Indigofera suffruticosa as a Source of Anti-Arboviral Phytoconstituents: Bioguided Fractionation, Isolation of 3-O-β-D-Glucosyl-β-Sitosterol, and In Vitro Evaluation Against ZIKV, CHIKV, and MAYV

Arboviruses transmitted by Aedes mosquitoes, including Zika (ZIKV), Chikungunya (CHIKV), and Mayaro (MAYV) viruses, lack approved antiviral therapies. This study evaluated the cytotoxicity and antiviral activity of ethanolic extracts and fractions from stems and leaves of Indigofera suffruticosa Mill. (Fabaceae), and isolated the active principles through bioguided fractionation. Extracts were obtained by cold maceration and partitioned into chloroform, ethyl acetate, and aqueous methanol fractions. Cytotoxicity was assessed by the MTT assay in MRC-5 and Vero cells, and antiviral activity was determined in simultaneous treatment-infection assays in Vero cells. The stem ethanolic extract showed potent anti-ZIKV activity (EC50 = 29.43 ± 1.43 µg/mL; SI > 13.59). The chloroform fraction displayed broad-spectrum efficacy, with EC50 = 89.48 ± 2.70 µg/mL (ZIKV; SI > 4.47), 37.05 ± 2.26 µg/mL (CHIKV; SI > 10.80), and 42.39 ± 3.60 µg/mL (MAYV; SI > 9.44). Bioguided fractionation afforded 3-O-β-D-glucosyl-β-sitosterol, which exhibited selective anti-alphavirus activity against CHIKV (EC50 = 151.76 µM; SI = 2.02) and MAYV (EC50 = 150.35 µM; SI = 2.03), but was inactive against ZIKV, indicating that additional constituents account for the anti-flaviviral effect. Indigofera suffruticosa is a promising source of broad-spectrum anti-arboviral agents, supporting further bioassay-guided isolation and mechanistic studies.