Despite the availability of effective vaccines, yellow fever outbreaks persist, highlighting the need for antiviral drugs. Background/Objectives: This study investigated Hippeastrum puniceum (Amaryllidaceae) as a potential source of antiviral compounds against wild-type yellow fever virus (wt-YFV). Methods/Results: The crude bulb extract of H. puniceum exhibited 58% protection against wt-YFV. Bioassay-guided fractionation of the extract by UHPLC-HRMS led to the annotation of six alkaloids (bulbisine, cathinone, trigonelline, tetrahydroharman-3-carboxylic acid, and 2,7-dimethoxyhomolycorine or 3-O-acetylnarcissidine) in active fractions, along with the amino acids arginine, asparagine, tryptophan, and glutamic acid. In silico ADMET analyses predicted favorable pharmacokinetic and toxicological profiles, supporting their potential as drug candidates. Six of the annotated compounds were evaluated in vitro for cytotoxicity and antiviral activity against wt-YFV. However, none showed significant antiviral activity when tested individually, suggesting that the observed antiviral effect may result from synergistic interactions between two or more compounds within active fractions. Conclusions: Our results underscore the importance of further investigations in vitro, particularly assays exploring the synergy among the annotated compounds against YFV. The integration of bioassay-guided fractionation of active plant extracts with computational analyses emerges as a promising strategy for the discovery of natural products with therapeutic potential against yellow fever, a reemerging disease.

Despite effective vaccines, yellow fever outbreaks persist, increasing the urgency to find antivirals for patient treatment. This study aimed to discover compounds with potential antiviral properties against the wild-type yellow fever virus (wt-YFV) in Hippeastrum puniceum, of the Amaryllidaceae family, through bioassay-guided fractionation of the crude bulb extract using ultra-high-performance liquid chromatography (UHPLC) coupled with high-resolution ESI-QTOF mass spectrometry and subsequent analysis of the pharmacokinetic and toxicological properties (ADMET) of the annotated compounds. In the active fractions against wt-YFV, six alkaloids were proposed such as bulbisine, cathinone, trigonelline, tetrahydroharman-3-carboxylic acid, and 2,7-dimethoxyhomolycorine (or 3-O-acetylnarcissidine). Co-occurring in these fractions were the amino acids arginine, asparagine, tryptophan, and glutamic acid. In silico ADMET analysis of the alkaloids predicted good absorption, distribution, excretion, favorable metabolization profiles, and non-mutagenic toxicity, indicating their potential for drug development. These findings justify further in vitro assessment of the compounds against YFV and in vivo testing in animal models. This strategy seems to be an efficient approach for discovering antiviral compounds against YFV. Such discoveries hold promise not only in combating YFV but also in addressing a spectrum of other flaviviruses.