The antileishmanial activity of novel oxygenated chalcones and their mechanism of action

Lin Zhaia,b, Ming Chena,b, Jens Blomb, Thor G. Theanderc, Søren Brøgger Christensend and Arsalan Kharazmi*,a

a Centre for Medical Parasitology, Department of Clinical Microbiology, University Hospital (Rigshospitalet), Copenhagen, Denmark b Department of Molecular Cell Biology, Institute for Medical Microbiology and Immunology, Copenhagen University, Copenhagen, Denmark c Statens Serum Institut, Institute for Medical Microbiology and Immunology, Copenhagen University, Copenhagen, Denmark d Department of Medicinal Chemistry, The Royal Danish School of Pharmacy, Copenhagen, Denmark


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our previous studies have shown that licochalcone A, an oxygenated chalcone, has antileishmanial and antimalarial activities, and alters the ultrastructure and function of the mitochondria of Leishmania spp. parasites. The present study was designed to investigate the antileishmanial activity and the mechanism of action of a group of new oxygenated chalcones. The tested oxygenated chalcones inhibited the in-vitro growth of Leishmania major promastigotes and Leishmania donovani amastigotes. Treatment of hamsters infected with L.donovani with intraperitoneal administration of two oxygenated chalcones resulted in a significant reduction of parasite load in the liver and the spleen compared with untreated control animals. The oxygenated chalcones also inhibited the respiration of the parasite and the activity of mitochondrial dehydrogenases. Electron microscopic studies illustrated that they altered the ultrastructure of the mitochondria of L. major promastigote. The data clearly indicate that this group of oxygenated chalcones has a strong antileishmanial activity and might be developed into a new antileishmanial drug. The antileishmanial activity of oxygenated chalcones might be the result of interference with function of the parasite mitochondria.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The World Health Organization (WHO) has identified leishmaniasis as a major and increasing public health problem, particularly in Africa, Asia and Latin America.1 The total number of infected people in the world is estimated to be 12 million, and 350 million live in areas where the diseases can be transmitted to man. Three million individuals suffer from various forms of leishmaniases, the number of new cases being in the order of 1.5 million each year, of which 500 000 are visceral leishmaniasis.2 In Europe, the disease is endemic in many of the southern countries, and imported leishmaniasis is a growing problem, and the emergence of AIDS-related leishmaniasis has added a new dimension to the problem.3,4 The presently used antileishmanial drugs are in general toxic, expensive and require long-term treatment.1 The large-scale clinical resistance to the drug of first choice, antimonial agents, has been reported in India and Sudan.3,5 Therefore, there is a great and urgent need for the development of new, effective and safe drugs for the treatment of leishmaniasis.

Our previous studies have shown that licochalcone A, an oxygenated chalcone, has strong antileishmanial activity.6,7 Recently, we reported that 2,4-dimethoxy-4' -butoxychalcone, a novel oxygenated chalcone, exhibited potent activity against human malaria parasite Plasmodium falciparum, in vitro, and rodent parasites Plasmodium berghei and Plasmodium yoelii, in vivo.8 Licochalcone A alters the ultrastructure of the parasite mitochondria and inhibits their function.9 The present study was designed to investigate the antileishmanial activity of a group of new oxygenated chalcones and their mechanism of action. The data indicate that the tested oxygenated chalcones inhibit the in-vitro growth of Leishmania major promastigotes and Leishmania donovani amastigotes, reduce the parasite load in the liver and the spleen of hamsters infected with L. donovani, and alter the ultrastructure and the function of the parasite mitochondria.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Oxygenated chalcones

The tested oxygenated chalcones were synthesized in our laboratory and were dissolved in 1% dimethyl sulphoxide (DMSO) in medium 199 to prepare a working solution of 1 g/mL. The structure of the tested oxygenated chalcones is shown in Figure 1.



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Figure 1. Chemical structure of the oxygenated chalcones.

 
Animals

Male golden hamsters (Mesocricetus auratus), with a body weight of approximately 50 to 60 g, were used in this study.

Parasite cultures

One strain of L. major (MHOM/IL/67/LRC-L137) and two strains of L. donovani (MHOM/KE/85/NLB 439 and MHOM/SD/00/1SD2D) were used. Promastigotes were cultured in medium 199 containing 10% heat-inactivated fetal calf serum. Incubation and growth of the parasite were carried out at 26°C. Promastigotes were harvested on day four of the culture and used. The axenic amastigotes of MHOM/SD/00/1SD2D were cultured using a modified method originally described by Doyle et al.10 Briefly, after 7 days of culture at 26°C, the promastigotes were centrifuged at 500g for 10 min, supernatant was removed and the pellet was resuspended in Schneider's Drosophila medium with 20% HFCS. The cultures were carried out at 37°C in a 95% air:5% CO2 atmosphere for 7 days.

Effect on promastigotes

The effect of oxygenated chalcones on promastigotes was assessed as described previously. 6 Promastigotes were incubated at 26°C in the presence of different concentrations of oxygenated chalcones, or the medium alone in 96-well flat-bottom microtitre plates (Nunc, Roskilde, Denmark). After 2 h, 1 µCi of 3H-thymidine (Amersham International plc, Amersham, UK) was added to each well. Parasites were harvested 18 h later and 3H-thymidine incorporation was measured. All cultures were performed in triplicate.

Effect on intracellular amastigotes

The study was performed on human peripheral blood monocyte-derived macrophages (MDM) as described previously.6 Briefly, 200 µL of a suspension containing 2x 106 human peripheral blood mononuclear cells were added to each well in 96 well flat-bottom microtitre plates. After 4 h and 3 days of incubation at 37°C in 5% CO2, the old medium was removed and the cells were washed three times and then replaced with fresh medium. After 6 days, 200 µL of 1 x 107/mL stationary phase L. donovani promastigotes were added to each well. After 24 h, the cultures were washed three times and incubated in the medium containing different concentrations of oxygenated chalcones or the medium alone. Three days after infection, macrophages were lysed by 0.01% sodium dodecyl sulphate (SDS) and the cultures were incubated ar 26°C and the amastigotes were left to transform into promastigotes. Parasite growth was determined by adding 1 µCi of 3H-thymidine to each well after 48 h incubation, the parasites were harvested 20 h later, and 3H-thymidine incorporation was measured.

Effect on L. donovani infection in hamsters

The in-vivo activity of 35m4ac and 24m4ac in hamsters infected with L. donovani was examined according to a method described previously.7 On day 0, hamsters received intracardial injections of 1x 109 stationary phase L. donovanipromastigotes in 0.1 mL PBS. One hour after inoculation, five of the hamsters were killed by carbon dioxide asphyxiation. The liver and the spleen from the hamsters were removed and weighed, and impression smears were made. The slides were fixed with absolute methanol, stained with Giemsa stain and examined by light microscopy. Parasite load in the liver and the spleen was determined according the method described by Stauberet al.11 The parasite load in the spleen was also estimated by a 3H-thymidine uptake method as described previously.7 Briefly, the tissues were cut into small pieces and homogenized. The supernatants containing the released parasites were cultured in 15 mL of RPMI 199 containing 10% HFCS in a 25 cm2, 50 mL culture flask (Nunc, Roskilde, Denmark) at 28°C. After 3 days of incubation, 1 mL of the culture supernatant was centrifuged at 1000 r.p.m. for 10 min, washed with medium three times, resuspended in 1 mL of fresh medium and then 200 µL of the culture solution was transferred to round-bottom microtitre plates. The cultures were then pulsed with 1 µCi of 3H-thymidine. After 18 h of incubation the cultures were harvested and 3H-thymidine incorporation was measured. From day one, groups of five hamsters were each injected intraperitoneally with 5 or 20 mg of 35m4ac or 24m4ac per kg body weight in 0.1 mL of PBS od for 6 consecutive days. Five hamsters received the same volume of PBS as negative control and another five hamsters received 400 mg of Pentostam (Sodium Stibogluconate Injection BP, The Wellcome Foundation Ltd., London, UK) per kg body weight as positive control. On day eight, all hamsters were killed by carbon dioxide asphyxiation. Their liver and spleen were removed and weighed, and impression smears were prepared. Parasite load in the liver and the spleen was determined. The parasite load in the spleen was also estimated by 3H-thymidine uptake method.

Ultrastructure studies

Electron microscopic studies were carried out to examine the effect of oxygenated chalcones (35m4ac, 24m4hc, 24m4ac and 34m4ac) on the ultrastructure of the promastigote form of the parasite as described previously.6 Briefly, a suspension of 3 x 106 L. major promastigotes per millilitre was incubated in the presence of either oxygenated chalcones or medium alone for 24 h at 26°C. After incubation, the promastigotes were centrifuged, resuspended in 1 mL of medium, fixed with 3% glutaraldehyde and embedded in Noble agar. After postfixation in OsO4, specimens were stained in 2% uranyl acetate and embedded in Vestopal W. The sections were poststained with magnesium uranyl acetate and lead citrate and were examined in a Philips 201 C electron microscope at 60 KV.

Respiration of the parasite (oxygen consumption and changes of carbon dioxide and pH)

A previously described method was used for this study.9 Briefly, a suspension of 4.95 mL of L. major promastigotes (2 x 10 6/mL) or L. donovani axenic amastigotes (5x 107/mL) were incubated at 26°C in the presence of 50µL of oxygenated chalcones in different concentrations or medium alone in sealed bottles. Oxygen consumption and changes of carbon dioxide and pH were measured at 2, 24, 48 and 72 h after incubation using an acid-base laboratory ABL4 (Radiometer, Copenhagen, Denmark).

Activity of mitochondrial dehydrogenases

The effect of oxygenated chalcones on the activity of parasite mitochondrial dehydrogenases was determined by an MTS method as described previously.12 In this method, the substrate, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulphonyl)-2H-tetrazolium (MTS), is converted into a soluble formazan-like dye-complex by the parasite mitochondrial dehydrogenase. Briefly, 5 x 104 promastigotes were seeded in 96-well flat-bottom microtitre plates and incubated with different concentrations of oxygenated chalcones or medium alone at 26°C. After 24 h, 25 µL MTS/PMS was added to each well and further incubated for 3 h at 37°C, where after the optical densities (ODs at 492 nm) were measured directly using a Titre-Tech 96-well scanner.

Statistical analysis

A paired two-tailed t-test was used for analysis of the data. Values of P < 0.05 were considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effect on the in-vitro growth of L. major promastigotes and on L. donovani amastigotes in human MDM

Figure 2 shows the effect of the oxygenated chalcones on the in-vitro growth of L. major promastigotes. All 10 oxygenated chalcones exhibited a clear concentration-dependent inhibitory effect. In comparison with the control, the oxygenated chalcones showed a significant inhibitory effect on the in-vitro growth of promastigotes at concentrations of 1.5 µM and above (P <0.05).



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Figure 2. Effect of 10 oxygenated chalcones on the in-vitro growth of L. major promastigotes. Promastigotes (3 x 106) were incubated in the presence of 1.5, 3, 15 and 30 µM of oxygenated chalcones for 2 h followed by 18 h of incubation in the presence of 3H-thymidine. The results are from six experiments and are given as percentage of inhibition (mean ± S.E.M.) as measured by 3H-thymidine incorporation. The mean level of incorporation of 3H-thymidine in control cultures was 20.5 kCPM.

 
Figure 3 shows that six oxygenated chalcones markedly inhibited the in-vitro growth of L. donovani amastigotes in human MDM in a concentration-dependent manner. In comparison with the control, the oxygenated chalcones showed a significant inhibitory effect on the in-vitro growth of amastigotes at concentrations of 1.5 µM and above (P < 0.05).



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Figure 3. Effect of six oxygenated chalcones on the intracellular parasite killing. Human MDMs infected with L. donovani promastigotes were incubated in the presence of 1.5, 3, 15 and 30 µM of oxygenated chalcones for 3 days followed by SDS lysis and further incubation. The proliferation of surviving parasites was measured by 3H-thymidine incorporation. The results are from five experiments and are given as percentage of inhibition (mean ± S.E.M.). The mean level of incorporation of 3H-thymidine in control cultures was 11.5 kCPM.

 
The Table shows that the IC50 values of 13 oxygenated chalcones on the in-vitro growth of L. major promastigotes and the IC50 values of six oxygenated chalcones on L. donovani amastigotes in human MDM. The IC50 values of the oxygenated chalcones on amastigotes is much smaller than that on promastigotes, indicating that the inhibitory effect of the oxygenated chalcones on amastigotes is stronger than that on promastigotes.


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Table. Effect of oxygenated chalcones on the in-vitro growth of L. major promastigotes and on L. donovani amastigotes in human MDM
 
Effect on L. donovani infection in hamsters

In the hamster model, the total number of L. donovani parasites (as determined by the method of Stauber et al.11) in the liver were reduced by 97% and 84% when animals received intraperitoneal administration of 20 and 5 mg of 35m4ac per kg of body weight for 6 days (P <0.05; Figure 4a). The parasite load in the liver was also reduced by 88% and 70% when animals received intraperitoneal administration of 20 and 5 mg of 24m4ac per kg of body weight. 35m4ac and 24m4ac also significantly reduced the parasite load in the spleen as determined by both Stauber' s method and 3H-thymidine uptake method (P < 0.05; Figure 4b and c). When the pentavalent antimony, Pentostam was administered intraperitoneally at a dosage of 400 mg per kg of body weight for 6 days, the parasite load in the liver was reduced by 94%.



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Figure 4. Effect of 35m4ac, 20mg/kg ( ) and 5mg/kg ( ); 24m4ac, 20mg/kg ( ) and 5 mg/kg ( ); pentostam, 400mg/kg ( ); and control ( ) given intraperitoneally once a day for six days on the parasite load in the livers and spleens of hamsters infected with L. donovani promastigotes. Parasite loads were measured by the method of Stauber et al. 11 (a and b) and by 3H-thymidine uptake (c). Results are means ± S.E.M. for five hamsters in each group.

 
Effect on the ultrastructure of the mitochondria of L. major promastigotes

Electron microscopic studies were carried out to determine the possible ultrastructural changes of the parasite mitochondria after incubation with the oxygenated chalcones. Figure 5 shows representative samples of the effect of 35m4ac, 24m4hc and 24m4ac on the ultrastructure of the mitochondria of L. major promastigotes. The unaffected control promastigotes contained slender and dense mitochondria (Figure 5a). After adding 30 µM of different oxygenated chalcones, the mitochondria showed different degrees of enlargement, and a disarray, with irregularity and a loss of the cristae (Figure 5b, c and d). These changes did not differ from the effect of 34m4ac (data not shown).



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Figure 5. Representative electron micrographs of mitochondrial profiles from L. major promastigotes incubated in medium alone (a), in 30 µM of 35m4ac (b), in 30 µM of 24m4hc (c) and 30 µM of 24m4ac (d). The mitochondria in the control experiment are dense and slender (a), whereas mitochondria incubated with the oxygenated chalcones (b- d) show different degrees of enlargement and edema of the matrix together with irregularity of the cristae. Small arrowheads denote the inner membrane and large arrowheads denote the cristae of the mitochondria. Bar, 0.2 µm.

 
Effect on the respiration of the parasite

Figure 6 shows that 24m4ac, 24m4hc, 35m4ac and 34m4ac inhibited the respiration of L. major promastigotes in a concentration-dependent manner, as revealed by inhibition of the oxygen consumption of the parasites, the accumulation of CO2 and the decline of pH in the parasite culture. Among the four oxygenated chalcones, 34m4ac exhibited the strongest inhibitory effect on the respiration of the promastigotes, and the other three oxygenated chalcones inhibited the respiration of the parasite to almost the same degree. Licochalcone A also exhibited a strong inhibitory effect on the oxygen consumption, the accumulation of CO2, and the decline of pH in L. donovani axenic amasitigotes (data not shown).



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Figure 6. Time study on the effect of 24m4ac, 24m4hc, 35m4ac and 34m4ac on the oxygen content and changes of carbon dioxide and pH on L. major pomastigotes. Data are from six experiments and are given as mean ± S.E.M. *KPA, kilopascal. Control, ({square}); 15mg/L, (•); 30mg/L, ({circ}).

 
Effect on the mitochondria dehydrogenase of the parasite

Figure 7 shows a concentration-dependent inhibitory effect of some oxygenated chalcones on the activity of the mitochondria dehydrogenase in L. major promastigotes. The P values for the effect of most oxygenated chalcones (except 24mc, 25m4ac and 26m4ac) on the activity of the mitochondria dehydrogenase at concentrations of 1.5 µM and greater in comparison with the effect of the control were <0.05. The P values for the effect of 24mc and 26m4ac on the enzyme activity at concentrations of 3 µM and greater in comparison with the effect of the control were <0.05. At concentrations of 15 and 30 µM, the P values for the effect of 25m4ac in comparison with the effect of the control were <0.05.



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Figure 7. Effect of 10 oxygenated chalcones on the activity of mitochondrial dehydrogenase of L. major promastigotes. Promastigotes were incubated in the presence of 1.5, 3, 15 and 30 µM of oxygenated chalcones for 24 h followed by 3 h of incubation in the presence of the substrate. The data are from six experiments and are given as percentage of control (mean ± S.E.M).

 
Comparison of the effect on the in-vitro growth and on the activity of the mitochondrial dehydrogenase of L. major promastigotes

Figure 8 shows the comparison of the effect of six oxygenated chalcones on the in-vitro growth ofL. major promastigotes measured by 3H-thymidine incorporation and on the activity of the mitochondrial dehydrogenase of the parasite. Licochalcone A, 24mc and 34m4ac inhibited the 3H-thymidine incorporation and the activity of the mitochondrial dehydrogenase to the same degree. The inhibitory effects of 24m4ac and 35m4ac on the 3H-thymidine incorporation were slightly stronger than that on the activity of the mitochondrial dehydrogenase. At concentrations of 15 and 30 µM, 24m4hc exhibited a significantly stronger inhibitory effect on 3H-thymidine incorporation than on the mitochondrial dehydrogenase.



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Figure 8. Comparison the inhibitory effect of some oxygenated chalcones on the in-vitro growth of L. major promastigotes measured by the 3H-thymidine incorporation (•) and on the activity of the parasite mitochondrial dehydrogenase ({circ}). The data are from six experiments and are given as mean ± S.E.M.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Previously, we have demonstrated that licochalcone A, an oxygenated chalcone, has strong antileishmanial activity.6,7 The inhibition of the parasite growth was shown to be associated with alteration of the ultrastructure and the function of the parasite mitochondria.9 Here we present data demonstrating that a group of new oxygenated chalcones inhibit the in-vitro growth of the extracellular and intracellular forms of Leishmania spp. parasites, reduce the parasite load in the liver and the spleen of hamsters infected with L. donovani, and alter the ultrastructure, the respiration and the activity of dehydrogenases of the parasite mitochondria.

Similar to licochalcone A, all the tested oxygenated chalcones showed inhibition of the in-vitro growth of L. major promastigotes measured by 3H-thymidine incorporation in a concentration-dependent manner. The six oxygenated chalcones also exhibited a clear concentration-dependent inhibitory effect on the in-vitro growth of L. donovaniamastigotes in human MDM. These data imply that the DNA synthesis in the parasites was inhibited. At low concentrations, the inhibitoryeffect of the oxygenated chalcones on amastigotes was stronger than that on promastigotes. This effect could be a consequence of a direct action of chalcones on amastigotes, or interference with defence mechanism of amastigotes in the hostile environment of the macrophage. The third possibility could be that chalcones activate macrophages to kill amastigotes. Nevertheless, the important aspect of this finding is that the intracellular amastigote form of the parasite which is present in the in-vivo environment is more susceptible to these compounds. Treatment of hamsters infected with L. donovani with intraperitoneal administration of 35m4ac and 24m4ac at dosages of 5 and 20 mg/kg of body weight per day for 6 consecutive days resulted in a significant reduction of parasite load in the liver and the spleen of treated animals.

The studies on the ultrastructure of the L. major promastigotes showed that four oxygenated chalcones, 35m4ac, 24m4hc, 24m4ac and 34m4ac, destroyed the mitochondria while the other organelles of the parasite appeared normal. In a concentration-dependent manner, oxygenated chalcones inhibited the respiration of L. major promastigotes and L. donovani axenic amasitigotes, as shown by inhibition of O2 consumption and CO 2 production by the parasites. Oxygenated chalcones also inhibited the activity of the mitochondrial dehydrogenase of L. major promastigotes in a concentration-dependent manner. The mitochondrial dehydrogenase activity measured in this assay includes the activity of many dehydrogenases in mitochondrion, such as malate dehydrogenase and succinate dehydrogenase, and has been shown to be mitochondria specific.12 These findings indicate that the enzymes of the parasite respiratory chain might be the target for oxygenated chalcones.

The understanding of the mechanism of action will help the development of oxygenated chalcones into new antileishmanial drugs. In order to elucidate the mechanism of action of the oxygenated chalcones further, we have compared the effect of six oxygenated chalcones on the in-vitro growth of L. major promastigotes measured by 3H-thymidine incorporation into DNA and on the activity of the mitochondrial dehydrogenase of the parasite. Three of the oxygenated chalcones inhibited 3H-thymidine incorporation and the mitochondrial dehydrogenase to the same degree, indicating that the effect on DNA replication could be secondary to the effect on the mitochondrial dehydrogenase. In the other three oxygenated chalcones there was a disassociation between the effect on 3H-thymidine incorporation and on mitochondrial dehydrogenase. These findings indicate that the inhibition of the mitochondrial dehydrogenase of the parasite was probably not the only mechanism responsible for the effect of these chalcones on DNA replication. It has been reported that the trypanocidal activity of quinones is probably caused by their effect on specific enzymes, involved in DNA synthesis, or cell membrane. 13,14 Quinones bind easily to proteinic blood components, with interaction of the quinone moiety with basic free NH 2 residues, leading to the fixation of quinone to the protein. Oxygenated chalcones also easily bind to protein, and they might bind to specific enzymes, influencing function of mitochondria or DNA synthesis and resulting in the death of parasite. Li et al. have shown that some chalcones inhibit cysteine proteinases in malaria parasites P. falciparum.15 Our preliminary results indicate that the oxygenated chalcones described in this communication do not act through the cysteine proteinases of Leishmania spp. parasites (data not shown ).

Studies on the energy metabolism pathway in the mitochondria and other biochemical aspects of the parasite are warranted to clarify the exact mechanism of action of the oxygenated chalcones. Further elucidation of the mechanism of action of the oxygenated chalcones is important for the development of these compounds into a new class of antiparasitic drugs and could also help to design new antiparasitic drugs which act on some specific targets only present in the parasite.

In conclusion, the oxygenated chalcones exhibit a strong antileishmanial activity both in vitro and in vivo. This activity might be the result of interference with the function of the parasite mitochondria.


    Acknowledgments
 
MTS was a generous gift from Dr K. Berg and Professor T. C. Owen. Expert technical assistance of Anne Asanovski, Elisabeth Brakti, Bibi Spliid and Shahin Khabaz Bovani is acknowledged. This work was supported by a grant from the European Commission INCO-DC programme and a grant from the Foundation Idella.


    Notes
 
* Correspondence address. Department of Clinical Microbiology, Rigshospitalet 7806, Tagensvej 20, DK-2200 Copenhagen, Denmark. Tel: +45-35-45-77-34; Fax: +45-35-45-68-31; E-mail: Kharazmi{at}inet.uni2.dk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . UNDP/WB/WHO (1989). The leishmaniasis. In Tropical Diseases: Progress in International Research, 1987–1988 . WHO special programme for research and training in tropical diseases ninth programme report. World Health Organization, Geneva.

2 . Modabber, F. (1993). Leishmaniasis. In Tropical Disease Research. Progress 1991–92. (UNDP/World Bank/WHO special programme for research and training in tropical diseases). pp. 77–87. World Health Organization, Geneva.

3 . Olliaro, P. L. & Bryceson, A. D. M. (1993). Practical progress and new drugs for changing patterns of leishmaniasis. Parasitology Today 9, 323–8.[ISI]

4 . Alvar, J., Gutierrez-Solar, B., Pachon, I., Calbacho, E., Ramirez, M., Valles, R. et al. (1996). AIDS and Leishmania infantum : new approaches for a new epidemiological problem. Clinics in Dermatology 14, 541–6.[ISI][Medline]

5 . UNDP/WB/WHO. (1990). Special programme for research and training in tropical diseases (TDR). Antimonials: large-scale failure in leishmaniasis ` alarming' . TDR News 34, 1–7.

6 . Chen, M., Christensen, S. B., Blom, J., Lemmich E., Nadelmann, L., Fich, K. et al. (1993). Licochalcone A, a novel antiparasitic agent with potent activity against human pathogenic protozoan species of Leishmania. Antimicrobial Agents and Chemotherapy37 , 2550–6.[Abstract]

7 . Chen, M., Christensen, S. B., Theander, T. G. & Kharazmi, A. (1994). Antileishmanial activity of licochalcone A in mice infected with Leishmania major and in hamsters infected with Leishmania donovani. Antimicrobial Agents and Chemotherapy38 , 1339–44.[Abstract]

8 . Chen, M., Christensen, S. B., Zhai, L., Rasmussen, M. H., Theander, T. G., Frøkjaer, S. et al. (1997). The novel oxygenated chalcones, 2,4-dimethoxy-4' -butoxychalcone, exhibits potent activity against human malaria parasite Plasmodium falciparum in vitro and rodent parasites Plasmodium berghei and Plasmodium yoelii in vivo. Journal of Infectious Diseases 176,1327 –33.[ISI][Medline]

9 . Zhai, L., Blom, J., Chen, M., Christensen, S. B. & Kharazmi, A. (1995). The antileishmanial agent licochalcone A interferes with the function of parasite mitochondria. Antimicrobial Agents and Chemotherapy 39, 2742–8.[Abstract]

10 . Doyle, P. S., Engel, J. C., Pimenta, P. F., da-Silva, P. P. & Dwyer, D.M. (1991). Leishmania donovani : long-term culture of axenic amastigotes at 37°C. Experimental Parasitology 73, 326–34.[ISI][Medline]

11 . Stauber, L. A., Franchino, E. M. & Grun, J. (1958). An eight-day method for screening compounds against Leishmania donovani : in the golden hamster. Journal of Protozool 5, 269–73.

12 . Berg, K., Zhai, L., Kharazmi, A. & Owen, T. C. (1994). The use of a water-soluble formazan-complex to quantitate the cell number and mitochondrial function of Leishmania major promastigotes. Parasitolgy Research 80, 235–9.

13 . Goijman, S. G. & Stoppani, A. O. M. (1985). Effects of ß -Lapachone, a peroxide-generating quinoe, on macromolecule synthesis and degradation in Trypanosoma cruzi. Archives of Biochemistry Biophysics . 240, 273–80.[ISI][Medline]

14 . Pinto, A. V., Pinto, C. N., Pinto, M. C. F. R., Rita, R. S., Pezzella, C. A. C. & Castro, S. L. (1997). Trypanocidal activity of synthetic heterocyclic derivatives of active quinones from Tabebuia sp. Arzneimittel-Forshung Drug Research 47, 74–9.[Medline]

15 . Li, R., Kenyon, G. L., Cohen, F. E., Chen, X., Gong, B., Dominguez, J. D. et al. (1995). In vitro antimalarial activity of chalcones and their derivatives. Journal of Medical Chemistry 38, 5031–7.

Received 9 July 1998; returned 20 November 1998; revised 14 December 1998; accepted 14 February 1999