Exploring American Elderberry Compounds for Antioxidant, Antiviral, and Antibacterial Properties Through High-Throughput Screening Assays Combined with Untargeted Metabolomics

American elderberry (Sambucus nigra subsp. canadensis) is a rapidly emerging new perennial crop for Missouri, recognized for its high level of bioactive compounds with significant health benefits, including antibacterial, antiviral, and antioxidant properties. A high-throughput screening assay combined with untargeted metabolomics analysis was utilized on American elderberry juice from 21 genotypes to explore and characterize these bioactive compounds. Our metabolomics study has identified 32 putative bioactive compounds in the American Elderberry juices. An array of high-throughput screening bioassays was conducted to evaluate 1) total antioxidant capacity, 2) activation of antioxidant response elements (ARE), 3) antiviral activity, and 4) antibacterial activity of the putatively identified compounds. Our results revealed that 14 of the 32 American elderberry compounds exhibited strong antioxidant activity. Four compounds (isorhamnetin 3-O-glucoside, kaempferol, quercetin, and naringenin) activated ARE activity and were found to be non-cytotoxic to cells. Notably, six of the 32 compounds demonstrated significant antiviral activity in an in vitro TZM-bl assay against two strains of HIV-1 virus, CXCR4-dependent NL4-3 virus and CCR5-dependent BaL virus. Luteolin showed the most potent anti-HIV activity against the NL4-3 virus (IC50 = 1.49 {micro}M), followed by isorhamnetin (IC50 = 1.67 {micro}M). The most potent anti-HIV compound against the BaL virus was myricetin (IC50 = 1.14 {micro}M), followed by luteolin (IC50 = 4.38 {micro}M). Additionally, six compounds were found to have antibacterial activity against gram-positive bacteria S. aureus, with cyanidin 3-O-rutinoside having the most potent antibacterial activity in vitro (IC50 = 2.9 {micro}M), followed by cyanidin 3-O-glucoside (IC50 = 3.7 {micro}M). These findings support and validate the potential health benefits of compounds found in American elderberry juices and highlight their potential for use in dietary supplements as well as innovative applications in health and medicine.