Es were performed to uncover probable mechanisms of action. two. Components and Strategies two.1. Chemical compounds All chemicals have been of analytical grade. Culture media had been purchased from Welgene, Inc. (Seoul, South Korea), fetal bovine serum (FBS) and penicillin/streptomycin from Gibco Inc. (Life Technologies, Billings, MT, USA), DRAQ5TM from BioStatus Ltd. (Loughborough, UK), benznidazole from Epichem Pty Ltd. (Perth, Australia), and falcarindiol from ChemSpace US Inc. (Monmouth Junction, NJ, USA). Further reagents/solvents had been obtained from VWR International (Leuven, Belgium). two.2. Sample Collection Specimens of Helichrysum italicum (Roth) G. Don subsp. picardii (Boiss Reuter) Franco (everlasting, Rezafungin Protocol Asteraceae family members; voucher code XBH32) had been collected in Ria Formosa coastal lagoon (37 01 54.0 N eight 02 12.1 W), south Portugal, in July 2015. Crithmum maritimum L. (sea fennel, Apiaceae family members; voucher code XBH33) was collected from Aljezur beach (37 20 30.7 N 8 51 06.0 W), south Portugal, in August of 2015. Botanist Dr. Manuel J. Pinto (National Museum of All-natural Rogaratinib Protocol History, University of Lisbon, Botanical Garden,Plants 2021, 10,3 ofPortugal) performed the taxonomical classification. Voucher specimens are kept in the herbarium of XtremeBio’s laboratory (CCMAR, University of Algarve, Portugal). Sea fennel plants have been divided into stems, leaves, and flowers, although only flowers in the everlasting have been utilised. Plant material was oven-dried for 3 days at 40 C till full dryness, then powdered and stored at -20 C until required. two.three. Preparation on the Extracts Water extracts were prepared similarly to decoctions, by boiling 1 g of dried biomass for five min in 200 mL of ultrapure water. Hydro-ethanolic extracts have been prepared similarly to tinctures, by homogenizing 20 g of dried biomass in 200 mL of 80 aqueous ethanol for any week. Extracts were filtered (Whatman n four), vacuum and/or freeze-dried, and stored in a cool, dark, and moisture-free atmosphere. To get the vital oils (EOs), fresh biomass (500000 g, based on biomass availability) was cut into smaller pieces and subjected to hydro-distillation inside a Clevenger-type apparatus for 3 h; EOs have been dried with sodium sulphate, filtered, weighed, and stored in sealed glass vials at -20 C till further use. 2.4. Fractionation on the Active Extract Soon after a key screening of the extracts’ anti-trypanosomal activity (described in Section 2.five), the active extract, sea fennel’s decoction from flowers, was fractionated: a 500 mL decoction was prepared and subjected to a sequential liquid iquid partition applying solvents of increasing polarity (hexane, dichloromethane, chloroform, and ethyl acetate, 150 mL every single; fractions 1 to four, respectively). All fractions, such as the water residue (fraction 5), were vacuum concentrated and/or freeze-dried and stored till assessment for anti-trypanosomal activity within a secondary screening (described in Section two.five). two.five. Evaluation of In Vitro Anti-Trypanosomal Activity All mammalian cell lines, namely human osteosarcoma, U2OS, and Macaca mulatta kidney epithelial, LLC-MK2, cells, previously obtainable in C.B. Moraes laboratory, had been cultured in DMEM medium supplemented with 10 heat-inactivated FBS, one hundred U/mL penicillin, and 100 mg/mL streptomycin within a humid atmosphere (five CO2 , 37 C). LLC-MK2 cultures maintained the T. cruzi mammalian cycle in vitro and these tissue-derived trypomastigote types had been used to infect U2OS cells inside the anti-trypanosomal assay. Two T. cru.
