8.5 cal/mol and bounded to RBD protein with four hydrogen bonds (Fig. 2B). Lig230 with − 8.5 kcal/mol and its position enabled to form only two hydrogen bonds (Fig. 2C). These compounds had remarkable molecular docking results, which make them good candidates to perform MD simulations. The other compounds revealed a close result and potential activity, but we confined the MD simulation only to the top three compounds.
Molecular Dynamic Simulation.
To validate the molecular docking results, molecular dynamic simulation, root mean square deviation (RMSD), root mean square fluctuation (RMSF), and radius of gyration (Rg) were performed. The RBD – ligand complexes results represented by Xmgrace graph plotting. From the start steps up to last, Lig434 is poorly stable during MD simulation. And its RMSD value reached 0.5 nm in some steps. In addition, Lig434 terminated the MD simulation with 0.34 nm. Lig68 stabilized after 80 ns with RMSD 0.28 nm. Finally, Lig230 elucidated high stabilization with RMSD 0.2 nm (Fig. 3A).
RMSF plotting graph showed a fluctuation and flexible residues of the RBD as a function of time (Fig. 3B). Eleven residues were showed with respectively high fluctuations, (333, 369, 417, 505, 511, and 519) related to RBD-Lig230 complexes, (333, 324, 444, 449, and 466) related to RBD-lig434 complex and (449) related to RBD-Lig 68 complex. All these residues showed vibration during MD simulation (Fig. 3C).
The radius of gyration (Rg) measures the compactness and folding of the RBD protein. The results of RBD-complexes were approximately matching the Rg of RBD alone which means the protein properly folded and compacted during the MD simulation.
Hydrogen bonds are an important indicator of intermolecular interaction between the RBD and the ligands compounds. Lig434 has a high tendency in the formation of highly variable hydrogen bonds and predicated to form 1–3 hydrogen bonds and a maximum of five hydrogen bonds was formed. Lig68 results showed 2–4 hydrogen formed during MD simulation and a maximum of five hydrogen bonds was observed, which is equal to the number of hydrogen bonds in the molecular docking results. In the case of Lig230, 1–3 hydrogen bonds and a maximum of three hydrogen bonds were formed. Until the end of 100 ns, Lig68 and Lig230 are still connected to RBD by 3 and 2 hydrogen bonds (Fig. 3D). The binding, stability, and strength of the interaction were analyzed by extracting the Lennard–Jones (LJ) and Coulomb (Coul) energies. Lig230 revealed the highest average of interaction energy during MD simulation (− 417.284 kJ/mol) followed by Lig434 (− 366.186 kJ/mol) and the lowest interaction energy was by Lig68 (− 352.5872 kJ/mol) (Table 2).
Binding pose of non-alkaloid compounds during MD Simulation. The MD simulation analysis was performed to determine the stability of the nonalkaloid compounds docked to the hACE2 receptor calculated from the molecular docking simulations. The virtually studied non-alkaloid compounds (Lig 434, Lig 68, and Lig 230) docked to the SARS-CoV-2 ACE2 receptor were screened for complex stability and formation of a satisfactory position of the ligand molecules through intermolecular interactions to achieve a global energy minimum during the simulation time (100 ns). The last trajectory (100 ns point) of the Protein-Ligands complexes was extracted to evaluate and visualize the deviation that occurred to the ligand’s positions. The position variation and intermolecular contact were proportionally related to each other, where the increased position variation was confronted with an increase in intermolecular contact. Comparing to the Molecular docking poses (initial configuration), all the ligands preserved their poses in the binding cavity which contains the critical residues in the RBD-hACE2 interaction process. The RMSD of the ligands in the 100 ns trajectory (Lig230: 0.20 Aº, Lig68: 0.28 Aº and Lig434: 0.34 Aº) indicates that the Lig230 faced the slightest conformational changes (Fig. 4).
Drug-likeness. To evaluate the oral bioavailability of these compounds, a drug-likeness analysis is done by applying the five rules of the Lipinski method. All the compounds had two violations, which basically found in the Molecular Weight and the number of N and O atoms (Table 3). These results suggest, the three compounds expected to confront permeability and solubility difficulties if they introduce by the oral route, which adds another route of exposure into consideration (inhalation, injection, transdermal route, etc).
The assessment of absorption, distribution, metabolism and excretion (ADME) have been performed for the top leading compounds (Lig 68, Lig 434 and Lig 230) using SwissADME web tool. Bioavailability Radar is shown (Fig. 5) to provide a quick assessment of drug-likeness.
Six physicochemical characteristics are taken into consideration: lipophilicity, size, polarity, solubility, flexibility and saturation. On each axis, a physicochemical range was specified using descriptors adapted from 37 and 38 and represented as a pink region within which the molecule's radar plot must completely lie to be considered drug-like. The results revealed that the Lig 68 and Lig 434 were not orally bioavailable, because too flexible and too polar. It can be concluded that the compound (Lig 230) has a good water solubility, which is necessary for oral bioavailability and absorption, as well as a good lipophilicity.