| Summary: | <i>Calendula officinalis</i> L. (Asteraceae), commonly known as English or pot marigold, is an herbaceous plant with edible flowers. In this study, UPLC-ESI-MS/MS analysis was used for tentative identification of compounds in marigold flower methanol extract (MFE). In addition, RP-HPLC-DAD analysis was used to quantify the flavonoids hesperidin and rutin in MFE. The antileishmanial potentials of the crude extract and compounds were evaluated against <i>Leishmania major</i> promastigotes and amastigotes. Further, in vivo 4-day antimalarial testing of the extract and compounds was carried out at doses of 25 mg kg<sup>−1</sup> per day using mice infected with ANKA strain of <i>Plasmodium berghei</i>, following standard procedure. Molecular docking studies were carried out to assess the binding mode of flavonoids against the vital targets of <i>L. major</i>, including pteridine reductase 1 and farnesyl diphosphate synthase enzymes. The in silico antimalarial potentials of flavonoids were evaluated against wild-type <i>Plasmodium falciparum</i> dihydrofolate reductase-thymidylate synthase and phosphoethanolamine methyltransferase enzymes. Twenty compounds were tentatively identified by UPLC-ESI-MS/MS analysis of MFE, of which, seven flavonoids, six saponins, three phenolic acids, three fatty acids, and a triterpene glycoside were identified. MFE phytochemical analysis revealed that hesperidin content was 36.17 mg g<sup>−1</sup> extract, that is, 9.9-fold their content of rutin (3.65 mg g<sup>−1</sup> extract). The method was validated to ensure reproducibility of the results. The tested samples exhibited antileishmanial potentials against <i>L. major</i> promastigotes, with IC<sub>50</sub> values of 98.62, 118.86, and 104.74 ng µL<sup>−1</sup> for hesperidin, rutin, and MFE, respectively. Likewise, hesperidin showed inhibitory potentials against <i>L. major</i> amastigote with an IC<sub>50</sub> value of 108.44 ± 11.2 µM, as compared to miltefosine. The mean survival time, parasitemia, and suppression percentages showed similar results for the three samples against ANKA strain of <i>P. berghei</i>. The docking studies showed good binding affinities of rutin and hesperidin with numerous H-bonding and van der Waals interactions. Marigold flowers are nutraceuticals, presenting important sources of bioactive flavonoids with potential against neglected tropical diseases.
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