This Issue is Dedicated to Professor Kurt Hostettmann On the Occasion of his 65th Birthday Volume 4. Issue 10. Pages 1319-1448. 2009 ISSN 1934-578X (printed); ISSN 1555-9475 (online) www.naturalproduct.us
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EDITOR-IN-CHIEF DR. PAWAN K AGRAWAL Natural Product Inc. 7963, Anderson Park Lane, Westerville, Ohio 43081, USA
[email protected] EDITORS PROFESSOR ALESSANDRA BRACA Dipartimento di Chimica Bioorganicae Biofarmacia, Universita di Pisa, via Bonanno 33, 56126 Pisa, Italy
[email protected] PROFESSOR DEAN GUO State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100083, China
[email protected] PROFESSOR J. ALBERTO MARCO Departamento de Quimica Organica, Universidade de Valencia, E-46100 Burjassot, Valencia, Spain
[email protected] PROFESSOR YOSHIHIRO MIMAKI School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan
[email protected] PROFESSOR STEPHEN G. PYNE Department of Chemistry University of Wollongong Wollongong, New South Wales, 2522, Australia
[email protected] PROFESSOR MANFRED G. REINECKE Department of Chemistry, Texas Christian University, Forts Worth, TX 76129, USA
[email protected] PROFESSOR WILLIAM N. SETZER Department of Chemistry The University of Alabama in Huntsville Huntsville, AL 35809, USA
[email protected] PROFESSOR YASUHIRO TEZUKA Institute of Natural Medicine Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan
[email protected] PROFESSOR DAVID E. THURSTON Department of Pharmaceutical and Biological Chemistry, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, UK
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HONORARY EDITOR PROFESSOR GERALD BLUNDEN The School of Pharmacy & Biomedical Sciences, University of Portsmouth, Portsmouth, PO1 2DT U.K.
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ADVISORY BOARD Prof. Berhanu M. Abegaz Gaborone, Botswana Prof. Viqar Uddin Ahmad Karachi, Pakistan Prof. Øyvind M. Andersen Bergen, Norway Prof. Giovanni Appendino Novara, Italy Prof. Yoshinori Asakawa Tokushima, Japan Prof. Lee Banting Portsmouth, U.K. Prof. Anna R. Bilia Florence, Italy Prof. Maurizio Bruno Palermo, Italy Prof. Josep Coll Barcelona, Spain Prof. Geoffrey Cordell Chicago, IL, USA Prof. Samuel Danishefsky New York, NY, USA Prof. Duvvuru Gunasekar Tirupati, India Prof. A.A. Leslie Gunatilaka Tucson, AZ, USA Prof. Stephen Hanessian Montreal, Canada Prof. Kurt Hostettmann Lausanne, Switzerland Prof. Martin A. Iglesias Arteaga Mexico, D. F, Mexico Prof. Jerzy Jaroszewski Copenhagen, Denmark Prof. Leopold Jirovetz Vienna, Austria Prof. Teodoro Kaufman Rosario, Argentina Prof. Norbert De Kimpe Gent, Belgium
Prof. Hartmut Laatsch Gottingen, Germany Prof. Marie Lacaille-Dubois Dijon, France Prof. Shoei-Sheng Lee Taipei, Taiwan Prof. Francisco Macias Cadiz, Spain Prof. Anita Marsaioli Campinas, Brazil Prof. Imre Mathe Szeged, Hungary Prof. Joseph Michael Johannesburg, South Africa Prof. Ermino Murano Trieste, Italy Prof. Virinder Parmar Delhi, India Prof. Luc Pieters Antwerp, Belgium Prof. Om Prakash Manhattan, KS, USA Prof. Peter Proksch Düsseldorf, Germany Prof. Satyajit Sarker Wolverhampton, UK Prof. Raffaele Riccio Salerno, Italy Prof. Monique Simmonds Richmond, UK Prof. Valentin Stonik Vladivostok, Russia Prof. Hiromitsu Takayama Chiba, Japan Prof. Karen Valant-Vetschera Vienna, Austria Prof. Peter G. Waterman Lismore, Australia Prof. Paul Wender Stanford, USA
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Natural Product Communications
A Protocol for HPLC-based Activity Profiling for Natural Products with Activities against Tropical Parasites
2009 Vol. 4 No. 10 1377 - 1381
Michael Adamsa, Stefanie Zimmermanna, b, Marcel Kaiserb, Reto Brunb and Matthias Hamburgera,* a
Institute of Pharmaceutical Biology, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland
b
Department of Medical Parasitology and Infection Biology, Swiss Tropical Institute, Socinstrasse 57, CH-4002 Basel, Switzerland
[email protected] Received: July 7th, 2009; Accepted: September 10th 2009
HPLC based activity profiling is an effective strategy to accelerate the discovery of new hits and leads from nature. It conveniently combines the superior separation power of HPLC micro scale compound separation with miniaturized biological screening methods, and on-line and off-line spectroscopy (PDA, MSn, HR-MS, NMR) for structure elucidation. We here describe a protocol for the discovery of natural products with antimalarial, antileishmanial and antitrypanosomal activity, from extracts libraries in 96-well format. Analytical gradient HPLC on a 3 x 150 mm column of 350 μg of extract, and collection of one-minute fractions into 96 deep-well microtiter plates, parallel evaporation of the micro-fractions, and a suitable dilution scheme permitted parallel activity profiling against three parasites from a single HPLC injection. The protocol was validated with extracts and positive controls such as Artemisia annua. Keywords: HPLC based activity profiling, Plasmodium, Trypanosoma, Leishmania.
Malaria, various forms of trypanosomiases and leishmaniases are insect transmitted tropical diseases caused by protozoal parasites. Due to increasing resistance to currently used antimalarial drugs there is a great need for the discovery of new agents with no cross-resistance to current drugs. There is also an urgent need to replace old drugs with severe side effects like arsenic containing melarsoprol used to treat Trypanosoma brucei rhodesiense (African sleeping sickness) and T. cruzi (Chagas disease). Successful natural products in the area of tropical parasitic diseases have been quinine from Cinchona succirubra Pav. ex Klotzsch (Rubiaceae), artemisinin from Artemisia annua L. (Asteraceae), and the avermectins from Streptomyces avermitilis [1,2], and numerous natural products have since been studied in vitro and in vivo [3].
complex mixtures which need to be separated to identify the actives substances. The most commonly used approach is preparative bioassay guided isolation. It is tedious, time-consuming and, hence, not suited for a medium-throughput setting. In contrast, HPLC-based activity profiling overcomes most of these limitations and provides effective means for early compound dereplication. Compared to classical bioassay guided isolation, the process of fractionation and testing is separated from the isolation process [4]. We have previously developed HPLC-based profiling protocols for the discovery of COX-2, 5-LOX, MAO-B and iNOS inhibitors, and GABAA-receptor agonists [5-8]. We here describe a profiling protocol for discovery of new antiplasmodial, antitrypanosomal and antileishmanial natural products.
To discover new hits and leads against tropical diseases thousands of compounds (or extracts) have to be tested in medium-throughput screens. Unlike pure compounds, extracts from natural sources are
When developing such protocols, methodological and technical issues need to be considered. These include, among others, sensitivity and signal-to-noise ratio of the bioassay, assay requirements with respect to
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solvents and additives. HPLC conditions have then to be tailored in a manner as to meet these specifications. Appropriate hardware is needed, such as fraction collectors for 96 deep-well plates, tubing with minimized dead volumes, and on-line detectors such as PDA, low- and high resolution MS, and access to a dedicated on- or off-line NMR spectrometer. Spectroscopic information on active HPLC peaks can be compared with available databanks. Known active substances are rapidly dereplicated, and unnecessary isolation of well known compounds can be avoided. Hence, the number of true “hits” can be increased. The starting point to this work was a screening of our in house extract library of 640 plant and fungal extracts against Plasmodium falciparum, Leishmania donovani, and Trypanosoma brucei rhodesiense, at test concentrations of 4.8 and 0.8 µg/mL. Based on these test concentrations, on the one hand, and the format of our liquid library (10 mg/mL in DMSO) on the other, we developed a protocol that accommodated these conditions (Figure 1). We calculated that the theoretical test concentration in the assays would be 2.5 µg /mL for Plasmodium (final test volume of 200 µL), and 5 µg/mL for the less sensitive Leishmania, and Trypanosoma parasites (final test volume of 100 µL) when injecting 350µg of extract and collecting 35 one-minute fractions, under the hypothetical assumption of an equal distribution of all contained components over the time of separation (35 min). Obviously the distribution of the substances will never be even, if the separation is successful. The test volumes described here were derived from established assay protocols to fully integrate with ongoing screening activities at the Swiss Tropical Institute. The amount of extract (350 μg) and solvent (35 μL DMSO per injection) were compatible with an analytical HPLC separation and with micro-fractionation into 96 deepwell plates (see Experimental). To validate the profiling protocol we first tested negative controls. Since DMSO solutions are used in the extract library, pure DMSO (35 μL) was injected and fractionated according to the protocol. In the antiplasmodial assay, none of the fractions showed activity that was > 5 % above the untreated controls (data not shown). The background noise in the T. b. rhodesiense test and the L. donovani tests was higher, but always below 25 %. The concentration of DMSO in the test was 0.25% for Trypanosoma and Leishmania, 0.125% for Plasmodium at the higher
Adams et al.
test concentrations, and a sixth of this at the lower concentrations. At these DMSO concentrations no inhibition of the parasites’ growth was to be expected. Here we show data for the positive control, artemisinin, for Plasmodium falciparum (IC50 = 0.001 µg/mL), and melarsoprol for T. b. rhodesiense (IC50 = 0.003 µg/mL). We injected these controls at amounts that the lower of the two final test concentrations equalled the IC50 of the compounds in this assay. The HPLC and activity profiles are shown in Figures 2 and 3. The positive controls indeed appeared as clear activity peaks matching the HPLC peak of the compounds. Sample from extract library (10 mg/ml in DMSO)
Online collection of spectroscopic data. HPLC hyphenated methods: HPLC-DAD, -MSn, - HRMS, ELSD
HPLC separation
35 x 60 sec. fractions collected in 96 well micro titer plate
Drying of microfraction at 10 mbar in GeneVacTM EZ 2 for 24 hours NMR in 1mm TXI microtubes stock solution of micro fraction 5 µL of DMSO and 95 µL PBS buffer
5 ug ml added to 195 µµL assay buffer with P. falciparum
5 ug ml added to 95 µL assay buffer with T. b. rhodosiense
Measurement of inhibition + measurement in 1:6 dilution by scintillation counter
Measurement of inhibition + measurement in 1:6 dilution by microplate fluorometer
5 ug ml added to 95 µL assay buffer with L. donovani
Measurement of inhibition + measurement in 1:6 dilution by microplate fluorometer
Activity profile of HPLC microfractions is established
Figure 1: Scheme of the HPLC based activity profiling protocol against P. falciparum, T. b. rhodesiense, and L. donovani.
Artemisinin had a retention time of 21.6 minutes, which is also where the anti-plasmodial activity was found. Melarsoprol with its retention time of 8.7 minutes was also active in this profiling assay against T. b. rhodesiense. As a next step we profiled an EtOAc extract of Artemisia annua, to test the applicability of the protocol for more complex extracts. A. annua contains artemisinin, which had already been used as a positive control (Figure 2). Again, the peak of activity corresponded with the HPLC peak of artemisinin (Figure 4). Due to the high potency of artemisinin and the fact that the compound elutes towards the end of fraction 21, activity in the profiling was found in fractions 21 and 22.
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Natural Product Communications Vol. 4 (10) 2009 1379
H
H
O O
O O
H
% Inhibition of Plasmodium falciparum
% Inhibition Plasmodium falciparum
O H
O O
0
5
10
15
20
25
O H
H
O O
30
Figure 2: Activity trace (in one minute fractions) of the positive control artemisinin against P. falciparum. The chromatogram is the ESI positive MS trace m/z 150 -1500. 9.6 ng of artemisinin was injected, leading to 0.006 µg/mL at the higher concentration (shown here) and 0.001 µg/mL at the lower test concentration, which is the IC50 of the substance. Separation parameters for all the following chromatograms: SunFire RP18 3.5 µm, 3 * 150 mm, A= H2O (+ 0.1% formic acid), B: AcCN (+ 0.1% formic acid) 90% - 0%A in 30 min, 100% B for 5 min, flow rate 0.5 mL/min, The chromatogram is the ESI positive MS trace m/z 150 1500.
0
5
10
15
20
25
30
Figure 4: Activity profile against Plasmodium falciparum (in minutes) of an EtOAc extract of Artemisia annua (10mg/mL stock solution, 35µL were injected) at the lower test concentration. The chromatogram is the ESI positive MS trace m/z 150 -1500.
OH O
O O OH
O
NH2 N
N S
H2N
N
NH
OH
As S
Figure 5: Example of HPLC – based activity profiling: Activity trace (in minutes) of an EtOAc extract of Pistacia atlantica (10mg/mL stock solution, 35µL were injected) and UV trace at 254nm of the one minute fractions tested against P. falciparum at the lower test concentration [9].
and characterized. The compound had an IC50 of 3.4 µM towards P. falciparum K1 strain [9].
Figure 3: Activity profile (in minutes) of the positive control melarsoprol against T. b. rhodesiense. 14.4 ng was injected, leading to 0.003 µg/mL at the lower test concentration, which is the IC50 of the substance. The higher test concentration is shown in grey, the lower one in white. The chromatogram is the PDA trace at 245 nm.
Once the basic methodology had been established we applied the protocol to extracts that had been active in the screening. One example is shown in Figure 5. In the initial screen an EtOAc extract of Pistacia atlantica DC. (Anacardiaceae) had been tested active against P. falciparum with 73.7% inhibition at a concentration of 4.9 µg/mL. In the HPLC profiling, the anti-plasmodial activity was confined to fraction 26. HPLC-MSn, LC-HRMS and off-line NMR (500 MHz, 1mm TXI probe) data collected for the fraction, when compared with the literature and to a natural product databank, suggested that the active substance was not yet known. 3-Methoxycarpachromene was subsequently isolated
As we have shown here, HPLC based activity profiling is an efficient way to quickly identify substances in complex biological extracts which exhibit antiplasmodial, antitrypanosomal and antileishmanial activity. The separation protocol was adapted to the specific needs of the test system. A single injection of 350 µg extract on an analytical HPLC column afforded sufficient material to be tested in a medium-throughput antiplasmodial assay in 96-well format. We have shown the testing of positive controls for Plasmodium falciparum und Trypanosoma b. rhodesiense (Figures 2, 3), known extracts (Figure 4) and the identification of a new antiplasmodial compound with this method (Figure 5). We currently profile various active extracts from our library screening with the aid of this protocol. Experimental General experimental procedures: Analytical grade solvents for extraction and HPLC grade solvents for
1380 Natural Product Communications Vol. 4 (10) 2009
chromatography were purchased from Scharlau (Barcelona, Spain). HPLC grade water was obtained by an EASY-pure II (Barnstead; Dubuque IA, USA) water purification system. Formic acid (98.0 – 100.0 %) was from Sigma-Aldrich (Buchs, Switzerland). Artemisinin was from Sigma Aldrich and melarsoprol from Sanofi-Aventis. Preparation of samples: Extracts that had been tested active in a previous screen and were available as 10 mg/mL DMSO solutions were used for further testing. Negative control was DMSO. Substances used as positive controls were prepared in a way that the lower of the two final test concentrations was the substances IC50. Generally substances were separated in the same equipment under exactly the same conditions as those used to obtain spectroscopic data with hyphenated techniques. The MS and NMR data were, however, not collected within the same run, because the mechanical post column splitter was not considered reliable enough to guarantee an ideal reproducible split ratio. The HR-MS data were recorded on a different machine with an identical HPLC setup linked to a microTOF system. HPLC separation and on-line spectroscopy: HPLC separations were carried out on an Agilent series 1100 system equipped with degasser, binary high pressure mixing pump, column thermostat and photodiode array (PDA) detector (all Agilent, Waldbronn, Germany). A Gilson 215 liquid handler with Gilson 819 injection module and 50 µL loop was used as autosampler (Gilson; Mettmenstetten, Switzerland). Separation conditions: SunFire RP-18 column (3.5 um, 3 x 150 mm i.d.; Waters GmbH, Eschborn, Germany), H2O (+0.1% FA) 90%-0% in 30 min, 100% MeCN (+0.1% FA) for 5 min. Flow rate was 0.5 mL/min, and injection volume was 35 µL (350 µg extract in DMSO). The HPLC was coupled to an Esquire 3000 Plus ion trap mass spectrometer equipped with an electrospray (ESI) interface (Bruker Daltonics; Bremen, Germany). High-resolution mass spectra were obtained on a microTOF ESI-MS system (Bruker Daltonics) connected to an Agilent 1100 series HPLC, as described above. MS 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. Low- and high-resolution mass spectra were recorded in positive and negative ion modes. Data acquisition
Adams et al.
and processing for all HPLC systems was performed using HyStar 3.0 software (Bruker Daltonics). Micro-fractionation and processing for bioassay: Sixty second fractions starting from minute 0.00 were collected on a FC 204 fraction collector (Gilson, Middleton, WI, USA) into deep well plates (ScreenMates 96 well, Matrix Technology, Hudson, USA) during the run and dried in a GeneVac EZ-2 Plus evaporator (Genevac Ltd, Ipswich, UK), at 40°C using the pre-installed “HPLC-fractions” vacuum settings. Typical evaporation time was 24 h. The dried microfractions were dissolved in 5 µL of DMSO and then diluted with 95 µL of PBS buffer (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4). This gave the stock solution for the higher test concentration (4.8 μg/mL), which could be used for any of the three assays. A 1:6 dilution in the corresponding parasite culture medium was prepared for the lower test concentration (0.8 μg/mL) Testing against Plasmodium falciparum strain K1: A modification of the [3H]-hypoxanthine incorporation assay was used for determining intraerythrocytic inhibition of parasite growth. The stock solution (5 µL) was transferred into 96 well plates (Costar, USA) containing 95 µL culture medium per well. Infected erythrocytes (100 µL per well with 2.5% hematocrit and 0.3% parasitemia) were added to each drug plate in duplicate. After 48 h incubation, 0.5 µCi of [3H]hypoxanthine in 50 μL medium was added and plates were incubated for an additional 24 h. Parasites were harvested onto glass-fiber filters and radioactivity was counted using a Betaplate liquid scintillation counter (Wallac, Zürich, Switzerland). The results were recorded as counts per minute (cpm) per well at each drug concentration and expressed as percentage of the untreated controls [10]. Testing against Trypanosoma brucei rhodesiense: Minimum Essential Medium supplemented according to Baltz et al. [11] with 2-mercaptoethanol and 15% heat-inactivated horse serum was used for determining the inhibition of parasite growth [12]. Stock solution (5 µL) was transferred to 96 well plates (Costar, USA) containing 50 µL culture medium per well. Bloodstream forms of T. brucei rhodesiense STIB 900 in 45 µL of medium were added to each well and the plate incubated at 37°C under a 5% CO2 atmosphere for 72 h. Resazurin solution (12.5 mg resazurin dissolved in 100 mL distilled water) (10 µL) was then added to each well
HPLC based activity profiling
Natural Product Communications Vol. 4 (10) 2009 1381
and incubation continued for a further 2–4 h. The plate was then read in a Spectramax Gemini XS microplate fluorometer (Molecular Devices Cooperation, Sunnyvale, CA, USA) using an excitation wavelength of 536 nm and emission wavelength of 588 nm [12]. Fluorescence development was measured and expressed as percentage of the control.
plate incubated at 37°C under a 5% CO2 atmosphere for 72 h. Resazurin solution (12.5 mg resazurin dissolved in 100 mL distilled water) (10 µL) was then added to each well and incubation continued for a further 2–4 h. The plate was then read in a Spectramax Gemini XS microplate fluorometer (Molecular Devices Cooperation, Sunnyvale, CA, USA) using an excitation wavelength of 536 nm and emission wavelength of 588 nm. Fluorescence development was measured and expressed as percentage of the control.
Testing against Leishmania donovani: Culture mediumSM at pH 5.4 supplemented with 10% heatinactivated FBS (50 μL), was added to each well of a 96-well microtiter plate (Costar, USA) [13]. Then 5 μL of the stock solution and axenically grown L. donovani amastigotes (strain MHOM/ET/67/L82) in 45 μL medium were added to each well and the
Acknowledgments - The authors are indebted to the Swiss National Science Foundation for funding (grant 316000-113109).
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Volatile Constituents of Ocimum minimum Herb Cultivated in Portugal Krystyna Skalicka-Woźniak, Agnieszka Ludwiczuk, Jarosław Widelski, Joao J. Filipe, Yoshinori Asakawa and Kazimierz Głowniak
1383
Volatile Components from Selected Tahitian Liverworts Agnieszka Ludwiczuk, Ismiarni Komala, André Pham, Jean-Pierre Bianchini, Phila Raharivelomanana and Yoshinori Asakawa
1387
Evaluation of Chemical Variability of Cured Vanilla Beans (Vanilla tahitensis and Vanilla planifolia) Christel Brunschwig, François Xavier Collard, Jean-Pierre Bianchini and Phila Raharivelomanana
1393
Composition and Biological Activity of Essential Oils from Protium confusum Ana I. Santana, Roser Vila, Alex Espinosa, Dionisio Olmedo, Mahabir P. Gupta and Salvador Cañigueral
1401
Review/Account How Can Phytochemists Benefit from Invasive Plants? Peihong Fan and Andrew Marston
1407
MS-based Plant Metabolomic Approaches for Biomarker Discovery Jean-Luc Wolfender, Gaetan Glauser, Julien Boccard and Serge Rudaz
1417
Decades of Phytochemical Research on African Biodiversity Sami A. Khalid
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Natural Product Communications 2009 Volume 4, Number 10 Contents Original Paper
Page
Secondary Metabolites from the Liverwort Ptilidium pulcherrimum Dong-Xiao Guo, Yu Du, Yan-Yan Wang, Ling-Mei Sun, Jian-Bo Qu, Xiao-Ning Wang and Hong-Xiang Lou
1319
Triterpenes from Warszewiczia coccinea (Rubiaceae) as Inhibitors of Acetylcholinesterase Angela I. Calderón, Johayra Simithy, Giuliana Quaggio, Alex Espinosa, Jose Luis López-Pérez and Mahabir P. Gupta
1323
Use of a Saponin Based Molluscicide to Control Pomacea canaliculata Snails in Southern Brazil R. San Martín, Claudio Gelmi, Jaime Vargas de Oliveira, José Luis Galo and Honorio Pranto
1327
The Effect of Eurycoma longifolia on Sperm Quality of Male Rats Kit-Lam Chan, Bin-Seng Low, Chin-Hoe Teh and Prashanta K. Das
1331
Two New Tropane Alkaloids from the Bark of Erythroxylum vacciniifolium Mart. (Erythroxylaceae) Emerson F. Queiroz, Boris Zanolari, Andrew Marston, David Guilet, Leila Burgener, Marçal de Queiroz Paulo and Kurt Hostettmann
1337
Characterization of Positional and Configurational Tropane Alkaloid Isomers by Combining GC with NPD, MS and FTIR Philippe Christen, Stefan Bieri and Orlando Muñoz
1341
Detection by UPLC/ESI-TOF-MS of Alkaloids in Three Lycopodiaceae Species from French Polynesia and Their Anticholinesterase Activity Raimana Ho, Niloufar Marsousi, Philippe Eugster, Jean-Pierre Bianchini and Phila Raharivelomanana
1349
Identification and Quantification of Flavonoids from Chuquiraga spinosa (Asteraceae) Amaya Landa, Raquel Casado and M. Isabel Calvo
1353
Fast Counter Current Chromatography of n-Butanolic Fraction from Senecio giganteus (Asteraceae) Nadjet Mezache, Séverine Derbré, Salah Akkal, Hocine Laouer, Denis Séraphin and Pascal Richomme
1357
Isolation and Activity of Two Antibacterial Biflavonoids from Leaf Extracts of Garcinia livingstonei (Clusiaceae) Adamu A. Kaikabo, Babatunde B. Samuel and Jacobus N. Eloff,
1363
HPLC Analysis and NMR Identification of Homoisoflavonoids and Stilbenoids from the Inter-bulb Surfaces of Scilla nervosa Merhatibeb Bezabih, Samson O. Famuyiwa and Berhanu M. Abegaz
1367
Ellagic Acid Derivatives from Syzygium cumini Stem Bark: Investigation of their Antiplasmodial Activity Claudia A. Simões-Pires, Sandra Vargas, Andrew Marston, Jean-Robert Ioset, Marçal Q. Paulo, An Matheeussen and Louis Maes
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A Protocol for HPLC-based Activity Profiling for Natural Products with Activities against Tropical Parasites Michael Adams, Stefanie Zimmermann, Marcel Kaiser, Reto Brun and Matthias Hamburger
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Continued inside back cover
