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Buxus sempervirens L. (European Box, Buxaceae) has been used in ethnomedicine to treat malaria. In the course of our screening of plant extracts for antiprotozoal activity, a CH2Cl2 extract from leaves of B. sempervirens showed selective in vitro activity against Plasmodium falciparum (IC50 = 2.79 vs. 20.2 µg/mL for cytotoxicity against L6 rat cells). Separation of the extract by acid/base extraction into a basic and a neutral non-polar fraction led to a much more active and even more selective fraction with alkaloids while the fraction of non-polar neutral constituents was markedly less active than the crude extract. Thus, the activity of the crude extract could clearly be attributed to alkaloid constituents. Identification of the main triterpene-alkaloids and characterization of the complex pattern of this alkaloid fraction was performed by UHPLC/+ESI-QTOF-MS analyses. ESI-MS/MS target-guided larger scale preparative separation of the alkaloid fraction was performed by ‘spiral coil-countercurrent chromatography’. From the most active subfraction, the cycloartane alkaloid O-tigloylcyclovirobuxeine-B was isolated and evaluated for antiplasmodial activity which yielded an IC50 of 0.455 µg/mL (cytotoxicity against L6 rat cells: IC50 = 9.38 µg/mL). O-tigloylcyclovirobuxeine-B is thus most significantly responsible for the high potency of the crude extract.

Buxus sempervirens L. (European Box, Buxaceae) has been used in ethnomedicine to treat malaria. In the course of our screening of plant extracts for antiprotozoal activity, a CH2Cl2 extract from leaves of B. sempervirens showed selective in vitro activity against Plasmodium falciparum (IC50 = 2.79 vs. 20.2 μg/mL for cytotoxicity against L6 rat cells). Separation of the extract by acid/base extraction into a basic and a neutral non-polar fraction led to a much more active and even more selective fraction with alkaloids while the fraction of non-polar neutral constituents was markedly less active than the crude extract. Thus, the activity of the crude extract could clearly be attributed to alkaloid constituents. Identification of the main triterpene-alkaloids and characterization of the complex pattern of this alkaloid fraction was performed by UHPLC/+ESI-QTOF-MS analyses. ESI-MS/MS target-guided larger scale preparative separation of the alkaloid fraction was performed by 'spiral coil-countercurrent chromatography'. From the most active subfraction, the cycloartane alkaloid O-tigloylcyclovirobuxeine-B was isolated and evaluated for antiplasmodial activity which yielded an IC50 of 0.455 μg/mL (cytotoxicity against L6 rat cells: IC50 = 9.38 μg/mL). O-tigloylcyclovirobuxeine-B is thus most significantly responsible for the high potency of the crude extract. [ABSTRACT FROM AUTHOR]

• Comparison of high performance- and spiral-coil countercurrent chromatography. • Recovery of bioactive neolignans by countercurrent chromatography in the gram range. • Off-line electrospray mass-spectrometry of neolignan targets in fractions. • Accurate measurement of compound retention volumes, peak widths and co-elution. • Partition ratio KD based projection of countercurrent chromatography. Nectandra leucantha (Lauraceae) is a tree indigenous to the tropical Atlantic forests of Brazil, one of the most biodiverse flora hotspots worldwide. This plant species contains high concentrations of neolignan and dehydrodieugenol derivatives that express significant in-vitro activities against various parasite strains. These activities are however responsible for severe tropical human infections, such as Leishmaniasis (Leishmania spp.) and Chagas disease (Trypanosoma cruzi), which have been classified by the World Health Organization (WHO) as Neglected Tropical Diseases (NTDs). In order to optimize the isolation process for these target metabolites, n -hexane extract of the leaves was separated by means of semi-preparative high performance countercurrent chromatography (HPCCC) and scale-up spiral-coil countercurrent chromatography (sp −CCC) systems. Several biphasic solvent mixtures were evaluated for their partitioning effects on neolignans, resulting in the selection of an optimized system n -hexane – ethylacetate – methanol – water (7:3:7:3, v/v/v/v). The chromatographic experiments on the HPCCC and sp- CCC were run in the head-to-tail mode with 500 mg and 16 g injections, respectively. For specific and multiple metabolite detection, the recovered CCC-fractions were off-line injected, in the sequence of recovery, to an electrospray mass-spectrometry (ESI-MS/MS) device. A projection of the single ion traces of the target compounds, in the positive ionization mode at a scan range of m/z 100–1500, located chromatographic areas where the co-elution effects occurred and pure target metabolites were present. Five major target neolignans were specifically detected, which enabled the accurate pooling of CCC-fractions for an optimum recovery of the metabolites. The direct comparison of the performance characteristics of the two CCC-devices, with very different mechanical designs was achieved by the conversion of the time axis into a partition ratio (K D) separation scale. As a result, the compound specific K D - elution values of the target neolignan were determined in high precision, while the comparison of the calculated separation factor (α) and resolution factor (R S) values revealed a superior separation performance for the HPCCC system. Also, the reproducibility of detected metabolites in the two CCC experiments was confirmed by small variations (ΔK D ±0.1). Neolignan target compounds with anti-parasite activities were successfully isolated in the 100 mg to 4 g range in a single lab-scale countercurrent chromatographic process step. [ABSTRACT FROM AUTHOR]