# Selective Control by _Pistacia vera_ L. and Its Carotenoid Zaxanthin on SARS-CoV-2 Virus
## 2 Results
### Inhibition of TMPRSS2 by Zexanthin upon Cells Treatment  
To acquire data on zeaxanthin's mechanistic activity, we tested the zeaxanthin inhibitory activity on cellular proteases TMPRSS2, which is involved in viral entry. First, we treated the A549 cells with or without zeaxanthin for two hours, and then we transduced treated cells with \(\alpha\) and o-SARS-CoV-2 pseudotype variants separately. We analyzed the levels of TMPRSS2 protein accumulation using western blot analysis. The findings show that treatment with zeaxanthin reduced the accumulation of TMPRSS2 protein alone or upon the transduction of the \(\alpha\)- SARS-CoV-2 pseudotype variant (Figure 6). The degree of the TMPRSS2 accumulation was less evident following the treatment and transduction with the o-SARS-CoV-2 pseudotype. This suggests that zeaxanthin treatment can reduce TMPRSS2 accumulation in A549 cells. Furthermore, transduction with the \(\alpha\)-SARS-CoV-2 pseudotype appears to have a more significant impact on TMPRSS2 protein levels. The above data can justify the failure to use TMPRSS2 by the omicron SARS-CoV-2 variant for entry in comparison with the alfa SARS-CoV-2 variant [21]. These data are supported by our previous experiments confirming efficient cathepsin-mediated entry by omicron SARS-CoV-2 pseudotype variant in a monkey kidney cell line [20].  
In Vitro Screening for 3 CL\({}^{\text{pro}}\) and PL\({}^{\text{pro}}\) Inhibition by Zexanthin  
Proteases have been identified in DNA or RNA viruses and enveloped and non-enveloped viruses. These enzymes can use different catalytic mechanisms involving Ser, Cys, or Asp residues to attack the cleavable peptide bond with a high degree of recognition of specific cleavage [15]. Given the role of zeaxanthin in the modulation of cellular protease TMPRSS2, we performed an in vitro screening for 3 CL\({}^{\text{pro}}\) and PL\({}^{\text{pro}}\). 3 CL\({}^{\text{pro}}\) (also known as Main Protease or MP\({}^{\text{pro}}\)) and PL\({}^{\text{pro}}\) are chymotrypsin-like proteases encoded in the SARS-CoV-2 genome and play essential roles in the virus's lifecycle. A luminescent protease assay was carried out to verify the anti-protease activity of zeaxanthin (Figure 5). Recombinant SARS-CoV-2 3 CL\({}^{\text{pro}}\) and PL\({}^{\text{pro}}\) were separately combined with luminogenic 3 CL\({}^{\text{pro}}\) substrate and zeaxanthin for 60 min at 37 \({}^{\circ}\)C. The reaction was stopped by adding Luciferin Detection Reagent, and the luminescence was recorded on a plate-reading luminometer. The GC376 was used as a positive control. The results expressed as RLU and percentage of inhibition of enzymatic activity (Figure 5, panels a and c) showed that the enzymatic activity of 3 CL\({}^{\text{pro}}\) was inhibited following incubation with zeaxanthin. Similarly, zeaxanthin significantly reduced the activity of papain-like protease PL\({}^{\text{pro}}\), as Figure 7b,d reports.  
Figure 6: Expression of TMPRSS2 protein in zeaxanthin-treated and SARS-CoV-2-transducted cells. After zeaxanthin treatment (10 \(\mu\)M), A549 cells were transduced or not with \(\alpha\)-SARS-CoV-2 and o-SARS-CoV-2 pseudoviruses, and accumulation of TMPRSS2 protein was evaluated. GAPDH was used as a housekeeping gene. Quantification of the TMPRSS2 bands was calculated by the ImageJ Version 1.54.