Investigation of some medicinal plants traditionally used for treatment of malaria in Kenya as potential sources of antimalarial drugs

Experimental Parasitology 127 (2011) 609–626

Contents lists available at ScienceDirect

Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr

Minireview

Investigation of some medicinal plants traditionally used for treatment of malaria in Kenya as potential sources of antimalarial drugs C.N. Muthaura a,⇑, J.M. Keriko b, S. Derese c, A. Yenesew c, G.M. Rukunga a a

Centre for Traditional Medicine and Drug Research, Kenya Medical Research Institute, P.O. Box 54840, Nairobi, Kenya Department of Chemistry, Jomo Kenyatta University of Agriculture and Technology, P.O. Box 62000, Nairobi, Kenya c Department of Chemistry, University of Nairobi, P.O. Box 30197, Nairobi, Kenya b

a r t i c l e

i n f o

Article history: Received 19 February 2010 Received in revised form 1 November 2010 Accepted 9 November 2010 Available online 21 November 2010 Keywords: Ethnopharmacology Kenyan antimalarial plants Antiplasmodial screening

a b s t r a c t Malaria is a major public health problem in many tropical and subtropical countries and the burden of this disease is getting worse, mainly due to the increasing resistance of Plasmodium falciparum against the widely available antimalarial drugs. There is an urgent need for discovery of new antimalarial agents. Herbal medicines for the treatment of various diseases including malaria are an important part of the cultural diversity and traditions of which Kenya0 s biodiversity has been an integral part. Two major antimalarial drugs widely used today came originally from indigenous medical systems, that is quinine and artemisinin, from Peruvian and Chinese ancestral treatments, respectively. Thus ethnopharmacology is a very important resource in which new therapies may be discovered. The present review is an analysis of ethnopharmacological publications on antimalarial therapies from some Kenyan medicinal plants. Ó 2010 Elsevier Inc. All rights reserved.

1. Malaria in Kenya Malaria constitutes one of the biggest health problems in tropical Africa and is slowly spreading to hitherto non-malaria areas (Trape, 2002). The emergence of resistant parasites, changes in climatic conditions over a large part of Africa, changes in land use and population migration (Foster, 1991; Ridley, 1997) are extending the areas of malaria transmission, which requires innovative strategies for malaria and the mosquito vector control. Malaria is also endemic in South-east Asia, in Central and South America and Oceania. After the African countries, India and Brazil are presently the regions of highest endemicity in the World (WHO, 1997). It is estimated that the malaria incidence range between 350 and 500 million cases with 90% of these being in tropical Africa (WHO, 2005). In Kenya, more than 90% of malaria is caused by Plasmodium falciparum (Khaemba et al., 1994) transmitted by Anopheles gambiae which is the most widespread in Africa and difficult to control. Each year, there are over 8.2 million malaria infections in Kenya (Jean-Marie, 2002) mostly due to inadequate medical care, failure to use insecticide treated nets and increased resistance of the parasites to drugs. The disease accounts for 30% of all the outpatient cases and 19% of all admissions, 5.1% of whom die, and 72 children below the age of 5 years die daily (DMS, 2006; WHO, 1996; Mouchet, 1999). ⇑ Corresponding author. Address: Kenya Medical Research Institute, P.O. Box 70174, 00400 Nairobi, Kenya. Fax: +254 20 2720030. E-mail address: [email protected] (C.N. Muthaura). 0014-4894/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2010.11.004

The disease is endemic in the lowlands, particularly the coastal strip and Lake Victoria basin where transmission is sufficiently intense. Both incidence and prevalence of infection reach more than 90% of the population within 10–12 weeks after the beginning of the rainy season (Hoffman et al., 1996). Malaria transmission patterns in Kenya are influenced by several factors such as rainfall, relative humidity, vector species, and intensity of biting, altitude and presence of susceptible new human hosts. Patterns of endemicity are described in terms of stable, unstable, epidemic and malaria-free zones (MoH, 1992); stable malaria occurs in zones which have continuously high transmission rate throughout the year and includes the coastal strip and western parts of Kenya. The population exhibits a high degree of immunity and epidemics are highly unlikely. Unstable malaria occurs where there is seasonal endemicity with one or two annual transmission peaks and includes parts of Eastern Province and some parts of the Rift Valley. Epidemic malaria occurs in highland areas bordering endemic zones. Since 1988 there has been sporadic highland epidemics and considerable child mortality reported in Uasin Gishu, Nandi, Kericho, Kisii and Nyamira districts. Malariafree zones include all land that lies at attitudes 1600 m above sea level, but due to the critical epidemiological situation of this disease very few areas are safe (MoH, 1992). A major impact of the disease was documented in the highlands of East Africa, where the spread of chloroquine (CQ) resistance was probably the only factor likely to explain the changing epidemiology of malaria in areas of low and unstable transmission, despite initial claims that it could be attributed to global warming (Shanks et al., 2000).

610

C.N. Muthaura et al. / Experimental Parasitology 127 (2011) 609–626

2. The flora and fauna of Kenyan biodiversity The vegetation and animal life of Kenya reflect the variety of its topography and climate, which also leads to a wide variation in malaria and subsequent disease epidemiology (Ochola, 2003). From the coastal (Kwale and Kilifi disticts) mangrove swamps and rain forest to the vast plains of the hinterland covered with grass giving way to typical tropical savanna and mountain forest. The highland areas to the west of the country are densely forested with bamboo, dense undergrowth and timber. The equatorial alpine climate around Mount Kenya (3000–5200 m above sea level), the second highest mountain in Africa, borders Meru district and influences the natural conditions in the district leading to a wide variety of microclimates and agroecological zones with a wide range of flora and fauna. Wildlife of great variety is to be found in Kenya, both in the sparsely populated areas and in the National Parks and Reserves that have been created for its protection. Forests occupy about 2–3% of Kenya’s land area and yet, they are reservoirs of biological diversity (genes, species and ecosystems). Many of the plant species have medicinal value, with an estimate of over 400 plant species used for management of common diseases in East Africa (Kokwaro, 1993; Gachathi, 1989). The forests like Kakamega, Aberdares, Mount Kenya and Arabuko Sokoke have exemplary display of flora and fauna. The biological diversity they carry is important because it contributes directly to the well being of Kenyans, especially those in the rural areas, and indirectly to the mainly agricultural economy. Harversting of medicinal plants and other forest products is strictly controlled by the Kenya Wildlife Service that oversees the conservation of the forests and its wildlife. However, human expansionist demands can be expected to wreak environmental deterioration and biotic destruction well into the next century. Kenya’s strategy for conservation of forests involves intensification of timber and other non-wood products outside forest areas (Njuguna et al., 2000). The Taita Hills, in the coastal region of Kenya (south-east Kenya, 03o200 S, 38o1500 E) is the northernmost extreme of the Eastern Arc Mountains, a biodiversity hotspot chain of mountains that run from south-eastern Kenya to southern Tanzania (Lovett and Wasser, 1993) and boosts an extremely high diversity of flora and fauna (Mittermeier et al., 1998). Indigenous cloud forest in the Taita Hills currently covers an area of 430 ha, reflecting 98% forest reduction over the last 200 years, mainly due to clearance for agricultural purposes (Myers et al., 2000). Forest clearance is less widespread at present, but despite the small size of the 12 remaining indigenous forest fragments, they are of global conservation importance, holding numerous rare and endemic plants and animals. Conservation effort is being put in place through a multi-disciplinary approach that seeks better legal and law enforcement co-ordination between the Government, Local Authorities, scientists and the local community (Githiru and Lens, 2004).

3. The role of herbal medicine for treatment of malaria The use of traditional and herbal remedies seems to be the alternative choice of treatment in countries where malaria is endemic (Sofowora, 1982; Rasoanaivo et al., 1992; Gessler et al., 1995). In the Third World, 80% of people are thought to rely on herbal remedies (Zirihi et al., 2005; WHO, 2002). Local medicinal plants continue to be used in the treatment of malaria and the evaluation of antimalarial activity of medicinal plants against P. falciparum has been extensively studied (O’Neill et al., 1985; Carvalho et al., 1991; Gakunju et al., 1995; Gessler et al., 1994; Basco et al., 1994; Andrade-Neto et al., 2003; Tran et al., 2003; Simonsen et al., 2001). Reviews for those studies have been undertaken in many countries: in South Africa (Pillay et al., 2008), in West Africa (Soh

and Benoit-Vical, 2007), in Brazil (Krettli et al., 2001) and elsewhere (Kaur et al., 2009). In Asia, Latin America and Africa, the extensive use of natural plants as primary health remedies, due to their pharmacological properties, is quite common (Conco, 1991; Phillipson et al., 1987). The Brazilian flora, just like the Kenyan which is transversed by the equator and rich in biodiversity offers a wide variety of bioactive substances (Brandão et al., 1992). Many people in these areas rely on traditional medicine for the treatment of many infectious diseases, including malaria (de Mesquita et al., 2007). SouthEast Asia is home to the most drug resistant parasites in the world, both P. falciparum and Plasmodium vivax (WHO, 2001) and traditional medicine is also widely practiced. Vietnamese traditional medicine is similar in many respects to the traditional Chinese school having incorporated Chinese and Indian teaching (Nghiem, 2002). In Africa herbal medicines are an important part of the culture and traditions of its people and its biodiversity has played major specific roles in the cultural evolution of human societies (Mugabe and Clark, 1998). Apart from their cultural significance, traditional medicines have been accessible and affordable and most people in Kenya especially in rural areas use traditional medicine and medicinal plants to treat many diseases including malaria (Njoroge and Bussmann, 2006). It is estimated that there is one traditional healer for every 200–400 people in Uganda, one of East African countries. This contrasts sharply with the availability of trained medical personnel for which the ratio is 1:20,000 or less (WHO, 2002a). The role of ethnopharmacology is to give direction on the plant species for selection as well as data for plant preparation, posology, effects and side effects which could provide specific targets for isolation of active compounds and pharmacological investigation in the quest for development of new pharmaceuticals (Cox and Balick, 1994). Recent work on African plants used in the treatment of malaria is very encouraging. It is striking how many different plants are reported by herbalists to cure malaria. When we compare antimalarial species being used in Kenya with those recently reported to be used in Ivory Coast, Ghana, Madagascar, and Sudan, only Azadirachta indica (Neem tree) is shared (Benoit-Vical et al., 1998; Addae-Kyereme et al., 2001; Rasoanaivo et al., 1999; El Tahir et al., 1999). However, the chemical composition of the various plant constituents is affected by the climatic conditions and the locality under which the plant species are growing (Gessler et al., 1995). The challenge will be to translate herbal medicine practice with these plants into an evidence-based monotherapy or combined therapy as suggested by Rasoanaivo et al. (1999). There is need therefore, to corroborate with traditional healers and clinicians for observational retrospective treatment-outcome and prospective clinical study of a traditional medicine. The administration of a traditional treatment (e.g. a plant preparation) as a decoction/concoction, and the systematic follow up of the outcome in a clinical study with the effect of a rapid and complete cure, without failure and or serious side effects, would lead to further research of the product with a view to isolating active constituents that would form the basis of a monotherapy or combination therapy. The use of medicinal herbs in traditional human societies has primarily been for treatment of active or acute conditions whereas in most instances, the reported use of medicinal herbs by animals appears to be prophylactic (Hart, 2005). During epidemics some medicinal preparations may be used as preventive and other preparations used as tonics to enhance better health. The traditional culture of the Maasai pastoralists of Kenya has outlived the present day development. They are known to ingest wild plant materials as foods, as regular ingredients of milk and meat based soups and as herbal medicines or as flavours and tonics for prevention or allevation of a range of common ailments (Johns

C.N. Muthaura et al. / Experimental Parasitology 127 (2011) 609–626

et al., 1999). Perhaps the most widespread prophylactic use of medicinal herbs in humans is the use of spices in food preparation. Evidence for the antimicrobial effects of spices in reducing the growth of ingested foodborne pathogens and in the preservation of meat, has been well documented (Billing and Sherman 1998). There are no available causal prophylaxis drugs to prevent malaria in endemic areas. Primaquine, the only drug specifically developed to inhibit the liver infection, has been curtailed by the associated toxicity, poor compliance, and increased risk of hemolysis when administered to persons with glucose-6-phosphate dehydrogenase deficiency (Carraz et al., 2006). The existence of medicinal plants used for prophylaxis also seems to be rare. Strychnopsis thouarsii an endemic plant in Madagascar has been used for malaria prophylaxis (Boiteau, 1986). Carraz et al. (2006) isolated a compound tazopsine from the plant that had activity against the erythrocytic and the initial pre-erythrocytic phase of malaria infection. However, on derivativation of tazopsine to achieve N-cyclopentyl-tazopsine (NCP-tazopsine), the latter was shown to be specifically active against the in vitro liver stage but inactive against the blood forms of the malaria parasite, placing this lead molecule into a novel category of antimalarial compounds with true causal prophylactic activity. Ampelozyziphus amazonicus is another plant used in some regions of the Amazon to prevent malaria infection (Brandão et al., 1992). Using newly established methods to screen for drugs that inhibit sporozoites and/or liver stage parasites, Andrade-Neto et al. (2008) demonstrated evident activity of its ethanolic root bark extract against pre-erythrocytic forms of Plasmodium berghei sporozoite cultures in hepatoma cells in vitro and in vivo. Demonstration of efficacy for prophylactic use has been problematic because the behaviour is based on reducing the severity or likelihood of adverse events in the future. In antimalarial drug discovery programs, routine screening for anti-pre-erythrocytic stage activity is seldom carried out, partly because these stages are clinically silent, but also because access to pre-erythrocytic parasites and purified infected hepatocytes is costly and restricted to a few laboratories. However, demonstration of efficacy for the treatment of acute infections has been straightforward because plant extracts or isolated compounds can be tested in vitro or in vivo using laboratory experimental models, which have been easier to develop (Hart, 2005). Traditional medicines are a potential rich source of new drugs against malaria and other infectious diseases and given the remarkable antimalarial properties of Cinchona bark that have been known for more than 300 years, resulting in the discovery of quinine (Camacho et al., 2000) and the more recent development of artemisinin derivatives has re-affirmed the potential of plant species to provide effective drugs for the treatment of malaria. Artemisinin, a sesquiterpene lactone was isolated from the herb Artemisia annua in China in 1971 and was highly unusual as it contained an endoperoxide moiety in contrast to known antimalarial drugs (Wright, 2005). Its discovery heralded a new era in antimalarial drug development as the compound and its synthetic derivatives such as artemether and artesunate were rapidly effective against parasites resistant to other antimalarials. The compounds had gametocyticidal activity, thus were able to reduce transmission to the mosquito vector (Wright and Warhurst, 2002). Despite the cost and adverse effects, a standard treatment for severe malaria in Africa is the intravenous administration of quinine (WHO, 2001) and resistance against the drug in Africa has not been reported (Le Bras et al., 2006). Resistance of P. falciparum to current antimalarial drugs (Trape, 2002), coupled with unavailability and unaffordability of these agents (Bathurst and Hentschel, 2006), in addition to lack of new therapeutic agents (Benoit-Vical, 2005; Mutabingwa, 2005) has led the Government of Kenya to

611

provide free and subsidized Artemisinin based combination therapy (ACT) in public health facilities and private pharmacies, respectively, as first line of treatment for uncomplicated malaria. However, hopes that artemisinins will have a major impact on malaria have been tempered by a recent study in which a number of clinical isolates of P. falciparum from malaria patients showed resistance to artemether (Jambou et al., 2005). The World Health Organization have called for an immediate halt to the marketing and sale of malaria medicines that contain only artemisinin or one of its derivatives (WHO, 2006) and recommended the use of ACTs to minimize the risk of resistance development. Even so, there is evidence that resistance to lumefantrine–artemether may be developing in Zanzibar after a short period of use (Sisowath et al., 2005). This underlines the urgent need for continued search of new antimalarial drugs from medicinal plants. It has long been recognized that natural product structures have the characteristics of high chemical diversity, biochemical specificity, and other molecular properties that make them favourable structures for drug discovery, and which serve to differentiate them from libraries of synthetic and combinatorial compounds (Clardy and Walsh, 2004). The new drug discovery approaches need to take into account some specific concerns, in particular, the requirement for new therapies to be inexpensive and simple to use, as well as the need to limit the cost of drug research in as much as the new therapy would be a drug of mass treatment, affordable to the poor who are most vulnerable to the disease. (Bathurst and Hentschel, 2006). A number of ethnobotanical surveys have described several plant species traditionally used for the management of malaria in Kenya (Muthaura et al., 2007c,d). However, only about 20% of the plants with claimed bioactivities have been subjected to bioassay screening (Houghton, 2001). Among the currently ongoing efforts is the discovery of new antimalarial leads from natural products (Rukunga et al., 2007; Murata et al., 2008; Yenesew et al., 2003; 2004; Oketch-Rabah et al., 2000). Kenya is rich in green tropical vegetation cover and its biodiversity nature and long history of traditional plant uses are claimed to possess medicinal value. The search for new bioactive plant products can follow three different ways of approach for the selection of medicinal plants: random, chemotaxonomical and ethnopharmacological where the chances for research success are greater (Trotter et al., 1982; Elisabetsky and Wannmacher, 1993). This review is an analysis of ethnopharmacological publications describing research into antimalarial therapies. 4. Methods and selection of articles The articles selected concerned studies on in vitro or in vivo antimalarial activity of medicinal plants from Kenya. The publications cited were sourced from databases such as PubMed and have used classical methodologies such as the continuous culture of P. falciparum strains (Trager and Jensen, 1976); the in vitro antimalarial activity tests using radio-isotopic methods (Desjardins et al., 1979); the in vivo antiplasmodial assays with the reference for blood schizonticidal activity of plant extracts using the classical 4-day suppressive test by Peters and Robinson (1992) on a rodent malaria model. The cytotoxicity concerned in vitro known cell-lines using standard protocols for example, 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Mosmann, 1983). MTT reduction is one of the most frequently used methods for measuring cell proliferation and cytotoxicity assays. The ratio of cytotoxicity to biological activity is defined as selectivity index (SI) and it is generally considered that biological efficacy is not due to the in vitro cytotoxicity when SI P 10 (Vonthron-Senecheau et al., 2003).

612

C.N. Muthaura et al. / Experimental Parasitology 127 (2011) 609–626

5. Antiplasmodial screening The development of a culture system for P. falciparum (Trager and Jensen, 1976) was quickly exploited to test antimalarial agents in vitro (Desjardins et al., 1979). The publication of O’Neill et al. (1985) was among the first report that demonstrated the use of an in vitro assay using P. falciparum for the bioassay-guided fractionation of plant extracts in the search for new antimalarial drugs. He determined the ratio of in vitro antimalarial activity and in vitro toxicity before any in vivo work. From this there followed many research papers (Weenen et al., 1990; Gakunju et al., 1995; Omulokoli et al., 1997; Muregi et al., 2003). These tools have enabled many scientific teams to evaluate the efficacy of Kenyan medicinal plants. The evaluations sought to establish the rationale for the use of medicinal plants traditionally used for treatment of malaria and the potential of their constituents as possible new antimalarial agents or leads to new antimalarial compounds. Ethnobotanical studies on some Kenyan plants with relevance to plants used for preparation of remedies against malaria have been undertaken (Muthaura et al., 2007c,d; Orwa et al., 2007; Njoroge and Bussmann, 2006) and several ethnobotanical screening programs have been conducted on Kenyan medicinal plants (Rukunga et al., 2009; Gathirwa et al., 2008; Muthaura et al., 2007a,b; Irungu et al., 2007; Muregi et al., 2003). In addition some plants with known antiplasmodial activity were evaluated in individual studies leading to isolation of antiplasmodial compounds (Rukunga et al., 2008; Yenesew et al., 2004; Kuria et al., 2002; Oketch-Rabah et al., 1997). Table 1 shows plant species that have been bioassayed for in vitro antiplasmodial properties in Kenya using tritiated [3H]-hypoxanthine incorporation method (Desjardins et al., 1979) and whose IC50 were less than 5 lg/ml, a value at which plant extracts were considered to be highly active with potential for isolation of active constituents (Clarkson et al., 2004). In vitro cytotoxicity was measured on known cell lines (viz. Vero E6 cells or African green monkey kidney cells and KB cells, human oral epidermoid cancer cell line) using standard protocols such as the MTT assay (Mosmann, 1983). Table 2 shows plant species that have been bioassayed for in vivo antimalarial properties using a murine model infected with P. berghei (Peters and Robinson 1992) and whose suppression of parasitaemia was greater than 30%, a value at which plant extracts were considered to be partially active (Muriithi et al., 2002; Carvalho et al., 1991). Several compounds with antiplasmodial properties were isolated through bioassay-guided fractionation of active plant extracts as well as through phytochemical analysis and some of them are illustrated in Table 3.

5.1. Herbal leads from Meru district Muthaura et al. (2007b), Kirira et al. (2006) and Gathirwa et al. (2007, 2008) in different studies evaluated ethnobotanically described antimalarial plants, collected in Meru district for their antiplasmodial properties. In the first study Muthaura et al. (2007b) evaluated 10 plants against CQ sensitive (D6) and resistant (W2) P. falciparum clones. Four of these (40%) were found to have IC50 <5 lg/ml for either CQ sensitive (D6) or resistant (W2) clones and 8 (80%) had IC50 <10 lg/ml. This is within the range comparable to antiplasmodial activity reported for ethanolic extracts of A. annua (IC50, 3.9 lg/ml; KI resistant strain) and A. indica (IC50, 4.1–7.2 lg/ml; FcBI resistant strain) in the in vitro microdilution assay (O’Neill et al., 1985; Benoit-Vical et al., 1996; Udeinya, 1993) from which potent antiplasmodial compounds (artemisinin and nimbolide, respectively) have been isolated. The four active extracts were the water stem bark extract of Boscia angustifolia A.

Rich. (Capparaceae) (IC50, 1.42 lg/ml), methanol whole plant extract of Schkuhria pinnata (Lam) O. Ktze (Compositae) (IC50, 1.30 lg/ml), methanol whole plant extract of Fuerstia africana T.C.E. Fries (Lamiaceae) (IC50, 0.98 lg/ml) and the water whole plant extract of Ludwigia erecta (L.) Hara (Onagraceae). (IC50, 0.93 lg/ml). B. angustifolia has been reportedly used for malaria and epilepsy in Mali and the methanol leaf extract had IC50 of 37.6 lg/ml against CQ sensitive (3D7) P. falciparum strain (Bah et al., 2007). The root bark of the plant is reported to have antibacterial activity against a range of organisms and the presence of alkaloids and saponins were demonstrated by Hassan et al. (2006). Koch et al. (2005) reported antiplasmodial activity of chloroform stem bark extract (IC50 > 10 lg/ml) for the same species collected in Kajiado district of Kenya. Polar constituents in the water extract seem to be more active than organic extracts since Muthaura et al. (2007b) reported a much lower activity for the methanol extract as compared to the water extract for the same species collected in Meru district. S. pinnata whole plant is reported to be used for malaria (Njoroge and Bussmann, 2006); leaf decoction has been used for malaria in Zimbabwe (Watt and Breyer-Brandwijk, 1962) and in Peru (Ramirez et al., 1988). Munoz et al. (2000) reported antimalarial activity and Pacciaroni et al. (1995) demonstrated the presence of sesquiterpenes lactones. Koch et al. (2005) have reported the use of F. africana for malaria by the Maasai and confirmed its antiplasmodial activity (IC50, 3.76 lg/ml; D6 sensitive strain) in addition to isolation of a previously known compound ferruginol, which exhibited antiplasmodial activity (IC50, 1.95 lg/ml; D6 strain) but was cytotoxic to KB cells. The decoction of L. erecta whole plant has been used for malaria in East Africa (Kokwaro, 1993). There is no literature information about this plant but the findings are interesting in that the results show high in vitro antiplasmodial activity for water extract, which was about five times more active than the methanol extract, but it was the latter that showed a higher suppression of parasitaemia in P. berghei infected mice. However, the authors observed that the water extracts were generally less active than the methanol extracts in antiplasmodial in vitro tests, albeit water is mostly the formulation that has been used in traditional medicine. On the other hand, the water extracts exhibited relatively high suppression of parasitaemia in P. berghei infected mice giving credence to consistent reports by traditional healers that these plants are effective in treating malaria in humans. The authors noted that the ratio of sensitivity for the two clones was much less than that of CQ for the four plant extracts suggesting no cross-resistance with the latter and consequently phytochemical studies of these extracts is underway in the authors0 laboratories in order to identify the active principles. The SI for the extracts were high suggestive of safer therapy. In the second study Kirira et al. (2006) evaluated 10 plants against CQ sensitive (NF54) and resistant (ENT30) P. falciparum strains. Two of these (20%) were found to have IC50 <5 lg/ml for CQ sensitive (NF54) strain and these were the methanol stem bark extract of Fagaropsis angolensis (Engl.) Dale (Rutaceae) and the methanol stem bark extract of Zanthoxylum usambarense (Engl.) Kokwaro (Rutaceae).They were not toxic in the brine shrimp nauplii assay, indicative of selective toxicity. Nitidine, the most common anti-malarial benzophenanthridine alkaloid in Zanthoxylum species (Gakunju et al., 1995), has been previously isolated from Z. usambarense (Kato et al., 1996) and F. angolensis (Khalid and Waterman, 1985). This may explain the observed antiplasmodial activity. Gathirwa et al. (2007, 2008) in two independent studies evaluated 10 ethnobotanically described antimalarial plants for in vitro antiplasmodial and in vivo antimalarial assays, singly as well as

613

C.N. Muthaura et al. / Experimental Parasitology 127 (2011) 609–626 Table 1 Plant extracts exhibiting high antiplasmodial activity (IC50 < 5 lg/ml) and their cytotoxic activity. Plant parts tested

Extraction solvent; antiplasmodial activity IC50 (lg/ml) and strains of Plasmodium falciparum used

Cytotoxicity IC50 (lg/ml) and cell-lines used cell-lines used

References Selectivity index: (cytotoxicity/ activity)

Root bark

CHCl3; 3.78 (D6)

>20 (KB)

>5.29

Koch et al. (2005)

Leaves

MeOH; 3.98 (W2) Water; 4.65 (W2)

594.85 (Vero E6)

149.46

Gutenbergia cordifolia Benth. Catharanthus roseus (L.) Don

Leaves Leaves

CHCl3; 4.40 (D6) MeOH; 4.65 (D6)

0.2 (KB) 167.52 (Vero E6)

0.045 36.03

Inner bark

CHCl3; 4.63 (D6)

>20 (KB)

>4.32

Canellaceae

Commiphora schimperi (O. Berg) Engl. Warburgia stuhlmannii Engl.

Gathirwa et al. (2007 Koch et al. (2005) Gathirwa et al. (2007) Koch et al. (2005)

Stem bark

MeOH; 1.81 (D6), 2.33 (W2)

233 (Vero E6)

128.7 (D6)

Capparaceae

W. ugandensis Sprague Boscia angustifolia A. Rich.

Stem bark Stem bark

CH2Cl2; 2.2 (NF54), 1.4 (KI) Water; 1.42 (D6), 4.77 (W2)

0.34 (L6) 6720.0 (Vero E6)

0.24 (K1) 4732.4 (D6)

Capparidaceae

B. salicifolia Oliv.

Stem bark

MeOH; 1.04 (D6) Water; 3.65 (D6)

304.92 (Vero E6)

293.19

Celastraceae

Maytenus arbutifolia (A. Rich.) Wilczek M. undata (Thunb.) Blakelock

Root

Water; 4 (M24), 4 (K67)

nd

nd

Leaves

Water; 0.95 (D6), 1.90 (W2)

3645.7 (Vero E6)

3837.6 (D6)

M. putterlickioides (Loes) Excell and Mendoca Schkuhria pinnata (Lam) O. Ktze Vernonia lasiopus O. Hoffm.

Root bark

MeOH; 4.41 (D6)

112.4 (Vero E6)

25.5

Whole plant

MeOH; 1.30 (D6)

1 61.5 (Vero E6)

124.2

Leaves

nd

nd

Cyperaceae

V. lasiopus O. Hoffm Cyperus articulatus L.

Root bark Rhizome

CHCl3; 1.2 (K39), 3.4 (V1/S), 1.7 (NF54), 3.6 (ENT30) EtOAc; 1.0 (K39), 1.6 (V1/S), 1.6 (NF54), 1.4 (ENT30) CH2Cl2; 4.9 (NF54), 4.7 (KI) MeOH; 4.84 (NF54)

>90 (L6) nd

>10.7 nd

Euphorbiaceae

C. robusta Pax, E, FZ.

Leaves

MeOH; 3.41 (D6)

460.29 (Vero E6)

134.98

Phyllanthus reticulatus Poir.

Leaves

Water; 1.7 (K67)

nd

nd

Suregada zanzibariensis Baill.

Leaves

Water; 1.5 (K67), 1.5 (ENT36),

nd

nd

Flueggea virosa (Willd.) Voigt

Leaves

MeOH; 2.28 (D6), 3.64 (W2)

682.6 (Vero E6)

299.3 (D6)

Fabaceae Lamiaceae

Acacia mellifera (Vahl) Benth. Fuerstia africana T.C.E. Fries

Inner bark Whole plant

CHCl3; 4.48 (D6) MeOH; 0.98 (D6), 2.40 (W2)

>20 (KB) 954.7 (Vero E6)

>4.46 974.2 (D6)

Leguminosae

F. africana T.C.E. Fries Erythrina abyssinica DC.

Leaf Roots

CHCl3; 3.76 (D6) Acetone; 0.49 (D6), 0.64 (W2)

>20 (KB) nd

>5.32 nd

Ekebergia capensis Sparrm. E. capensis Sparrm.

Inner bark Stem bark

>20 (KB) nd

>5.04 nd

Turraea robusta Gurke

Root bark

CHCl3; 3.97 (D6) CHCl3; 3.9 (K39), EtOAc; 4.7 (K39), 4.9 (ENT30), MeOH; 4.6 (K39), Water; 3.9 (K39) MeOH; 2.09 (D6)

24.38 (Vero E6)

11.83

nd nd

nd nd

3283.6 (Vero E6)

3530.7 (D6)

nd nd

nd nd

nd nd

nd nd

Root bark Root bark Stem bark Root bark

MeOH; 2.4 (NF54), 3.5 (KI) MeOH; 4.8 (ENT22), 4.0 (ENT30), 1.8 (KIL9) MeOH; 4.10 (D6), Water; 0.93 (D6), 1.61 (W2) MeOH; 4.68 (NF54) MeOH; 3.14 (ENT30), Water; 2.32 (ENT30) MeOH; 3.20 (NF54) MeOH; 0.78 (K39), 0.7 (K67), 1.46 (M24), CHCl3; 3.53 (D6) CHCl3; 4.15 (D6) CH2Cl2; 5.6 (NF54), 4.4 (KI) MeOH; 4.0 (V1/S)

>20 (KB) >20 (KB) 32.8 (L6) nd

>5.67 >4.82 7.5 nd

Root bark

CHCl3; 3.49 (D6)

>20 (KB)

>5.73

Family

Plant species

Amaranthaceae Sericocomopsis hildebrandtii Schinz Asteraceae Artemisia afra Jacq.

Apocynaceae Burseraceae

Compositae

Meliaceae

Mimosaceae

T. robusta Guerke Root bark Albizia gummifera (JF Gmel.) C.A. Sm. Stem bark

Onagraceae

Ludwigia erecta (L.) Hara

Whole plant

Rutaceae

Fagaropsis angolensis (Engl.) Dale Zanthoxylum chalybeum Engl.

Stem bark Root bark

Z. usambarense (Engl.) Toddalia asiatica (L.) Lam.

Stem bark Root bark

´ erit. Rhamnus prinoides L´H Clematis brachiata Thunb. Harrisonia abyssinica Oliv Clerodendrum myricoides (Hochst.) Vatke Zygophyllaceae Balanites aegyptiaca (L.) Del.

Rhamnaceae Ranunculaceae Simaroubaceae Verbenaceae

nd = not done. CQ sensitive strains: D6, NF54, K39, K67, M24. CQ resistant strains: W2, KI, V1/S, ENT30, ENT36, KIL9.

Muthaura et al. (2007a) Irungu et al. (2007) Muthaura et al. (2007b) Gathirwa et al. (2007) Gakunju et al. (1995) Muthaura et al. (2007a) Muthaura et al. (2007a) Muthaura et al. (2007b) Muregi et al. (2003)

Irungu et al. (2007) Rukunga et al. (2009) Gathirwa et al. (2007) Omulokoli et al. (1997) Omulokoli et al. (1997) Muthaura et al. (2007a) Koch et al. (2005) Muthaura et al. (2007b) Koch et al. (2005) Yenesew et al. (2003a) Koch et al. (2005) Muregi et al. (2004) Gathirwa et al. (2008) Irungu et al. (2007) Ofulla et al. (1995) Muthaura et al. (2007b) Kirira et al. (2006) Rukunga et al. (2009) Kirira et al. (2006) Gakunju et al. (1995) Koch et al. (2005) Koch et al. (2005) Irungu et al. (2007) Muregi et al. (2004) Koch et al. (2005)

614

C.N. Muthaura et al. / Experimental Parasitology 127 (2011) 609–626

Table 2 In vivo antimalarial activity of plants exhibiting suppression of parasitaemia >30% in P. berghei infected mice. Family

Plant species

Plant part

Parasite strain

Dose mg/kg/day (route)

Extract

% Chemo suppression

References

Amaranthaceae Anacardiaceae

Cyathula schimperana Igifashi Rhus natalensis Krauss

Root bark Leaves

P. berghei – ANKA

Stem bark

Lannea schweinfurthii (Engl.) Engl.

Stem bark

(ip) (ip) (ip) (ip) (ip) (oral) (oral) (ip) (ip) (oral) (oral) (ip) (ip) (ip) (ip) (oral) (ip) (ip) (ip) (ip) (oral) (oral)

MeOH MeOH Water MeOH Water MeOH Water MeOH

500 100 100 100 100 100 100 100 100 500 500 100 100 100 100 100 100 100 100 100 100 100 100 500 100 100 100 100 500 500 500 500 100 100 100 100 500

(oral) (ip) (ip) (ip (ip) (ip) (ip) (ip) (ip) (oral) (oral) (ip) (ip) (ip) (ip) (ip) (ip) (ip) (ip) (ip) (ip) (ip) (ip) (oral) (ip) (ip) (oral) (oral) (oral) (oral) (oral) (oral) (ip) (ip) (ip) (ip) (oral)

Water MeOH Water MeOH Water MeOH Water MeOH Water Water MeOH MeOH MeOH Water Water MeOH Water MeOH Water MeOH Water MeOH Water MeOH MeOH Water MeOH Water MeOH MeOH MeOH MeOH MeOH Water MeOH Water Water

45.08 56.24 83.15 63.49 66.51 42.04 40.21 91.37 83.08 51.66 48.43 34.6 42.36 77.45 70.25 33.3 52.95 84.95 86.50 43.75 33 41.5 36.6 49 76.29 47.74 78.66 45.31 46.74 45.38 49.9 64.22 54 59.3 60.12 40.45 71.69 42.35 70.91 35.15 53.13 88.04 42.20 31.70 61.85 43.16 33.3 78.2 63.81 52.24 48.83 31.7 48.8 34.1 43.9 36.49 21.55 65.28 49.64 51 33

Gathirwa et al. (2007)

Sclerocarya birrea (A. Rich.) Hochst.

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 500 100 100 100 100 500 500

500 (oral)

MeOH

Muregi et al. (2007b)

500 (oral) 500 (oral)

MeOH Water

34.1 43.9 48.1 42 33

100 (ip) 500 (oral)

Water MeOH

39.02 59.3 37.0

Muthaura et al. (2007b) Muregi et al. (2007b)

Apocynaceae

Catharanthus roseus (L.) Don

Leaves

Asteraceae

Artemisia afra Jacq.

Leaves

Caesalpinaceae Canellaceae

Caesalpinia volkensii Harms Warburgia stuhlmannii Engl.

Seed Stem bark

Capparidaceae

Boscia salicifolia Oliv.

Stem bark

Celastraceae

Maytenus acuminata (L.f.) Loes. M. acuminata (L.f.) Loes M. heterophylla (Eckl. and Zeyh.) M. undata (Thunb.) Blakelock

Root bark Root bark Leaves Root bark Leaves

M. putterlickioides (Loes.) Excell and Mendoca

Root bark

Sphaeranthus suaveolens (Forsk.) DC.

Whole plant

Schkuhria pinnata (Lam) O. Ktze

Whole plant

Vernonia lasiopus O. Hoffm. V. lasiopus O. Hoffm. Boscia angustifolia A. Rich. Clutia abyssinica Jaub and Spach

Root bark Root bark Stem bark Leaves

C. robusta Pax, E, FZ. Flueggea virosa (Willd.) Voigt

Leaves Leaves

Guttiferae

Harunganamadagascariensis Poir

Leaves

Lauraceae

Ocotea usambarensis Engl.

Stem bark

Lamiaceae

Fuerstia africana T.C.E. Fries

Whole plant

Meliaceae

Ekebergia capensis Sparrm. Turraea robusta Guerke

Stem bark Root bark

P. berghei – NK65 P. berghei – ANKA

Mimosaceae Moraceae

Albizia gummifera (JF Gmel.) C.A. Sm. Ficus sur Forssk.

P. berghei – NK65

Olacaceae

Ximenia americana L.

Stem bark Root bark Stem bark Leaves Root bark

Onagraceae

Ludwigia erecta (L.) Hara

Whole plant

Rhamnaceae

Rhamnus prinoides L´ Hérit

Root bark Leaves Root bark Leaves Root bark Root bark Leaves Leaves Root bark Leaves

Compositae

Euphorbiaceae

´ erit R. prinoides L´H R. staddo A. Rich. R. staddo A.Rich. Rubiaceae Rutaceae

Vangueria acutiloba Robyns Toddalia asiatica (L.) Lam.

P. berghei – NK65 P. berghei – ANKA

P. berghei – NK65

P. berghei – ANKA

P. berghei – NK65 P. berghei – ANKA

P. berghei – ANKA

P. berghei – NK65

P. berghei – ANKA P. berghei – NK65

MeOH Water MeOH Water MeOH Water MeOH MeOH Water MeOH Water Water MeOH

Gathirwa et al. (2008)

Gathirwa et al. (2007)

Muregi et al. (2007b) Muthaura et al. (2007a) Gathirwa et al. (2007) Muregi et al. (2007a) Muregi et al. (2007b) Muregi et al. (2007a) Muthaura et al. (2007a)

Muthaura et al. (2007b)

Muregi et al. (2007a) Muregi et al. (2007b) Muthaura et al. (2007b)

Gathirwa et al. (2007) Muthaura et al. (2007a)

Muthaura et al. (2007b)

Muregi et al. (2007b) Gathirwa et al. (2008)

Muregi et al. (2007b)

Gathirwa et al. (2007) Muthaura et al. (2007b) Muregi et al. (2007a)

Muregi et al. (2007a)

615

C.N. Muthaura et al. / Experimental Parasitology 127 (2011) 609–626 Table 2 (continued) Family

Plant species

Plant part

Parasite strain

Dose mg/kg/day (route)

Extract

% Chemo suppression

References

Verbenaceae

Clerodendrum eriophyllum Guerke

Root bark

P. berghei – ANKA

Root bark

P. berghei – NK65

MeOH Water MeOH

90.13 61.54 31.7

Muthaura et al. (2007b)

C. myricoides (Hochst.) Vatke

100 (ip) 100 (ip) 500 (oral)

Muregi et al. (2007b)

ip = intraperitoneal. CQ sensitive strain: P. berghei – ANKA. CQ resistant strain: P. berghei – NK65.

in combination with other extracts. In the first study the methanol and water leaves extract of Artemisia afra Jacq. (Asteraceae), the methanol and water stem bark extract of Boscia salicifolia Oliv. (Capparidaceae), the methanol leaves extract of Catharanthus roseus (L.) G. Don (Apocynaceae) and the methanol leaves extract of Clutia robusta Pax, E, FZ. (Euphorbiaceae) gave IC50s <5 lg/ml for either CQ sensitive (D6) or resistant (W2) P. falciparum clones. All the extracts showed a SI > 10. The pounded leaves of B. salicifolia form a remedy for fever in cattle and the stem bark has been used for backache (Kokwaro, 1993; Beentje, 1994). In Zimbabwe, Kazembe and Nkomo (2010) have confirmed its traditional use as a mosquitocide while Walter and Séquin (1990) reported the presence of flavonol glycosides from the leaves. C. roseus root bark is reportedly used to treat gastrointestinal problems among the Luo of Kenya (Johns et al., 1995; Kokwaro, 1993) and malaria or fever in Brazil (Brandão et al., 1985). The leaves of this species are the only source of the indolomonoterpenic alkaloids, vincristin (leurocristine) and vinblastin (vincaleucoblastine), which are anticancer agents (Ferreres et al., 2010). Weenen et al. (1990) reported insignificant antiplasmodial activity of C. robusta from Tanzania, and the Maasai use it for wound healing (Beentje, 1994). Of particular interest was the A. afra which grows in upland bushes and forest edges in Kenya. Its closely related species, A. annua, is known to contain potent antimalarial molecule artemisinin, which proved to be active against CQ sensitive and resistant P. falciparum (Phillipson and Wright, 1991). A. afra is reported to show activity against P. falciparum (Kraft et al., 2003; Van Zyl and Viljoen, 2003; Clarkson et al., 2004). It was interesting to note that the methanol and water extracts showed twofold activity for the resistant strain W2 (3.98 and 4.65 lg/ml, respectively) compared to the sensitive strain D6 (9.04 and 11.23 lg/ml, respectively) and that it was the less polar extract that was more active. The constituents of an extract may have different mechanisms of action. An alkaloidal fraction from Cissampelos andromorpha DC. (Menispermaceae), a Brazilian medicinal plant, was found to be active against CQ-resistant (K1) but inactive against CQ sensitive (Palo Alto) P. falciparum strains (Fischer et al., 2004). In vivo parasitaemia suppression of P. berghei in mice for the methanol and water extracts was significant (77% and 70%, respectively). Hot water extracts of A. annua had no activity on rodent malaria parasite P. berghei in contrast with ether extracts, which led to isolation of artemisinin (Wright, 2005), casting doubt to the similarity of the active constituents in the two plant species. Indeed, there are no reports that A. afra contains artemisinin or any of its derivatives and this was confirmed by a recent study by Van der Kooy et al. (2008) who found out that artemisinin is an important chemical marker to differentiate between the two species. Kraft et al. (2003) demonstrated the activity of a lipophilic (petrol ether/ethylacetate 1:1) extract of A. afra against a CQ-sensitive (PoW) and resistant (Dd2) P. falciparum strains (IC50, 8.9 and 15.3 lg/ml, respectively). Several flavanoids and sesquiterpene lactones were isolated from this plant through bioassay-guided fractionation; with acacetin (1), genkwanin (2) and 7-methoxyacacetin (3) exhibiting the best in vitro antiplasmodial activity

(IC50, 4.3–12.6 lg/ml). The exact mechanism of antimalarial action of flavonoids is unclear but some flavonoids have been shown to inhibit the influx of L-glutamine and myoinositol into infected erythrocytes (Elford, 1986). Exiguaflavanone A and exiguaflavanone B from A. indica exhibited similar in vitro antiplasmodial activities (IC50, 4.6 and 7.0 l lg/ml, respectively) (Chanphen et al., 1998). In contrast to artemisinin from A. annua none of the compounds isolated from A. afra showed extraordinary activity, and the authors concluded that the activity of A. afra was due to the complex mixture of substances, which may act additively or synergistically (Kraft et al., 2003). 5.2. Herbal leads from Kwale district Five ethnobotanically described antimalarial plants, collected in Kwale district of the Kenyan Coast were evaluated against CQ sensitive (D6) and resistant (W2) P. falciparum clones (Muthaura et al., 2007a). Four of these (80%) were found to have IC50 <5 lg/ml for either CQ sensitive (D6) or resistant (W2) clones and these were the methanol leaves extract of Flueggea virosa (Willd.) Voigt (Euphorbiaceae), methanol stem bark extract of Warburgia stuhlmannii Engl. (Canellaceae), water leaves extract of Maytenus undata (Thunb.) Blakelock (Celastraceae) and methanol root bark extract of Maytenus putterlickioides (Loes.) Excell and Mendoca (Celastraceae). The methanol extracts of the Maytenus species were cytotoxic to Vero E6 cells in comparison to the other extracts. These species are widely used in folk medicine as antitumour, antiasthmatic and for stomache problems. Dihydroagarofuran sesquiterpene alkaloids mayteine, putterine A and putterine B have been isolated from the root bark extract of M. putterlickioides (Schaneberg et al., 2001). Quinone methide triterpenoids are a diverse group of secondary metabolites from a related species Maytenus ilicifolia (Buffa Filho et al., 2002), which have revealed potential anti-tumour and anti-microbial activities (Bavovada et al., 1990). The various biological activities have been attributed to those diverse secondary metabolites among them maytansinoids (Reider and Roland, 1984). W. stuhlmannii is found on the Kenyan Coast and a dichloromethane stem bark extract from its close variety found inland, Warburgia ugandensis, had a similar antiplasmodial activity (IC50s 1.4 and 2.2 lg/ml for CQ sensitive (NF54) and resistant (KI) P. falciparum strains, respectively (Irungu et al., 2007). A cytotoxic sesquiterpene, characterized as muzigadial, was isolated from W. ugandensis. It showed in vitro trypanocidal activity and was highly toxic in the brine shrimp assay (Olila et al., 2001). M. undata and F. virosa are reported in South Africa having good in vitro antiplasmodial activity (Clarkson et al., 2004) and bergenin, an isocoumarin isolated from methanol leaves extract of F. virosa has been reported to be antiprotozoal (Nyasse et al., 2004). The in vivo antimalarial results in mice for the four plant extracts were much lower than would be expected from their high activity in vitro. The results may not necessarily correlate as reported by Gessler et al. (1995). There is, however, good correlation in all of the clinically used antimalarial drugs and Peters’ 4-day test is therefore regarded as a good screen for in vivo activity. It could be that crude extracts with their complex mixture of several

616

C.N. Muthaura et al. / Experimental Parasitology 127 (2011) 609–626

Table 3 Antiplasmodial activity of constituents from some Kenyan medicinal plants. Constituents

Plant species (family)

Cytototoxicity Chemical structure Antimalarial CC50 (lg/ml) and activity IC50 in lg/ml (a) or (celllines used) lM (b) and (strains of Plasmodium falciparum used)

References

OCH 3

Flavonoids: acacetin (1)

Artemisia afra Jacq.(Asteraceae)

5.5a (PoW), 12.6 (Dd2)

nd

HO

O

Kraft et al. (2003)

H

OH

O

OH

O

H3CO

H

a

5.5 (PoW), 8.1 (Dd2)

Genkwanin (2)

O

OH

OCH3

H3CO

O H

4.3a (PoW), 7.0 (Dd2)

7-Methoxyacacetin (3)

O

OH

OCH

3

OH

H

HO

Flavonones: 5-deoxyabyssinin II (19)

Erythrina abyssinica DC.(Leguminosae)

13.6b (D6), 13.3 (W2)

O Prenyl

nd

Yenesew et al. (2004)

H

O

H

Prenyl OH

Prenyl

Abyssinin III (20)

5.8b (D6), 5.2 (W2)

HO

O OH H

O

OH

Prenyl OH

H

Abyssinone IV (21)

5.4b (D6), 5.9 (W2)

HO

O Prenyl H

H

O

Prenyl H

Abyssinone V (22)

4.9b (D6), 6.1 (W2)

HO

OH

O Prenyl H

OH

O Prenyl H

Sigmoidin A (23)

5.8b (D6), 5.9 (W2)

HO

OH

O OH Prenyl

OH

O

617

C.N. Muthaura et al. / Experimental Parasitology 127 (2011) 609–626 Table 3 (continued) Constituents

Plant species (family)

Cytototoxicity Antimalarial Chemical structure CC50 (lg/ml) and activity a IC50 in lg/ml ( ) or (celllines used) lM (b) and (strains of Plasmodium falciparum used)

Terpenoids: diterpene ajugarin-1 (17)

Ajuga remota Benth (Labiatae)

23.0a (FCA20/GHA)

References

O

>88 (A431)

Kuria et al. (2002)

O

O OAc AcO O

Triterpenoid limonoid -deacetoxy-7-oxogedunin (4)

Ekebergia capensis Sparm.(Meliaceae)

6a (FCR-3)

nd

Murata et al. (2008)

O

O O O

O

OH

18 (FCR-3), 7 (K1)

2-Hydroxymethyl-2,3,22,23tetrahydroxy-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene (5)

Tritepene ergosterol5,8-endoperoxide (18)

HO

a

OH

OH

Ajuga remota Benth (Labiatae)

8.2a (FCA20/ GHA)

OH

>23 (A431)

Kuria et al. (2002) O H O HO

Sesquiterpenes: mustakone (6)

Cyperus articulatus L. (Cyperaceae)

0.14a (NF54), 0.25 (ENT30)

nd

Rukunga et al. (2008)

1.07a (NF54), 1.92 (ENT30)

Corymbolone (7)

CH 3

Spermine alkaloids: budmunchiamine K1 (12)

6-Hydroxybudmunchiamine K2 (13)

Albizia gummifera J.F. Gmel C.A. Sm (Mimosaceae)

0.09a (NF54), 0.73 (ENT30)

nd

CH 3 N

N

CH 3 (CH2)

(CH2) 8

5

H N

N

CH 3

H

0.15a (NF54), 0.91 (ENT30)

Rukunga et al. (2007)

O

CH3

CH3

N

N

(CH )

(CH 2)8

25

CH3 N

N

CH 3

H

OH O

(continued on next page)

618

C.N. Muthaura et al. / Experimental Parasitology 127 (2011) 609–626

Table 3 (continued) Constituents

Plant species (family)

Cytototoxicity Antimalarial Chemical structure CC50 (lg/ml) and activity a IC50 in lg/ml ( ) or (celllines used) lM (b) and (strains of Plasmodium falciparum used)

References

CH3

H

N

N

(CH2)5

(CH 2)8

0.10a (NF54), 0.93 (ENT30)

5-Normethylbudmunchiamine K3 (14)

CH3 H N

N

CH 3

H

0.17a (NF54), 0.88 (ENT30)

6-Hydroxy-5-normethylbudmunchiamineK4 (15)

O

CH3

H

N

N

(CH )5

(CH2)8

2

CH3 N

N

CH3

H

OH O

H

CH 3

N

N

(CH )5

(CH2)8

2

0.12a (NF54), 0.89 (ENT30)

9-Normethylbudmunchiamine K5 (16)

CH3 H N

N

CH3

H

O

O

Alkaloid: nitidine (10)

Toddalia asiatica (L.) Lam. (Rutaceae)

0.042a – 0.052 (S – R)

nd

MeO

O

Gakunju et al. (1995)

X

N MeO CH3 H O O

Coumarins: 20 epicycloisobrachycoumarinone epoxide (8)

Vernonia brachycalyx O. Hoffm.(Asteraceae)

160b (3D7), 54 (Dd2)

313 (Human lymphocytes) O

O

OketchRabah et al. (1997)

O

H CH3 O O

111b (3D7), 54 (Dd2)

Cycloisobrachycoumarinone epoxide (9)

1810 (Human lymphocytes) O

O

O

OMe

5,7-dimethoxy-8-(31-hydroxy31-methyl-11-butene)coumarin (11)

Toddalia asiatica (L.) Lam. (Rutaceae)

16.2a (K39), 8.8 (VI/S)

nd MeO

O

OH

O

OketchRabah et al. (2000)

619

C.N. Muthaura et al. / Experimental Parasitology 127 (2011) 609–626 Table 3 (continued) Constituents

Plant species (family)

Cytototoxicity Antimalarial Chemical structure CC50 (lg/ml) and activity a IC50 in lg/ml ( ) or (celllines used) lM (b) and (strains of Plasmodium falciparum used)

References

HO OH O

Phenylanthraquinones: joziknipholone A (24) and

Bulbine frutescens (Jacq.) Rowley (Asphodelaceae)

0.14a (K1)

16.3 (L6), 10 (L5178y)

Me

P

Bringmann et al. (2008)

O

H HO OH

Me

P

O

O H

S

O

HO

OMe

HO

O

O

HO

P OMe

HO

O

HO

Me

Me

HO OH O

Me

P H

B (25)

0.23a (K1)

17.4 (L6), 8.7 (L5178y)

O

HO OH

Me

P

O

O O

H

S

HO

OMe

HO Me O

O

HO

M

HO

Me

O

OMe

HO

nd = not done. CQ sensitive strains: PoW, K39, NF54, 3D7, FCA/20GHA, D6, FCR-3, HB3, S. CQ resistant strains: Dd2, ENT30, V1/S, KI, W2, R.

compounds could explain their high in vitro activity due to potential synergism but the fragile chemical structure could be upset due to biotransformation of the constituents or poor bioavailability of the active constituents in vivo, and the results may also be encumbered by other host factors. 5.3. Herbal leads from Kajiado district (Maasai community) The Maasai maintain a rich traditional culture and herbal remedies are an integral part of their culture. Some elder Maasai are to be seen hawking herbal concoctions (miti ni dawa) for enhancing better health in some of the towns outside Maasailand. Koch et al. (2005) evaluated 21 ethnobotanically described antimalarial plants, collected from the Maasai community of Kajiado district in southwestern Kenya for antiplasmodial and cytotoxic properties. Nine of the plants (42%) prepared in chloroform had IC50 <5 lg/ ml against CQ sensitive (D6) P. falciparum clone. These were: root bark extract of Balanites aegyptiaca (L.) Del. (Zygophyllaceae), root bark extract of Sericocomopsis hildebrandtii Schinz (Amaranthaceae), inner bark extract of Commiphora schimperi (O. Berg) Engl. (Burseraceae), inner bark extract of Acacia mellifera (Vahl) Benth. (Fabaceae), leaf extract of Fuerstia africana T.C.E. Fries (Lamiaceae),

leaf extract of Gutenbergia cordifolia Benth. (Asteraceae), root bark extract of Clematis brachiata Thunb. (Ranunculaceae), inner bark extract of Ekebergia capensis Sparrm. (Meliaceae) and root bark ex´ erit. (Rhamnaceae). The cytotoxicities tract of Rhamnus prinoides L´H of the 9 plant extracts except that of G. cordifolia on KB cells showed IC50 >20 lg/ml and thus considered to have good SI for the parasites (Likhitwitayawuid et al., 1993). Muregi et al. (2004) found similar in vitro antiplasmodial activity for E. capensis collected fom Kisii district in Kenya. A limonoid compound (4), (Oxogedunin) (Bevan et al., 1963) and a triterpenoid (5), (2-hydroxymethyl-2,3,22,23-tetrahydroxy-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene) (Nishiyama et al., 1996, 1999) both isolated from the plant showed potent IC50 activities as low as 6 and 7 lM, respectively (Murata et al., 2008). Limonoids are produced by species of Meliaceae. One well known representative from this family is A. indica, the Neem tree, widely used as an antiplasmodial plant in Asia. Rochanakij et al. (1985) identified nimbolide as the active antimalarial principle of the Neem tree (EC50, 0.95 ng/ml, P. falciparum K1). Gedunin (IC50, 720 ng/ml, P. falciparum D6) and its dihydro derivative were also found to be active in vitro (IC50, 2630 ng/ml) (MacKinnon et al., 1997). Their antimalarial activities may be related to the presence

620

C.N. Muthaura et al. / Experimental Parasitology 127 (2011) 609–626

of reactive groups on the ring like the carbonyl group and unsaturation in C-1/C-2 positions. Benoit-Vical et al. (2003) found similar in vitro antiplasmodial activity between iridal, a triterpenoidic compound extracted from Iris germanica L. and an extract of A. indica on P. falciparum, but there was no correlation between CQ and iridal sensitivity against P. falciparum, indicating that the modes of action of iridal and CQ should be different. Compound (5) showed strong in vivo parasitemia suppression of 52.9%, at a dose of 500 mg/kg (which was unusually high concentration for a pure compound) against a CQ-tolerant (NK65) P. berghei strain, consistent with in vitro activity (IC50) of 18 lM and 7 lM against CQ-sensitive (FCR-3) and resistant (K1) P. falciparum strains, respectively an indication of no cross resistance with CQ (Murata et al., 2008). The total methanol extract of E. capensis showed in vivo parasitaemia suppression of 33% at the same dose and parasite strain (Muregi et al., 2007b). B. aegyptiaca was previously reported as not possessing antiplasmodial activity (Weenen et al., 1990) however, Koch et al. (2005) reported good activity. The discrepancy may be attributed to plant parts tested, extraction procedures, georeference and seasonal variation; factors which can account for the differences in observed antiplasmodial activities between the various studies. Antileishmanial and larvicidal properties have been reported (Wiesman and Chapagain, 2006). Saponins have been isolated from various parts of B. aegyptiaca, C. schimperi, Carissa edulis, C. brachiata and Clerodendrum myricoides which may play part in antiplasmodial activity of these plants (Chhabra et al., 1984; Reed, 1986; Johns et al., 1999; Mohamed et al., 2002; Speroni et al., 2005). 5.4. Herbal leads from Kilifi and Tharaka districts Rukunga et al. (2009) evaluated 12 ethnobotanically described antimalarial plants, used traditionally for anti-malarial therapy in Kilifi and Tharaka districts against CQ sensitive (NF54) and resistant (ENT30) P. falciparum strains using 3[H]-hypoxanthine in vitro assay. Two of these (17%) were found to have IC50 <5 lg/ ml for either CQ sensitive (NF54) or resistant (ENT30) strains and these were the methanol and water root bark extract of Zanthoxylum chalybeum Engl. (Rutaceae) and the methanol rhizome extract of Cyperus articulatus L. (Cyperaceae). Both plants were most often cited by all the traditional healers interviewed as the most effective antimalarial remedies among the Kilifi and Tharaka people. Both the water and methanol extracts of Z. chalybeum exhibited higher antiplasmodial activity for the resistant strain than for the sensitive strain suggesting a different mode of action for the constituents as compared to CQ. Traditionally the stem bark, root bark and leaves of the plant is reported to be used for malaria (Kokwaro, 1993; Gessler et al., 1994, 1995; Beentje, 1994) and species of Z. chalybeum from different regions of Tanzania are reported to exhibit high in vitro antiplasmodial activity (Gessler et al., 1994). Quinoline alkaloids from the root bark have been reported (Kato et al., 1996). The leaves of C. articulatus are reportedly used in Guinea for cerebral malaria (Akendengue, 1992). The roots have antiplasmodial activity and traditionally have been used for treatment of malaria in Nigeria (Etkin, 1997). In Kenya, the rhizomes were used to prepare traditional medicine for fever, convulsions and malaria (Kokwaro, 1993). The extracts from the rhizomes have been shown to selectively inhibit NMDA receptor-mediated neurotransmission and also have anticonvulsant properties (Bum et al. 2001; 1996). Two Sesquiterpenes, mustakone (6) and corymbolone (7) isolated from the chloroform extract of the rhizomes of C. articulatus exhibited significant antiplasmodial properties. Mustakone was approximately ten times more active than corymbolone against the CQ sensitive (NF54) P. falciparum strain (Rukunga et al., 2008). The presence of the a,b-unsaturated ketone function may probably

contribute to the observed high activity, a phenomenon that has been observed in some other sesquiterpenes isolated from the closely related species C. rotundus (Weenen et al., 1990). Some eudesmane sesquiterpenes, from the ethyl acetate leaves extract of Melampodium camphoratum have been reported to show activity in the hemin degradation assay (Chaturvedula et al., 2004). The authors concluded that the results of this study suggested that mustakone can be used as a ‘‘marker compound’’ in the standardization of herbal medicines for malaria prepared from C. articulatus. One of the plants from Kilifi, screened by Kirira et al. (2006) among others from Meru district above, was A. indica or Neem (Meliaceae), a plant growing in the coastal region of Kenya and widely used around the world for malaria treatment, but showed weak antiplamodial activity (IC50 > 250 lg/ml) for water and methanol leaves extracts against CQ sensitive (NF54) and resistant (ENT30) P. falciparum strains. Similar findings were reported by Ofulla et al. (1995) for water leaves extract of A. indica (IC50 > 250 lg/ml) collected elsewhere in Kenya. Studies in other countries showed good in vitro antiplasmodial activity against P. falciparum on extracts from Sudan (El Tahir et al., 1999), the Ivory Coast (Benoit-Vical et al., 1996), India (Dhar, 1998) and Thailand (Rochanakij et al., 1985). Tests showed that the cytotoxicity of crude Neem extracts was lower than synthesized molecules (Badam et al., 1987). In vivo studies in mice with P. berghei have been uniformly disappointing (Ekanem, 1971; Obih and Makinde, 1985). Many reasons could explain these poor in vivo activities. It may be that mice do not metabolise Neem extracts in the same way as humans. Murine Plasmodium have different properties and sensitivities compared with human P. falciparum and the 4day suppressive test may not be sufficient to evaluate plant extracts (Willcox and Bodeker, 2004). 5.5. Herbal leads from Kisii district Muregi et al. (2003, 2004) in two separate studies evaluated 22 ethnobotanically described antimalarial plants, collected in Kisii district against CQ sensitive (K39 and NF54) and resistant (ENT30 and V1/S) P. falciparum strains. In the first and second study, one (4.5%) and two (9%) plant species, respectively showed IC50s <5 lg/ml for either CQ sensitive or resistant P. falciparum strains. These were Vernonia lasiopus O. Hoffm. (Compositae), Ekebergia capensis Sparrm. (Meliaceae) and C. myricoides (Hochst.) Vatke (Verbenaceae). V. lasiopus was particularly active, with chloroform and ethyl acetate extracts exhibiting IC50 <4 lg/ml for all the strains tested. Irungu et al. (2007) reported a similar finding (IC50 < 5 lg/ml) for the dichloromethane root bark extract of V. lasiopus against CQ resistant (KI) P. falciparum strain. The leaf infusion is traditionally used for malaria in Uganda (Katuura et al., 2007; Hamill et al., 2000). V. brachycalyx, a closely related species has been used for malaria by the Maasai, the Kipsigis, and other tribes in East Africa for treatment of parasitic diseases (Beentje, 1994). Two isomeric 5-methylcoumarins, 20 -epicycloisobrachycoumarinone epoxide (8) and cycloisobrachycoumarinone epoxide (9), with some antiplasmodial and antileishmanial activities have been isolated from V. brachycalyx root extracts through bioactivityguided fractionation (Oketch-Rabah et al., 1997) as well as 16,17dihydrobrachycalyxolide which was also antiplasmodial and antileishmanial but inhibited human lymphocytes at the same concentration, indicating that the antiprotozoal activity was due to general toxicity (Oketch-Rabah et al., 1998). Oxygenated sesquiterpene lactones are the most abundant secondary metabolites of the genus Vernonia, and artemisinin, which is effective against multi-drug resistant strains of P. falciparum and isolated from A. annua belong to the same class of compounds (Oketch-Rabah, 1996; Trigg, 1989). Muregi et al. (2007b) investigated the in vivo activity of some of the plants above and demonstrated a

C.N. Muthaura et al. / Experimental Parasitology 127 (2011) 609–626

parasitaemia suppression of 59.3% for V. lasiopus methanol root bark extract against mice infected with P. berghei strain NK65. 5.6. The Pokot and the antimalarial leads The Pokot traditionally are herders and inhabit a highland plateau west of Rift Valley in Kenya. They are endowed with a rich cultural heritage and herbal remedies are widely practiced due to inaccessibility of modern medicine. A Pokot herbalist aged 90 years described the use of 14 plants for the treatment of malaria and fever. Gakunju et al. (1995) went onto evaluate their antiplasmodial activity and to isolate an antimalarial compound nitidine (10) from one of the active plant extracts, Toddalia asiatica (L.) Lam. (Rutaceae). Of the 14 ethnopharmacologically described plants two (14%) had IC50 <5 lg/ml and those were T. asiatica and Maytenus arbutifolia (A Rich.) Wilczek (Celastraceae). The active fraction of T. asiatica, with a mean IC50 of 51.6 ng/ml against CQ resistant (ItD12, FCR3, FCB) strains, was more active than quinine against Kenyan parasites (IC50, ca. 120 ng/ml) (Pasvol et al., 1991). In addition to being antiplasmodial, nitidine is a well-known cytotoxic agent which has received considerable attention as a potential anticancer drug following the discovery of potent antileukemic activity in mice (Messmer et al., 1972). Subsequent chemical development of this compound has further improved its potency against CQ resistant P. falciparum isolates in vitro (Waigh, 2002). Oketch-Rabah et al. (2000) isolated a coumarin derivative, 5,7-dimethoxy-8-(30 -hydroxy-30 -methyl-10 -butene)coumarin (11) from the same plant which exhibited antiplasmodial activity. The in vivo antimalarial activity of the methanolic root bark extract showed 59.3% parasitaemia suppression of P. berghei in mice at a dose of 500 mg/kg/day (Muregi et al., 2007b). T. asiatica is widespread in Kenya and field interviews with local herbalists most often cite this plant as important in traditional therapy against malaria (Muthaura et al., 2007c,d; Orwa et al., 2007; Katuura et al., 2007). 5.7. Herbal leads from other places in Kenya Omulokoli et al. (1997) evaluated four ethnobotanically described antimalarial plants, collected in Coast and Western Provinces of Kenya against CQ sensitive (K67) and resistant (ENT36) P. falciparum strains. Two of the plants Phyllanthus reticulatus Poir. and Suregada zanzibariensis Baill. both in Euphorbiaceae family showed IC50 <5 lg/ml for the CQ sensitive (K67) and resistant (ENT36) P. falciparum strains. Phytochemical screening revealed classes of compounds such as alkaloids, steroids, and triterpenoids in the two plant extracts (Omulokoli et al., 1997) and also flavonoid glycosides (Lam et al., 2007). Irungu et al. (2007) evaluated 14 ethnobotanically described plants, traditionally used for antimalarial therapy in Eastern, Central and Coast Provinces of Kenya for their ability to inhibit the in vitro proliferation of CQ sensitive (NF54) and resistant (K1) P. falciparum strains using 3[H]-hypoxanthine in vitro assay. Four of these (28.5%) were found to have IC50 <5 lg/ml for either CQ sensitive (NF54) or resistant (KI) strains and these were the dicloromethane stem bark extract of Harrisonia abyssinica Oliv (Simaroubaceae), methanol root bark extract of Turrea robusta Guerke (Melianthaceae), dichloromthane stem bark extract of W. ugandensis Sprague (Canellaceae) and dichloromethane stem bark extract of V. lasiopus O. Hoffm (Compositae). The family Simaroubaceae is known to contain quassinoids which are heavily oxygenated lactones, possessing a wide spectrum of biological activities, and among the antiplasmodial compounds isolated is simalikalactone D from Simaba guianesis with IC50 <1.7 ng/ml (Cabral et al., 1993). The activity of these compounds is due to the methylene– oxygen bridge. Ofulla et al. (1995) evaluated 4 ethnobotanically described antimalarial plants from Kenya against CQ resistant (ENT22, ENT30,

621

KIL9) P. falciparum isolates. One of them, Albizia gummifera methanol stem bark extract was found to have IC50 <5 lg/ml for all the isolates. Bioassay-guided fractionation of the methanol extract led to isolation of five bioactive known spermine alkaloids namely: Budmunchiamine K(1) (12), 6-Hydroxybudmunchiamine K(2) (13), 5-Normethylbudmunchiamine K(3) (14), 6-Hydroxy-5-normethybudmunchiamine K(4) (15) and 9-Normethybudmunchiamine K(5) (16) (Rukunga and Waterman, 1996). These alkaloids exhibited activities against CQ sensitive (NF54) and resistant (ENT30) with IC50s ranging from 0.09 to 0.91 lg/ml. With the exception of (15), the alkaloids were further evaluated for in vivo activity against P. berghei infected mice and showed suppression of parasitaemia ranging from 43% to 72% (Rukunga et al., 2007). Similar bioactive spermine alkaloids, budmunchiamines L4 and L5 (IC50, 14.0 and 15.0 lM, respectively) have been isolated from the methanol stem bark and leaves extracts of Albizia adinocephala. The extracts were found to inhibit the malarial enzyme plasmepsin II (Ovenden et al., 2002). Alkaloids have been successfully used for the treatment of parasitic infections. The outstanding example is quinine from Cinchona succirubra (Rubiaceae) used for the treatment of malaria for more than three centuries. Kuria et al. (2001) picked out Ajuga remota as the most commonly used herbal medicine to treat malaria in Kenya during field trips to herbalists’ practices in an area about 200 miles around Nairobi but similarly to Muregi et al. (2004) findings, the antiplasmodial activity was moderate (IC50 > 10 lg/ml). In a follow up study Kuria et al. (2002) isolated ajugarin-1 (17) and ergosterol-5,8endoperoxide (18) from A. remota and evaluated their in vitro antiplasmodial activity. Ajugarin-1 was moderately active, with IC50 of 23.0 lM, against the CQ sensitive (FCA20/GHA) P. falciparum strain. Ergosterol-5,8-endoperoxide was about 3 times as potent. Both ajugarin-1 and ergosterol-5,8-endoperoxide did not exhibit cytotoxicity against A431 (skin carcinoma) cell line. Ajugarin-1 is interesting in view of its unusual chemical structure (epoxide ring) when compared to conventional antimalarial compounds. Such a compound can be considered as a lead compound to synthesize new pharmaceutically important derivatives with possibly higher antimalarial activities. Ergosterol-5,8-endoperoxide has been found to inhibit the growth of protozoan parasite of Trypanosomatidae family such as Trypanosome cruzi and various Leishmania species by interfering with the integrity of the cell membrane (Liñares et al., 2006). The broad action of ergosterol endoperoxide against several protozoal parasites and mycobacteria opens up new possibilities for application in the treatment of several diseases. Possibly the mechanism of action of ergosterol endoperoxide could involve the formation of reactive oxygen species, that causes disruption of the parasite membrane (Correa et al., 2006). Among the five Erythrina species present in Kenya, E. abyssinica is the most widely used in traditional medicine where it has been used for treatment of malaria and microbial infections (Kokwaro, 1993). The antiplasmodial activities of the stem and root bark extracts of E. abyssinica have been reported (Yenesew et al., 2003a, 2004). The acetone extract of the roots were found to have IC50 < 5 lg/ml for either CQ sensitive (D6) or resistant (W2) P. falciparum strain. Compounds isolated which showed antiplasmodial activity included chalcones, isoflavonoids and flavonones, with the latter showing higher antipasmodial activities. Some of the active flavonones were 5-deoxyabyssinin II (19), Abyssinin III (20), Abyssinone IV (21), Abyssinone V (22) and Sigmoidin A (23) (Yenesew et al., 2004). And from the roots of South African plant species Bulbine frutescens (Jacq.) Rowley (Asphodelaceae) widely cultivated in other parts of the world for aesthetic purposes, phenylanthraquinone knipholone compounds with remarkable antiplasmodial activities have been reported (Abegaz et al., 2002). Further work by Bringmann et al. (2008) led to isolation of two unprecedented dimeric

622

C.N. Muthaura et al. / Experimental Parasitology 127 (2011) 609–626

phenylanthraquinones, namely joziknipholones A (24) and B (25), that showed high antiplasmodial activity against CQ resistant (K1) P. falciparum strain. The antiplasmodial activity appears to be associated intrinsically with the complete molecular array of a phenylanthraquinone including the stereogenic axis (Abegaz et al., 2002). The two compounds exhibited low antitumoural and cell cytotoxicity activities against murine leukemic lymphoma L5178y cells and rat skeletal myoblast (L6) cells, respectively. 5.8. Herbal leads and synergism In a study for in vitro antiplasmodial activity of some plants used in Kisii, against malaria and their CQ potentiation effects, Muregi et al. (2003) observed that the ethyl acetate leaves extract of V. lasiopus with CQ against the multi-drug resistant (V1/S) P. falciparum isolate showed synergistic effects. Cepharanthine, a bisbenzylisoquinoline alkaloid isolated from Stephania erecta was shown to synergize with CQ by significantly reducing the IC50 for the CQ resistant (W2) clone but not low enough to the level of the sensitive (D6) clone (Tamez et al., 2005). In the in vitro antiplasmodial combination tests between different extracts for some plants from Meru, Gathirwa et al. (2008) showed that interaction was mainly additive, while in the in vivo antimalarial tests some synergistic interactions were observed resulting in unusually high parasitaemia suppression for the extracts (p > 0.05) when compared to CQ. This may explain why traditional healers use a concoction of different plants in preparation of herbal remedies. Synergistic interactions between the components of individual or mixtures of plant extracts are considered to be a vital part of their therapeutic efficacy; however until recently there has been very little evidence to demonstrate what herbal practitioners have always believed. The ‘‘isobole method’’ of Berenbaum (1989) seems to be one of the most practicable experimentally and also the most demonstrative method among all those so far proposed for the proof of synergy effects. For example over the last two decades it has been suggested that the efficacy of A. annua plant extract derives from a synergistic effect and that it is a combination of constituents in the plant which produce the total antiplasmodial activity. Several polymethoxyflavones such as casticin, artemetin, chrysosplenetin, chrysosplenol-D and circilineol (Bhakuni et al., 2001) may contribute to the in vitro activity of artemisinin against P. falciparum (Bhakuni et al., 2001; Elford et al., 1987; Phillipson, 1999; Liu et al., 1992). However, data concerning the mechanism related to the synergistic effects have been scarce mainly due to the complexity of very many compounds in a plant extract. Bilia et al. (2002) demonstrated that the synergism of the flavonoids is related to the assisted activation of artemisinin before its interaction with hemin. In Mali, Nauclea latifolia was associated with Mitragyna inermis or Guiera senegalensis and Feretia apodanthera with M. inermis (Azas et al., 2004). In modern drug therapy synergistic interaction is to be preferred because the magnitude of the drug efficacy is many times enhanced with a minimal risk of selecting mutant, resistant parasite populations (White and Olliaro, 1996). Resistance development by infective agents to traditional therapies may be difficult due to the activities of a complex mixture of many constituents either in a single plant or in a mixture of plants. Drug combination has been the way forward to counter parasite resistance in antimalarial chemotherapy (White and Olliaro, 1996) and to minimize the risk of resistant development (Anne et al., 2001; Olliaro and Taylor, 2003). 6. Conclusions The ethnopharmacology approach used in search for new antimalarial compounds appears to be predictive, and whereas

all the crude extracts are used daily for malaria treatment and show high in vitro activity, only a few extracts showed good in vivo efficacy, although in vivo assays reported are few in comparison to the in vitro assays. The cytotoxicity and toxicity tests in animal models are even fewer albeit folk medicine has been in use for centuries without apparent serious side effects. However, information in this area may be lacking due to non-reporting and the possible potential genotoxic effects that follow prolonged use of the more popular herbal remedies are a cause for alarm. In vitro screening programmes, using the ethnobotanical approach, are important in validating the traditional use of herbal remedies and for providing leads in the search fornew active principles. Using bioassay-guided fractionation, several compounds having biological activity have been isolated and identified. The number of active plant extracts and compounds identified from only a small sampling of Kenya’s medicinal flora, using a series of in vitro tests, further emphasises the potential areas for future work. Ideally, extracts and compounds effective at the blood stage of the malaria parasite should have strong in vitro antiplasmodial and in vivo antimalarial activities with good selectivity for the malaria parasites in cytotoxic assays. No clinical trials have been reported in the country for even the more popular medicinal plants like A. indica, whose products are to be found dotting many supermarket shelves in both upmarket and downmarket trading areas. The significance of the reported research lies not only in serving to promote the value of the Kenyan flora and the quest for new antimalarial molecules but also the need for traditional healthcare. There is a growing awareness on the usefulness of traditional foods and natural products and it may not be long, before a draft policy which is already under discussion is implemented through an act of parliament, for integration of traditional medicine with allopathic medicine in public health facilities. This would lead to well-controlled clinical trials that would be invaluable for locating new antimalarial and other drugs.

Acknowledgments This work received financial support from UNICEF/UNDP/World Bank/WHO special programme for Research and Training in Tropical Diseases (TDR). We thank the Director, KEMRI for allowing publication of this study.

References } hlbacher, J., Abegaz, B.M., Bezabih, M., Msuta, T., Brun, R., Menche, D., Mu Bringmann, G., 2002. Gaboroquinones A and B and 40 -O-demethylknipholone40 -O-b-D-glucopyranoside, phenylanthraquinones from the roots of Bulbine frutescens. Journal of Natural Products 65, 1117–1121. Addae-Kyereme, J., Croft, S.L., Kendrick, H., Wright, C.W., 2001. Antiplasmodial activities of some Ghanaian plants traditionally used for fever/malaria treatment and of some alkaloids isolated from Pleiocarpa mutica; in vitro antimalarial activity of pleiocarpine. Journal of Ethnopharmacology 76, 99–103. Akendengue, B., 1992. Medicinal plants used by the fang traditional healers in Equatorial Guinea. Journal of Ethnopharmacology 37, 165–173. Andrade-Neto, V.F., Brandão, M.G.L., Stehmann, J.R., Oliveira, L.A., Krettli, A.U., 2003. Antimalarial activity of Cinchona-like plants used to treat fever and malaria in Brazil. Journal of Ethnopharmacology 87, 253–256. Andrade-Neto, V.F., Brandão, M.G.L., Nogueira, F., Rosário, V.E., Krettli, A.U., 2008. Ampelozyziphus amazonicus Ducke (Rhamnaceae), a medicinal plant used to prevent malaria in the Amazon Region, hampers the development of Plasmodium berghei sporozoites. International Journal for Parasitology 38, 1505–1511. Anne, R., Françoise, B., Odile, D., Bernard, M., 2001. From classical antimalarial drugs to new compounds based on the mechanism of action of artemisinin. Pure and Applied Chemistry 73, 1173–1188. Azas, N., Laurencin, N., Delmas, F., Di Giorgio, C., Gasquet, M., Laget, M., TimonDavid, P., 2004. Synergistic in vitro anti-malarial activity of plant extracts used as traditional herbal remedies in Mali. Parasitology Research 88, 165–171. Badam, L., Deolankar, R.P., Kulkarni, M.M., Nagsampgi, B.A., Wagh, U.V., 1987. In vitro antimalarial activity of neem leaf and seed extracts. Indian Journal of Malariology 24, 111–117.

C.N. Muthaura et al. / Experimental Parasitology 127 (2011) 609–626 Bah, S., Jäger, A.K., Adsersen, A., Diallo, D., Paulsen, B.S., 2007. Antiplasmodial and GABA (A)-benzodiazepine receptor binding activities of five plants used in traditional medicine in Mali, West Africa. Journal of Ethnopharmacology 110, 451–457. Basco, L.K., Mitaku, S., Skaltsounis, A.L., Ravelomanaintsoa, N., Tillequin, F., Koch, M., Le Bras, J., 1994. In vivo activities of acridone alkaloids against Plasmodium falciparum. Antimicrobial Agents and Chemotherapy 5, 1169–1171. Bathurst, I., Hentschel, C., 2006. Medicines for malaria venture: sustaining antimalarial drug development. Trends in Parasitology 22, 301–307. Bavovada, R., Blaskom, G., Shienm, H.L., Pezzutom, J.M., Cordell, G.A., 1990. Spectral assignment and cytotoxicity of 22-hydroxytingenone from Glyptopetalum sclerocarpum. Planta Medica 56, 380–382. Beentje, H.J., 1994. Kenya Trees, Shrubs, and Lianas. National Museums of Kenya, Nairobi, Kenya. Benoit-Vical, F., Imbert, C., Bonfils, J.P., Sauvaire, Y., 2003. Antiplasmodial and antifungal activities of iridal, a plant triterpenoid. Phytochemistry 62, 747–751. Benoit-Vical, F., Valentin, A., Pelissier, Y., Diafouka, F., Marion, C., Kone-Bamba, D., Kone, M., Mallie, M., Yapo, A., Bastide, J.M., 1996. In vitro antimalarial activity of vegetal extracts used in West African traditional medicine. American Journal of Tropical Medicine and Hygiene 54, 67–71. Benoit-Vical, F., 2005. Ethnomedicine in malaria treatment. Investigational Drugs 8, 45–52. Benoit-Vical, F., Valentin, A., Cournac, V., Pélissier, Y., Mallie, M., Bastide, J.M., 1998. In vitro antiplasmodial activity of stem and root extracts of Nauclea latifolia S.M. (Rubiaceae). Journal of Ethnopharmacology 61, 173–178. Berenbaum, M.C., 1989. What is synergy? Pharmcological Reviews 41, 93–141. Bevan, C.W.L., Powell, J.W., Taylor, D.A.H., 1963. Extracts of the genera Khaya, Guarea, Carapa, and Cedrela. Journal of Chemical Society (C) 980, 982. Bhakuni, R.S., Jain, D.C., Sharma, R.P., Kumar, S., 2001. Secondary metabolites of Artemisia annua and their biological activity. Current Science 80, 35–48. Bilia, A.R., Lazari, D., Messori, L., Taglioli, V., Temperini, C., Vincieri, F.F., 2002. Simple and rapid physico-chemical methods to examine action of antimalarial drugs with hemin: its application to Artemisia annua constituents. Life Sciences 70, 769–778. Billing, J., Sherman, P.W., 1998. Antimicrobial functions of spices: why some like it hot. Quarterly Review of Biology 73, 4–38. Boiteau, P., 1986. Précis de matiére médicale Malgache. Agence de Coopération Culturelle et Technique, Paris. p. 141. Brandão, M.G.L., Grandi, T.S.M., Rocha, E.M.M., Sawyer, D.R., Krettli, A.U., 1992. Survey of medicinal plants used as antimalarials in the Amazon. Journal of Ethnopharmacology 36, 175–182. Brandão, M., Botelho, M., Krettli, E., 1985. Antimalarial experimental chemotherapy using natural products. Cienceia Culture 37, 1152–1163. Bringmann, G., Mutanyatta-Comar, J., Maksimenka, K., Wanjohi, J.M., Heydenreich, } ller, W.E.G., Peter, M.G., Midiwo, J.O., Yenesew, A., 2008. M., Brun, R., Mu Joziknipholones A and B: the first dimeric phenylanthraquinones, from the roots of Bulbine frutescens. Chemistry – A European Journal 14, 1420–1429. Buffa Filho, W., Corsino, J., Bolzani, V., da Silva, M., Furlan, M., Pereira, A.M.S., Franca, S.C., 2002. Quantitative determination of cytotoxic friedo-nor-oleanane derivatives from five morphological types of Maytenus ilicifolia (Celestraceae) by reverse-phase high-performance liquid chromatography. Phytochemical Analysis 13, 75–78. Bum, E.N., Schmutz, M., Meyer, C., Rakotonirina, A., Bopelet, M., Portet, C., Jeker, A., Rakotonirina, S.V., Olpe, H.R., Herrling, P., 2001. Anticonvulsant properties of the methanolic extract of Cyperus articulatus (Cyperaceae). Journal of Ethnopharmacology 76, 145–150. Bum, E.N., Meier, C.L., Urwyler, S., Wang, Y., Herrling, P.L., 1996. Extracts from rhizomes of Cyperus articulatus (Cyperaceae) displace [3H]CGP39653 and [3H]glycine binding from cortical membranes and selectively inhibit NMDA receptor-mediated neurotransmission. Journal of Ethnopharmacology 54, 103– 111. Cabral, J.A., McChesney, J.D., Milhous, W.K., 1993. A new antimalarial quassinoid from Simaba guianensis. Journal of Natural Products 56, 1954–1961. Camacho, M.D.R., Croft, S.L., Phillipson, J.D., 2000. Natural products as sources of antiprotozoal drugs. Current Opinion in Anti-infective Investigational Drugs 2, 47–62. Carvalho, L.H., Brandão, M.G., Santos-Filho, D., Lopes, J.L., Krettli, A.U., 1991. Antimalarial activity of crude extracts from Brazilian plants studied in vivo in Plasmodium berghei-infected mice and in vitro against Plasmodium falciparum in culture. Brazilian Journal of Medical and Biological Research 24, 1113–1123. Carraz, M., Jossang, A., Franetich, J.F., Siau, A., Ciceron, L., Hannoun, L., Sauerwein, R., Frappier, F., Rasoanaivo, P., Snounou, G., Mazier, D., 2006. A plant-derived morphinan as a novel lead compound active against malaria liver stages. Public Library of Science Medicine 3, 2392–2402. Chaturvedula, V.S., Farooq, A., Schilling, J.K., Malone, S., Derveld, I., Werkhoven, M.C., Wisse, J.H., Ratsimbason, M., Kingston, D.G., 2004. New eudesmane derivatives from Melampodium camphoratum from the suriname rainforest. Journal of Natural Products 67, 2053–2057. Chanphen, R., Thebtaranonth, Y., Wanauppathamkul, S., Yuthavong, Y.J., 1998. Antimalarial principles from Artemisia indica. Journal of Natural Products 61, 1146–1147. Chhabra, S.C., Uiso, F.C., Mshiu, E.N., 1984. Phytochemical screening of Tanzanian medicinal plants, I. Journal of Ethnopharmacology 11, 157–179. Clardy, J., Walsh, C., 2004. Lessons from natural molecules. Nature 432, 829–837. Clarkson, C., Maharaj, V.J., Crouch, N.R., Grace, O.M., Pillay, P., Matsabisa, M.G., Bhagwandhin, N., Smith, P.J., Folb, P.I., 2004. In vitro antiplasmodial activity of

623

medicinal plants native to or naturalized in South Africa. Journal of Ethnopharmacology 92, 177–191. Conco, W.Z., 1991. Zulu traditional medicine: its role in modern society. Community Health 5, 8–13. Correa, E., Cardona, D., Quiñones, W., Torres, F., Franco, A.E., Vélez, I.D., Robledo, S., Echeverri, F., 2006. Leishmanicidal activity of Pycnoporus sanguineus. Phytotherapy Research 20, 497–499. Cox, P.A., Balick, M.J., 1994. The ethnobotanical approach to drug discovery. Scientific American 270, 60–65. de Mesquita, M.L., Grellier, P., Mambu, L., de Paula, J.E., Espindola, L.S., 2007. In vitro antiplasmodial activity of Brazilian cerrado plants used as traditional remedies. Journal of Ethnopharmacology 110, 165–170. Desjardins, R.E., Canfield, C.J., Haynes, J.D., Chulay, J.D., 1979. Quantitative assessment of antimicrobial activity in vitro by a semi-automated microdilution technique. Antimicrobial Agents and Chemotherapy 16, 710–718. Dhar, R., 1998. Inhibition of the growth and development of asexual and sexual stages of drug-sensitive and resistant strains of the human malaria parasite Plasmodium falciparum by Neem (Azadiracta indica) fractions. Journal of Ethnopharmacology 61, 31–39. DMS, 2006. Director of Medical Services. Ministry of Health, Republic of Kenya. Malaria Fact Sheet. Ekanem, O., 1971. Has Azadirachta indica (Dongoyaro) any antimalarial activity? Nigerian Medical Journal 8, 8–11. Elford, B.C., 1986. L-Glutamine influx in malaria-infected erythrocytes: a target for antimalarials? Parasitology Today 2, 309–312. Elford, B.C., Roberts, M.F., Phillipson, J.D., Wilson, R.J., 1987. Potentiation of the antimalarial activity of qinghaosu by methoxylated flavones. Transactions of the Royal Society of Tropical Medicine and Hygiene 81, 434–436. Elisabetsky, E., Wannmacher, L., 1993. The status of ethnopharmacology in Brazil. Journal of Ethnopharmacology 38, 137–143. El Tahir, A., Satti, G.M.H., Khalid, S.A., 1999. Antiplasmodial activity of selected Sudanese medicinal plants with emphasis on Maytenus senegalensis (Lam.) excell. Journal of Ethnopharmacology 64, 227–233. Etkin, N.L., 1997. Antimalarial plants used by Hausa in Northern Nigeria. Tropical Doctor 27, 12–16. Ferreres, F., Pereira, D.M., Valentão, P., Oliveira, J.M.A., Faria, J., Gaspar, L., Sottomayor, M., Andrade, P.B., 2010. Simple and reproducible HPLC–DAD– ESI–MS/MS analysis of alkaloids in Catharanthus roseus roots. Journal of Pharmaceutical and Biomedical Analysis 51, 65–69. Fischer, D.C.H., Gualda, N.C.D.A., Bachiega, D., Carvalho, C.S., Lupo, F.N., Bonotto, S.V., Alves, M.D.O., Yogi, A., Di Santi, S.M., Avila, P.E., Kirchgatter, K., Hrihorowitsch Moreno, P.R., 2004. In vitro screening for antiplasmodial activity of isoquinoline alkaloids from Brazilian plant species. Acta Tropica 92, 261–266. Foster, S.D., 1991. Pricing, distribution and use of antimalarial drugs. Bulletin of the World Health Organization 69, 349–363. Gachathi, F.N., 1989. Kikuyu botanical dictionary of plant names and uses. AMREF, Print. Department Nairobi. Gakunju, D.M.N., Mberu, E.K., Dossaji, S.F., Gray, A.I., Waigh, R.D., Waterman, P.G., Watkins, W.M., 1995. Potent anti-malarial activity of the alkaloid nitidine, isolated from a Kenyan herbal remedy. Antimicrobial Agents and Chemotherapy 39, 2606–2609. Gathirwa, J.W., Rukunga, G.M., Njagi, E.N., Omar, S.A., Mwitari, P.G., Guantai, A.N., Tolo, F.M., Kimani, C.W., Muthaura, C.N., Kirira, P.G., Ndunda, T.N., Amalemba, G., Mungai, G.M., Ndiege, I.O., 2008. The in vitro anti-plasmodial and in vivo antimalarial efficacy of combinations of some medicinal plants used traditionally for treatment of malaria by the Meru community in Kenya. Journal of Ethnopharmacology 115, 223–231. Gathirwa, J.W., Rukunga, G.M., Njagi, E.N.M., Omar, S.A., Guantai, A.N., Muthaura, C.N., Mwitari, P.G., Kimani, C.W., Kirira, P.G., Tolo, F.M., Ndunda, T.N., Ndiege, I.O., 2007. In vitro anti-plasmodial and in vivo anti-malarial activity of some plants traditionally used for the treatment of malaria by the Meru community in Kenya. Journal of Natural Medicine 61, 261–268. Gessler, M.C., Mysuya, D.E., Nkunya, M.H.H., Mwasumbi, L.B., Schar, A., Heinrich, M., Tanenr, M., 1995. Traditional healers in Tanzania: the treatment of malaria with plant remedies. Journal of Ethnopharmacology 48, 131–144. Gessler, M.C., Nkunya, M.H.H., Mwasumbi, L.B., Heinrich, M., Tanner, M., 1994. Screening Tanzanian medicinal plants for antimalarial activity. Acta Tropica 56, 65–77. Githiru, M., Lens, L., 2004. Using scientific evidence to guide the conservation of a highly fragmented and threatened Afrotropical forest. Oryx 38, 404– 409. Hamill, F.A., Apio, S., Murbiru, N.K., Mosango, M., Bukenya-Ziraba, R., Maganyi, O.W., Soejarto, D.D., 2000. Traditional herbal drugs of Southern Uganda I. Journal of Ethnopharmacology 70, 281–300. Hart, B.L., 2005. The evolution of herbal medicine: behavioural perspectives. Animal Behaviour 70, 975–989. Hassan, S.W., Umar, R.A., Lawal, M., Bilbis, L.S., Muhammad, B.Y., Dabai, Y.U., 2006. Evaluation of antibacterial activity and phytochemical analysis of root extracts of Boscia angustifolia. African Journal of Biotechnology 5, 1602–1607. Hoffman, S.L., Gardner, M.J., Doolan, D.L., Hedstrom, R.C., Wang, R., Sedegah, M., Gramzinski, R.A., Aguiar, J.C., Wang, H., Margalith, M., Hobart, P., 1996. DNA vaccines against malaria: Immunogenicity and protection in a rodent model. Journal of Pharmaceutical Sciences 85, 1294–1300. Houghton, P.J., 2001. Old yet new-pharmaceuticals from plants. Journal of Chemical Education 78, 175–184.

624

C.N. Muthaura et al. / Experimental Parasitology 127 (2011) 609–626

Irungu, B.N., Rukunga, G.M., Mungai, G.M., Muthaura, C.N., 2007. In vitro antiplasmodial and cytotoxicity activities of 14 medicinal plants from Kenya. South African Journal of Botany 73, 204–207. Jambou, R., Legrand, E., Niang, M., Khim, N., Lim, P., Volney, B., Ekala, M.T., Bouchier, C., Este Esterre, P., Fandeur, T., Mercereau-Puijalan, O., 2005. Resistance of Plasmodium falciparum field isolates to in-vitro artemether and point mutations of the SERCA-type PfATPase6. Lancet 366, 1960–1963. Jean-Marie, K., 2002. Changing national malaria treatment protocols in Africa. Press Dossier. Johns, T., Mahunnah, R.L., Sanaya, P., Chapman, L., Ticktin, T., 1999. Saponins and phenolic content in plant dietary additives of a traditional subsistence community, the Batemi of Ngorongoro District, Tanzania. Journal of Ethnopharmacology 66, 1–10. Johns, T., Faubert, G.M., Kokwaro, J.O., Mahunnah, R.L.A., Kimanani, E.K., 1995. Antigiardial activity of gastrointestinal remedies of the Luo of East Africa. Journal of Ethnopharmacology 46, 17–23. Katuura, E., Waako, P., Tabuti, J.R.S., Bukenya-Ziraba, R., Ogwal-Okeng, J., 2007. Antiplasmodial activity of extracts of selected medicinal plants used by local communities in Western Uganda for treatment of malaria. African Journal of Ecology 45, 94–98. Kato, A., Moriyasu, M., Ichimaru, M., Nishiyama, Y., 1996. Isolation of alkaloidal constituents of Zanthoxylum usambarense and Zanthoxylum chalybeum using ion-pair HPLC. Journal of Natural Products 59, 316–318. Kaur, K., Jain, M., Kaur, T., Jain, R., 2009. Antimalarials from nature. Bioorganic and Medicinal Chemistry 17, 3229–3256. Kazembe, T.C., Nkomo, S., 2010. Mosquitocides of Argeratum conyzoides, Boscia salicifolia and Grewia monticola. Indian Journal of Traditional Knowledge 9, 394– 397. Khalid, S.A., Waterman, P.G., 1985. 6-Hydroxymethyldihydronitidine from Fagaropsis angolensis. Journal of Natural Products 48, 118–119. Khaemba, B.M., Mutani, A., Bett, M.K., 1994. Studies of anopheles mosquitoes transmitting malaria in a newly developed highland urban area: a case study of Moi University and its environs. East African Medical Journal 71, 159–164. Kirira, P.G., Rukunga, G.M., Wanyonyi, A.W., Muregi, F.M., Gathirwa, J.W., Muthaura, C.N., Omar, S.A., Tolo, F., Mungai, G.M., Ndiege, I.O., 2006. Antiplasmodial activity and toxicity of extracts of plants used in traditional malaria therapy in Meru and Kilifi districts of Kenya. Journal of Ethnopharmacology 106, 403–407. Koch, A., Tamez, P., Pezzuto, J., Soejarto, D., 2005. Evaluation of plants used for antimalarial treatment by the Maasai of Kenya. Journal of Ethnopharmacology 101, 95–99. Kokwaro, J.O., 1993. Medicinal plants of East Africa. Kenya Literature Bureau, Nairobi. Kraft, C., Jenett-Siems, K., Siems, K., Jakupovic, J., Mavi, S., Bienzle, U., Eich, E., 2003. In vitro antiplasmodial evaluation of medicinal plants from Zimbabwe. Phytotherapy Research 17, 123–128. Krettli, A.U., Andrade-Neto, V.F., Brandão, M.G., Ferrari, W.M., 2001. The search for new antimalarial drugs from plants used to treat fever and malaria or plants randomly selected. Memórias do Instituto Oswaldo Cruz 96, 1033–1042. Kuria, K.A.M., Muriuki, G., Masengo, W., Kibwage, I., Hoogmartens, J., Laekeman, G.M., 2001. Antimalarial activity of Ajuga remota Benth (Labiatae) and Caesalpinia volkensii Harms (Caesalpiniaceae): in vitro confirmation of ethnopharmacological use. Journal of Ethnopharmacology 74, 141–148. Kuria, K.A.M., Chepkwony, H., Govaerts, C., Roets, E., Busson, R., DeWitte, P., Zupko, I., Hoornaert, G., Quirynen, L., Maes, L., Janssens, L., Hoogmartens, J., Laekeman, G., 2002. The Antiplasmodial activity of isolates from Ajuga remota. Journal of Natural Products 65, 78–793. Lam, S.H., Wang, C.Y., Chen, C.K., Lee, S.S., 2007. Chemical investigation of Phyllanthus reticulatus by HPLC-SPE-NMR and conventional methods. Phytochemical Analysis 18, 251–255. Le Bras, J., Musset, L., Clain, J., 2006. Antimalarial drug resistance. Medecine et Maladies Infectieuses 36, 401–405. Likhitwitayawuid, K., Angerhofer, C.K., Chai, H., Pezzuto, J.M., Cordell, G.A., Ruangrungsi, N., 1993. Cytotoxic and antimalarial alkaloids from the bulbs of Crinum amabile. Journal of Natural Products 56, 1331–1338. Liñares, G.E., Ravaschino, E.L., Rodriguez, J.B., 2006. Progresses in the field of drug design to combat tropical protozoan parasitic diseases. Current Medicinal Chemistry 13, 335–360. Liu, K.C.S.C., Yang, S.L., Roberts, M.F., Elford, B.C., Phillipson, J.D., 1992. Antimalarial activity of Artemisia annua flavonoids from whole plants and cell cultures. Plant Cell Reports 11, 637–640. Lovett, J.C., Wasser, S.K. (Eds.), 1993. Biogeography and Ecology of the Rainforests of Eastern Africa. Cambridge University Press, Cambridge. MacKinnon, S., Durst, T., Arnason, J.T., Angerhofer, C., Pezzuto, J., SanchezVindas, P.E., Poveda, L.J., Gbeassor, M., 1997. Antimalarial activity of tropical Meliaceae extracts and gedunin derivatives. Journal of Natural Products 60, 336–341. Messmer, W.M., Tin-Wa, M., Fong, H.H.S., Bevelle, C., Farnsworth, N.R., Abraham, D.J., Trojanek, J., 1972. Fagaronine, a new tumor inhibitor isolated from Fagara zanthoxyloides Lam. (Rutaceae). Journal of Pharmaceutical Sciences 61, 1858– 1859. Mittermeier, R.A., Myers, N., Thomsen, J.B., 1998. Biodiversity hotspots and major tropical wilderness areas: approaches to setting conservation priorities. Conservation Biology 12, 516–520. MoH, 1992. Kenya National Plan of Action for Malaria Control. Ministry of Health, Republic of Kenya. pp. 16-18.

Mohamed, A.M., Wolf, W., Spiess, W.E., 2002. Physical, morphological and chemical characteristics, oil recovery and fatty acid composition of Balanites aegyptiaca Del. kernels. Plant Foods Human Nutrition 57, 179–189. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65, 55–63. Mouchet, J., 1999. Vectors and environmental factors in malaria. Transfusion and Clinical Biology 6, 35–43. Mugabe, J., Clark, N. (Eds.), 1998. Managing Biodiversity: The National systems of Conservation and Innovation in Africa. Acts Press, Nairobi. Munoz, V., Sauvain, M., Bourdy, G., Arrazola, S., Callapa, J., Ruiz, G., Choque, J., Deharo, E., 2000. A search for natural bioactive compounds in Bolivia through a multidisciplinary approach. Part III. Evaluation of the antimalarial activity of plants used by Altenos Indians. Journal of Ethnopharmacology 71, 123–131. Murata, T., Miyase, T., Muregi, F.W., Naoshima-Ishibashi, Y., Umehara, K., Warashina, T., Kanou, S., Mkoji, G.M., Terada, M., Ishih, A., 2008. Antiplasmodial triterpenoids from Ekebergia capensis. Journal of Natural Products 71, 167–174. Muregi, F.W., Ishih, A., Suzuki, T., Kino, H., Amano, T., Mkoji, G.M., Miyase, T., Terada, M., 2007a. In vivo antimalarial activity of aqueous extracts from Kenyan medicinal plants and their chloroquine (CQ) potentiation effects against a blood-induced CQ-resistant rodent parasite in mice. Phytotherapy Research 21, 337–343. Muregi, F.W., Ishih, A., Miyase, T., Suzuki, T., Kino, H., Amano, T., Mkoji, G.M., Terada, M., 2007b. Antimalarial activity of methanolic extracts from plants used in Kenyan ethnomedicine and their interactions with chloroquine (CQ) against a CQ-tolerant rodent parasite, in mice. Journal of Ethnopharmacology 111, 190– 195. Muregi, F.W., Chhabra, S.C., Njagi, E.N., Lang’at-Thoruwa, C.C., Njue, W.M., Orago, A.S., Omar, S.A., Ndiege, I.O., 2004. Anti-plasmodial activity of some Kenyan medicinal plant extracts singly and in combination with chloroquine. Phytotherapy Research 18, 379–384. Muregi, F.W., Chhabra, S.C., Njagi, E.N., Lang’at-Thoruwa, C.C., Njue, W.M., Orago, A.S., Omar, S.A., Ndiege, I.O., 2003. In vitro antiplasmodial activity of some plants used in Kisii, Kenya against malaria and their chloroquine potentiation effects. Journal of Ethnopharmacology 84, 235–239. Muriithi, M.W., Abraham, W.R., Addae-Kyereme, J., Scowen, I., Croft, S.L., Gitu, P.M., Kendrick, H., Njagi, E.N., Wright, C.W., 2002. Isolation and in vitro antiplasmodial activities of alkaloids from Teclea trichocarpa: in vivo antimalarial activity and X-ray crystal structure of normelicopicine. Journal of Natural Products 65, 956–959. Mutabingwa, T.K., 2005. Artemisinin-based combination therapies (ACTs): best hope for malaria treatment but inaccessible to the needy! Acta Tropica 95, 305– 315. Muthaura, C.N., Rukunga, G.M., Chhabra, S.C., Omar, S.A., Guantai, A.N., Gathirwa, J.W., Tolo, F.M., Mwitari, P.G., Keter, L.K., Kirira, P.G., Kimani, C.W., Mungai, G.M., Njagi, E.N., 2007a. Antimalarial activity of some plants traditionally used in treatment of malaria in Kwale district of Kenya. Journal of Ethnopharmacology 112, 545–551. Muthaura, C.N., Rukunga, G.M., Chhabra, S.C., Omar, S.A., Guantai, A.N., Gathirwa, J.W., Tolo, F.M., Mwitari, P.G., Keter, L.K., Kirira, P.G., Kimani, C.W., Mungai, G.M., Njagi, E.N., 2007b. Antimalarial activity of some plants traditionally used in Meru district of Kenya. Phytotherapy Research 21, 860–867. Muthaura, C.N., Rukunga, G.M., Chhabra, S.C., Mungai, G.M., Njagi, E.N., 2007c. Traditional antimalarial phytotherapy remedies used by the Kwale community of the Kenyan Coast. Journal of Ethnopharmacology 114, 377–386. Muthaura, C.N., Rukunga, G.M., Chhabra, S.C., Mungai, G.M., Njagi, E.N.M., 2007d. Traditional phytotherapy of some remedies used in treatment of malaria in Meru district of Kenya. South African Journal of Botany 73, 402–411. Myers, N., Mittermeier, R.A., Mittermeier, C.G., da Fonseca, A.B., Kent, J., 2000. Biodiversity hotspots for conservation priorities. Nature 403, 853–858. Nghiem, V.D., 2002. Indigenous peoples in Vietnam and their knowledge of biodiversity in traditional health care. Biodiversity 3, 22–23. Nishiyama, Y., Moriyasu, M., Ichimaru, M., Tachibana, Y., Kato, A., Mathenge, S.G., Nganga, J.N., Juma, F.D., 1996. Acyclic triterpenoids from Ekebergia capensis. Phytochemistry 42, 803–807. Nishiyama, Y., Moriyasu, M., Ichimaru, M., Kato, A., Mathenge, S.G., Nganga, J.N., Juma, F.D., 1999. Absolute configurations of two acyclic triterpenoids from Ekebergia capensis. Phytochemistry 52, 1593–1596. Njoroge, G.N., Bussmann, R.W., 2006. Diversity and utilization of antimalarial ethnophytotherapeutic remedies among the Kikuyus (Central Kenya). Journal of Ethnobiology and Ethnomedicine 2, 8. Njuguna, P.M., Holding, C., Munyasya, C., 2000. In: A.B. Temu, G. Lund, R.E. Malimbwi, G.S. Kowero, K. Klein, Y. Malande, I. Kone (Eds.), On-farm Woody Biomass Surveys (1993 and 1998): A Case Workshop held in Arusha, Tanzania, 1999. The African Academy of Sciences, pp. 54–77. Nyasse, B., Nono, J., Sonke, B., Denier, C., Fontaine, C., 2004. Trypanocidal activity of bergenin, the major constituent of Flueggea virosa, on Trypanosoma brucei. Pharmazie 59, 492–494. Obih, P.O., Makinde, J.M., 1985. Effect of Azadirachta indica on Plasmodium berghei in mice. African Journal of Medicine and Medical Sciences 14, 51–54. Ochola, S., 2003. National Malaria guidelines. In: Proceedings of the Second National Congress on Quality Improvement in Health Care, Medical Research and Traditional Medicine, Nairobi, Kenya. Ofulla, A.V.O., Chege, G.M.K., Rukunga, G.M., Kiarie, F.K., Githure, J.I., Kofi-Tsekpo, W.M., 1995. In vitro antimalarial activity of extracts of Albizia gummifera, Aspilia

C.N. Muthaura et al. / Experimental Parasitology 127 (2011) 609–626 mossambicensis, Melia azedarach, and Azadirachta indica against Plasmodium falciparum. Africa Journal of Health Sciences 2, 309–311. Oketch-Rabah, H.A., Mwangi, J.W., Lisgarten, J., Mberu, E.K., 2000. A new antiplasmodial coumarin from Toddalia asiatica roots. Fitoterapia 71, 636–640. Oketch-Rabah, H.A., Brøgger Christensen, S., Frydenvang, K., Dossaji, S.F., Theander, T.G., Cornett, C., Watkins, W.M., Kharazmi, A., Lemmich, E., 1998. Antiprotozoal properties of 16,17-dihydrobrachycalyxolide from Vernonia brachycalyx. Planta Medica 64, 559–562. Oketch-Rabah, H.A., Lemmich, E., Dossaji, S.F., Theander, T.G., Olsen, C.E., Cornett, C., Kharazmi, A., Christensen, S.B., 1997. Two new antiprotozoal 5methylcoumarins from Vernonia brachycalyx. Journal of Natural Products 60, 458–461. Oketch-Rabah, H.A., 1996. Antimalarial and antileishmarial compounds from Kenyan medicinal plants. Ph.D. Thesis. Department of Medicinal Chemistry, The Royal Danish School of Pharmacy, Denmark, pp. 80–82. Olila, D., Opuda-Asibo, J., Olwa-Odyek, 2001. Bioassay-guided studies on the cytotoxic and in vitro trypanocidal activities of a sesquiterpene (Muzigadial) derived from a Ugandan medicinal plant (Warburgia ugandensis). African Health Sciences 1, 12–15. Olliaro, P.L., Taylor, W.R.J., 2003. Antimalarial compounds: from bench to bedside. Journal of Experimental Biology 206, 3753–3759. Omulokoli, E., Khan, B., Chhabra, S.C., 1997. Antiplasmodial activity of four Kenyan medicinal plants. Journal of Ethnopharmacology 56, 133–137. O’Neill, M.J., Bray, D.H., Boardman, P., Phillipson, J.D., Warhust, D.C., 1985. Plants as sources of antimalarial drugs. Part 1: in vitro test method for the evaluation of crude extracts from plants. Planta Medica 61, 394–397. Orwa, J.A., Mwitari, P.G., Matu, E.N., Rukunga, G.M., 2007. Traditional healers and the management of malaria in Kisumu district, Kenya. East African Medical Journal 84, 51–55. Ovenden, S.P.B., Cao, S., Leong, C., Flotow, H., Gupta, M.P., Buss, A.D., Butler, M.S., 2002. Spermine alkaloids from Albizia adinocephala with activity against Plasmodium falciparum plasmepsin II. Phytochemistry 60, 175–177. Pacciaroni, A.D.V., Sosa, V.E., Espinar, L.A., Oberti, J.C., 1995. Sesquiterpene lactones from Schkuhria pinnata. Phytochemistry 39, 127–131. Pasvol, G.C., Newton, R.J.C., Winstanley, P.A., Watkins, W.M., Peshu, N.M., Were, J.B.O., Marsh, K., Warrell, D.A., 1991. Quinine treatment of severe falciparum malaria in African children: randomized comparison of three regimens. American Journal of Tropical Medicine and Hygiene 45, 702–713. Peters, W., Robinson, B.L., 1992. The chemotherapy of rodent malaria XLVII: studies on pyronaridine and other Mannich base antimalarials. Annals of Tropical Medicine and Parasitology 86, 455–465. Phillipson, J.D., 1999. New drugs from nature – it could be yew. Phytotherapy Research 13, 2–8. Phillipson, J.D., Wright, C.W., 1991. Can ethnopharmacology contribute to the development of antimalarial agents? Journal of Ethnopharmacology 32, 155– 165. Phillipson, J.D., O’Neill, M.J., Wright, C.W., Bray, D.H., Warhurst, D.C., 1987. Plants as sources of antimalarial and amoebicidal compounds. In: Leeuwenberg, A.J.M. (Ed.), Medicinal and Poisonous Plants of the Tropics. Centre for Agricultural Publishing and Documentation, Wageningen, Netherlands, pp. 70–79. Pillay, P., Maharaj, V.J., Smith, P.J., 2008. Investigating South African plants as a source of new antimalarial drugs. Journal of Ethnopharmacology 119, 438–454. Ramirez, V.R., Mostacero, L.J., Garcia, A.E., Mejia, C.F., Pelaez, P.F., Medina, C.D., Miranda, C.H., 1988. Vegetales Empleados en Medicina Tradicional Norperuana. Banco Agrario Del Peru and Nacl Univ Trujillo, Trujillo, Peru. p. 54. Rasoanaivo, P., Ratsimamanga-Uverg, S., Ramanitrahasimbola, D., Rafatro, H., Rakoto-Ratsimamanga, A., 1999. Screening of plant extracts from Madagascar for research on antimalarial activity and potentiating effect of chloroquine. Journal of Ethnopharmacology 64, 117–126. Rasoanaivo, P., Petitjean, A., Ratsimamanga-Urverg, S., Rakoto-Ratsimamanga, A., 1992. Medicinal plants used to treat malaria in Madagascar. Journal of Ethnopharmacology 37, 117–127. Reed, J.D., 1986. Relationships among soluble phenolics, insoluble proanthocyanidins, and fiber in East African browse species. Journal of Range Management 39, 5–7. Reider, P.J., Roland, D.M., 1984. The Alkaloids, vol. XXIII. Academic Press, New York, pp. 71–156. Ridley, R.G., 1997. Plasmodium: drug discovery and development an industrial perspective. Experimental Parasitology 87, 293–304. Rochanakij, S., Thebtaranonth, Y., Yenjai, C., Yuthavong, Y., 1985. Nimbolide, a constituent of Azadirachta indica, inhibits Plasmodium falciparum in culture. Southeast Asian Journal of Tropical Medicine and Public Health 16, 66–72. Rukunga, G.M., Gathirwa, J.W., Omar, S.A., Muregi, F.W., Muthaura, C.N., Kirira, P.G., Mungai, G.M., Kofi-Tsekpo, W.M., 2009. Anti-plasmodial activity of the extracts of some Kenyan medicinal plants. Journal of Ethnopharmacology 121, 282–285. Rukunga, G.M., Muregi, F.W., Omar, S.A., Gathirwa, J.W., Muthaura, C.N., Peter, M.G., Heydenreich, M., Mungai, G.M., 2008. Anti-plasmodial activity of the extracts and two sesquiterpenes from Cyperus articulatus. Fitoterapia 79, 188–190. Rukunga, G.M., Muregi, F.W., Tolo, F.M., Omar, S.A., Mwitari, P., Muthaura, C.N., Omlin, F., Lwande, W., Hassanali, A., Githure, J., Iraqi, F.W., Mungai, G.M., Kraus, W., Kofi-Tsekpo, W.M., 2007. The antiplasmodial activity of spermine alkaloids isolated from Albizia gummifera. Fitoterapia 78, 455–459. Rukunga, G.M., Waterman, P.G., 1996. New macrocyclic spermine (budmunchiamine) alkaloids from Albizia gummifera: with some observations on the structure–activity relationships of the budmunchiamines. Journal of Natural Products 59, 850–853.

625

Schaneberg, B.T., Green, K.D., Sneden, A.T., 2001. Dihydroagarofuran sesquiterpene alkaloids from Maytenus putterlickioides. Journal of Natural Products 64, 624– 626. Shanks, G.D., Biomondo, K., Hay, S.I., Snow, R.W., 2000. Changing patterns of clinical malaria since 1965 among a tea estate population located in the Kenyan highlands. Transactions of the Royal Society of Tropical Medicine and Hygiene 94, 253–255. Simonsen, H.T., Nordskjold, J.B., Smitt, U.W., Nyman, U., Palpu, P., Joshi, P., Varughese, G., 2001. In vitro screening of Indian medicinal plants for antiplasmodial activity. Journal of Ethnopharmacology 74, 195–204. Sisowath, C., Stromberg, J., Martensson, A., Msellem, M., Obondo, C., Bjorkman, A., Gil, J.P., 2005. In vitro selection of Plasmodium falciparum pfmdr1 86N coding alleles by artemether-lumefantrine (Coartem). Journal of Infectious Diseases 191, 1014–1017. Speroni, E., Cervellati, R., Innocenti, G., Costa, S., Guerra, M.C., Dall Acqua, S., Govoni, P., 2005. Anti-inflammatory, anti-nociceptive and antioxidant activities of Balanites aegyptiaca (L.) delile. Journal of Ethnopharmacology 98, 117–125. Sofowora, A., 1982. Medicinal Plants and Traditional Medicine in Africa. John Wiley and Sons Ltd., Chichester, New York/Brisbane, Toronto/Singapore. p. 177. Soh, P.N., Benoit-Vical, F., 2007. Are West African plants a source of future antimalarial drugs? Journal of Ethnopharmacology 114, 130–140. Tamez, P.A., Lantvit, D., Lim, E., Pezzuto, J.M., 2005. Chemosensitizing action of cepharanthine against drug-resistant human malaria, Plasmodium falciparum. Journal of Ethnopharmacology 98, 137–142. Trager, W., Jensen, J.B., 1976. Human malaria parasites in continuous culture. Science 193, 673–675. Tran, Q.L., Tezuka, Y., Ueda, J.Y., Nguyen, N.T., Maruyama, Y., Begum, K., Kim, H.S., Wataya, Y., Tran, Q.K., Kadota, S., 2003. In vitro antiplasmodial activity of antimalarial medicinal plants used in Vietnamese traditional medicine. Journal of Ethnopharmacology 86, 249–252. Trape, J.F., 2002. Combating malaria in Africa. Trends in Parasitology 18, 224–230. Trigg, P.I., 1989. In: Wagner, H., Hikino, H., Farnsworth, N.R. (Eds.), Economic and Medicinal Plant Research, vol. 3. Academic Press, London, pp. 19–55. Trotter, R.T., Logan, M.H., Rocha, J.M., Boneta, J.L., 1982. Ethnography and bioassay: combined method for a preliminary screen of home remedies for potential pharmacological activity. Journal of Ethnopharmacology 8, 113–119. Udeinya, I.J., 1993. Antimalarial activity of Nigerian neem leaves. Transactions of the Royal Society of Tropical Medicine and Hygiene 87, 471. Van der Kooy, F., Verpoorte, R., Meyer, J.J.M., 2008. Metabolomic quality control of claimed anti-malarial Artemesia afra herbal remedy and A. Annua plant extracts. South African Journal of Botany 74, 186–189. Van Zyl, R.L., Viljoen, A.M., 2003. Antimalarial activity and essential oil composition of South African medicinal aromatic plants. South African Journal of Botany 69, 265. Vonthron-Senecheau, C., Weniger, B., Quattara, M., Tra Bi, F., Kamenan, A., Lobstein, A., Brun, R., Anton, R., 2003. In vitro antiplasmodial activity and cytotoxicity of ethnobotanically selected Ivorian plants. Journal of Ethnopharmacology 87, 221–225. Waigh, R., 2002. Medicinal chemistry at the University of Strathclyde. The Newsletter for the Society for Medicines Research (SMR) 8, 6–7. Walter, A., Séquin, U., 1990. Flavonoids from the leaves of Boscia salicifolia. Phytochemistry 29, 2561–2563. Watt, J.M., Breyer-Brandwijk, M.G., 1962. The Medicinal and Poisonous Plants of Southern and Eastern Africa, second ed. E. + S. Livingstone Ltd., London. Weenen, H., Nkunya, M.H.H., Bray, D.H., Mwasubi, L.B., Kinabo, L.S., Kilimali, V.A., 1990. Antimalarial activity of Tanzanian medicinal plants. Planta Medica 56, 368–370. White, N.J., Olliaro, P.L., 1996. Strategies for the prevention of anti-malarial drug resistance. Rationale for combination chemotherapy for malaria. Parasitology Today 12, 399–401. Wiesman, Z., Chapagain, B.P., 2006. Larvicidal activity of saponin containing extracts and fractions of fruit mesocarp of Balanites aegyptiaca. Fitoterapia 77, 420–424. Willcox, M., Bodeker, G., 2004. Traditional herbal medicines for malaria. British Medical Journal 329, 1156–1159. World Health Organisation, 2002. Traditional Medicine: Planning for Cost-effective Traditional Health Services in the New Century – A Discussion Paper. Centre for Health Development. World Health Organization, 2002a. WHO Traditional Medicine Strategy 2002–2005. Geneva. World Health Organisation, 2001. The Use of Antimalarial Drugs. Report of a WHO Informal Consultation 2001. Geneva 33, p. 141. World Health Organization, 1997. World malaria situation in 1994. WHO Weekly Epidemiological Record 22, 161–167. World Health Organisation, 1996. WHO Weekly Epidemiological Record 71, 17–24. World Health Organization, 2006. WHO Calls for an Immediate Halt to Provision of Single-drug Artemisinin Malaria Pills. News release WHO/2. World Health Organization, 2005. Roll back Malaria; Working Document. Geneva, Switzerland. Wright, C.W., 2005. Traditional antimalarials and the development of novel antimalarial drugs. Journal of Ethnopharmacology 100, 67–71. Wright, C.W., Warhurst, D.C., 2002. The mode of action of artemisinin and its derivatives. In: Wright, C.W. (Ed.), Artemisia. Taylor and Francis, London, pp. 249–288. Yenesew, A., Derese, S., Midiwo, J.O., Oketch-Rabah, H.A., Lisgarten, J., Palmer, R., Heydenreich, M., Peter, M.G., Akala, H., Wangui, J., Liyala, P., Waters, N.C., 2003a.

626

C.N. Muthaura et al. / Experimental Parasitology 127 (2011) 609–626

Anti-plasmodial activities and X-ray crystal structures of rotenoids from Millettia usaramensis subspecies usaramensis. Phytochemistry 64, 773–779. Yenesew, A., Derese, S., Irungu, B., Midiwo, J.O., Waters, N.C., Liyala, P., Akala, H., Heydenreich, M., Peter, M.G., 2003b. Flavonoids and isoflavonoids with antiplasmodial activities from the root bark of Erythrina abyssinica. Planta Medica 69, 658–661.

Yenesew, A., Induli, M., Derese, S., Midiwo, J.O., Heydenreich, M., Peter, M.G., Akala, H., Wangui, J., Liyala, P., Waters, N.C., 2004. Anti-plasmodial flavonoids from the stem bark of Erythrina abyssinica. Phytochemistry 65, 3029–3032. Zirihi, G.N., Mambu, L., Guede-Guina, F., Bodo, B., Grellier, P., 2005. In vitro antiplasmodial activity and cytotoxicity of 33 West African plants used for treatment of malaria. Journal of Ethnopharmacology 98, 281–285.