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

ABSTRACT 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.