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The antimalarial activity of Vernonia amygdalina Del (Compositae)
TRANSACTIONS OFTHE ROYALSOCIETYOFTROPICALMEDICINEAND HYGIENE(2000)
The antimalarial activity of Vernonia amygda/ina Del (Compositae) S. C. Masaba Depamnent of Zoology, Makerere University, P.O. Box 7062, Kampala, Uganda Keywords: malaria, Plasmodium fahparum, herbal medicine, plant extracts, Venzonia amygdalina, antimalarial activity, in vitro
94,694-695
cells were centrifuged once more at 800 g for 5 min. The synchronized parasites were resuspended in the culture medium and washed twice with the medium. The tests were done in triplicate in 96-well culture plates (Nucleon, Denmark) with initial cultures of 0.5% parasitaemia and 1% haematocrit. Concentrations of the plant extracts used were 10, 50, 100 and 200 I*g/mL. Blood suspension (50 pL) was transferred to each test well including the control wells which had no drug. After shaking to myx the extract and the cells, the plates were incubated in a candle jar at a temperature of 37.5”C for 48 h. Thick blood smears were made and stained with Giemsa stain and the parasites counted. Results
Introduction
Malaria affects millions of people in the tropics with an estimated annual mortality rate of between 1 and 2 million, most of whom are children under the age of 5 years. The people who are the most vulnerable to malaria are usually the poor with little or no accessto modem medical services. Such people make up the bulk of the estimated 80% of the world’s population that relies on herbal medicine to manage disease (KIRBY, 1996). The malaria situation has also been compounded by the emergence of multidrug-resistant forms of Plasmodium falciparum in much of the malarious parts of the world. The need to urgently develop alternative antimalarials that are not only effective against resistant malaria but also relatively inexpensive for those who need them most is understandable. One area that seemsto offer promising prospects concerns the use of plant extracts. Extracts of the Chinese plant Artemisia annua are now being used to treat multidrug-resistant malaria (BUNNAG et al., 1992). Vemonia amygdalina is a plant that is widely used in Africa, including Uganda, to treat a multiplicity of diseases including schistosomiasis, amoebic dysentery, and gastrointestinal problems (HUFFMAN et al., 1996). Although any part ofthe plant can be used, there seemsto be a preference for the leaves which are quite bitter in taste, the bitterness being taken as a sign of potency. Chimpanzees are also known to chew the pith of the young shoots as a way of treating parasitic infections (HUFFMAN & SEIFU, 1989; KOSHIMIZU et al., 1994). The purpose of this study was (1) to investigate, in vitro, the antimalarial activity of V. amygdalina, and (2) to compare the bioactivities of its aqueous and acetonewater extracts. Materials
and Methods
Two extraction methods were used. One method involved boiling 20 g of tiesh green leaves at 100°C in 500 mL of water for 20 min. This is the usual method of extraction bv the indigenous users in Uganda. The decoction was cooled anh filtered. In the second method 20 e: of fresh leaves were dried in an oven at 50°C for 3 days. The dried leaves were crushed to fine particles in a mortar and extracted with 50150 by volume acetonewater mixture (500 mL of each) in a soxhlet at a temperature of 6O’C. The extracts were depigmented and evaporated over a hot plate to dryness. The antimalarial activities of the 2 extracts were tested on an isolate of Plasmodium falciparum originally collected from a schoolchild and preserved in liquid nitrogen. The isolate was known to be sensitive to chloroquine, having been tested using WHO in-vitro microtechnique. Cultures
in 5% sterilized
sorbital.
Discussion
This study has demonstrated the presence of antimalarial activity in the acetone-water &d aqueous extracts of V. amwdaha. However. the antimalarial activitv of the aqueous extract was much lower than that of-the acetone extract. In his study of the Nigerian neem trees, UDEINYA (1993) did not detect any antimalarial activity in the aqueous extracts even though the acetone extract showed a high antimalarial activity. In its traditional use, V. amygdalina and indeed many other herbs are simply boiled in water and the decoction is taken. In the caseof V. amygdalina, up to 100-200 mL of the decoction may be taken by an adult. It is therefore tempting to speculate that herbal decoctions are generally low in bioactivity and this may explain why they are usually consumed in relatively large quantities. V. amygdalina is known here locally as ‘bitter leaf and its medicinal nature is supposed to be due to the bitter taste. The bioactive compounds of V. amygdahna are sesquiterpene lactones. These compounds are present in
the standard were carried
out according to the me&d of TRAGER & JENSEN (1976), with slight modifications. The RPM1 1640 medium was supplemented with 25 mM HEPES buffer, 5% NaHCO, and 5% human (AB) serum. The complete medium was sterilized through 0.22~pm Millipore filters. Parasitized blood was centrifuged at 800 g for 5 min and after discarding the supematant the parasites were synchronized
Parasite growth was expressed as a percentage of the growth in the control wells and plotted on a graph against the concentrations of the extracts. Antimalarial activity was detected in both the aqueous and the acetone-water extracts of V. amygdalina. However, as shown in the Figure, the acetone-water extract showed more bioactivity than the aqueous extract. The acetone-water extract at 10, 50, 100 and 200 @mL produced 51.8%, 5.8%, 0.7% and no growth, respectively (compared with the control well without drug) after 48 h incubation; the corresponding values for the aqueous extract at the same concentrations were 88.5%, 49.4%, 17.5% and 5.9%, respectively. Regression analysis to determine the concentrations of the 2extracts thaiwould be needed to inhibit growth in 50% of the oarasites aave concentrations of 25.5 ue/mL for the acetbne-wat& extract and 76.7 pg/mL $0; the aqueous extract.
After
5 min the
0
100 Concentration
260 (@nL.)
Figure. Activity in vitm of (a) acetone-water and m) aqueous extracts of F’emoniaamygdalina on l? falcipancm. The activity is expressed as a percentage of the growth (after 48 h) in control wells without drug. See the text for experimental details.
ANTIMALARIAL
ACTIVITY OF VERNONIA
695
AMYGDALINA
the leaves and the pith of young shoots and are not only bitter tasting but also show significant bioactivity against a number of protozoan and helminth parasites, and antitumour and antibacterial activities @SARA et al., 1993; TADESSE et al., 1993; OHIGASHI
et al., 1994).
Acknowledgement I am grateful to Mr D. C. J. Obbo for his assistance with the laboratory work. This study was supported by the United Nations Small Grants Programme For Africa: Social and Economic Aspects of Tropical Diseases. References Bunnag, D., Karbwang, J. & Harinasuta, T. (1992). Artemether in the treatment of multidrug resistant falciparum malaria. Southeast AsianJournal of Tropical Medicine and Public Health, 23,762-767. Huffman, M. A. & Seifu, M. (1989). Observations on the illness and consumption of possible medicinal plant Vernonia arnygdalina (Del), by a wild chimpanzee in the Mahele Mountains National Park, Tanzania. Primates, 30, 51-63. Huffman, M. A., Koshimizu, K. & Ohigashi, H. (1996). Ethnobotany and zoopharmacognosy of Vernonia amygdalina, a medicinal plant used by humans and chimpanzees. In: Compositae: BioZogy end Utilization, Volume 2, Caligari, P. D. S. & Hind, D. J. N. (editors). pp. 351-360. Jisaka, M., Ohigashi, H., Takegawa, K., Huffman, M. A. & Koshimizu, K. (1993). Antitumor and antimicrobial activities of bitter sesquiterpene lactones of Vemonia amygdalina, a
1 Announcement
possible medicinal plant used by wild chimpanzee. Bioscience, Biotechnology, Biochemistry, 57,833-834. Kirby, G. C. (1996). Medicinal plants and the control of protozoa1 disease, with particular reference to malaria. Transactions of the Royal Society of Tropical Medicine and Hygiene, 90, 605-609. Koshimizu, K., Ohigashi, J. & Huffman, M. A. (1994). Use of Vernonia amygdalina by wild chimpanzee: possible roles of its bitter and related constituents. Physiology and Behaviour, 56, 1209-1216. Ohigashi, H., Huffman, M. A., Izutsu, D., Koshimizu, K., Kawanaka,M., Sugiyama,H.,Kirby,G. C., Warhurst,D. C., Allen, D., Delmas, F., Elias, R. & Balansard, G. (1994). Toward the chemical ecology of medicinal plant use in chimpanzees: the case of Vernonia amygdalina, a plant used by chimpanzees possibly for parasite-related diseases. Journal of Chemical Ecology, 20, 541-553. Tadesse, A., Gabre-Hiot, A., Asres, K., Djote, M. & Frommel, D. (1993). The in vitro activity of Vemoniu amygdalina on Letihmania aethiopica. Ethiopian Medical Journal, 31, 183189. Trager, W. &Jensen, J. R. (1976). Human malarial parasites in continuous culture. Science, 193, 673-675. Udeinya, I. J. (1993). Antimalarial activity of Nigerian neem leaves. Transactionsof the Royal Sociey of TropicalMedicine and Hygiene, 87,47 1. Received 3 Februa y 2000; revised 14 March for publication 9 May 2000
2000; accepted
1 The Tropical
Health
and Education
Trust
(THET)
Since THET was established in 1988, it has actively supported training for health care in tropical countries, where resources are limited. THET works with those who are responsible for such training so that their goals can be reached, as they prepare their staff and students for the tasks that they will be called on to do, relevant to the needs of their local community. THET achieves this strategy through collaborative links between a hospital or medical/nursing school in the United Kingdom and its counterpart overseas. These institutional links are made up of individual links, each of which is carefully planned over a number of years. Precise goals are set so that progress, which is closely monitored, can be used as a basis for additional funding. Links cover many disciplines from nursing, clinical medicine, community health, laboratory and library services, and management, to fieldwork by students. We respond to requests to establish these links and, while we do not prescribe what the pattern of a link should be, we are only prepared to seek funding for those that are relevant and needed, and that directly or indirectly contribute to the health care of poor people. THET does not fund individuals, except when newly acquired personal skills contribute to an institution and the service that it provides. Trustees: Professor Eldryd Parry (chairman), Professor Roy Anderson, FRS, Professor Brian Greenwood, FRS, Miss Thelma Holland, Mrs Helen Parry, Mr John Rennie FRCS, Mr Richard Southwell, QC and Professor David Warrell. For further information please contact THET, Euston House, 24 Eversholt Street, London NW1 lAD, UK; phone +44 (0)20 7611 870516, fax +44 (0)20 7611 068314, e-mail [email protected]
The antimalarial activity of Vernonia amygda/ina Del (Compositae) S. C. Masaba Depamnent of Zoology, Makerere University, P.O. Box 7062, Kampala, Uganda Keywords: malaria, Plasmodium fahparum, herbal medicine, plant extracts, Venzonia amygdalina, antimalarial activity, in vitro
94,694-695
cells were centrifuged once more at 800 g for 5 min. The synchronized parasites were resuspended in the culture medium and washed twice with the medium. The tests were done in triplicate in 96-well culture plates (Nucleon, Denmark) with initial cultures of 0.5% parasitaemia and 1% haematocrit. Concentrations of the plant extracts used were 10, 50, 100 and 200 I*g/mL. Blood suspension (50 pL) was transferred to each test well including the control wells which had no drug. After shaking to myx the extract and the cells, the plates were incubated in a candle jar at a temperature of 37.5”C for 48 h. Thick blood smears were made and stained with Giemsa stain and the parasites counted. Results
Introduction
Malaria affects millions of people in the tropics with an estimated annual mortality rate of between 1 and 2 million, most of whom are children under the age of 5 years. The people who are the most vulnerable to malaria are usually the poor with little or no accessto modem medical services. Such people make up the bulk of the estimated 80% of the world’s population that relies on herbal medicine to manage disease (KIRBY, 1996). The malaria situation has also been compounded by the emergence of multidrug-resistant forms of Plasmodium falciparum in much of the malarious parts of the world. The need to urgently develop alternative antimalarials that are not only effective against resistant malaria but also relatively inexpensive for those who need them most is understandable. One area that seemsto offer promising prospects concerns the use of plant extracts. Extracts of the Chinese plant Artemisia annua are now being used to treat multidrug-resistant malaria (BUNNAG et al., 1992). Vemonia amygdalina is a plant that is widely used in Africa, including Uganda, to treat a multiplicity of diseases including schistosomiasis, amoebic dysentery, and gastrointestinal problems (HUFFMAN et al., 1996). Although any part ofthe plant can be used, there seemsto be a preference for the leaves which are quite bitter in taste, the bitterness being taken as a sign of potency. Chimpanzees are also known to chew the pith of the young shoots as a way of treating parasitic infections (HUFFMAN & SEIFU, 1989; KOSHIMIZU et al., 1994). The purpose of this study was (1) to investigate, in vitro, the antimalarial activity of V. amygdalina, and (2) to compare the bioactivities of its aqueous and acetonewater extracts. Materials
and Methods
Two extraction methods were used. One method involved boiling 20 g of tiesh green leaves at 100°C in 500 mL of water for 20 min. This is the usual method of extraction bv the indigenous users in Uganda. The decoction was cooled anh filtered. In the second method 20 e: of fresh leaves were dried in an oven at 50°C for 3 days. The dried leaves were crushed to fine particles in a mortar and extracted with 50150 by volume acetonewater mixture (500 mL of each) in a soxhlet at a temperature of 6O’C. The extracts were depigmented and evaporated over a hot plate to dryness. The antimalarial activities of the 2 extracts were tested on an isolate of Plasmodium falciparum originally collected from a schoolchild and preserved in liquid nitrogen. The isolate was known to be sensitive to chloroquine, having been tested using WHO in-vitro microtechnique. Cultures
in 5% sterilized
sorbital.
Discussion
This study has demonstrated the presence of antimalarial activity in the acetone-water &d aqueous extracts of V. amwdaha. However. the antimalarial activitv of the aqueous extract was much lower than that of-the acetone extract. In his study of the Nigerian neem trees, UDEINYA (1993) did not detect any antimalarial activity in the aqueous extracts even though the acetone extract showed a high antimalarial activity. In its traditional use, V. amygdalina and indeed many other herbs are simply boiled in water and the decoction is taken. In the caseof V. amygdalina, up to 100-200 mL of the decoction may be taken by an adult. It is therefore tempting to speculate that herbal decoctions are generally low in bioactivity and this may explain why they are usually consumed in relatively large quantities. V. amygdalina is known here locally as ‘bitter leaf and its medicinal nature is supposed to be due to the bitter taste. The bioactive compounds of V. amygdahna are sesquiterpene lactones. These compounds are present in
the standard were carried
out according to the me&d of TRAGER & JENSEN (1976), with slight modifications. The RPM1 1640 medium was supplemented with 25 mM HEPES buffer, 5% NaHCO, and 5% human (AB) serum. The complete medium was sterilized through 0.22~pm Millipore filters. Parasitized blood was centrifuged at 800 g for 5 min and after discarding the supematant the parasites were synchronized
Parasite growth was expressed as a percentage of the growth in the control wells and plotted on a graph against the concentrations of the extracts. Antimalarial activity was detected in both the aqueous and the acetone-water extracts of V. amygdalina. However, as shown in the Figure, the acetone-water extract showed more bioactivity than the aqueous extract. The acetone-water extract at 10, 50, 100 and 200 @mL produced 51.8%, 5.8%, 0.7% and no growth, respectively (compared with the control well without drug) after 48 h incubation; the corresponding values for the aqueous extract at the same concentrations were 88.5%, 49.4%, 17.5% and 5.9%, respectively. Regression analysis to determine the concentrations of the 2extracts thaiwould be needed to inhibit growth in 50% of the oarasites aave concentrations of 25.5 ue/mL for the acetbne-wat& extract and 76.7 pg/mL $0; the aqueous extract.
After
5 min the
0
100 Concentration
260 (@nL.)
Figure. Activity in vitm of (a) acetone-water and m) aqueous extracts of F’emoniaamygdalina on l? falcipancm. The activity is expressed as a percentage of the growth (after 48 h) in control wells without drug. See the text for experimental details.
ANTIMALARIAL
ACTIVITY OF VERNONIA
695
AMYGDALINA
the leaves and the pith of young shoots and are not only bitter tasting but also show significant bioactivity against a number of protozoan and helminth parasites, and antitumour and antibacterial activities @SARA et al., 1993; TADESSE et al., 1993; OHIGASHI
et al., 1994).
Acknowledgement I am grateful to Mr D. C. J. Obbo for his assistance with the laboratory work. This study was supported by the United Nations Small Grants Programme For Africa: Social and Economic Aspects of Tropical Diseases. References Bunnag, D., Karbwang, J. & Harinasuta, T. (1992). Artemether in the treatment of multidrug resistant falciparum malaria. Southeast AsianJournal of Tropical Medicine and Public Health, 23,762-767. Huffman, M. A. & Seifu, M. (1989). Observations on the illness and consumption of possible medicinal plant Vernonia arnygdalina (Del), by a wild chimpanzee in the Mahele Mountains National Park, Tanzania. Primates, 30, 51-63. Huffman, M. A., Koshimizu, K. & Ohigashi, H. (1996). Ethnobotany and zoopharmacognosy of Vernonia amygdalina, a medicinal plant used by humans and chimpanzees. In: Compositae: BioZogy end Utilization, Volume 2, Caligari, P. D. S. & Hind, D. J. N. (editors). pp. 351-360. Jisaka, M., Ohigashi, H., Takegawa, K., Huffman, M. A. & Koshimizu, K. (1993). Antitumor and antimicrobial activities of bitter sesquiterpene lactones of Vemonia amygdalina, a
1 Announcement
possible medicinal plant used by wild chimpanzee. Bioscience, Biotechnology, Biochemistry, 57,833-834. Kirby, G. C. (1996). Medicinal plants and the control of protozoa1 disease, with particular reference to malaria. Transactions of the Royal Society of Tropical Medicine and Hygiene, 90, 605-609. Koshimizu, K., Ohigashi, J. & Huffman, M. A. (1994). Use of Vernonia amygdalina by wild chimpanzee: possible roles of its bitter and related constituents. Physiology and Behaviour, 56, 1209-1216. Ohigashi, H., Huffman, M. A., Izutsu, D., Koshimizu, K., Kawanaka,M., Sugiyama,H.,Kirby,G. C., Warhurst,D. C., Allen, D., Delmas, F., Elias, R. & Balansard, G. (1994). Toward the chemical ecology of medicinal plant use in chimpanzees: the case of Vernonia amygdalina, a plant used by chimpanzees possibly for parasite-related diseases. Journal of Chemical Ecology, 20, 541-553. Tadesse, A., Gabre-Hiot, A., Asres, K., Djote, M. & Frommel, D. (1993). The in vitro activity of Vemoniu amygdalina on Letihmania aethiopica. Ethiopian Medical Journal, 31, 183189. Trager, W. &Jensen, J. R. (1976). Human malarial parasites in continuous culture. Science, 193, 673-675. Udeinya, I. J. (1993). Antimalarial activity of Nigerian neem leaves. Transactionsof the Royal Sociey of TropicalMedicine and Hygiene, 87,47 1. Received 3 Februa y 2000; revised 14 March for publication 9 May 2000
2000; accepted
1 The Tropical
Health
and Education
Trust
(THET)
Since THET was established in 1988, it has actively supported training for health care in tropical countries, where resources are limited. THET works with those who are responsible for such training so that their goals can be reached, as they prepare their staff and students for the tasks that they will be called on to do, relevant to the needs of their local community. THET achieves this strategy through collaborative links between a hospital or medical/nursing school in the United Kingdom and its counterpart overseas. These institutional links are made up of individual links, each of which is carefully planned over a number of years. Precise goals are set so that progress, which is closely monitored, can be used as a basis for additional funding. Links cover many disciplines from nursing, clinical medicine, community health, laboratory and library services, and management, to fieldwork by students. We respond to requests to establish these links and, while we do not prescribe what the pattern of a link should be, we are only prepared to seek funding for those that are relevant and needed, and that directly or indirectly contribute to the health care of poor people. THET does not fund individuals, except when newly acquired personal skills contribute to an institution and the service that it provides. Trustees: Professor Eldryd Parry (chairman), Professor Roy Anderson, FRS, Professor Brian Greenwood, FRS, Miss Thelma Holland, Mrs Helen Parry, Mr John Rennie FRCS, Mr Richard Southwell, QC and Professor David Warrell. For further information please contact THET, Euston House, 24 Eversholt Street, London NW1 lAD, UK; phone +44 (0)20 7611 870516, fax +44 (0)20 7611 068314, e-mail [email protected]