Comparative antiplasmodial, leishmanicidal and antitrypanosomal activities of several biflavonoids.

Abstract

The antiplasmodial, leishmanicidal and antitrypanosomal activities of eight natural biflavonoids were estimated in vitro on a chloroquine-resistant strain of Plasmodium falciparum, axenically grown leishmania donovani amastigotes and Trypanosoma cruzi trypomastigotes and Trypanosoma brucei rhodesiense bloodstream forms. Lanaroflavone showed the highest antiplasmodial activity (I[C.sub.50] = 0.48 [micro]M), isoginkgetin was the most active leishmanicidal compound (I[C.sub.50] = 1.9 [micro]M), whereas ginkgetin (I[C.sub.50] = 11 [micro]M) and isoginkgetin (I[C.sub.50] = 13 [micro]M) showed the best antitrypanosomal activity in our assays. The cytotoxicity and the selectivity indices for the most active compounds were also estimated. Lanaroflavone exhibited a high selectivity index value (SI = 159), indicating selective antiplasmodial activity.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Biflavonoids; Antiplasmodial activity; Leishmanicidal activity; Antitrypanosomal activity; Selectivity index

Introduction

Protozoal diseases, and particularly malaria, leishmaniasis, Chagas disease and African trypanosomiasis, represent major causes of mortality in various tropical and subtropical regions. The situation is compounded by increasing treatment failures by current drugs. With the increase in the resistance of Plasmodium falciparum to more and more chemotherapeutic agents (Wellems and Plowe, 2001), morbidity and mortality due to malaria remain an important health problem in many developing countries. The different forms of leishmaniasis require expensive treatments, and the currently used medicines, pentavalent antimonials and/or pentamidine salts, show toxicity together with numerous side effects (Barrett and Gilbert, 2002). Trypanosoma cruzi is a protozoan that causes Chagas disease (American trypanosomiasis), an illness that affects approximately 16 million people in tropical and sub-tropical Americas. The drugs currently in use against this disease, nifurtimox and benznidazole, present several side effects and have limited efficacy (Kirchhoff, 2003). African trypanosomiasis is a vector-borne parasitic disease which is causing major health and economic problems in rural sub-Saharan Africa. The drugs currently used to treat sleeping sickness are far from satisfactory because of major side effects, increasing resistance and the unaffordable price for African countries (De Koning, 2001).

In an effort to discover new lead compounds for infectious diseases, several research groups screen plant extracts to detect secondary metabolites with relevant biological activities. Biflavonoids have recently come to the forefront of the polyphenol family for their antiviral and antituberculosis activities (Lin et al., 1999, 2001). In recent pharmacological studies, they are also reported to possess antinociceptive, antiinflammatory and cytotoxic activities (Bittar et al., 2000; Kim et al., 2000; Lin et al., 2000). Biflavonoids are characterized by two flavonoid monomeric units (flavone or flavanone) covalently linked either with C-C or C-O-C bonds. The monomeric units may be of the same or different structural types. Only few reports are available in relation with their antiprotozoal activity, but recently antiplasmodial activity was established for two compounds of this class (Ahmed et al., 2001; Suarez et al., 2003; Nunome et al., 2004). The aim of the present study was to examine eight biflavonoids for their antiplasmodial, leishmanicidal, and antitrypanosomal activities. Toxicity of the different compounds was also estimated on L-6 rat skeletal myoblast cells.

Materials and methods

Cupressuflavone, bilobetin, sciadopitysin and amentoflavone-4',4'",7,7"-tetramethyl ether were obtained commercially (LGC Promochem, Molsheim, France). The other examined compounds were isolated from plant species: Lanaroflavone was isolated by bioguided fractionation from the methanol extract of the aerial part of Campnosperma panamense Standl. collected in May 2000 in Southern Colombia (Weniger et al., 2001); Amentoflavone was isolated from Viburnum pichinchense Benth. leaves collected in 1996 near Medellin, Colombia (Lobstein et al., 2003); Ginkgetin and isoginkgetin were isolated from Ginkgo biloba L. leaves from the Ginkgo plantation S.C.A., Domaine de Saint-Jean d'Illac, France (Joyeux et al., 1995).

All tested compounds were dissolved in 10% DMSO and working stock solutions of 10 mg/ml were prepared in culture medium. Stock solutions were then further diluted in culture medium, as indicated for each test.

Antiplasmodial activity

Quantitative assessment of antimalarial activity in vitro was determined by means of the microculture radioisotope technique based upon the method previously described by Desjardins et al. (1979) and modified by Ridley et al. (1996). The assay uses the uptake of [[.sup.3]H]hypoxanthine by parasites as an indicator of viability. Continuous in vitro cultures of asexual erythrocytic stages of P. falciparum were maintained following the methods of Trager and Jensen (1976). Compounds were tested on K1 strain (multidrug pyrimethamine/chloroquine-resistant strain; Thaithong and Beale, 1981). Initial concentration of the compounds was 20 [micro]g/ml diluted with two-fold dilutions to make seven concentrations, the lowest being 0.32 [micro]g/ml. After 48 h incubation of the parasites with the compounds at 37[degrees]C, [[.sup.3]H]hypoxanthine (Amersham, UK) was added to each well and the incubation was continued for another 24 h at the same temperature. Chloroquine (Sigma C6628) and artemisinin (Sigma 36, 159-3) were used as positive references. The values given in Table 1 are means of two independent assays; each assay was run in duplicate.

Leishmanicidal activity

Fifty microlitres of culture medium, a 1:1 mixture of SM medium (Cunningham, 1977) and SDM-79 medium (Brun and Schonenberger, 1979) at pH 5.4 supplemented with 10% heat-inactivated fetal bovine serum (FBS), was added to each well of a 96-well microtiter plate (Costar, USA). Serial drug dilutions in duplicates were prepared covering a range from 30 to 0.041 [micro]g/ml. Then [10.sup.5] axenically grown Leishmania donovani amastigotes (strain MHOM/ET/67/L82) in 50 [micro]l medium were added to each well and the plate incubated at 37[degrees]C under a 5% C[O.sub.2] atmosphere for 72 h. Ten microlitres of resazurin solution (12.5 mg resazurin dissolved in 100 ml distilled water) were 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 (Raz et al., 1997). Fluorescence development was measured and expressed as percentage of the control. Miltefosin (Zentaris GmbH, Germany) was used as a positive reference.

Antitrypanosomal activity (T. cruzi)

Rat skeletal myoblasts (L-6 cells) were seeded in 96-well microtiter plates at 2000 cells per well per 100 [micro]l in RPMI 1640 medium with 10% FBS and 2 mM L-glutamine. After 24 h, 5000 trypomastigotes of T. cruzi Tulahuen strain C2C4 containing the [beta]-galactosidase (Lac Z) gene (Buckner et al., 1996) were added in 100 [micro]l per well with two times of a serial drug dilution covering a range from 30 to 0.041 [micro]g/ml. The plates were incubated at 37 [degrees]C in 5% C[O.sub.2] for 4 days. After 96h, the minimum inhibitory concentration (MIC) was determined microscopically. For measurement of the I[C.sub.50], the substrate CPRG/Nonidet was added to the wells. The colour reaction which developed during the following 2-4 h was read photometrically at 540 nm. Each experiment was run in duplicate and the result is the mean of these. Benznidazole (Roche) was used as a positive reference.

Antitrypanosomal activity (Trypanosoma brucei rhodesiense)

The assays were performed according to the procedures described by Freiburghaus et al. (1996). Working stock solutions of 180 [micro]g/ml in serum containing culture medium according to Baltz et al. (1985) were prepared. Hundred microlitres of the diluted compounds were pipetted in duplicate into the first row of a 96-well microtiter plate (Costar, USA). With complete culture medium, three-fold serial dilutions were prepared. After the addition of T. brucei rhodesiense bloodstream form trypanosomes from axenic culture, the concentrations of the compounds ranged from 90 to 0.13 [micro]g/ml. The total number of trypanosomes in each well was 2 x [10.sup.3]/100 [micro]l. The plate was then incubated for 72 h in a humidified atmosphere at 37[degrees]C in 5% C[O.sub.2]. Ten microlitres of resazurin solution (12.5 mg resazurin dissolved in 100 ml distilled water) were then added to each well and incubation continued for a further 2-4h. 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 (Raz et al., 1997). Fluorescence development was measured and expressed as percentage of the control. Melarsoprol (Arsobal) was used as a positive reference.

Cytotoxicity

The cytotoxicity assay of the examined compounds was done following the method of Page et al. (1993) with the modification of Ahmed et al. (1994). Cell line L-6 (rat skeletal muscle myoblasts) were seeded in 96-well Costar microtiter plates at 2 x 103 cells/100 [micro]l, 50 [micro]l per well in MEM supplemented with 10% heat inactivated FBS. A three-fold serial dilution ranging from 90 to 0.13 [micro]g/ml of compounds in test medium was added. Plates with a final volume of 100 [micro]l per well were incubated at 37[degrees]C for 72 h in a humidified incubator containing 5% C[O.sub.2]. Resazurin was added as viability indicator according to Ahmed et al. (1994). After an additional 2 h of incubation, the plate was measured with a fluorescence scanner using an excitation wavelength of 536 nm and an emission wavelength of 588 nm (SpectraMax GeminiXS, Molecular Devices). Podophyllotoxin (Polysciences Inc., USA) was used as a positive reference.

Calculations of IC[5.sub.0] values

For antiplasmodial activity, the compounds concentrations at which the parasite growth (= [[.sup.3]H]hypoxanthine uptake) was inhibited by 50% (I[C.sub.50]) was calculated by linear interpolation between the two concentrations above and below 50% (Huber and Koella, 1993). For leishmanicidal, antitrypanosomal and cytotoxic activities, data were transferred into the graphic programme Softmax Pro (Molecular Devices) which calculated I[C.sub.50] values from the sigmoidal inhibition curve.

Results and discussion

All the tested biflavonoids have a C-C interflavonyl linkage, with the exception of lanaroflavone 1, which has a C-4'"-O-C-8 linkage (Fig. 1). The substitution at C-4'" seems to have high relevance regarding antiplasmodial activity, since the I[C.sub.50] reported for compounds 4-7, which all bear a methoxyl group at C-4'", are in the low micromolar range, whereas cupressuflavone 2 and amentoflavone 3, bearing both a free hydroxyl group at C-4'", are inactive. For biflavonoids having a C-C interflavonyl linkage, there is also a tendency to increase the antimalarial activity with increasing numbers of methoxyl groups in the molecule. Compounds 1, 2 and 3, are apigenin dimers but, interestingly, they show marked differences of activity. Lanaroflavone 1, a biapigenin with a C-4'"-O-C-8 interflavonoid linkage is the most active compound in our antiplasmodial assay, whereas cupressuflavone 2 and amentoflavone 3, both with C-C interflavonoid linkages, are inactive.

Compounds 4-6 were found to be the most active in the leishmanicidal assay against L. donovani amastigotes. The increase of the leishmanicidal activity with increasing numbers of methoxyl groups in the molecule is not observed, as compounds 7 and 8, which bear 3 and 4 methoxyl groups, respectively, show only moderate activity. On the other hand, compounds 2 and 3, which bear no methoxyl groups, are inactive, and compound 1 shows only moderate activity. Four out of the eight examined biflavonoids show moderate antitrypanosomal activity against T. b. rhodensiense (1<I[C.sub.50]<20[micro]M). Two of them (5,6) show good antitrypanosomal activity against T. cruzi, with I[C.sub.50] values only 6-8 fold greater than the reference compound benznidazole.

[FIGURE 1 OMITTED]

We have also assessed the cytotoxicity of the biflavonoids against L6 cells in order to determine the ratio of cytotoxicity to antiprotozoal activity (selectivity index, SI). Considering leishmanicidal activity, the determined S[I.sup.b] values (ratio of cytotoxicity to leishmanicidal activity) are smaller than 10, indicating probable non selective cytotoxicity for all tested compounds. On the other hand, the ratio of cytotoxicity to antiplasmodial activity for lanaroflavone 1 (S[I.sup.a] = 159) appears to be of particular interest, since this compound shows at the same time good antimalarial activity and no significant cytotoxicity. These promising results suggest that the evaluation of antiprotozoal activity should be extended to other structural types of biflavonoids.

Acknowledgement

C.V-S is supported by an Agence Universitaire de la Francophonie (AUF) fellowship.

References

Ahmed, S.A., Gogal, R.M., Walsh, J.E., 1994. A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [[.sup.3]H]thymidine incorporation assay. J. Immunol. Methods 170, 211-224.

Ahmed, M.S., Galal, A.M., Ross, S.A., Ferreira, D., ElSohly, M.A., Ibrahim, A.-R.S., Mossa, J.S., El-Feraly, F.S., 2001. A weakly antimalarial biflavanone from Rhus retinorrhoea. Phytochemistry 58 (4), 599-602.

Baltz, T., Baltz, D., Giroud, C., Crokett, J., 1985. Cultivation in semi-defined medium of animal infective forms of Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambiense. EMBO J. 4, 1273-1277.

Barrett, M.P., Gilbert, I.H., 2002. Perspectives for new drugs against trypanosomiasis and leishmaniasis. Curr. Top. Med. Chem. 2 (5), 471-482.

Bittar, M., De Souza, M.M., Yunes, R.A., Lento, R., Delle Monache, F., Cechinel Filho, V., 2000. Antinociceptive activity of 13,II8-binaringenin, a biflavonoid present in plants of the Guttiferae. Planta Med. 66, 84-86.

Buckner, F.S., Verlinde, C.L., La Flamme, A.C., Van Voorhis, W.C., 1996. Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing [beta]-galactosidase. Antimicrob. Agents Chemother. 40, 2592-2597.

Brun, R., Schonenberger, M., 1979. Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Acta Trop. 36, 289-292.

Cunningham, I., 1977. New culture medium for maintenance of tsetse tissues and growth of trypanosomatids. J. Protozool. 24, 325-329.

De Koning, H.P., 2001. Transporters in African trypanosomes: role in drug action and resistance. Int. J. Parasitol. 31, 512-522.

Desjardins, R.E., Canfield, C.J., Haynes, J.D., Chilay, J.D., 1979. Quantitative assessment of antimalarial activity in vitro was determined by a semi-automated microdilution technique. Antimicrob. Agents Chemother. 16, 710-718.

Freiburghaus, F., Kaminsky, R., Nkunya, M.H.H., Brun, R., 1996. Evaluation of African medicinal plants for their in vitro trypanocidal activity. J. Ethnopharmacol. 55, 1-11.

Huber, W., Koella, J.C., 1993. A comparison of the three methods of estimating E[C.sub.50] in studies of drug resistance of malaria parasite. Acta Trop. 55, 257-261.

Joyeux, M., Lobstein, A., Anton, R., Mortier, F., 1995. Comparative antilipoperoxidant, antinecrotic and scavenging properties of terpenes and biflavones from Ginkgo and some flavonoids. Planta Med. 61, 126-129.

Kim, H.P., Son, K.H., Chang, H.W., Kang, S.S., 2000. Effects of naturally occurring flavonoids on inflammatory responses and their action mechanisms. Nat. Prod. Sci. 6, 170-178.

Kirchhoff, L.V., 2003. Changing Epidemiology and Approaches to Therapy for Chagas Disease, Curr. Infect. Dis. Rep. 5, 59-65.

Lin, Y.M., Flavin, M.T., Schure, R., Chen, F.C., Sidwell, R., Barnard, D.L., Huffman, J.H., Kern, E.R., 1999. Antiviral activities of biflavonoids. Planta Med. 61.

Lin, L.-C., Kuo, Y.-C., Chou, C.-J., 2000. Cytotoxic biflavonoids from Selaginella delicatula. J. Nat. Prod. 63, 627-630.

Lin, Y.M., Flavin, M.T., Cassidy, C.S., Mar. A., Chen, F.C., 2001. Biflavonoids as novel antituberculosis agents. Bioorg. Med. Chem. Lett. 11, 2101-2104.

Lobstein, A., Weniger, B., Malecot, V., Um, B.H., Alzate, F., Anton, R., 2003. Polyphenolic content of two Colombian Viburnum species (Caprifoliaceae). Biochem. Syst. Ecol. 31, 95-97.

Nunome, S., Ishiyama, A., Kobayashi, M., Otoguro. K., Kiyohara, H., Yamada, H., Omura, S., 2004. In vitro antimalarial activity of biflavonoids from Wikstroemia indica. Planta Med. 70, 76-78.

Page, C., Page, M., Noel, C., 1993. A new fluorimetric assay for cytotoxicity measurements in vitro. Int. J. Oncol. 3, 473-476.

Raz, B., Iten, M., Grether, Y., Kaminsky, R., Brun, R., 1997. The Alamar BlueR assay to determine drug sensitivity of African trypanosomes (T. b. rhodesiense and T. b. gambiense) in vitro. Acta Trop. 68, 139-147.

Ridley, R.G., Hofheinz, W., Matile, H., Jaquet, C., Dorn, A., Masciadri, R., Jolidon, S., Richter, W.F., Guenzi, A., Girometta, M.A., Urwyler, H., Huber, W., Thaithong, S., Peters, W., 1996. 4-Aminoquinoline analogs of chloroquine with shortened side chains retain activity against chloroquine-resistant Plasmodium falciparum. Antimicrob. Agents Chemother. 40, 1846-1854.

Suarez, A.I., Diaz, B.M., Delle Monache, F., Compagnone, R.S., 2003. Biflavonoids from Podocalyx loranthoides. Fitoterapia 74 (5). 473-475.

Thaithong, S., Beale, G.H., 1981. Resistance of 10 Thai isolates of Plasmodium falciparum to chloroquine and pyrimethamine by in vitro tests. Trans. R. Soc. Trop. Med. Hygiene 75, 271-273.

Trager, W., Jensen, J.B., 1976. Human malaria parasites in continuous culture. Science 193, 673-675.

Wellems, T.E., Plowe, C.V., 2001. Chloroquine-resistant malaria. J Infeet. Dis. 184, 770-776.

Weniger, B., Robledo. S., Arango, G.J., Deharo, E., Aragon, R., Munoz, V., Callapa, J., Lobstein, A., Anton, R., 2001. Antiprotozoal activities of Colombian plants. J. Ethnopharmacol. 78, 193-200.

B. Weniger (a), C. Vonthron-Senecheau (a), M. Kaiser (b), R. Brun (b), R. Anton (a,*)

(a) Pharmacognosie et Biomolecules Naturelles Actives, UMR no 7081, Faculte de Pharmacie, Universite Louis Pasteur Strasbourg, BP 60024, 67401, Illkirch Cedex, France

(b) Swiss Tropical Institute, Socinstrasse 57, CH-4002 Basel, Switzerland

Received 26 July 2004; accepted 19 October 2004

*Corresponding author. Tel.: + 33 0390244241; fax: + 33 0390244311.

E-mail address: [e-mail].

Table 1. In vitro antiprotozoal activities, cytotoxic activity and
selectivity indexes of the examined biflavonoids

                         Antiprotozoal activity I[C.sub.50] ([micro]M)
                         Antiplasmodial  Leishmanicidal
Compounds                activity (a)    activity (b)

Lanaroflavone (1)          0.48            7.2
Cupressusflavone (2)     >30             >50
Amentoflavone (3)        >30             >50
Bilobetin (4)              6.7             2.7
Ginkgetin (5)              2.0             2.8
Isoginkgetin (6)           3.5             1.9
Sciadopitysin (7)          1.4             8.2
Amentoflavone             20             >50
4',4'",7,7"-tetramethyl
ether (8)
Standards
Chloroquine                0.19          --
Artemisinin                0.007         --
Miltefosine              --                0.47
Benznidazole             --              --
Melarsoprol              --              --
Podophyllotoxin          --              --

                         Antiprotozoal activity I[C.sub.50] ([micro]M)
                         Antitrypanosomal  Antitrypanosomal
Compounds                activity (c)      activity (d)

Lanaroflavone (1)        >50                27
Cupressusflavone (2)     >50               >50
Amentoflavone (3)        >50               >50
Bilobetin (4)            >50                 8.9
Ginkgetin (5)             11                 7.0
Isoginkgetin (6)          13                 3.5
Sciadopitysin (7)        >50                 7.0
Amentoflavone            >50               >50
4',4'",7,7"-tetramethyl
ether (8)

Standards
Chloroquine              --                --
Artemisinin              --                --
Miltefosine                                --
Benznidazole               1.69            --
Melarsoprol              --                  0.005
Podophyllotoxin          --                --

                         Cytotoxic activity (e)
                         I[C.sub.50]
Compounds                ([micro]M)   Selectivity index (SI)

Lanaroflavone (1)          70          159    9.8
Cupressusflavone (2)     >150          nd     nd
Amentoflavone (3)        >150          nd     nd
Bilobetin (4)              26            4.0  9.8
Ginkgetin (5)               9            4.1  3.3
Isoginkgetin (6)           11            3.2  5.8
Sciadopitysin (7)          68           49    8.2
Amentoflavone            >150          nd     nd
4',4'",7,7"-tetramethyl
ether (8)

Standards
Chloroquine              --            --     --
Artemisinin              --            --     --
Miltefosine              --            --     --
Benznidazole                           --     --
Melarsoprol              --            --     --
Podophyllotoxin             0.048      --     --

Data shown are values from two replicate experiments.
SI: selectivity index, ratio of cytotoxic activity on L6 cells to
antiplasmodial activity against K1 strain of P. falciparum (SI (a)) and
to leishmanicidal activity against L. donovani amastigotes (SI (b))
(a) P. falciparum K1 resistant strain.
(b) L. donovani axenic amastigotes.
(c) T. cruzi Tulahuen strain trypomastigotes.
(d) T. brucei rhodensiense STIB 900 strain trypomastigotes.
(e) L6 cells.