In silico studies of anti-oxidative and hot temperament-based phytochemicals as natural inhibitors of SARS-CoV-2 Mpro.

Main protease (Mpro) of SARS-CoV-2 is considered one of the key targets due to its role in viral replication. The use of traditional phytochemicals is an important part of complementary/alternative medicine, which also accompany the concept of temperament, where it has been shown that hot medicines cure cold and cold medicines cure hot, with cold and hot pattern being associated with oxidative and anti-oxidative properties in medicine, respectively. Molecular docking in this study has demonstrated that a number of anti-oxidative and hot temperament-based phytochemicals have high binding affinities to SARS-CoV-2 Mpro, both in the monomeric and dimeric deposited states of the protein. The highest ranking phytochemicals identified in this study included savinin, betulinic acid and curcumin. Complexes of savinin, betulinic acid, curcumin as well as Nirmatrelvir (the only approved inhibitor, used for comparison) bound to SARS-CoV-2 Mpro were further subjected to molecular dynamics simulations. Subsequently, RMSD, RMSF, Rg, number of hydrogen bonds, binding free energies and residue contributions (using MM-PBSA) and buried surface area (BSA), were analysed. The computational results suggested high binding affinities of savinin, betulinic acid and curcumin to both the monomeric and dimeric deposited states of Mpro, while highlighting the lower binding energy of betulinic acid in comparison with savinin and curcumin and even Nirmatrelvir, leading to a greater stability of the betulinic acid-SARS-CoV-2 Mpro complex. Overall, based on the increasing mutation rate in the spike protein and the fact that the SARS-CoV-2 Mpro remains highly conserved, this study provides an insight into the use of phytochemicals against COVID-19 and other coronavirus diseases.

A Novel Molecular Approach for Enhancing the Safety of Ozone in Autohemotherapy and Insights into Heme Pocket Autoxidation of Hemoglobin.

Major ozone autohemotherapy (MAH) is a popular clinical practice for treating a variety of pathological conditions due to the mild and controlled oxidative stress produced by the reaction of ozone gas with other biological components. Previous studies have shown that blood ozonation leads to structural changes in hemoglobin (Hb); therefore, in the present study, the molecular effects of ozonation on Hb of a healthy individual were assessed by ozonating whole blood samples with single doses of ozone at 40, 60, and 80 µg/mL or double doses of ozone at 20 + 20, 30 + 30, and 40 + 40 µg/mL ozone to investigate whether ozonating once versus twice (but with the same final ozone concentration) would have varying effects on Hb. Additionally, our study aimed to verify whether using a very high ozone concentration (80 + 80 µg/mL), despite mixing it with blood in two steps, would result in Hb autoxidation. The pH, oxygen partial pressure, and saturation percentage of the whole blood samples were measured through a venous blood gas test, and the purified Hb samples were analyzed using several techniques including intrinsic fluorescence, circular dichroism and UV-vis absorption spectroscopies, SDS-polyacrylamide gel electrophoresis, dynamic light scattering, and a zeta potential analyzer. Structural and sequence analyses were also used to study the Hb heme pocket autoxidation sites and the residues involved. The results showed that the oligomerization and instability of Hb can be reduced if the ozone concentration to be used in MAH is divided into two doses. Indeed, our study demonstrated that two-step ozonation with 20, 30, and 40 µg/mL of ozone instead of single-dose ozonation with 40, 60, and 80 µg/mL of ozone reduced the potential adverse effects of ozone on Hb including protein instability and oligomerization. Moreover, it was found that for certain residues, their orientation or displacement leads to the entry of excess water molecules into the heme moiety, which can contribute to Hb autoxidation. Additionally, the autoxidation rate was found to be higher in alpha globins compared to beta globins.