# Selective Control by _Pistacia vera_ L. and Its Carotenoid Zaxanthin on SARS-CoV-2 Virus
## 2 Results
### Docking and Molecular Dynamics Simulations  
The crystallographic structures of the RBDs in complex with ACE2 were used to investigate the interactions between the spike proteins of the alpha and omicron variants and zexanathin. A detailed analysis of the two-dimensional interaction diagram (Figure 3) revealed that zexanathin engages in hydrophobic interactions with the RBD alpha variant (Figure S4). Specifically, the isoprene units of zexanathin interact with Ala386, Ala387, and Pro389, while the aliphatic ring interacts with His34, Pro321, and Met383 (Figure 3a,b). Additionally, two hydrogen bonds were identified between the hydroxyl groups at the terminus of zexanathin's aliphatic chain and the residues Lys26 and Met323. Figure 1: In vitro pseudovirus binding assay of NRRE and RURE using \(\alpha\)-SARS-CoV-2 and o-SARS-CoV-2 pseudotyped particles on A549 cells. (**a**) Schematic diagram of the binding assay. (**b,c**) The luciferase activity was performed in A549 cells 72 h post-infection with or without NRRE (0.6 mg/mL) or RURE (0.6 mg/mL) using \(\alpha\)-SARS-CoV-2. (**d,e**) The luciferase activity was performed in A549 cells 72 h post-infection with or without NRRE (0.6 mg/mL) or RURE (0.6 mg/mL) using o-SARS-CoV-2 pseudotyped particles. Experiments were performed in triplicates, and the data presented the RLU of three independent experiments (n = 3). Error bars indicate standard deviation (SD). The asterisks (** and ***) indicate the significance of \(p\)-values less than 0.01 and 0.0001, respectively, compared to untreated infected control. Figure 3: Interaction profile of the docked poses (**a**) and 2D diagram interaction profile (**b**) of zeaxanthin/\(\alpha\)-RBD complex and zeaxanthin/\(\alpha\)-RBD complex (**c**,**d**). Figure 2: In vitro pseudovirus binding assay using zeaxanthin against \(\alpha\)-SARS-CoV-2 and o-SARS-CoV-2 pseudotypes. (**a**) Schematic diagram of the binding assay. (**b**,**c**) The luciferase activity was performed in A549 cells 72 h post-infection with or without zeaxanthin (10 \(\upmu\)M) using \(\alpha\) and o-SARS-CoV-2 S-pseudotyped particles. Experiments were performed in triplicates, and the data presented the RLU of three independent experiments (n = 3). Error bars indicate standard deviation (SD). The asterisks (***) indicate the significance of \(p\)-values less than 0.001 compared to untreated infected \(\alpha\) and o-SARS-CoV-2 pseudotyped particles. Molecular dynamics (MD) simulations were conducted over 200 ns to elucidate further zeaxanthin's different behavior with the alpha and omicron variants. For the zeaxanthin/\(\alpha\)-RBD complex, the root-mean-square deviation (RMSD) of the C\(\alpha\) atoms exhibited an initial variation of up to 2.6 A within the first 50 ns, after which it stabilized for the remainder of the (Figure S5a). In contrast, the RMSD of the ligand itself displayed significant fluctuations, reaching up to 14 A within the first 15 ns and stabilizing between 10 and 12 A for the rest of the simulation. Throughout the MD simulation, the interactions between zeaxanthin and the \(\alpha\)-RBD were predominantly hydrophobic, while hydrogen bonds were notably unstable, frequently disappearing over extended periods (Figure S5b). This behavior may explain the in vitro observations, suggesting that zeaxanthin forms transient and inherently unstable interactions with the \(\alpha\)-RBD. In the case of the o-RBD variant, the best docking pose of zeaxanthin revealed that the ligand was deeply buried within a subunit adjacent to the ACE2 interaction domain (Figure S6). The interaction diagram (Figure 3c,d) highlighted extensive hydrophobic interactions with residues Pro321, Met383, Phe356, Ala386, Ala387, Pro389, Pro321, Leu29, Val93, and stable hydrogen bonds with Met323, Aspe30, and Lys26 at distances of 1.86, 2.98, and 2.34 A, respectively. Analysis of the MD trajectories for the zeaxanthin/o-RBD complex showed a distinct behavior compared to the \(\alpha\)-variant.