Isolation of antiplasmodial naphthylisoquinoline alkaloids from Ancistrocladus sp. through bioassay-guided fractionation

Abstract

Ancistrocladus is the only genus in the Ancistrocladaceae family and together with the closely related Dioncophyllaceae, they produce naphthylisoquinoline (NIQ) alkaloids. This class of compounds exhibits a very interesting large spectrum of bioactivities. Some of these compounds have shown very promising antiplasmodial activity. Leaves of an as yet unidentified Ancistrocladus sp collected in the region of Bonsolerive (Northwestern part of DRC), were investigated for their antiplasmodial activity. They were air dried at room temperature and ground. The ground plant material was extracted sequentially with n-hexane, dichloromethane, ethyl acetate, methanol, and water. Five extracts were obtained and all, except for the n-hexane extract, were assayed for their antiplasmodial activity using the Malaria SYBR Green I based assay. The results of the antiplasmodial screening of extracts showed that the methanol extract was the most active one (100 % inhibition at 10 and 20 μg/mL) and was thus chosen for further research. UPLC QTOF MS was used to chemically profile the sequentially extracted material and to identify naphthylisoquinoline alkaloids in the extracts. The methanol extract was fractionated using silica gel column chromatography to produce 18 fractions. TLC and UPLC QTOF MS were used to target fractions mainly containing naphthylisoquinoline alkaloids and based on this, five fractions were selected for screening against Plasmodium falciparum NF54 strains. They all exhibited good antiplasmodial activity (˃ 95 % inhibition at 5 and 10 μg/mL). One fraction was further fractionated using preparative HPLC and four sub-fractions collected. They were screened against P. falciparum NF54 strains. One sub-fraction showed an excellent activity with more than 82 % inhibition at 1 μg/mL. Three compounds were isolated from the active fractions and one compound from a non-screened fraction using preparative HPLC and LC-MS-SPE-NMR. Through NMR and ECD, the structures of three compounds were fully elucidated as ealamine A (43), ancistrocladinium A (44) and ancistrocyclinone A (45). The structure of one compound was elucidated with only the relative configuration determined (compound 46). The presence of ealamine A (43), which is a 7,8'-coupled hybrid-type NIQ (R configured at C-3 with oxygen function at C-6), in this plant material has a geo- and chemotaxonomic importance. Such NIQs are mostly present in Ancistrocladus plants from the Northwestern Congo Basin. The results of antiplasmodial screening of compounds revealed that ancistrocladinium A (44) and ealamine A (43) showed good activity (IC50 ˂ 10 μM) against the chloroquine-sensitive (NF54) and chloroquine-resistant (K1 and Indochina clones W2) strains with no cross-resistance towards drug resistant P. falciparum parasites (RI values < 1). This study reports for the first time the activity of ancistrocladinium A and ealamine A against the W2 strain of P. falciparum. Ancistrocyclinone A and compound 46 showed no or only minimal antiplasmodial activity. The inhibitory potential of ancistrocladinium A (44) and ealamine A (43) were less effective than that of some NIQs previously isolated from other Ancistrocladus species. Further studies will be carried beyond the scope of this work to isolate and identify additional NIQs for the purpose of identification of the species (understanding the NIQs pattern of the species) and identification of more potent antiplasmodial NIQs.