Multilevel Residual Complexity Analysis Reveals Origin of Nanomolar Antiviral Bioactives of "Isoquercitrin"

The remarkable reported in vitro and in vivo antiviral activity of a commercial, naturally derived, isoquercitrin sample (IQC90) against Ebola (EBOV), Zika virus (ZIKV), and SARS-CoV-2 could not be confirmed with a greater purity isoquercitrin (IQC). To resolve this discrepancy, IQC90 was subjected to a two-step, quantitative bioassay-guided fractionation employing countercurrent separation and gel filtration monitored by inhibition of syncytium formation in HEK293 cells transfected with SARS-CoV-2 spike protein and ACE2. This process revealed the IQC90 antiviral activity to be due to a new family of 21-hydroxyoleanane-3-O-oligosaccharides, named dicitriosides, present at <1 mol %, rather than IQC. The two dominant dicitriosides, the hexoside, dicitrioside A₁ (1), and the pentoside, dicitrioside B₁ (2), inhibited syncytia formation with an IC₅₀ = 0.530 μM; 25-fold more active than IQC90 (IC₅₀ = 12.8 μM). Beyond anti-SARS-CoV-2 activity, dicitrioside B₁ (2) also prevented EBOV infection of Vero E6 cells, supporting the conclusion that the dicitriosides inherit the promising potential of IQC90 as antiviral leads for clinical translation. Ultrahigh field 1.1 GHz NMR spectroscopy, particularly 1D selective TOCSY experiments and nuclear genotyping via quantum-mechanical spin analysis, enabled structure elucidation and provided definitive reference points for the dicitriosides as complex oligoglycoside esters.