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Antiplasmodial activity of isoflavones from Andira inermis
Journal of Ethnopharmacology 73 (2000) 131 – 135 www.elsevier.com/locate/jethpharm
Antiplasmodial activity of isoflavones from Andira inermis Carola Kraft a, Kristina Jenett-Siems a,*, Karsten Siems b, Mahabir P. Gupta c, Ulrich Bienzle d, Eckart Eich a a
Institut fu¨r Pharmazie (Pharmazeutische Biologie), Freie Uni6ersita¨t Berlin, Ko¨nigin-Luise Str. 2 -4, D-14195 Berlin, Germany b AnalytiCon AG, Potsdam, Germany c Centro de In6estigationes Farmacognosticas de la Flora Panamen˜a (CIFLORPAN), Facultad de Farmacia, Uni6ersidad de Panama, and Smithsonian Tropical Research Institute, Panama City, Panama d Institut fu¨r Tropenmedizin, Medizinische Fakulta¨t Charite´, Humboldt Uni6ersita¨t zu Berlin, Berlin, Germany Received 28 February 2000; received in revised form 22 May 2000; accepted 30 May 2000
Abstract The stem bark and seeds of Andira inermis, Fabaceae, are employed as a purgative, vermifuge, and febrifuge. In particular, the powdered bark is claimed to be efficacious in intermittent fever. Bioassay-guided fractionation of lipophilic extracts from the stems and leaves yielded six isoflavones: biochanin A, calycosin, formononetin, genistein, pratensein, and prunetin. Calycosin (3%,7-dihydroxy-4%-methoxyisoflavone) and genistein (4%,5,7-trihydroxyisoflavone) have been shown to possess in vitro activity against the chloroquine-sensitive strain poW and the chloroquine-resistant clone Dd2 of Plasmodium falciparum. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Andira inermis; Fabaceae; Isoflavones; Calycosin; Genistein; Antiplasmodial activity; Plasmodium falciparum
1. Introduction
Part 2 in the series ‘Herbal remedies traditionally used against malaria’, presented in part at the 2nd Meeting of the Deutsche Pharmazeutische Gesellschaft (Landesgruppe BerlinBrandenburg): ‘Der wissenschaftliche Nachwuchs stellt sich vor’; July 5th, 1999, Berlin, Germany; and at the joint meeting of the American Society of Pharmacognosy, Association Franc¸aise pour l’Enseignement et la Recherche en Pharmacognosie, Gesellschaft fu¨r Arzneipflanzenforschung, and Phytochemical Society of Europe: ‘2000 Years of Natural Product Research — Past, Present and Future’ — July 26–30, 1999, Amsterdam, The Netherlands. * Corresponding author. Fax: + 49-30-83853729.
Malaria is still the most important parasitic disease in the world, causing 2–3 million deaths every year (WHO, 1997). The rising resistance of Plasmodium spp., especially Plasmodium falciparum, to known antimalarials such as chloroquine makes the search for new antimalarial drugs increasingly important. Therefore, we investigated several medicinal plants, which are traditionally used as antimalarial or antipyretic remedies by the indigenous population in Latin America (Duke, 1975; Hirschhorn, 1981; Joly et al., 1987; Gupta et al., 1993). During a screening
0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 0 ) 0 0 2 8 5 - 3
132
C. Kraft et al. / Journal of Ethnopharmacology 73 (2000) 131–135
program, we obtained lipophilic and hydrophilic extracts of these plants with antiplasmodial activity and we selected the most active ones for further investigation (Jenett-Siems et al., 1999). Andira inermis (W. Wright) H.B.K. (Fabaceae) is a tall tree characterised by a thick trunk and red-purple flowers, native from southern Mexico to northern South America. As a febrifuge, the bark is boiled in milk, sweetened water or performed in pills. It is also used as a purgative, vermifuge, or for dermal irritations. The seeds have similar uses. Large doses of the stem bark or seeds caused vomiting and violent diarrhoea; in some cases they were even fatal (Hirschhorn, 1981; Morton, 1981). From this species, several isoflavones and related structures have been reported (Cocker et al., 1962; Lock de Ugaz et al., 1991). The lipophilic extracts of stems and leaves showed moderate in vitro activity against P. falciparum in this screening program and were further analysed by bioassay-guided fractionation to isolate the active compounds.
2. Material and methods
2.1. General For fractionation, silica gel 60 (70 – 230 mesh) was utilised. Preparative high performance liquid chromatography (HPLC) was performed on a Knauer Eurochrom 2000 equipped with a Nucleosil P 300 C-18 (10 mm) column. Mass spectra were determined with a Finnigan MAT CH7A (220°C, ionisation 70 eV) and 1H-NMR spectra were obtained using acetone-D6 as a solvent with a Bruker AVANCE DPX 400 (400 MHz, TMS as internal standard). The erythrocytes were harvested with an Inotech cell harvester and the b-radiation of the incorporated [3H]hypoxanthine was measured with a Wallac 1450 MicroBeta plus liquid scintillation counter.
Llano-Carti), Panama, during March 1997. Voucher specimens (FLORPAN 2758) were identified by Professor M.D. Correa A. and deposited at the Herbarium of the University of Panama.
2.3. Extraction and isolation In the screening program, the air-dried stem bark and leaves (20 g) were extracted three times for 2 h with 150 ml petroleum ether/ethyl acetate (1:1) at room temperature. Then the plant material was air dried again and treated three times with 150 ml methanol/water (8:2) to yield the hydrophilic extracts. The solvents were evaporated under reduced pressure at 40°C. For further investigations, the crushed stems (600 g) were air dried and extracted three times with 2 l of petroleum ether/ethyl acetate (1:1) for 24 h at room temperature. The solvents were evaporated under reduced pressure at 40°C. The oily residue (3.3 g) was subjected to column chromatography on silica gel 60 (45 g) and eluted with cyclohexane, cyclohexane/ethyl acetate mixtures, and methanol to yield seven fractions, which were tested against P. falciparum. Fraction 5 and 6, which were eluted with cyclohexane/ ethyl acetate (8:2 and 7:3, respectively), proved to be most active. Further separation of fraction 5 (150 mg) by HPLC at a 5 ml/min flow rate of methanol/water (55:45) yielded the compounds 1 (2.4 mg), 2 (6.1 mg), and 3 (6.6 mg). Fraction 6 was separated with methanol/water (45:55) to give the compounds 4 (8.3 mg), 5 (8.5 mg), and 6 (1.6 mg). Compound 2 (prunetin): EI-MS: m/z (rel. int.)= 284 (M+, 100), 166 (35), 138 (22), 118 (15), 110 (10), 95 (13); 1H-NMR (400 MHz, acetone-D6): d (ppm)= 3.93 (3 H, s, CH3O-7), 6.36 (1 H, d, J= 2.3 Hz, H-6), 6.55 (1 H, d, J= 2.4 Hz, H-8), 6.91 (2 H, d, J=8.5 Hz, H-3%,5%), 7.47 (2 H, d, J =8.5 Hz, H-2%,6%), 8.22 (1 H, s, H-2), 13.4 (1 H, s, OH-5).
2.4. Antiplasmodial bioassay 2.2. Plant material Leaves and stems of A. inermis were collected in the area of Panama City (at the road El
In this study, the chloroquine-sensitive strain of P. falciparum poW (IC50 = 0.015 mM) and the chloroquine-resistant clone Dd2 (IC50 = 0.14 mM)
C. Kraft et al. / Journal of Ethnopharmacology 73 (2000) 131–135
were used. They 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. Crude extracts and isolated compounds were dissolved in DMSO (1 mg/50 ml) and diluted in RPMI 1640 medium (Trager and Jensen, 1976). The tests were performed in 96-well microtiter plates (Desjardins et al., 1979) which contained 150 ml of a parasitized red blood suspension (A+, 2.5% haematocrit, 0.5% parasitaemia). The samples were added to give final concentrations between 1.56 and 100 mg/ml. After incubation in a candle jar for 24 h, 0.5 mCi [3H]hypoxanthine (1 mCi/ml, ICN) was added to each well and the plate was incubated for another 18 h. Incorporation of [3H]hypoxanthine was measured by liquid scintillation after harvesting the cells on glass fibre filters with the cell harvester. All tests were performed in triplicate. The percentage of growth inhibition was calculated as follows: (1− [cpm in drug treated cultures/cpm in untreated cultures])×100. The concentration at which growth was inhibited by 50% (IC50) was estimated by interpolation. Extracts with IC50 values above 50 mg/ml were considered to be inactive (O’Neill et al., 1985).
133
3. Results The isolated compounds 1–6 (Fig. 1) turned out to be isoflavones. Compound 1, isolated from both stems and leaves was identified as formononetin by comparison with an authentic sample. According to the spectral data (1H-NMR, EI-MS), compound 2 proved to be prunetin. To the best of our knowledge, no 1H-NMR values for prunetin have been published so far, therefore they are given in Section 2.3. The isoflavones 3, 4, 5, and 6 were identified as biochanin A, calycosin, genistein, and pratensein, respectively, by comparison of 1H-NMR and EI-MS with published data (Asres et al., 1985; Kobayashi et al., 1985). Prunetin (2), calycosin (4), and pratensein (6) were detected for the first time from the genus Andira. The lipophilic extracts of A. inermis exhibited IC50 values from 56.0 mg/ml (leaves) to 108.7 mg/ml (stems) against P. falciparum in vitro (Table 1). The hydrophilic extracts proved to be inactive. For all tested substances the antiparasitic activity against the chloroquine-sensitive strain poW was higher (factor two) than against the chloroquine-resistant clone Dd2. Calycosin (IC50 4.2 mg/ml for poW; 9.8 mg/ml for Dd2) and genistein (IC50 2.0 mg/ml for poW; 4.1 mg/ml for
Fig. 1. Isoflavones isolated from A. inermis.
C. Kraft et al. / Journal of Ethnopharmacology 73 (2000) 131–135
134
Table 1 Antiplasmodial activity of lipophilic extracts (petroleum ether/ ethyl acetate 1:1) and isolated isoflavones from A. inermis against P. falciparum Extract/compound
Mean IC50 values (mg/ml)a poW
Leaf extract Stem extract Formononetin (1) Prunetin (2) Biochanin A (3) Calycosin (4) Genistein (5) Pratensein (6) Quinine sulphate Chloroquine a
56 82.0 \50 27.8 46.8 4.2 2.0 45 0.035 0.008
Dd2 58.6 108.7 \50 \50 \50 9.8 4.1 \50 0.092 0.073
Performed in triplicate.
Dd2) proved to be most active. The other four compounds were considered to be inactive.
4. Discussion Although the lipophilic extracts of A. inermis showed only moderate antiplasmodial activity compared to those known for other plant remedies (O’Neill et al., 1985; Jenett-Siems et al., 1999) they afforded two active isoflavones. This is the first report on antiplasmodial activity of isoflavones against P. falciparum. The low concentration of calycosin and genistein ( B 0.01% w/w) may be the explanation for the low antiplasmodial activity of the crude extract. It is known that calycosin also proved to be active against Giardia intestinalis, a potent protozoan agent for enteric diseases (ElSohly et al., 1999). During the preparation of this paper a patent application was published (Khan et al., 1999), which claimed a method of treating giardiasis and/or malaria comprising the use of isoflavones, but mentioning no data, proving their antiplasmodial activity against P. falciparum. In our study, we have focused on the investigation of the lipophilic extract. The indigenous population, e.g. in Brazil (Morton, 1981), prefer to boil the stem bark with milk. This procedure covers the bitter taste and probably causes the extraction of
lipophilic constituents. Interestingly, hydrophilic extracts (methanol/water 8:2) from A. inermis did not show antiplasmodial activity in our test system. The evaluation of extracts obtained with water or milk is still in progress and might be interesting with regard to the ethnobotanical use of this species.
Acknowledgements The authors are indebted to C. Guerra, for his support in collecting the plant material, and to Professor M.D. Correa A., Herbarium of the University of Panama, Republic of Panama, for her identification of the species. We thank Drs D.S. Kim and B. Kleuser, Institut fu¨r Pharmazie, Freie Universita¨t Berlin, Germany, for their advice concerning determination of radioactivity. This study was supported by a grant from the Deutsche Pharmazeutische Gesellschaft to K. Jenett-Siems and by Charite´ Grant Nr. 98-700. CIFLORPAN acknowledges the Fundation Natura and the Organization of American States (OAS) for financial support.
References Asres, K., Mascagni, P., O’Neill, M.J., Phillipson, J.D., 1985. Isoflavonoids from Bolusanthus speciosus (Bolus) Harms Leguminosae. Zeitschrift fu¨r Naturforschung 40c, 617 – 620. Cocker, W., Dahl, T., Dempsey, C., McMurry, T.B.H., 1962. Extractives from woods. Part I. Extractives from Andira inermis (Wright) H.B.K., Journal of the Chemical Society, 4906 – 4909. Desjardins, R.E., Canfield, C.J., Haynes, J.D., Chulay, J.D., 1979. Quantitative assessment of antimalarial activity in 6itro by a semiautomated microdilution technique. Antimicrobial Agents and Chemotherapy 16, 710 – 718. Duke, J.A., 1975. Ethnobotanical observations on the Cuna Indians. Economic Botany 29, 278 – 293. ElSohly, H.N., Joshi, A.S., Nimrod, A.C., 1999. Antigiaridial isoflavones from Machaerium aristulatum. Planta Medica 65, 490. Gupta, M.P., Correa, M.D., Solis, P.N., Jones, A., Galdames, C., Guionneau-Sinclair, F., 1993. Medicinal plant inventory of Cuna Indians: Part 1. Journal of Ethnopharmacology 40, 77 – 109. Hirschhorn, H.H., 1981. Botanical remedies of South and Central America, and the Caribbean: an archival analysis. Part 1. Journal of Ethnopharmacology 4, 129 – 158.
C. Kraft et al. / Journal of Ethnopharmacology 73 (2000) 131–135 Jenett-Siems, K., Mockenhaupt, F.P., Bienzle, U., Gupta, M.P., Eich, E., 1999. In vitro antiplasmodial activity of Central American medicinal plants. Tropical Medicine and International Health 4, 611–615. Joly, L.G., Guerra, S., Septimo, R., Solis, P.N., Correa, M.D., Gupta, M.P., Levy, S., Sandberg, F., 1987. Ethnobotanical inventory of medicinal plants used by Guaymi Indians in Western Panama. Journal of Ethnopharmacology 20, 145– 171. Khan, I.A., Avery, M.A., Goins, D.K., Walker, L.A., Burandt, C.L., 1999. Isoflavones for treating giardiasis and malaria. PCT Int. Appl. WO 99 49,862 (CL A61K31/35), 7 Oct 1999, US Appl. 50,734, 30 Mar 1998; 16 pp. Kobayashi, M., Noguchi, H., Sankawa, U., 1985. Formation of isoflavones by callus culture of Glycyrrhiza uralensis
.
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with different production pattern. Chemical and Pharmaceutical Bulletin 33, 3811 – 3816. Lock de Ugaz, O., Costa, J., Sanchez, L., Ubillas Sanchez, R.P., Tempesta, M.S., 1991. Flavonoids from Andira inermis. Fitoterapia 62, 89 – 90. Morton, J.F., 1981. Atlas of Medicinal Plants of Middle America. Thomas, Springfield, IL, pp. 275 – 276. O’Neill, M.J., Bray, D.H., Boardmann, P., Phillipson, J.D., Warhurst, D.C., 1985. Plants as sources of antimalarial drugs part 1. In 6itro test method for the evaluation of crude extracts from plants. Planta Medica 51, 394 – 398. Trager, W., Jensen, J.B., 1976. Human malaria parasites in continuous culture. Science 193, 673 – 675. WHO, 1997. World malaria situation in 1994. Weekly Epidemiological Record 72, 269 – 292.
Antiplasmodial activity of isoflavones from Andira inermis Carola Kraft a, Kristina Jenett-Siems a,*, Karsten Siems b, Mahabir P. Gupta c, Ulrich Bienzle d, Eckart Eich a a
Institut fu¨r Pharmazie (Pharmazeutische Biologie), Freie Uni6ersita¨t Berlin, Ko¨nigin-Luise Str. 2 -4, D-14195 Berlin, Germany b AnalytiCon AG, Potsdam, Germany c Centro de In6estigationes Farmacognosticas de la Flora Panamen˜a (CIFLORPAN), Facultad de Farmacia, Uni6ersidad de Panama, and Smithsonian Tropical Research Institute, Panama City, Panama d Institut fu¨r Tropenmedizin, Medizinische Fakulta¨t Charite´, Humboldt Uni6ersita¨t zu Berlin, Berlin, Germany Received 28 February 2000; received in revised form 22 May 2000; accepted 30 May 2000
Abstract The stem bark and seeds of Andira inermis, Fabaceae, are employed as a purgative, vermifuge, and febrifuge. In particular, the powdered bark is claimed to be efficacious in intermittent fever. Bioassay-guided fractionation of lipophilic extracts from the stems and leaves yielded six isoflavones: biochanin A, calycosin, formononetin, genistein, pratensein, and prunetin. Calycosin (3%,7-dihydroxy-4%-methoxyisoflavone) and genistein (4%,5,7-trihydroxyisoflavone) have been shown to possess in vitro activity against the chloroquine-sensitive strain poW and the chloroquine-resistant clone Dd2 of Plasmodium falciparum. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Andira inermis; Fabaceae; Isoflavones; Calycosin; Genistein; Antiplasmodial activity; Plasmodium falciparum
1. Introduction
Part 2 in the series ‘Herbal remedies traditionally used against malaria’, presented in part at the 2nd Meeting of the Deutsche Pharmazeutische Gesellschaft (Landesgruppe BerlinBrandenburg): ‘Der wissenschaftliche Nachwuchs stellt sich vor’; July 5th, 1999, Berlin, Germany; and at the joint meeting of the American Society of Pharmacognosy, Association Franc¸aise pour l’Enseignement et la Recherche en Pharmacognosie, Gesellschaft fu¨r Arzneipflanzenforschung, and Phytochemical Society of Europe: ‘2000 Years of Natural Product Research — Past, Present and Future’ — July 26–30, 1999, Amsterdam, The Netherlands. * Corresponding author. Fax: + 49-30-83853729.
Malaria is still the most important parasitic disease in the world, causing 2–3 million deaths every year (WHO, 1997). The rising resistance of Plasmodium spp., especially Plasmodium falciparum, to known antimalarials such as chloroquine makes the search for new antimalarial drugs increasingly important. Therefore, we investigated several medicinal plants, which are traditionally used as antimalarial or antipyretic remedies by the indigenous population in Latin America (Duke, 1975; Hirschhorn, 1981; Joly et al., 1987; Gupta et al., 1993). During a screening
0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 0 ) 0 0 2 8 5 - 3
132
C. Kraft et al. / Journal of Ethnopharmacology 73 (2000) 131–135
program, we obtained lipophilic and hydrophilic extracts of these plants with antiplasmodial activity and we selected the most active ones for further investigation (Jenett-Siems et al., 1999). Andira inermis (W. Wright) H.B.K. (Fabaceae) is a tall tree characterised by a thick trunk and red-purple flowers, native from southern Mexico to northern South America. As a febrifuge, the bark is boiled in milk, sweetened water or performed in pills. It is also used as a purgative, vermifuge, or for dermal irritations. The seeds have similar uses. Large doses of the stem bark or seeds caused vomiting and violent diarrhoea; in some cases they were even fatal (Hirschhorn, 1981; Morton, 1981). From this species, several isoflavones and related structures have been reported (Cocker et al., 1962; Lock de Ugaz et al., 1991). The lipophilic extracts of stems and leaves showed moderate in vitro activity against P. falciparum in this screening program and were further analysed by bioassay-guided fractionation to isolate the active compounds.
2. Material and methods
2.1. General For fractionation, silica gel 60 (70 – 230 mesh) was utilised. Preparative high performance liquid chromatography (HPLC) was performed on a Knauer Eurochrom 2000 equipped with a Nucleosil P 300 C-18 (10 mm) column. Mass spectra were determined with a Finnigan MAT CH7A (220°C, ionisation 70 eV) and 1H-NMR spectra were obtained using acetone-D6 as a solvent with a Bruker AVANCE DPX 400 (400 MHz, TMS as internal standard). The erythrocytes were harvested with an Inotech cell harvester and the b-radiation of the incorporated [3H]hypoxanthine was measured with a Wallac 1450 MicroBeta plus liquid scintillation counter.
Llano-Carti), Panama, during March 1997. Voucher specimens (FLORPAN 2758) were identified by Professor M.D. Correa A. and deposited at the Herbarium of the University of Panama.
2.3. Extraction and isolation In the screening program, the air-dried stem bark and leaves (20 g) were extracted three times for 2 h with 150 ml petroleum ether/ethyl acetate (1:1) at room temperature. Then the plant material was air dried again and treated three times with 150 ml methanol/water (8:2) to yield the hydrophilic extracts. The solvents were evaporated under reduced pressure at 40°C. For further investigations, the crushed stems (600 g) were air dried and extracted three times with 2 l of petroleum ether/ethyl acetate (1:1) for 24 h at room temperature. The solvents were evaporated under reduced pressure at 40°C. The oily residue (3.3 g) was subjected to column chromatography on silica gel 60 (45 g) and eluted with cyclohexane, cyclohexane/ethyl acetate mixtures, and methanol to yield seven fractions, which were tested against P. falciparum. Fraction 5 and 6, which were eluted with cyclohexane/ ethyl acetate (8:2 and 7:3, respectively), proved to be most active. Further separation of fraction 5 (150 mg) by HPLC at a 5 ml/min flow rate of methanol/water (55:45) yielded the compounds 1 (2.4 mg), 2 (6.1 mg), and 3 (6.6 mg). Fraction 6 was separated with methanol/water (45:55) to give the compounds 4 (8.3 mg), 5 (8.5 mg), and 6 (1.6 mg). Compound 2 (prunetin): EI-MS: m/z (rel. int.)= 284 (M+, 100), 166 (35), 138 (22), 118 (15), 110 (10), 95 (13); 1H-NMR (400 MHz, acetone-D6): d (ppm)= 3.93 (3 H, s, CH3O-7), 6.36 (1 H, d, J= 2.3 Hz, H-6), 6.55 (1 H, d, J= 2.4 Hz, H-8), 6.91 (2 H, d, J=8.5 Hz, H-3%,5%), 7.47 (2 H, d, J =8.5 Hz, H-2%,6%), 8.22 (1 H, s, H-2), 13.4 (1 H, s, OH-5).
2.4. Antiplasmodial bioassay 2.2. Plant material Leaves and stems of A. inermis were collected in the area of Panama City (at the road El
In this study, the chloroquine-sensitive strain of P. falciparum poW (IC50 = 0.015 mM) and the chloroquine-resistant clone Dd2 (IC50 = 0.14 mM)
C. Kraft et al. / Journal of Ethnopharmacology 73 (2000) 131–135
were used. They 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. Crude extracts and isolated compounds were dissolved in DMSO (1 mg/50 ml) and diluted in RPMI 1640 medium (Trager and Jensen, 1976). The tests were performed in 96-well microtiter plates (Desjardins et al., 1979) which contained 150 ml of a parasitized red blood suspension (A+, 2.5% haematocrit, 0.5% parasitaemia). The samples were added to give final concentrations between 1.56 and 100 mg/ml. After incubation in a candle jar for 24 h, 0.5 mCi [3H]hypoxanthine (1 mCi/ml, ICN) was added to each well and the plate was incubated for another 18 h. Incorporation of [3H]hypoxanthine was measured by liquid scintillation after harvesting the cells on glass fibre filters with the cell harvester. All tests were performed in triplicate. The percentage of growth inhibition was calculated as follows: (1− [cpm in drug treated cultures/cpm in untreated cultures])×100. The concentration at which growth was inhibited by 50% (IC50) was estimated by interpolation. Extracts with IC50 values above 50 mg/ml were considered to be inactive (O’Neill et al., 1985).
133
3. Results The isolated compounds 1–6 (Fig. 1) turned out to be isoflavones. Compound 1, isolated from both stems and leaves was identified as formononetin by comparison with an authentic sample. According to the spectral data (1H-NMR, EI-MS), compound 2 proved to be prunetin. To the best of our knowledge, no 1H-NMR values for prunetin have been published so far, therefore they are given in Section 2.3. The isoflavones 3, 4, 5, and 6 were identified as biochanin A, calycosin, genistein, and pratensein, respectively, by comparison of 1H-NMR and EI-MS with published data (Asres et al., 1985; Kobayashi et al., 1985). Prunetin (2), calycosin (4), and pratensein (6) were detected for the first time from the genus Andira. The lipophilic extracts of A. inermis exhibited IC50 values from 56.0 mg/ml (leaves) to 108.7 mg/ml (stems) against P. falciparum in vitro (Table 1). The hydrophilic extracts proved to be inactive. For all tested substances the antiparasitic activity against the chloroquine-sensitive strain poW was higher (factor two) than against the chloroquine-resistant clone Dd2. Calycosin (IC50 4.2 mg/ml for poW; 9.8 mg/ml for Dd2) and genistein (IC50 2.0 mg/ml for poW; 4.1 mg/ml for
Fig. 1. Isoflavones isolated from A. inermis.
C. Kraft et al. / Journal of Ethnopharmacology 73 (2000) 131–135
134
Table 1 Antiplasmodial activity of lipophilic extracts (petroleum ether/ ethyl acetate 1:1) and isolated isoflavones from A. inermis against P. falciparum Extract/compound
Mean IC50 values (mg/ml)a poW
Leaf extract Stem extract Formononetin (1) Prunetin (2) Biochanin A (3) Calycosin (4) Genistein (5) Pratensein (6) Quinine sulphate Chloroquine a
56 82.0 \50 27.8 46.8 4.2 2.0 45 0.035 0.008
Dd2 58.6 108.7 \50 \50 \50 9.8 4.1 \50 0.092 0.073
Performed in triplicate.
Dd2) proved to be most active. The other four compounds were considered to be inactive.
4. Discussion Although the lipophilic extracts of A. inermis showed only moderate antiplasmodial activity compared to those known for other plant remedies (O’Neill et al., 1985; Jenett-Siems et al., 1999) they afforded two active isoflavones. This is the first report on antiplasmodial activity of isoflavones against P. falciparum. The low concentration of calycosin and genistein ( B 0.01% w/w) may be the explanation for the low antiplasmodial activity of the crude extract. It is known that calycosin also proved to be active against Giardia intestinalis, a potent protozoan agent for enteric diseases (ElSohly et al., 1999). During the preparation of this paper a patent application was published (Khan et al., 1999), which claimed a method of treating giardiasis and/or malaria comprising the use of isoflavones, but mentioning no data, proving their antiplasmodial activity against P. falciparum. In our study, we have focused on the investigation of the lipophilic extract. The indigenous population, e.g. in Brazil (Morton, 1981), prefer to boil the stem bark with milk. This procedure covers the bitter taste and probably causes the extraction of
lipophilic constituents. Interestingly, hydrophilic extracts (methanol/water 8:2) from A. inermis did not show antiplasmodial activity in our test system. The evaluation of extracts obtained with water or milk is still in progress and might be interesting with regard to the ethnobotanical use of this species.
Acknowledgements The authors are indebted to C. Guerra, for his support in collecting the plant material, and to Professor M.D. Correa A., Herbarium of the University of Panama, Republic of Panama, for her identification of the species. We thank Drs D.S. Kim and B. Kleuser, Institut fu¨r Pharmazie, Freie Universita¨t Berlin, Germany, for their advice concerning determination of radioactivity. This study was supported by a grant from the Deutsche Pharmazeutische Gesellschaft to K. Jenett-Siems and by Charite´ Grant Nr. 98-700. CIFLORPAN acknowledges the Fundation Natura and the Organization of American States (OAS) for financial support.
References Asres, K., Mascagni, P., O’Neill, M.J., Phillipson, J.D., 1985. Isoflavonoids from Bolusanthus speciosus (Bolus) Harms Leguminosae. Zeitschrift fu¨r Naturforschung 40c, 617 – 620. Cocker, W., Dahl, T., Dempsey, C., McMurry, T.B.H., 1962. Extractives from woods. Part I. Extractives from Andira inermis (Wright) H.B.K., Journal of the Chemical Society, 4906 – 4909. Desjardins, R.E., Canfield, C.J., Haynes, J.D., Chulay, J.D., 1979. Quantitative assessment of antimalarial activity in 6itro by a semiautomated microdilution technique. Antimicrobial Agents and Chemotherapy 16, 710 – 718. Duke, J.A., 1975. Ethnobotanical observations on the Cuna Indians. Economic Botany 29, 278 – 293. ElSohly, H.N., Joshi, A.S., Nimrod, A.C., 1999. Antigiaridial isoflavones from Machaerium aristulatum. Planta Medica 65, 490. Gupta, M.P., Correa, M.D., Solis, P.N., Jones, A., Galdames, C., Guionneau-Sinclair, F., 1993. Medicinal plant inventory of Cuna Indians: Part 1. Journal of Ethnopharmacology 40, 77 – 109. Hirschhorn, H.H., 1981. Botanical remedies of South and Central America, and the Caribbean: an archival analysis. Part 1. Journal of Ethnopharmacology 4, 129 – 158.
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