A bioactivity-leveling approach for component-specific evaluation in natural product extracts: Optimizing SARS-CoV-2 antiviral screening by controlling for pheophorbide A

The emergence of SARS-CoV-2 has intensified the search for novel antiviral agents, with plants representing a promising source due to the documented antiviral properties. Pheophorbides, chlorophyll breakdown products, have been demonstrated to impede viral entry of enveloped viruses. However, the frequent occurrence of pheophorbide A in plant extracts presents a challenge for antiviral screening, as it may obscure the discovery of novel antiviral compounds. The objective of this study was therefore leveling out the influence of pheophorbide A in an in vitro virus pretreatment assay, to prioritize extracts and fractions and support the identification of additional antiviral compounds in plant extracts. Using different experimental derived examples, the limitations and advantages of the strategy presented are discussed. Assay-derived protective effects were interpreted in relation to pheophorbide A quantification, allowing six out of 15 plant species to be prioritized for further investigations. Qualitative comparison of metabolite profiles of fractions with the extent of protective effect supported the prioritization process and led to the identification of candidate compounds potentially contributing to the observed protective effect in Primula veris L. beyond pheophorbide A. Overall, the results highlight the potential value of the proposed strategy for revealing bioactivity beyond known predominant effectors and suggest an applicability in natural product–based screening approaches.