Screening and HPLC-Based Activity Profiling for New Antiprotozoal Leads from European Plants

Based on a survey of remedies used in Renaissance Europe to treat malaria, we prepared and screened a library of 254 extracts from 61 plants for antiplasmodial activity in vitro. HPLC-based activity profiling was performed for targeted identification of active constituents in extracts. One of the most remarkable results was the identification of onopordopicrin, a germacranolide sesquiterpene lactone isolated from Arctium nemorosum as a potent inhibitor of P. falciparum with an IC50 of 6.9 μM. It was tested similarly against Trypanosoma brucei rhodesiense, the parasite which causes African sleeping sickness. With an IC50 of 0.37 μM, onopordopicrin was one of the most potent natural products reported so far. Cytotoxicity was determined against rat myoblast L6 cells (IC50: 3.06).

chamber at 254 and 366 nm. Spots were also visualised with anis aldehyde-sulphuric acid reagent, which was prepared according to Wagner and Bladt [21].

HPLC ESI-MS
For micro fractionation and analysis of extracts an HPLC system consisting of a 1100 series low-pressure mixing pump with degasser module, column oven, and a 1100 series PDA detector (all Agilent, Waldbronn, Germany) was used. A Gilson 215 liquid handler with Gilson 819 injection module and 50 µl loop served as autosampler (Gilson; Mettmenstetten, Switzerland). The HPLC was coupled to an Esquire 3000 Plus ion trap mass spectrometer equipped with an electrospray (ESI) interface (Bruker Daltonics; Bremen, Germany). The MS parameters were as follows: Spectra were recorded under ion charge control conditions (ICCD 30 000) at a scan speed of 30 000 m/z/s with a Gauss filter with of 0.2 m/z. Nitrogen was used as a drying gas a flow rate of 10 L/min and as nebulising gas at a pressure of 30 psi. The nebulizer temperature was set 300 ° C. In the positive ion mode spectra were detected from 150-1500 m/z.

MPLC
A Büchi Sepacore system consisting of a control unit C-620, a fraction collector C-660, an UV photometer C-635, and two pump modules C-605 was used, with the following method. The column consisted of a cartridge (Büchi, ø 40 x150 mm) containing pressed silica gel (Silica gel 60, 0.040-0.063 mm, Merck, Darmstadt, Germany). A gradient system was used consisting of A (heptane) and B (ethyl acetate), starting at 100 % A and 0% B, and leading to 70% A and 30% B in 33 minutes, then to 20 % A and 80 % B in 31.5 minutes. The flow rate was 30 mL/min. Fractions were collected every 30 seconds. The sample was dissolved in A:B 1:1 at a concentration of 50 mg/mL and the injection volume was 10 ml.

Semi-preparative HPLC
Semi-preparative HPLC was done on an Agilent 1100 series HPLC system consisting of an 1100 series quaternary low-pressure mixing pump with degasser module, column oven, and a 1100 series PDA detector with a 1000 µL loop.) using a SunFire prep RP-18 column (5 µm, 10 x 150 mm, Waters GmbH, Eschborn, Germany). A gradient starting at 85% A (H 2 0 + 0.1% formic acid) and 15% B (acetonitrile + 0.1% formic acid) and leading to 40% A and 60% B in 15 minutes, then to 100 % B in another 5 minutes. Finally the column was flushed with 100% B for 7 minutes. The flow rate was 5 mL/min. The sample was dissolved in MeOH at a concentration of 50 mg/mL and the injection volume was 300 μl.

Preparative HPLC
Preparative HPLC was done on a SCL-10, HPLC system from Shimadzu (Kyoto, Japan).A SunFire ™ prep C18 OBD ™ (5 µm, 30x 150 mm, Waters, Ireland) was used. The gradient was isocratic for 30 min and consisted of acetonitrile:H 2 0 1: 1 at a flow rate of 30 ml/min. UV data were recorded from 220 to 500 nm. The samples were dissolved in acetonitrile at a concentration of 100 mg/ml and the injection volume was 300 μl.

High resolution Mass Spectrometry (micrOTOF)
High-resolution mass spectra were obtained on a micrOTOF ESI-MS system (Bruker Daltonics) connected to an Agilent 1100 series HPLC. Data acquisition and processing was performed using HyStar 3.0 software (Bruker Daltonics). Conditions for LC-TOF MS were as follows: spectra were recorded in the range of m/z 150-1500 in positive mode. Nitrogen was used as a nebulising gas at a pressure of 2.0 bar and as a drying gas at a flow rate of 9.0 L/min (dry gas temperature 240 °C). Capillary voltage was at 4500 V, endplate offset at -500 V, hexapole at 250.0 Vpp, skimmer 1 at −50 V and skimmer 2 at −22.5 V. Instrument calibration was performed using a reference solution of sodium formiate 0.1 % in isopropanol / water (1:1) containing 5 mM sodium hydroxide. Typical mass accuracy was ±2 ppm. The spectra were recorded in negative and positive mode in the range of m/z 150-1500.

NMR
NMR data were acquired at target temperature 18°C on a Bruker Avance III TM 500 MHz spectrometer (Bruker, Fällanden, Switzerland) operating at 500.13 MHz for 1 H, and 125.77 MHz for 13 C. A 1mm TXI microprobe with a z-gradient was used for 1 H-detected experiments; 13 C-NMR spectra were recorded with a 5 mm BBO probe head with z-gradient. NMR experiments were done as previously described [22]. For processing and evaluation Topspin 2.0 was used.

a. In vitro test against Trypanosoma brucei rhodesiense
Trypanosoma brucei rhodesiense (STIB 900) were grown in axenic medium as previously described [23].The compounds were tested using a modified Alamar Blue assay protocol [24] to determine the 50% inhibitory concentration (IC 50 ). Serial threefold drug dilutions were prepared in 96-well micro titer plates and 50 μl of T. b. rhodesiense STIB 900 bloodstream forms were added to each well except for the negative controls. Melarsoprol (Arsobal®, Sanofi-Aventis, Meyrin, Switzerland) was used as a reference drug. After 70 h of incubation Alamar blue marker (12.5 mg resazurin dissolved in 100 mL distilled water) was added. The plates were then incubated for an additional 2 to 5 h. A Spectramax Gemini XS micro plate fluorescence reader (Molecular Devices Cooperation, Sunnyvale, CA) with an excitation wavelength of 536 nm and an emission wavelength of 588 nm was used to read the plates. The IC 50 values were calculated from the sigmoidal growth inhibition curves using Softmax Pro software (Molecular Devices).

b. In vitro testing against Plasmodium falciparum
A modification of the [ 3 H]-hypoxanthine incorporation assay was used to determine the intra-erythrocytic antiplasmodial activity (Des Jardins 1979) of the extract library and purified compounds in 96 well plates. Chloroquine (Sigma-Aldrich) and artesunate (Mepha, Switzerland) were used as standard drugs. Briefly, infected human red blood cells in RPMI 1640 medium (100 µL per well with 2.5% haematocrit and 0.3% parasitaemia) were exposed to twofold serial drug dilutions in 96-well micro titer plates. After 48 h incubation, 0.5 μCi [ 3 H]-hypoxanthine was added to each well. The plates were incubated for further 24 h before being harvested using a Betaplate cell harvester (Wallac, Zürich, Switzerland) The radioactivity was counted with a Betaplate liquid scintillation counter (Wallac) as counts per minute per well at each drug concentration and compared to the untreated controls. IC 50 values were calculated from sigmoidal inhibition curves using Microsoft Excel. All assays were run in duplicate and repeated three times [25].

c. In vitro cytotoxicity testing
Cytotoxicity was assessed using a similar Alamar Blue assay protocol [23] whereby 4000 rat myoblast cells/well were seeded in RPMI 1640 medium. All following steps were according to the T. b. rhodesiense protocol. Podophyllotoxin (Sigma-Aldrich) was used as the reference drug.