# Selective Control by _Pistacia vera_ L. and Its Carotenoid Zaxanthin on SARS-CoV-2 Virus
## 3 Discussion
These results reinforce the notation that sequence differences in the spike protein's RBD significantly influence the binding efficiency of zeaxanthin across SARS-CoV-2 variants. It is well known that SARS-CoV-2 entry into respiratory cells is facilitated by the interaction between the viral spike protein and the ACE2 (angiotensin-converting enzyme 2) on the surface of lung cells, followed by membrane fusion that allows the viral nucleocapsid to enter the cell. TMPRSS2 (Transmembrane Serine Protease 2) plays a crucial role in the entry process of SARS-CoV-2 into human cells but less in the omicron variant. TMPRSS2 cleaves the S protein at specific sites, activating it. The activation of the spike protein by TMPRSS2 is essential for the virus to bind to the host cell receptor ACE2 (Angiotensin-Converting Enzyme 2) and subsequently fuse with the cell membrane. Building on these data, we identified a carotenoid, zeaxanthin, present in small quantities in NRRE and RURE, as a potential key factor responsible for the observed antiviral activity following pistachio extract treatment. Our investigation into the effect of zeaxanthin on the cellular protease TMPRSS2 revealed that it inhibited the entry of the \(\alpha\)-variant but not the o-variant. This was supported by reduced TMPRSS2 levels observed by western blot analysis in the presence of zeaxanthin. Our results showed that zeaxanthin displays a potent inhibitor effect on TMPRSS2, specifically hampering the \(\alpha\)-SARS-CoV-2 variant, which used it for entry, without affecting the o-SARS-CoV-2 variant (Figure 6). The data seem useless, given that the omicron variant is currently the most widespread worldwide. Nevertheless, as mentioned in [15], all the prominent animal viruses, herpesvirusives, HCV, alphaviruses, flaviviruses, HIV-1, and picornaviruses contain proteases that play crucial roles in their replication and thus, are essential targets for discovering potent antiviral drugs. Therefore, our data indicate for the first time that zeaxanthin can act as a natural inhibitor of TMPRSS2. This finding adds zeaxanthin to a growing list of natural compounds with potential anti-viral properties through the inhibition of key proteases involved in viral entry [23; 24]. In particular, the homoharringtonine, a plant alkaloid derived from Cephalotaxus for-tune, and the halofuginone, a halogenated derivative of febrifugine, the active component of the medicinal herb Dichroa febrifuga, were identified as inhibitors of TMPRSS2 [24]. Furthermore, we found that zeaxanthin significantly inhibited the activity of both viral proteases, 3 CL\({}^{\text{pro}}\) and PL\({}^{\text{pro}}\) (Figure 5). These data underscore zeaxanthin's potential as a broad-spectrum protease inhibitor, a quality that is particularly valuable in the fight against rapidly mutating viruses like SARS-CoV-2. Indeed, once inside, the viral RNA is translated into a polyprotein, which undergoes autocleavage to release the 3C-like main protein protease (3 CL\({}^{\text{pro}}\)). This protease cleaves the polyprotein to produce other proteins essential for viral replication. Thus, PL\({}^{\text{pro}}\) and 3 CL\({}^{\text{pro}}\) play crucial roles in coronavirus infection and are prime targets for antiviral drug development. Our data indicate that pistachio extracts and zeaxanthin possess significant antiviral properties against SARS-CoV-2. The different inhibition observed between \(\alpha\)- and \(\alpha\)-variants suggests that these compounds may offer a targeted approach in combating specific SARS-CoV-2 new emergent variants. Mutations of the Omicron variant compared to the alpha variant play a key role in the zeaxanthin interaction, as demonstrated by MD simulation studies. However, further research is crucial to fully elucidate the involved molecular mechanisms and to evaluate the clinical relevance of these findings.