Ace c18

HPLC assays were performed using a Shimadzu liquid chromatography system (Shimadzu Scientific Instruments Inc., Columbia, Maryland, USA) with an ACE C18 (100 × 4.6 mm) (Aberdeen, Scotland) column. Mobile phases used for HPLC contained acetonitrile/water 60:40 (flavone A) and 70:30 (flavone B) with 0.2% acetic acid and 0.05% triethylamine. Detection wavelength was at 245 nm with a temperature of 30 °C. Flow rate was 0.4 mL/min with run times of 11 and 10 min, respectively. LC solutions program was used to collect and analyze the data.

All starting materials were purchased from Sigma-Aldrich (St. Louis, MO, USA), Alfa-Aesar (Ward Hill, MA, USA), and TCI (Nihonbashi, Japan) and were used without further purification. Reactions were performed under an atmosphere of dry nitrogen. An Initiator microwave system (Biotage, Uppsala, Sweden) was used for microwave-assisted reaction. LC-MS was performed on a system consisting of an electrospray ionization (ESI) source in a LCMS-2020 liquid chromatography-mass spectrometer system (Shimadzu, Kyoto, Japan; column: Shim-pack GIS, 100 × 3.0 mm, 3 μm ODS). A Teledyne ISCO flash purification system (Lincoln, NE, USA) with various prepacked silica gel cartridges was used for flash column chromatography. ¹H- and ¹³C-NMR spectra were recorded in the indicated solvent on an AVANCE III HD (400 and 100 MHz for ¹H and ¹³C, respectively) spectrometer (Bruker, Billerica, MA, USA). Chemical shifts are reported as δ values in parts per million downfield from TMS (δ 0.0) as the internal standard in CD₃OD, DMSO-d₆ or CDCl₃. The purity of the compounds was evaluated on a Shimadzu reverse-phase analytical HPLC system (column: Ace C18, 150 × 4.6 mm, 3 μm). Purities of all compounds that were subjected to biological assay were >95%.

The pre-fractionation of crude venom was conducted using liquid-liquid partition with solvents of increasing polarity (n-hexane, ethyl acetate and butane). The hexane fraction (SaFrHex) and ethyl acetate fraction of S. annulatus (SaFrAcEt) were analyzed by reversed-phase ultra-performance liquid chromatography (RP-UPLC) using a binary UPLC system (20A Prominence, Shimadzu Co., Japan), in a C18 column (ACE C18, 5 μm, 100 Å, 250 mm × 4.6 mm). These fractions were diluted in methanol and applied in the column (10 μL). The column was eluted at a constant flow rate of 1 mL.min⁻¹ in a gradient of methanol (solvent B) and water (solvent A). Elution of the fractions were monitored in a range of 200–800 nm, using a photodiode array detector. The fractions with the same retention time were pooled. The biological activity was detected using in vitro incubation of fractions of L. (L.) infantum promastigotes and Trypanosoma cruzi trypomastigotes for 24 hours.