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Workshop Plantas Medicinais e Fitoterapêuticas nos Trópicos. IICT /CCCM, 29, 30 e 31 de Outubro de 2008 Antimalarial activity of some plants traditionally used in Mozambique _______________________________________________________________________________________________________ ANTIMALARIAL ACTIVITY OF SOME PLANTS TRADITIONALLY USED IN
MOZAMBIQUE
Cátia Ramalhete*, Dinora Lopes**, Silva Mulhovo***, Virgílio E. Rosário **, Maria José
U. Ferreira*
*Med.UL, Faculty of Pharmacy, University of Lisbon, Av. das Forças Armadas, 1600-083 Lisbon, Portugal; ** CMDT.LA, Institute of Hygiene and Tropical Medicine, UNL, R. da Junqueira 96, 1349-008 Lisbon, *** Polytechnic Institute of Gaza (ISPG), Chokwe, Mozambique. * Corresponding author. Tel.: 351-21-7946475; fax: +351-21-7946470; e-mail: mjuferreira@ff.ul.pt
Abstract
Developing countries, where malaria is endemic, depend strongly on traditional medicine as a source for
inexpensive treatment of this disease. However, scientific data to validate the antimalarial properties of these
herbal remedies are scarce. Consequently, it is important that antimalarial medicinal plants are investigated,
in order to establish their efficacy and to determine their potential as sources of new antimalarial drugs.
In this study, we evaluated the claimed antimalarial properties of fifty eight crude extracts from fifteen plants
used in traditional medicine against malaria and fever, mainly from Southern African regions. The air-dried
powdered plant parts (roots, leaves, seeds or bark) or whole plants were extracted, sequentially, with solvents
of increased polarity (n-hexane, dichloromethane, ethyl acetate and methanol). Schizontocidal activity was
measured using a standard in vitro assay, with 3D7 Plasmodium falciparum strain. From the 58 extracts
tested, two of them showed a significant activity (IC50 < 5 µg/mL), and 34.5 % showed a moderate activity
(10 < IC50 < 50 µg/mL). A bioassay-guided fractionation of the most active extracts is ongoing.
Key words: Traditional medicine; Medicinal plants; Malaria; Antimalarial; Plasmodium falciparum.
1. INTRODUCTION
Malaria is a major parasitic disease in the world, especially in Africa. It is responsible for 500 million new cases and 2 to 3 millions deaths every year, mostly among children under five years and pregnant women (WHO, 2008). Plasmodium falciparum the most widespread etiological agent for human malaria has become increasingly resistant to standard antimalarials e.g. chloroquine and antifolates. Consequently, new drugs or drug combinations are urgently needed today for the treatment of malaria. These drugs should have novel modes of action or be chemically different from the drugs in current use. In Africa and elsewhere, plant extracts are still widely used in the treatment of malaria and other ailments, and up to 80% of the African population use traditional medicines for primary health care (WHO, 2002). Since little scientific data exist to validate antimalarial properties of these medicinal plants, it is important that their claimed Workshop Plantas Medicinais e Fitoterapêuticas nos Trópicos. IICT /CCCM, 29, 30 e 31 de Outubro de 2008 Antimalarial activity of some plants traditionally used in Mozambique _______________________________________________________________________________________________________ antimalarial properties are investigated, in order to establish their efficacy and determine their potential as sources of new antimalarial drugs (such as, artemisinin isolated from In the present work, we have evaluated the in vitro antimalarial activity of fifteen plants uses in traditional medicine against malaria and/or fever. 2. MATERIAL AND METHODS
2.1. Plant materials
Plants were obtained from Mozambique and Portugal. The information of the plant material is summarized in Table 1. The species collected in Mozambique were identified locally and voucher specimens (Table 1) have been deposited at the herbarium of Instituto de Investigação Agronómica of Mozambique The remaining species were collected in Portugal and identified by Dr. Teresa Vasconcelos from the Instituto Superior de Agronomia, University of Lisbon, Portugal, where voucher specimens (Table 1) were 2.2. Preparation of extracts
Different plant parts (roots, leaves, seeds, and bark) or the entire plant, from selected species (Table 1), were dried at room temperature. Crude plant extracts were prepared by submitting 15-50g of air-dried powdered plant material to a sequential extraction procedure with 150-500 mL of n-hexane, dichloromethane (DCM), ethyl acetate (EtOAc), and methanol (MeOH) for 48 h, at room temperature. After filtration, the extracts were fully dried, under reduced pressure at 40-45 ºC, by using a Büchi rotatory evaporator, and then stored at low temperature (4ºC) until their use in antimalarial assays. 2.3. In vitro culture of Plasmodium falciparum and antimalarial activity
The in vitro activity against Plasmodium falciparum intraerythrocytic stages of crude extracts was evaluated by means of the Mark III test, as developed by the WHO (WHO, 2001). Briefly, the 3D7 P. falciparum strain (susceptible to chloroquine) was cultivated in recently collected erythrocytes as host cells in RPMI 1640 medium (Gibco) containing 25 mM HEPES (Sigma) and 6.8 M hypoxanthine (Sigma) supplemented with 0.5% AlbuMAX II (Invitrogen). Cultures were kept at 37ºC under an atmosphere of 5% O2, 3–5% CO2, and N2. Drug-sensitivity assays were carried out on 96-well micro-plates. Crude extracts were dissolved in ethanol or dimethyl sulfoxide (DMSO) and diluted in RPMI. The micro-plates were titrated with two-fold serial dilutions of each extract as well Workshop Plantas Medicinais e Fitoterapêuticas nos Trópicos. IICT /CCCM, 29, 30 e 31 de Outubro de 2008 Antimalarial activity of some plants traditionally used in Mozambique _______________________________________________________________________________________________________ as the control. The strain was plated (in duplicate) using a synchronized culture at ring- stage parasites, with a parasitaemia between 0.6–0.8%. Each well received 10 µL of parasite-loaded erythrocytes, 5% haematocrit, and 90 µL of the different drug dilutions. The plates were incubated at 37ºC for 20-24 h, after confirmation of presence of mature schizonts in control wells, without drug. At the end of this period, the blood from each well was harvested, and a thick film was prepared. The films were fixated with acetone, and stained for 60 min in Giemsa stain at a dilution of 1 vol-% in buffered H2O (pH 6.8). Three independent optical-microscopy readings of the number of schizonts with three or more nuclei were carried out in 200 parasitized red cells for each dilution and duplicate. Growth inhibition was expressed as percent of the number of schizonts for each concentration, compared with untreated controls. Mean IC50 values were calculated from dose-response curves (percentage of schizonts vs. logarithm of drug concentration) by linear interpolation. Table 1. Plant material data
Plant species
Plant part
aAcacia karroo Hayne
bAloe parvibracteata Schonland
cBridelia cathartica Bertol.f.
dCassia abbreviate Oliv.
cCassia occidentalis L.
eCrossopteryx febrifuga (Afzel. ex G. Don) Rubiaceae
Benth
fLeonotis leonurus (L.) R.Br
dMomordica balsamina L.
bParkinsonia aculeata L.
aPittosporum tobira (Thunb.) W.T. Aiton
bPlumbago auriculata Lam.
fSenna didymobotrya Fresen.
aSchefflera actinophylla (Endl.) Harms
cTabernaemontana elegans Strapt.
cTrichilia emetica Vahl
Workshop Plantas Medicinais e Fitoterapêuticas nos Trópicos. IICT /CCCM, 29, 30 e 31 de Outubro de 2008 Antimalarial activity of some plants traditionally used in Mozambique _______________________________________________________________________________________________________ Plants collected in: aGarcia da Horta Garden (Lisbon, Portugal, January 2006), b Parque botânico da Tapada da Ajuda (Lisbon, Portugal. August, 2005), c Maputo (Mozambique, March 2006), d Gaza (Mozambique, January 2006), e Inhambane (Mozambique, April 3. RESULTS AND DISCUSSION
The main goal of this work was to investigate the potential antimalarial properties of some plants used in traditional medicine, mostly in Mozambique, against malaria and/ or fever, and providing scientific validation for their use. Therefore, selection of plants was carried out based mainly on an ethnobotanical approach (JANSEN and MENDES, 1982, 1983, 1990, 1991; MADUREIRA, 2002). Some species, namely Aloe parvibracteata, Pittosporum tobira, Plumbago auriculata and Schefflera actinophylla
were selected based on a quimiotaxonomic approach (CLARKSON, 2004). Fifty eight extracts from fifteen plants species, belonging to several families, were screened for their potential antimalarial properties against cloroquine-sensitive P. falciparum strain (3D7). The extracts were prepared by sequentially extracting the plant material with n-hexane, dichloromethane, ethyl acetate and methanol (see experimental section). The results are summarized in Table 2. The antimalarial activity of extracts was defined according to the IC50 values obtained. An extract showing an IC50 value ≤ 5 µg/mL was classified as highly active. Extracts with IC50 values ≥ 10 µg/mL and ≤ 50 µg/mL were considered moderately active and those with IC50 values > 50 µg/mL inactive. As can be observed in Table 2, the lowest IC50 values were found for the ethyl acetate extracts of Momordica balsamina (IC50 = 1.0 ± 0.1 µg/mL) and Pittosporum tobira (IC50 = 4.8 ± 1.8 µg/mL). Significant IC50 values were also found for the n-hexane extracts of Cassia occidentalis (IC50 19.3 ± 2.0 µg/mL), and Parkisonia aculeata (IC50 24.5 ± 2.9 µg/mL). The remaining species have showed a moderate/week antiplasmodial Momordica balsamina Linn. (Cucurbitaceae) a climber extensively cultivated in many tropical and subtropical regions of the world has been used in sub-Saharan Africa as food (FLYMEN, 2007). This plant is traditionally used in Mozambique to treat vomiting believed to be associated with fever (BANDEIRA, 2001; VELE, 2000). Previous studies on this plant have resulted in the isolation and identification of two phenylpropanoid esters, rosmarinic acid and five pimarane diterpenes (DE TOMMASI, Workshop Plantas Medicinais e Fitoterapêuticas nos Trópicos. IICT /CCCM, 29, 30 e 31 de Outubro de 2008 Antimalarial activity of some plants traditionally used in Mozambique _______________________________________________________________________________________________________ 1991 and 1996). Antimalarial activity for Momordica balsamina (CLARKSON, 2004; BENOIT-VICAL, 2006) and other species of Momordica genus, was previously described (KAOU, 2008; KÜLTÜR, 2007; FROELICH, 2007; NGUYEN-POUPLIN, 2007; WAAKO, 2005; MÉNAN, 2006; HOUT, 2006). Table 2. In vitro antimalarial activity (IC50 values) of the plant extracts against
Plasmodium falciparum 3D7.
Plant Species
IC50 (µg/ mL)
n-hexane DMC
Acacia karroo
Aloe parvibracteata
Bridelia cathartica
Cassia abbreviata
Cassia occidentalis
Crossopteryx febrifuga
Leonotis leonurus
Momordica balsamina
Parkinsonia aculeata
Pittosporum tobira
Plumbago auriculata
Senna didymobotrya
Schefflera actinophylla
Tabernaemontana elegans
Trichilia emetica
Pittosporum tobira is an ornamental shrub or small tree common on the Mediterranean coast. In previous studies, this plant was reported to contain some terpenoids, sesquiterpenoids, and carotonoids, but no antimalarial activity was Workshop Plantas Medicinais e Fitoterapêuticas nos Trópicos. IICT /CCCM, 29, 30 e 31 de Outubro de 2008 Antimalarial activity of some plants traditionally used in Mozambique _______________________________________________________________________________________________________ 2001; FUJIWARA, 2001). A saponin mixture, obtained from the leaves of this specie also shown for Pittosporum viridiflorum (CLARKSON, 2004) corroborating the interest of Pittosporum genus as a potential source of antimalarials. Like Pittosporum tobira (Thumb.) W.T. Aiton, Acacia karroo Hayne, Aloe parvibracteata Schonland, Crossopteryx febriguga (Afzel. ex G. Don) Benth, Plumbago auriculata Lam., Schefflera actinophylla (Endl.) Harms, Tabernaemontana elegans Strapt. and Trichilia emetica (seeds) were also screened for the first time for their antimalarial activity in this work. Among these plants, Acacia karroo (IC50 = 60 ± 12.3 µg/mL), Crossopteryx febrifuga (IC50 = 44.4 ± 3.1 µg/mL), and Tabernaemontana elegans (IC50 = 26.9 ± 5.3 µg/mL), widely used to treat malaria in developing countries, namely Mozambique, revealed moderate or no significant activity. However, it is important to note that these plants are frequently used to treat fever, generally associated to malaria. Therefore, an explanation for their lack of in vitro antimalarial inactivity could be that these plants may act as antipyretics or may enhance the immune system, rather than having direct antiparasitic activity (PHILLIPSON and WRIGHT, 1991). Another explanation is that these plants could contain prodrugs non-active by themselves. In this case, these precursors of the active compounds have to be metabolized in vivo into active antimalarials, a major limitation in this study. As illustrated by the results, a number of plants did not display an effective antimalarial activity, though some of these had previously been described as having antimalarial activity. For example, the ethanol extract from the stem of Bridelia cathartica Bertol.f (Marracuene, Mozambique) caused a 50% inhibition of parasite growth at an incubation concentration of 0.05 µg/mL (JURG, 1991). In addition, extracts (DCM/MeOH; 1:1) from twigs of Senna didymobotrya, Parkinsonia aculeata as welll as from Leonotis leonurus showed a significant antimalarial activity against P. falciparum D10 strain (IC50 < 10 µg/mL) (CLARKSON, 2004). Furthermore, the ethanol extract from the leaves of Cassia occidentalis, showed an high in vitro antimalarial activity against a P. falciparum chloroquine-sensitive strain (IC50 < 3 The regions and periods of the year of plant collection are known to play an important role in the variation of the type of compounds found in plants as well as their Workshop Plantas Medicinais e Fitoterapêuticas nos Trópicos. IICT /CCCM, 29, 30 e 31 de Outubro de 2008 Antimalarial activity of some plants traditionally used in Mozambique _______________________________________________________________________________________________________ concentration. Therefore, they may be responsible for the different results obtained in This study has highlighted two promising plants for further antimalarial investigations. The determination of their active compounds is on going. ACKNOWLEDGMENTS
We are grateful to Dr. Teresa Vasconcelos (ISA, University of Lisbon, Portugal) for plants identification. We would also like to thank Dr. Catarina Arruda and Dr. Guedes de Sousa, from the Portuguese Embassy in Mozambique, as well as the Portuguese Office of International Affairs for plants transport. This work was supported by the Science and Technology Foundation, Portugal (FCT, grant 5. REFERENCES
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