PlantaMeci. 56 (1990) 371
Antimalarial Compounds Containing an a, f'-Unsaturated Carbonyl Moiety from Tanzanian Medicinal Plants1 H. Weenen23, M. H. H. Nkunya2. D. H. Bray4, L. B. Mwasumbi5, L. S. Kinabo2, and V.A. E. B. Kilimali6,
andf. B. P. A. Wnberg7 Part 2 in the series: Antimalarial activity of Tanzanian plants. For Part 1, see Ref. (13) Department of Chemistry, University of Dares Salaam, P.O. Box 35061, Dares Salaam, Tanzania Present address of author to whom all correspondence should be addressed: Quest International, P.O. Box 2, 1400 CA Bussum, the Netherlands Department of Medical Protozoology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, U.K. Department of Botany, University of Dares Salaam, P.O. Box 35060, Dares Salaam, Tanzania 6 National Institute for Medical Research, Amani Research Centre, P.O. Box 4, Amani, Tanzania Laboratory for Organic Chemistry, Agricultural University Wageningen, Dreijenplein 8, 6703 BC Wageningen, the Netherlands 2
Abstract Pure compounds were isolated from plant extracts with antimalarial activity. The extracts were ob-
tained from the tubers of Cyperus rotundus L.
(Cyperaceae), the rootbark of Zanthoxylum gilletii (De Wild) Waterm. (Rutaceae), and the rootbark of Margaritaria discoidea (Baill.) Webster (Euphorbiaceae). The most active compounds included (IC50 within brackets): u-cyperone (1) (5.5 .tg/ml), N-isobutyldeca-2,4dienamide (2) (5.4 .tg/ml), and securinine (3) (5.4 .tgIml). A mixture of autoxidation products of 3-selinene was found to be the most active antimalarial substances obtained from C. rotundus (5.6 .tg/ml.
A large-scale screening of plants from all over the world for anti-malarial activity was reported by Spenceretal. in 1947 (3). However, avian malaria parasites were used for the antimalarial testing, and no follow up has
been reported on the isolation and testing of pure compounds.
Recently a semi-automated antimalarial testing method was developed, using in vitro uptake of[3H]-
hypoxanthine by Plasmodium falciparum parasites as an indicator of growth (4, 5). Several quassinoids obtained from Simaroubaceae plants have been tested for their activity in inhibiting growth of P. falciparum, and were found to possess potent activity (6—10). Unfortunately, the majority
of quassinoids showed relatively high mammalian cytotoxocity. However, preliminary studies on the structure-ac-
Key words Tanzanian, medicinal plants, antimalarial activity, Cyperus rotundus, Zanthoxylum gilletii, Mar-
garitaria discoidea, a-cyperone, N-isobutyldeca-2,4dienamide, securinine.
Introduction Malaria afflicts over 400 million people, and kills about two million every year (1). Drugs presently in use include quinoline derivatives, suipha drugs, and antifolates. However, resistance is developing quickly against most of them; moreover, their use is hampered by undesired side-effects. Hence novel antimalarial drugs are urgently required.
The continuing clinical use of quinine isolated from Cinchona species, and the recent discovery of the antimalarial substance artemisinin from the Chinese herb Artemisia annua L. (Compositae) (2), has stimulated our interest in medicinal plants as sources of new antimalarial drugs.
tivity relationships of these compounds indicate that the structural requirements for the activity and toxicity are different(6, 11). Khalid et al. have also used the radiometric assay to test compounds which they isolated from Sudanese traditional medicinal plants (12). Of 21 compounds tested, the limonoid gedunin was found to be most potent (IC50 approx: 1 !.LM).
In this study, we have investigated some extracts which we had previously found to possess in vitro an-
timalarial activity (13). From these fractions compounds were isolated, identified, and their IC50 values determined.
Materials and Methods Plant materials All plants were collected from Dar es Salaam, Tanga, and Mwanza regions. Voucher specimens were deposited at the herbarium, Botany Department, University of Dares Salaam.
Downloaded by: Universite Laval. Copyrighted material.
Received: June 22, 1989
H. Weenenetal.
372 Planta Med. 56(1990)
Results and Discussion
CR3 H3
O.J:I:II;tI1:IIIJ..t:.CHz
CH3
CR3
2
1
When the dichloromethane extract of C. rotundus tubers was fractionated into ten fractions by preparative silica gel TLC, using petrol/ethyl acetate (4/1, v/v) as the eluent, the least polar fraction was found to be the one
with the highest activity (IC50 c S .tg/ml). The
dichioromethane extract was therefore reextracted with petrol, to exclude the more polar substances, and fractionated by column chromatography. Using silica gel and silver nitrate impregnated silica gel, five sesquiterpenes were obtained and tested for antimalarial activity (Table 1). Of these a-cyperone (1) showed the highest activity, with an IC50 of 5.5 .sg/ml. Humulene, cu-selinene, -selinene, and cyperene showed no antimalarial activity at concentrations below
HèY
50g/ml.
All plant materials were dried, pulverized and extracted either consecutively with petroleum ether (h.p. 40—60°C), dichioromethane and methanol, 2 x 48 h for each solvent, or with methanol alone. The concentrated extracts were tested for antimalarial activity, and some of the most active fractions were fractionated by chromatography into pure compounds, which were identified spectroscopically, and tested for antimalarial activity as well. The crude extracts were fractionated by column chromatography
using silica gel (400 mesh) and were eluted with a gradient of hexane and ethyl acetate. The semi-purified fractions were further purified by column chromatography using silica gel, silver nitrate impregnated silica gel, alumina and/or fractogel (PVA 500, Merck), by preparative TLC (silica gel and silanized silica gel), and whenever possible by recrystallization. Spectroscopic data and melting points of all known compounds were in agreement with literature data.
However, the activity of )3-selinene ap-
peared to fluctuate, depending on its history. When investigating this more closely, we observed that 3-selinene was
rather unstable, and decomposed to compounds which contain a hydroperoxide group, as indicated by KI/starch on TLC (15). A mixture of the decomposition products was observed to show antimalarial activity with an IC50 as low as
5.56 g/ml. As expected, the decomposition of 3-selinene could be inhibited by the addition of 0.1% vitamin E. The tertiary C-H a to the exocyclic double bond is particularly prone to homolytic cleavage, as the resulting tertiary allylic radical is highly stabilized. We assume a hydroperoxide is formed at this position via an autoxidation reaction, which
will decompose to more stable products. Similar by-
droperoxides have previously been isolated from Alpinia japonica (Thunb.) Miq. (Zingiberaceae) (16), and the fact Antimalarial testing that both C-S epimers were isolated suggests that their forIsolated compounds were assessed for in vitro mation was also due to autoxidation, rather than an enactivity against Plasmodiurn flaciparum strain K1, which origi- zymatic process. Since the autoxidized 3-selinene sample
nated in Thailand, and which is multidrug resistant (14). The
technique used measured the ability of the extracts and pure compounds to inhibit the incorporation of [311]-hypoxanthine into the malaria parasites. Precise details of the protocol are described in references (4) and (5). Compounds were tested in duplicate at 12 concentrations in threefold dilutions starting from 50 .tg/ml.
was a mixture, some of its individual components may have a much lower IC50. We are presently studying the nature and the activity of the hydroperoxides in more detail. How-
ever their potential as antimalarial drugs is questionable, since they are not very stable. Zanthoxylum gilletii gave two compounds with an IC50 of less than 50 zg/ml. Among these N-isobutyldeca-2,4-dienamide (2) was most active (IC50 = 5.37 .tg/ml), while fagaramide was only slightly active (IC50 = 12.34 jig/mi).
source
Cyperusrotundus
compound
a.cyperone(1) a-selinene 3-selinene 3-selinene (autoxidized) cyperene humulene
Zanthoxylumgilletii
Margarifaria discoidea
N-isobutyldeca-2,4-dienamide (2) fagaramide lupeol sesamin 4,7,8-trimethoxyfuro[2,3-bJquinoline securinine (3)
IC55
95%
(jig/mI)
confidence interval
5.50
(4.09—7.39)
> 50 > 50 5.56
>50
(4.65—6.34)
> 50 5.37 12.34
> 50
(3.72—7.73) (9.23—16.51)
>50 >50 5.35
(4.32—6.63)
Table 1 Antimalarial activity of compounds from Tanzanian medicinal plants.
Downloaded by: Universite Laval. Copyrighted material.
Extraction and isolation procedures
Antimalarial Compounds Containing an a, /3-Unsaturated Carbonyl Moiety from Tanzanian Medicinal Plants
decomposed after isolation, and could therefore not be tested.
Interestingly, all four compounds which were found to possess significant antimalarial activity and which were identified, contain an a,f3-unsaturated carbonyl
moiety. Their antimalarial properties can possibly be
References 1 Stürcher, D. (1987) Parasitology Today 5 (2), 39. 2 Liu, J. M., Ni, M. Y.. FanY. F., et al. (1979)Acta Chim. Sinica 37,129. Spencer, C. F., Koninszy, F. R., Rogers, E. F., et al. (1947) Lloydia 10, 145. Desjardins, R. E., Canfield, C. J., Haynes, J. D., Chulay, J. D. (1979) Antimicrob. Agents Chemother. 16, 710. O'Neill, M. J., Bray, D. H., Boardman, P., Phillipson, J. D., Warhurst, D. C. (1985) Planta Med. 394. 6 O'Neill, M. J., Bray, D. H., Boardman, P., Phillipson, J. D., Warhurst, D. C., Peters, W., SuiTness, M. (1986) Antimicrob. Agents Chemother.
'
30, 101. Chan, K. L., O'Neill, M. J., Phillipson, J. D., Warhurst, D. C. (1986) Planta Med. 105.
explained by the tendency of the nucleic acids of the parasites to react with the a,13-unsaturated carbonyl moiety, in a Michael addition fashion. Although Lantana camara L. was one of the most active plants in our screening, it was not investigated further, because it is reported to contain toxic principles (17). We are however presently studying the chemical com-
position of Hoslundia opposita Vahl., as well as some Uvaria species, which will be reported separately.
Acknowledgements We are grateful to Dr. H. Wijnberg, Dr. M. Posthumus (University of Wageningen), and to Prof. P. Waterman (University of Strathclyde) for providing spectra, and for their helpful suggestions. We thank Dr. 0. MotI (Czechoslovak Academy of Science) for sending us an authentic sample of cyperene. Part of this
research was supported by a grant from the University of Dar es Salaam.
Pavanand, K., Nutaleul, W., Dechatiwongse, T., Yoshihira, K.,
° 12 13 14 15 16 17
Yongvanitchit, K., Scovill, J. P., Flippen-Anderson, J. L., Gilardi, R., George, C., Kanchanapee, P., Webster, H. K. (1986) Planta Med. 108. Fandeur, T., Moretti, C., Polonsky, J. (1985) Planta Med. 20. Trager,W., Polonsky, J. (1981)Arn. J. Trop. Med. Hyg. 30,531—537. Bray, D. H., O'Neill, M. J., Phillipson, J. D., Warhurst, D.C. (1987) J. Pharmac. Pharmacology 39 (Suppl) 85. Khalid, S. A., Farouk, A., Geary, T. G., Jensen, J. B. (1986) J. Ethnopharmacology 15, 201. Weenen, H., Nkunya, M. H. H., Bray, D. H., Mwasumbi, L. B., Kinabo, L. S., Kilimali, V.A. E. B. (1990) Planta Med. 56, 368. Thaitong, S., Beale, G. H., Chutmongkonkul, M. (1983) Trans. Roy. Soc. Trop. Med. Hyg. 77, 228. Porter, N. A., Wolf, R. A., Weenen, H. (1980) Lipids 15, 163. Itokawa, H., Morita, H., Watanabe, K. (1987) Chem. Pharm. Bull. 35,
1460. Sharma, 0. P. (1984) Vet. Hum. Toxicol. 26(6), 488.
Downloaded by: Universite Laval. Copyrighted material.
From Margaritaria discoidea only one stable compound was isolated, i.e. the alkaloid securinine (3), which showed an IC50 of 5.35 ig/ml. Another isomeric alkaloid isolated from the same plant, allosecurinine, quickly
Planta Med. 56(1990) 373
Antimalarial Compounds Containing an a, f'-Unsaturated Carbonyl Moiety from Tanzanian Medicinal Plants1 H. Weenen23, M. H. H. Nkunya2. D. H. Bray4, L. B. Mwasumbi5, L. S. Kinabo2, and V.A. E. B. Kilimali6,
andf. B. P. A. Wnberg7 Part 2 in the series: Antimalarial activity of Tanzanian plants. For Part 1, see Ref. (13) Department of Chemistry, University of Dares Salaam, P.O. Box 35061, Dares Salaam, Tanzania Present address of author to whom all correspondence should be addressed: Quest International, P.O. Box 2, 1400 CA Bussum, the Netherlands Department of Medical Protozoology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, U.K. Department of Botany, University of Dares Salaam, P.O. Box 35060, Dares Salaam, Tanzania 6 National Institute for Medical Research, Amani Research Centre, P.O. Box 4, Amani, Tanzania Laboratory for Organic Chemistry, Agricultural University Wageningen, Dreijenplein 8, 6703 BC Wageningen, the Netherlands 2
Abstract Pure compounds were isolated from plant extracts with antimalarial activity. The extracts were ob-
tained from the tubers of Cyperus rotundus L.
(Cyperaceae), the rootbark of Zanthoxylum gilletii (De Wild) Waterm. (Rutaceae), and the rootbark of Margaritaria discoidea (Baill.) Webster (Euphorbiaceae). The most active compounds included (IC50 within brackets): u-cyperone (1) (5.5 .tg/ml), N-isobutyldeca-2,4dienamide (2) (5.4 .tg/ml), and securinine (3) (5.4 .tgIml). A mixture of autoxidation products of 3-selinene was found to be the most active antimalarial substances obtained from C. rotundus (5.6 .tg/ml.
A large-scale screening of plants from all over the world for anti-malarial activity was reported by Spenceretal. in 1947 (3). However, avian malaria parasites were used for the antimalarial testing, and no follow up has
been reported on the isolation and testing of pure compounds.
Recently a semi-automated antimalarial testing method was developed, using in vitro uptake of[3H]-
hypoxanthine by Plasmodium falciparum parasites as an indicator of growth (4, 5). Several quassinoids obtained from Simaroubaceae plants have been tested for their activity in inhibiting growth of P. falciparum, and were found to possess potent activity (6—10). Unfortunately, the majority
of quassinoids showed relatively high mammalian cytotoxocity. However, preliminary studies on the structure-ac-
Key words Tanzanian, medicinal plants, antimalarial activity, Cyperus rotundus, Zanthoxylum gilletii, Mar-
garitaria discoidea, a-cyperone, N-isobutyldeca-2,4dienamide, securinine.
Introduction Malaria afflicts over 400 million people, and kills about two million every year (1). Drugs presently in use include quinoline derivatives, suipha drugs, and antifolates. However, resistance is developing quickly against most of them; moreover, their use is hampered by undesired side-effects. Hence novel antimalarial drugs are urgently required.
The continuing clinical use of quinine isolated from Cinchona species, and the recent discovery of the antimalarial substance artemisinin from the Chinese herb Artemisia annua L. (Compositae) (2), has stimulated our interest in medicinal plants as sources of new antimalarial drugs.
tivity relationships of these compounds indicate that the structural requirements for the activity and toxicity are different(6, 11). Khalid et al. have also used the radiometric assay to test compounds which they isolated from Sudanese traditional medicinal plants (12). Of 21 compounds tested, the limonoid gedunin was found to be most potent (IC50 approx: 1 !.LM).
In this study, we have investigated some extracts which we had previously found to possess in vitro an-
timalarial activity (13). From these fractions compounds were isolated, identified, and their IC50 values determined.
Materials and Methods Plant materials All plants were collected from Dar es Salaam, Tanga, and Mwanza regions. Voucher specimens were deposited at the herbarium, Botany Department, University of Dares Salaam.
Downloaded by: Universite Laval. Copyrighted material.
Received: June 22, 1989
H. Weenenetal.
372 Planta Med. 56(1990)
Results and Discussion
CR3 H3
O.J:I:II;tI1:IIIJ..t:.CHz
CH3
CR3
2
1
When the dichloromethane extract of C. rotundus tubers was fractionated into ten fractions by preparative silica gel TLC, using petrol/ethyl acetate (4/1, v/v) as the eluent, the least polar fraction was found to be the one
with the highest activity (IC50 c S .tg/ml). The
dichioromethane extract was therefore reextracted with petrol, to exclude the more polar substances, and fractionated by column chromatography. Using silica gel and silver nitrate impregnated silica gel, five sesquiterpenes were obtained and tested for antimalarial activity (Table 1). Of these a-cyperone (1) showed the highest activity, with an IC50 of 5.5 .sg/ml. Humulene, cu-selinene, -selinene, and cyperene showed no antimalarial activity at concentrations below
HèY
50g/ml.
All plant materials were dried, pulverized and extracted either consecutively with petroleum ether (h.p. 40—60°C), dichioromethane and methanol, 2 x 48 h for each solvent, or with methanol alone. The concentrated extracts were tested for antimalarial activity, and some of the most active fractions were fractionated by chromatography into pure compounds, which were identified spectroscopically, and tested for antimalarial activity as well. The crude extracts were fractionated by column chromatography
using silica gel (400 mesh) and were eluted with a gradient of hexane and ethyl acetate. The semi-purified fractions were further purified by column chromatography using silica gel, silver nitrate impregnated silica gel, alumina and/or fractogel (PVA 500, Merck), by preparative TLC (silica gel and silanized silica gel), and whenever possible by recrystallization. Spectroscopic data and melting points of all known compounds were in agreement with literature data.
However, the activity of )3-selinene ap-
peared to fluctuate, depending on its history. When investigating this more closely, we observed that 3-selinene was
rather unstable, and decomposed to compounds which contain a hydroperoxide group, as indicated by KI/starch on TLC (15). A mixture of the decomposition products was observed to show antimalarial activity with an IC50 as low as
5.56 g/ml. As expected, the decomposition of 3-selinene could be inhibited by the addition of 0.1% vitamin E. The tertiary C-H a to the exocyclic double bond is particularly prone to homolytic cleavage, as the resulting tertiary allylic radical is highly stabilized. We assume a hydroperoxide is formed at this position via an autoxidation reaction, which
will decompose to more stable products. Similar by-
droperoxides have previously been isolated from Alpinia japonica (Thunb.) Miq. (Zingiberaceae) (16), and the fact Antimalarial testing that both C-S epimers were isolated suggests that their forIsolated compounds were assessed for in vitro mation was also due to autoxidation, rather than an enactivity against Plasmodiurn flaciparum strain K1, which origi- zymatic process. Since the autoxidized 3-selinene sample
nated in Thailand, and which is multidrug resistant (14). The
technique used measured the ability of the extracts and pure compounds to inhibit the incorporation of [311]-hypoxanthine into the malaria parasites. Precise details of the protocol are described in references (4) and (5). Compounds were tested in duplicate at 12 concentrations in threefold dilutions starting from 50 .tg/ml.
was a mixture, some of its individual components may have a much lower IC50. We are presently studying the nature and the activity of the hydroperoxides in more detail. How-
ever their potential as antimalarial drugs is questionable, since they are not very stable. Zanthoxylum gilletii gave two compounds with an IC50 of less than 50 zg/ml. Among these N-isobutyldeca-2,4-dienamide (2) was most active (IC50 = 5.37 .tg/ml), while fagaramide was only slightly active (IC50 = 12.34 jig/mi).
source
Cyperusrotundus
compound
a.cyperone(1) a-selinene 3-selinene 3-selinene (autoxidized) cyperene humulene
Zanthoxylumgilletii
Margarifaria discoidea
N-isobutyldeca-2,4-dienamide (2) fagaramide lupeol sesamin 4,7,8-trimethoxyfuro[2,3-bJquinoline securinine (3)
IC55
95%
(jig/mI)
confidence interval
5.50
(4.09—7.39)
> 50 > 50 5.56
>50
(4.65—6.34)
> 50 5.37 12.34
> 50
(3.72—7.73) (9.23—16.51)
>50 >50 5.35
(4.32—6.63)
Table 1 Antimalarial activity of compounds from Tanzanian medicinal plants.
Downloaded by: Universite Laval. Copyrighted material.
Extraction and isolation procedures
Antimalarial Compounds Containing an a, /3-Unsaturated Carbonyl Moiety from Tanzanian Medicinal Plants
decomposed after isolation, and could therefore not be tested.
Interestingly, all four compounds which were found to possess significant antimalarial activity and which were identified, contain an a,f3-unsaturated carbonyl
moiety. Their antimalarial properties can possibly be
References 1 Stürcher, D. (1987) Parasitology Today 5 (2), 39. 2 Liu, J. M., Ni, M. Y.. FanY. F., et al. (1979)Acta Chim. Sinica 37,129. Spencer, C. F., Koninszy, F. R., Rogers, E. F., et al. (1947) Lloydia 10, 145. Desjardins, R. E., Canfield, C. J., Haynes, J. D., Chulay, J. D. (1979) Antimicrob. Agents Chemother. 16, 710. O'Neill, M. J., Bray, D. H., Boardman, P., Phillipson, J. D., Warhurst, D. C. (1985) Planta Med. 394. 6 O'Neill, M. J., Bray, D. H., Boardman, P., Phillipson, J. D., Warhurst, D. C., Peters, W., SuiTness, M. (1986) Antimicrob. Agents Chemother.
'
30, 101. Chan, K. L., O'Neill, M. J., Phillipson, J. D., Warhurst, D. C. (1986) Planta Med. 105.
explained by the tendency of the nucleic acids of the parasites to react with the a,13-unsaturated carbonyl moiety, in a Michael addition fashion. Although Lantana camara L. was one of the most active plants in our screening, it was not investigated further, because it is reported to contain toxic principles (17). We are however presently studying the chemical com-
position of Hoslundia opposita Vahl., as well as some Uvaria species, which will be reported separately.
Acknowledgements We are grateful to Dr. H. Wijnberg, Dr. M. Posthumus (University of Wageningen), and to Prof. P. Waterman (University of Strathclyde) for providing spectra, and for their helpful suggestions. We thank Dr. 0. MotI (Czechoslovak Academy of Science) for sending us an authentic sample of cyperene. Part of this
research was supported by a grant from the University of Dar es Salaam.
Pavanand, K., Nutaleul, W., Dechatiwongse, T., Yoshihira, K.,
° 12 13 14 15 16 17
Yongvanitchit, K., Scovill, J. P., Flippen-Anderson, J. L., Gilardi, R., George, C., Kanchanapee, P., Webster, H. K. (1986) Planta Med. 108. Fandeur, T., Moretti, C., Polonsky, J. (1985) Planta Med. 20. Trager,W., Polonsky, J. (1981)Arn. J. Trop. Med. Hyg. 30,531—537. Bray, D. H., O'Neill, M. J., Phillipson, J. D., Warhurst, D.C. (1987) J. Pharmac. Pharmacology 39 (Suppl) 85. Khalid, S. A., Farouk, A., Geary, T. G., Jensen, J. B. (1986) J. Ethnopharmacology 15, 201. Weenen, H., Nkunya, M. H. H., Bray, D. H., Mwasumbi, L. B., Kinabo, L. S., Kilimali, V.A. E. B. (1990) Planta Med. 56, 368. Thaitong, S., Beale, G. H., Chutmongkonkul, M. (1983) Trans. Roy. Soc. Trop. Med. Hyg. 77, 228. Porter, N. A., Wolf, R. A., Weenen, H. (1980) Lipids 15, 163. Itokawa, H., Morita, H., Watanabe, K. (1987) Chem. Pharm. Bull. 35,
1460. Sharma, 0. P. (1984) Vet. Hum. Toxicol. 26(6), 488.
Downloaded by: Universite Laval. Copyrighted material.
From Margaritaria discoidea only one stable compound was isolated, i.e. the alkaloid securinine (3), which showed an IC50 of 5.35 ig/ml. Another isomeric alkaloid isolated from the same plant, allosecurinine, quickly
Planta Med. 56(1990) 373
