Diégina Araújo Fernandes¹, Brendo Araujo Gomes¹, Simony Carvalho Mendonça¹, Thamirys Silva da Fonseca¹, Mariana Freire Campos¹, Larissa Esteves Carvalho Constant¹, Alice Santos Rosa², Thamara Kelcya Fonseca Oliveira¹, Amanda Resende Tucci ², Júlia Nilo Henrique Lima², Vivian Neuza Santos Ferreira², Milene Dias Miranda², Diego Allonso¹, Gilda Guimarães Leitão¹, Suzana Guimarães Leitão¹

COVID-19 has resulted in over 778 million cases and more than 7 million deaths globally by June 2025. Although no longer classified as a pandemic, the continuous emergence of SARS-CoV-2 variants reinforces the urgent need for new antiviral strategies. Natural products are important sources of bioactive compounds in this context. Ampelozizyphus amazonicus and Sarcomphalus joazeiro (Rhamnaceae) are traditionally used in Brazil for respiratory ailments, making them relevant targets for bioprospecting. This study evaluated six extracts obtained from different parts of these species through ethanolic and aqueous extraction methods. The investigation began with in vitro antiviral assays using Calu-3 cells infected with SARS-CoV-2, followed by cytotoxicity testing via LDH release to assess safety and selectivity. After confirming antiviral activity, complementary mechanistic assays were conducted to explore the inhibition of key viral targets, including the Spike(RBD):ACE2 interaction and the viral proteases 3CL_pro and PL_pro. Chemical profiling of the extracts was performed using UHPLC-APCI/ESI(+/-)-MS². The data were processed with MZmine v2.53, applying intensity thresholds, deconvolution (Local Minimum Search), deisotoping, and alignment. Chemometric analyses correlated chemical features with biological responses, revealing bioactive markers linked to antiviral potential. All extracts maintained cell viability above 80% at 200 μg·mL⁻¹, supporting a favorable safety profile. At 50 μg·mL⁻¹, aqueous extract of A. amazonicus wood (SARLE) achieved >80% viral inhibition, aqueous extract of A. amazonicus bark (SARFLORA) >75%, and ethanolic extract of A. amazonicus bark (SETMA) >60%. Intermediate effects were observed for S. joazeiro leaf extract (SJFE, 56%) and A. amazonicus wood extract (SETLE, 35%). The ethanolic extract of S. joazeiro bark (SJCE) showed no antiviral activity in cell-based assays. In the mechanistic studies, SETMA exhibited the most potent inhibition of viral targets, reaching 92% inhibition of Spike(RBD):ACE2 binding, 81% of 3CLpro, and 82% of PLpro. SARFLORA and SETLE displayed moderate inhibition (47.3% to 56.9%), while SJCE and SJFE showed 20% inhibition for Spike, 51% for 3CL_pro, and 45% for PL_pro. SARLE did not inhibit any of the targets in this assay. Chemometric analyses consistently identified three molecular ions linked to the antiviral activity: [M–H]⁻ m/z 459.3, 551.2, and 597.1. These were annotated as C- and O-glycosylated flavonoids, known for their pharmacological versatility but not yet fully explored as multitarget antivirals. The ion m/z 597.1, corresponding to 3’-5’-di-C-glucopyranosyl-phloretin, was prioritized for further in silico docking studies, confirming its potential interaction with Spike(RBD), 3CLpro, and PLpro, thus supporting a multitarget inhibition profile. In conclusion, this work provides solid evidence of the antiviral potential of glycosylated flavonoids from A. amazonicus, reinforcing the need for ongoing isolation, characterization, and mechanistic validation. These findings contribute to the development of novel therapeutic options against SARS-CoV-2 and future coronavirus threats.

Agradecimentos: We acknowledge the support of CAPES, CNPq, and FAPERJ for funding projects and scholarships that made this work possible. Thanks are due to the Biosafety Level 3 (BSL3) laboratory facility in Pavilhão Leonidas Deane, Instituto Oswaldo Cruz. Fiocruz, RJ; and Andre Sampaio from Farmanguinhos, platform RPT11M, for kindly donating the Calu-3 cells.