Moringa oleifera potential for the treatment and prevention of COVID-19 involving molecular interaction, antioxidant properties and kinetic mechanism

The COVID-19 pandemic, caused by the emergence of the SARS-CoV-2 virus in 2019, has led to a global state of emergency declared by the World Health Organization (WHO). The search for effective therapies against the virus is ongoing, and traditional medicine is emerging as a promising alternative, as many plants with known antiviral potential have been utilized during the pandemic. In this study, we investigated the inhibition of cysteine proteases (Mpro, PLpro, and papain) by bioactive compounds from Moringa oleifera leaves, with the ultimate goal of developing a novel treatment. We conducted a virtual screening based on the structure of the Mpro and PLpro enzymatic targets of SARS-CoV-2, for the selection of compounds with higher inhibitory potential. We then identified 14 bioactive compounds with high binding energy against Mpro and PLpro using AutoDock. Gamma-sitosterol was particularly promising since it showed higher interaction energy with Mpro (-8.6Kcal/mol) and PLpro (-7.4Kcal/mol). Phytochemicals in Moringa oleifera leaves were extracted using aqueous and hydromethanolic solvent system. It was found that polyphenols (4.20-5.01 mg GAE/g DM) and flavonoids (95.86-135.41 mg QE/ g DM) were the major constituents in the extracts. Antioxidant activity in the extracts, particularly DPPH scavenging activity (63.46-123.58 mg TE/g DM) and FRAP activity (48.14-49.66 mg TE/g DM), was high suggesting a potential for the prevention of oxidation and inflammation. Global and multiple alignment were conducted between Mpro, PLpro and papain, and results showed high conservation of the amino acid residues in the active site of the enzymes, suggesting a similar molecular catalysis mechanism. The extracts showed inhibitory activity against papain with the IC50 of 7.5 mg/mL for the hydromethanolic extract and 12.5 mg/mL for the aqueous extract. It was found in kinetic studies that hydromethanolic extract was a competitive inhibitor of papain, while aqueous extract was a hyperbolic non-competitive inhibitor, suggesting the presence of compounds with different interaction profiles to the enzymes. This study demonstrates that Moringa oleifera leaf extracts have substantial antioxidant activity and inhibit the model cysteine protease papain in vitro. These data, together with in silico docking against Mpro and PLpro, identify candidate phytochemicals that merit further evaluation; however, papain inhibition is a preliminary biochemical filter and does not by itself establish antiviral activity against SARS-CoV-2.