In vitro antiplasmodial activity of prenylated chalcone derivatives of hops (Humulus lupulus) and their interaction with haemin

Sonja Frölich1, Carola Schubert1, Ulrich Bienzle2 and Kristina Jenett-Siems1,*

1 Institut für Pharmazie (Pharmazeutische Biologie), Freie Universität Berlin, Königin-Luise-Str. 2–4, D-14195 Berlin, Germany; 2 Institut für Tropenmedizin, Medizinische Fakultät der Charité, Humboldt-Universität, Spandauer Damm 130, D-13086 Berlin, Germany


* Corresponding author. Tel: +49-30-838-53720; Fax: +49-30-838-53729; Email: kjsiems{at}zedat.fu-berlin.de

Received 20 September 2004; returned 2 February 2005; revised 9 February 2005; accepted 20 February 2005


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: There is an urgent need to discover new antimalarials, due to the spread of chloroquine resistance and the limited number of available drugs. Chalcones are one of the classes of natural products that are known to possess antiplasmodial properties. Therefore, the in vitro antiplasmodial activity of the main hop chalcone xanthohumol and seven derivatives was evaluated. In addition, the influence of the compounds on glutathione (GSH)-dependent haemin degradation was analysed to determine its contribution to the antimalarial effect of chalcones.

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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
When blood stages of the malarial parasite Plasmodium falciparum enter human erythrocytes, they feed from enzymatic degradation of haemoglobin. Haemoglobin is ingested from the host cell and digested inside the parasite's food vacuole.1 The by-product of this digestion is toxic haemin, or ferriprotoporphyrin IX, which is detoxified by forming an insoluble polymer, malaria pigment or haemozoin.2 Recently, an alternative haem detoxification mechanism has been described. Non-polymerized haemin exits the food vacuole into the parasite's cytosol, where it is degraded by glutathione (GSH).3 Quinoline antimalarials—like chloroquine—have been shown to interfere with both detoxification pathways, thus leading to parasite death.4,5

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
RPMI 1640 medium was purchased from Gibco-BRL. GSH and diethylenetriamine-penta-acetic acid (DETEPAC) were obtained from Lancaster. Haemin, HEPES, Na phosphates, NaHCO3 and DMSO were obtained from Roth. [3H]Hypoxanthine was purchased from Amersham. Flat-bottomed 96-well plates were obtained from Neolab.

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 GSH–haemin interaction assay

The GSH–haemin 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 {Delta} 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The in vitro antiplasmodial activity was evaluated against the chloroquine-sensitive strain poW and the multiresistant clone Dd2 using a [3H]hypoxanthine-incorporation assay.19 Of the eight compounds tested (Figure 1), four were active with IC50 values < 25 µM against at least one of the two strains (Table 1). The main hop chalcone, xanthohumol (1), was most active, revealing IC50 values of 8.2 µM (poW) and 24.0 µM (Dd2), respectively. 2'',3''-Dihydroxanthohumol (2) gave an IC50 value of 12.9 µM against poW, and also two pyrano-derivatives (3, 4), where the prenyl residue forms an additional ring, possessed antiplasmodial activity with IC50 values of 16.4 and 23.7 µM (poW), respectively. 6'-Desmethylxantohumol (6), on the other hand, displayed only a moderate effect [IC50 values: 42.4 µM (poW); 92.1 µM (Dd2)]. Compounds 5 (2',4',4-trimethylxanthohumol) and 7 (2',4',4-trimethyl-6'-desmethylxanthohumol) were inactive, showing IC50 values > 100 µM against both strains, whereas the flavanone derivative 8 revealed moderate activity against the multiresistant clone Dd2 (IC50 value 55.3 µM), but no effect against poW (IC50 value > 100 µM).



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Figure 1. Chemical structures of chalcone and flavanone derivatives evaluated in this study.

 

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Table 1. IC50 values of chalcone derivatives tested against poW and Dd2 strains of P. falciparum

 
In the haemin-degradation assay, compounds 1, 2 and 4 displayed > 60% inhibition at a concentration of 11 µM, compared with 82% for chloroquine (Figure 2). Compounds 6 and 8 were weakly active, inhibiting haemin degradation by 36 and 24%, respectively, whereas the remaining derivatives showed no inhibition.



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Figure 2. GSH–haemin interaction assay. The effect of chalcone derivatives (11 µM) and chloroquine (11 µM) as reference compound on the interaction of 1 mM GSH with 5.7 µM haemin is given as % inhibition of haemin degradation compared with drug-free control. Values are the means of four assays ± SD.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this work, we evaluated the in vitro antiplasmodial activity of the major hop chalcone xanthohumol (1) as well as seven natural or semi-synthetic derivatives. The best activity was displayed by xanthohumol (1) itself. This is only the second time that prenylated natural chalcones were proven to possess antiplasmodial properties, but in contrast to 1, the prenyl residue of licochalcone A, which was isolated from Chinese liquorice roots,14 is attached to ring A of the chalcone skeleton. Nevertheless, the presence of the double bond in the prenyl side chain is irrelevant for the bioactivity, as indicated by the nearly identical potency of xanthohumol and its 2'',3''-dihydro derivative (2). Additionally, the two pyrano-derivatives 3 and 4, where the prenyl residue is cyclized to an additional ring, also show antiplasmodial activity. Compared with 1, desmethylxanthohumol (6), which only differs by the lack of the methyl residue at the C-6'-hydroxy group, is four to five times less active. This might be due to its higher hydrophilicity, which makes it more difficult to reach the site of action inside the parasite. Interestingly, the semi-synthetic methylated derivatives 5 and 7 are totally inactive, thus, as synthetic 2',4'-dimethoxychalcones revealed good antiplasmodial activity in an extensive structure–activity relationship study,15 the methylation of the hydroxy group at position 4 in particular seems to be less favourable.

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.


    Acknowledgements
 
We are indebted to Prof. R. Hänsel, Freie Universität Berlin, for providing the chalcone derivatives. Financial support by the DPhG to K. J.-S. is acknowledged.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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3 . Ginsburg, H., Famin, O., Zhang, J. et al. (1998). Inhibition of glutathione-dependent degradation of heme by chloroquine and amodiaquine as a possible basis for their antimalarial mode of action. Biochemical Pharmacology 56, 1305–13.[CrossRef][ISI][Medline]

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5 . Zhang, J., Krugliak, M. & Ginsburg, H. (1999). The fate of ferriprotorphyrin IX in malaria infected erythrocytes in conjunction with the mode of action of antimalarial drugs. Molecular and Biochemical Parasitology 99, 129–41.[CrossRef][ISI][Medline]

6 . Dorn, A., Vippagunta, S. R., Matile, H. et al. (1998). An assessment of drug-haematin binding as a mechanism for inhibition of haematin polymerisation by quinoline antimalarials. Biochemical Pharmacology 55, 727–36.[CrossRef][ISI][Medline]

7 . Basilico, N., Pagani, E., Monti, D. et al. (1998). A microtitre-based method for measuring the haem polymerization inhibitory activity (HPIA) of antimalarial drugs. Journal of Antimicrobial Chemotherapy 42, 55–60.[Abstract]

8 . Steele, J. C. P., Phelps, R. J., Simmonds, M. S. J. et al. (2002). Two novel assays for the detection of haemin-binding properties of antimalarials evaluated with compounds isolated from medicinal plants. Journal of Antimicrobial Chemotherapy 50, 25–31.[Abstract/Free Full Text]

9 . Wohlfart, R. (1993). Humulus. In Hagers Handbuch der Pharmazeutischen Praxis, 5th edn, vol. 5 (Hänsel, R., Keller, K., Rimpler, H. et al., Eds), pp. 447–58. Springer, Berlin, Heidelberg, Germany.

10 . Miranda, C. L., Stevens, J. F., Helmrich, A. et al. (1999). Antiproliferative and cytotoxic effects of prenylated flavonoids from hop (Humulus lupulus) in human cancer cell lines. Food Chemistry and Toxicology 37, 271–85.[CrossRef]

11 . Miranda, C. L., Stevens, J. F., Ivanov, V. et al. (2000). Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro. Journal of Agriculture and Food Chemistry 48, 3876–84.[CrossRef]

12 . Milligan, S. R., Kalita, J. C., Heyerick, A. et al. (1999). Identification of a potent phytoestrogen in hops (Humulus lupulus L.) and beer. Journal of Clinical Endocrinology and Metabolism 84, 2249–52.[Abstract/Free Full Text]

13 . Gerhäuser, C., Alt, A., Heiss, E. et al. (2002). Cancer chemopreventive activity of xanthohumol, a natural product derived from hop. Molecular Cancer Therapeutics 1, 959–69.[Abstract/Free Full Text]

14 . Chen, M., Theander, T. G., Christensen, S. B. et al. (1994). Licochalcone A, a new antimalarial agent, inhibits in vitro growth of the human malaria parasite Plasmodium falciparum and protects mice from P. yoelii infection. Antimicrobial Agents and Chemotherapy 38, 1470–5.[Abstract]

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16 . Hänsel, R. & Schulz, J. (1988). Desmethylxanthohumol: Isolierung aus Hopfen und Cyclisierung zu Flavanonen. Archiv der Pharmazie (Weinheim) 321, 37–40.

17 . Wohlfart, R. (1982). Beiträge zum Nachweis sedativ-hypnotischer Wirkstoffe in Humulus lupulus L (Cannabaceae). Thesis, Institut für Pharmazie, Freie Universität Berlin.

18 . Trager, W. & Jensen, J. B. (1976). Human malaria parasites in continuous culture. Science 193, 673–5.[ISI][Medline]

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20 . Miranda, C. L., Stevens, J. F., Helmrich, A. et al. (1999). Antiproliferative and cytotoxic effects of prenylated flavonoids from hops (Humulus lupulus) in human cancer cell lines. Food and Chemical Toxicology 37, 271–85.[CrossRef][ISI][Medline]

21 . Milligan, S. R., Kalita, J. C., Pocock, V. et al. (2000). The endocrine activities of 8-prenylnaringenin and related hop (Humulus lupulus L.) flavonoids. Journal of Clinical Endocrinology & Metabolism 85, 4912–5.[Abstract/Free Full Text]

22 . Li, R., Kenyon, G. L., Cohen, F. et al. (1995). In vitro antimalarial activity of chalcones and their derivatives. Journal of Medicinal Chemistry 38, 5031–7.[ISI][Medline]





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