Dual Assay Validation of Rosmarinus officinalis Extract as an Inhibitor of SARS-CoV-2 Spike Protein: Combining Pseudovirus Testing, Yeast Two-Hybrid, and UPLC-Q Exactive Orbitrap-MS Profiling.

Introduction: This study evaluates the effectiveness of Traditional Chinese Medicine (TCM) extracts in blocking the interaction between the SARS-CoV-2 Spike protein and human ACE2 receptor, utilizing a dual-method approach to explore the antiviral potential of natural compounds.

Objectives: This work aims to evaluate the capability of TCM extracts in inhibiting the SARS-CoV-2 Spike protein and ACE2 receptor interaction using advanced biochemical assays.

Methods: A dual-method screening approach was utilized, beginning with a pseudovirus assay to assess the inhibition capabilities of TCM extracts in vitro, followed by a split-ubiquitin yeast two-hybrid (Y2H) system to validate interactions in live cells. Active compounds were characterized and quantified using UPLC-Q-Exactive-Orbitrap-MS.

Results: Among the 91 TCM extracts tested, Rosmarinus officinalis exhibited the most potent inhibition in both pseudovirus and Y2H assays, significantly reducing viral entry and disrupting the Spike-ACE2 interaction. Comprehensive chemical profiling via UPLC-Q-Exactive-Orbitrap-MS identified 132 compounds, including phenolics, flavonoids, and terpenoids.

Conclusion: This research validates the use of TCM extracts in viral inhibition strategies, demonstrating the utility of integrating traditional remedies with modern scientific approaches to discover new therapeutic agents.