1 Institut für Pharmazie (Pharmazeutische Biologie), Freie Universität Berlin, Königin-Luise-Str. 24, D-14195 Berlin, Germany; 2 Institut für Tropenmedizin, Medizinische Fakultät der Charité, Humboldt-Universität, Spandauer Damm 130, D-13086 Berlin, Germany
Received 20 September 2004; returned 2 February 2005; revised 9 February 2005; accepted 20 February 2005
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Abstract |
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Methods: In vitro antiplasmodial activity was evaluated against the chloroquine-sensitive strain poW and the multiresistant clone Dd2 using a [3H]hypoxanthine-incorporation assay. Inhibition of GSH-dependent haemin degradation was analysed by a multiwell plate assay at 11 µM.
Results: Of the eight compounds tested, four possessed activity with IC50 values<25 µM against at least one of the two strains of Plasmodium falciparum. The main hop chalcone, xanthohumol, was most active with IC50 values of 8.2±0.3 (poW) and 24.0 ± 0.8 µM (Dd2). Three of these compounds were additionally active in the haemin-degradation assay.
Conclusions: The results demonstrate for the first time the ability of chalcone derivatives to interfere with the haemin-degradation process of P. falciparum. This effect might contribute to their antiplasmodial activity. Nevertheless, as one compound showed inhibition of P. falciparum without being able to interact with GSH-dependent haemin degradation, other modes of action must add to the observed antiparasitic activity of hop chalcones.
Keywords: Plasmodium falciparum , xanthohumol , chalcones , GSH-dependent haemin degradation
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Introduction |
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In order to test the ability of compounds to interfere with either pathway, different in vitro assays have been developed, thus allowing the possible modes of action of antiplasmodial compounds to be investigated.68
The hops plant, Humulus lupulus L., of the family Cannabinaceae is a large, dioecious climber often cultivated. Secretory glands on the surface of the female flowers contain a volatile oil and also a resin consisting of bitter compounds, such as humulones and lupulones. In addition, polyphenols like flavonoids and chalcones are present, with, in particular, the prenylated chalcone derivative xanthohumol (XN) as a main constituent.9
Therapeutically, hop cones are used as mild sedatives, normally in combination with valerian (Valeriana officinalis L.). Numerous biological activities of different hop constituents have been reported, e.g. antimicrobial, antioxidative and cytotoxic activities.10,11 8-Prenylnaringenin has been identified as a potent phytoestrogen.12 Recently, the possible cancer chemopreventive activity of xanthohumol has been described, based on its ability to modulate the activity of enzymes involved in carcinogen metabolism and detoxification.13
As different chalcones are known to possess antiparasitic properties,14,15 we were prompted to evaluate the in vitro antiplasmodial activity of the main hop chalcone, xanthohumol, and seven natural or semi-synthetic derivatives, against two different strains of P. falciparum, as well as their interaction with GSH-dependent haemin degradation.
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Materials and methods |
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Test compounds were kindly supplied by Prof. Dr Rudolf Hänsel, Institut für Pharmazie, Freie Universität Berlin and analysed for structure and impurities.16,17 The purity of the substances (>95%) was checked by HPLC and thin layer chromatography (TLC).
In vitro antiplasmodial assay
P. falciparum strains poW (IC50 of chloroquine = 0.015 µM) and Dd2 (IC50=0.14 µM) were maintained in continuous culture in human red blood cells (A+) diluted to 5% haematocrit in RPMI 1640 medium supplemented with 25 mM HEPES, 30 mM NaHCO3 and 10% human A+ serum.18 Extracts and substances were dissolved in DMSO (20 mg/mL) and diluted in medium to final concentrations between 100 and 1.56 µg/mL. Antiplasmodial activity tests were performed in 96-well culture plates (CorningTM, Sigma-Aldrich) as described by Desjardins et al.19 Briefly, aliquots of 150 µL of parasitized culture (2.5% haematocrit, 0.5% parasitaemia) were exposed to two-fold dilutions of test substances. After incubation in a candle jar for 24 h, 0.5 µCi of [3H]hypoxanthine (1 mCi/mL) was added to each well and the plates incubated for a further 18 h. Cells were harvested onto glass fibre filters (Wallac) with a cell harvester (Inotech) and incorporated radioactivity was determined by a liquid scintillation counter (1450 Microbeta plus). All tests were performed in triplicate. The percentage of growth inhibition was calculated as: [1(cpm in drug treated cultures/cpm in untreated cultures)]x100. The concentration at which growth was inhibited by 50% (IC50) was estimated by interpolation.
Multiwell plate GSHhaemin interaction assay
The GSHhaemin interaction assay was performed as described by Steele et al.8 In brief, three stock solutions were prepared: 1 mM DETEPAC in 10 mM Na phosphate pH 7.0; 2 mM haemin in DMSO (prepared fresh daily); 100 mM GSH, 1 mM DETEPAC, 10 mM Na phosphate pH 6.8. For the experiments, working solutions were as follows: A, 4 vol. of DETEPAC/phosphate stock +1 vol. of ethanol; B, 5 µL of haemin stock solution per mL of solution A; and C, 0.15 mL of GSH stock solution per mL of solution A.
Assays were carried out in 96-well (400 µL) flat-bottomed plates. Solution A (100 µL) was added, followed by drug (2 µL of 2 mM drug stock in DMSO) or solvent control in eight parallel samples. Solution B (200 µL) was then added to all wells followed by 50 µL of solution C. Final concentrations of drug and haemin were 11 and 5.7 µM, respectively. The absorbance at 360 nm (A360) was measured after 1 and 30 min with a plate reader (Spectrafluor Tecan) to determine the A360. The effect of the haemin-binding compounds was evaluated as the percentage decrease compared with control absorbance. Mean and SD for the eight parallel samples of at least three independent experiments were calculated.
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Results |
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Discussion |
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When comparing our results with those obtained with human cells, P. falciparum is slightly more sensitive to XN (1) than different cancer cell lines and also macrophages.13,20 Real cytotoxic activity can only be observed at 100 µM, although antiproliferative effects become visible at lower concentrations depending on the cell line used. In the case of XN, this cytotoxicity is not due to an oestrogen-mimicking activity, since in contrast to the flavanone derivative 8-prenylnaringenin, XN does not display oestrogenic effects;21 instead, inhibition of DNA synthesis is discussed.20 Nevertheless, in order to develop XN as a new antiplasmodial lead compound, efforts to separate antiplasmodial and cytotoxic bioactivities have to be undertaken.
The exact mechanism of action of antiplasmodial chalcones is not known, although they are often considered to be cysteine protease inhibitors.22 Nevertheless, natural congeners characterized by a carbonyl moiety in position 9 and a free hydroxy group in position 2' might also be able to form complexes with haemin, thus interfering with its detoxification pathways. Therefore, we evaluated the influence of compounds 18 on GSH-dependent haemin degradation. Interestingly, the active compounds 1, 2 and 4 showed high inhibitory activity of > 60% in this assay, whereas the inactive derivatives 5 and 7 did not inhibit haemin degradation. Low activity of < 40% was observed for compounds 6 and 8, which is also in agreement with the antiplasmodial findings. However, compound 3 is an exception, in so far as it is active against P. falciparum but does not inhibit haemin degradation.
Those compounds possessing a 2'-methoxy group (5, 7) or a 2',3'-pyrano ring system (3) instead of a free hydroxy group obviously were not able to interact with haemin, stressing the importance of this structural feature for this type of bioactivity. Desmethylxanthohumol (6) and the flavanone derivative 8, on the other hand, were only weakly active despite their free hydroxy group. In the case of cyclized chalcones such as flavanones and flavones, binding to haemin might not be possible via this structural feature because of the lesser flexibility of the skeleton. Finally, desmethylxanthohumol (6) is more easily isomerized to the analogous flavanone derivative in aqueous solutions,16 thus explaining its poor activity.
Our results demonstrate for the first time the ability of chalcone derivatives to interfere with the haemin degradation process of P. falciparum. This effect might contribute to their antiplasmodial activity. Nevertheless, as compound 3 showed inhibition of P. falciparum without being able to interact with GSH-dependent haemin degradation, other modes of action must contribute to the observed antiparasitic activity of hop chalcones. Thus, studies evaluating their possible inhibition of cysteine proteases are currently under way.
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Acknowledgements |
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References |
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