In vitro antiplasmodial, antitrypanosomal and antileishmanial activities of selected medicinal plants from Ugandan flora: Refocusing into multi-component potentials.

ETHNOPHARMACOLOGICAL RELEVANCE: Seven medicinal plants from Ugandan flora, namely Entada abyssinica, Khaya anthotheca, Vernonia amygdalina, Baccharoides adoensis, Schkuhria pinnata, Entandropragma utile and Momordica foetida, were selected in this study. They are used to treat conditions and infections ranging from inflammations, pains and fevers to viruses, bacteria, protozoans and parasites. Two of the plants, V. amygdalina and M. foetida, are also used as human food or relish, while others are important in ethnoveterinary practices and in zoopharmacognosy in the wild. The aim of this study was to evaluate the in vitro antiplasmodial, antitrypanosomal and antileishmanial activities, along with cytotoxicity of the multi-component extracts of these plants. MATERIALS AND METHODS: Different parts of the plants were prepared and serially extracted with hexane, petroleum ether, dichloromethane, ethyl acetate, methanol and double distilled water. Solvent free extracts were assayed for in vitro inhibition against four reference parasite strains, Plasmodium falciparum (K1), Trypanosoma brucei rhodesiense (STIB 900), Trypanosoma cruzi (Talahuen C2C4) and Leishmania donovani (MHOM-ET-67/L82) using standard methods. Toxicity was assessed against L6 skeletal fibroblast and mouse peritoneal macrophage (J774) cells and selectivity indices (SIs) calculated for the most active extracts. RESULTS: The strongest activities, demonstrating median inhibitory concentration (IC50) values ≤ 2 µg/ml, were observed for the dichloromethane and petroleum ether extracts of K. anthotheca, B. adoensis and S. pinnata. Overall, IC50 values ranged from < 1 µg/ml to > 90 µg/ml. Out of 22 extracts demonstrating IC50s < 20 µg/ml, seven were against T. b. rhodesiense (IC50: 1.6-16.2 µg/ml), six against T. cruzi (IC50: 2.1-18.57 µg/ml), none against L. donovani (IC50: falling > 3.3 and >10 µg/ml), and nine against P. falciparum (IC50: 0.96 µg/ml to 4.69 µg/ml). Selectivity indices (SI) calculated for the most active extracts ranged from <1.00 to 94.24. However, the B. adoensis leaf dichloromethane extract (a) was equipotent (IC50 = 3.3 µg/ml) against L. donovani and L6 cells respectively, indicating non-specific selection. Trypanosome and Plasmodium parasites were comparatively more sensitive to the test extracts. CONCLUSIONS: The benefits achieved from the seven tested plant species as traditional ethnomedicinal and ethnoveterinary therapies or in zoopharmacognosy against infections and conditions of animals in the wild are strongly supported by results of this study. The synergy of plant extracts, so achieved by concerted actions of the ligands, produces adequate perturbation of targets in the four parasite genera, resulting in the strong potencies exhibited by low IC50 values. The total inhibitory effect, achieved as a sum of perturbations contributed by each participating compound in the extract, minimises toxic effects of the compounds as seen in the high SI's obtained with some extracts. Those extracts demonstrating SI ≥ 4 form promising candidates for further cell-based and system pharmacology studies.

Antiplasmodial potential of traditional phytotherapy of some remedies used in treatment of malaria in Meru-Tharaka Nithi County of Kenya.

ETHNOPHARMACOLOGICAL RELEVANCE: Medicinal plants play a major role in many communities across the world, in the treatment and prevention of disease and the promotion of general health. The aim of the study was to escalate documentation from an earlier study of medicinal plants, traditionally used to combat malaria by the Ameru community of Imenti Forest area and Gatunga in Eastern Region of Kenya, and validate their ethnopharmacological claims by evaluating their antiplasmodial efficacies. MATERIALS AND METHODS: The study was carried out in Meru County at Imenti Forest Game Reserve and in Tharaka Nithi County at Gatunga. Traditional health practitioners (THP) were interviewed with a standard questionnaire to obtain information on medicinal plants traditionally used for management of malaria. Group interviews were also held among THPs and members of the community. The antiplasmodial activities of the crude extracts against chloroquine sensitive (D6) and resistant (W2) Plasmodium falciparum were determined using the semi-automated micro-dilution technique that measures the ability of the extracts to inhibit the incorporation of (G-3H) hypoxanthine into the malaria parasite. RESULTS: Ninety nine (99) species in eighty one (81) genera and forty five (45) families were documented and evaluated for in vitro antiplasmodial activity. Compositae, Fabaceae, Meliceae, Rubiaceae, Rutaceae and Verbenaceae had the highest number of species mentioned in treatment of malaria in Meru/Tharaka Nithi study area. Twenty four (24.2%) species showed antiplasmodial efficacy of IC50 ≤ 5 µg/ml and were considered to have potential for isolation of antimalarial compounds. Eight plant (8) species with moderate antiplasmodial activity namely; Cordia africana, Commiphora africana, Elaeodendron buchananii, Gomphocarpus semilunatus, Tarena graveolens, Plectranthus igniarius, Acacia senegal and Ziziphus abyssinica were documented from this region for the first time for the treatment of malaria. The antiplasmodial activity of MeOH root bark extract of Maytenus obtusifolia was very promising (IC50 < 1.9 µg/ml) and this is the first report on traditional use of M. obtusifolia for treatment of malaria and antimalarial activity. CONCLUSIONS: The results seem to indicate that ethnopharmacological inquiry used in search for new herbal remedies as predictive and could be used as the basis for search of new active principles. Eight plant (8) species are documented from this region for the first time for the treatment of malaria. This is the first report on traditional use of M. obtusifolia for treatment of malaria and evaluation of its antiplasmodial activity.

Antiplasmodial potential of traditional antimalarial phytotherapy remedies used by the Kwale community of the Kenyan Coast.

ETHNOPHARMACOLOGICAL RELEVANCE: In Kenya, 22 million people are at risk of malaria, 70% of them are in rural areas and most of these people use traditional plant based medicines to treat malaria. The aim of the study was to escalate documentation, from an earlier study of medicinal plants, traditionally used to treat malaria by the Digo community of Kwale County, taking cognizance of their pharmacological information by evaluating their antiplasmodial efficacies. MATERIALS AND METHODS: The study was carried out in Kwale County at Shimba Hills Game Reserve and adjoining part of Kinango. Traditional health practitioners (THP) were interviewed with a standard questionnaire to obtain information on medicinal plants traditionally used for management of malaria. Group interviews were also held among THPs and members of the community. The plant samples collected were tested for antiplasmodial activity against chloroquine sensitive (D6) and resistant (W2) Plasmodium falciparum using the ability of extracts, prepared from the plant species, to inhibit the incorporation of [G-3H] hypoxanthine into the malaria parasites. RESULTS: Fifty seven (57) species in forty eight (48) genera and thirty (30) families were documented and evaluated for in vitro antiplasmodial activity. Apocynaceae, Euphorbiaceae, and Rubiaceae families had each about 12% of the plant species reported as antimalarial remedy and represented the species that are most commonly used. Twelve species (21.1%) showed antiplasmodial efficacy of IC50<5µg/ml and these were Boscia salicifolia, Cissampelos mucronata, Clerodendrum myricoides, Commiphora schimperi, Flueggea virosa, Maytenus undata, Maytenus senegalensis, Maytenus putterlickioides, Vernonia amygdalina, Warburgia stuhlmannii, Zanthoxylum chalybeum and Tabernaemontana pachysiphon. CONCLUSIONS: These results seem to indicate that ethnopharmacological inquiry used in search for new herbal remedies as predictive and could form the basis of an ethnopharmacopoeia and search for new active principles. This is the first report on traditional use of T. pachysiphon for malaria and its antiplasmodial activity.

Traditional herbal antimalarial therapy in Kilifi district, Kenya.

AIM OF STUDY: To identify plant species used by the traditional health practitioners (THPs) in treatment of malaria, carry out cytotoxicity and efficacy evaluation of the identified plants and to evaluate combination effects. MATERIALS AND METHODS: Thirteen plants were selected through interviews with traditional healers. In vitro antiplasmodial testing was done by measuring ability of the test sample to inhibit the incorporation of radio-labelled hypoxanthine into the malaria parasite. The extracts were tested singly and then in combination using the standard fixed ratio analysis to evaluate synergism. In vivo bioassay was done in mice using Peter's 4-days suppressive test and cytotoxicity evaluated in vitro using Vero E6 cells. RESULTS: Of the plants tested in vitro, 25% were highly active (IC(50)<10 µg/ml), 46% moderately active (IC(50) 10-50 µg/ml), 16% had weak activity of 50-100 µg/ml while 13% were not active IC(50) >100 µg/ml. Methanolic extracts of Azadirachta indica, Premna chrysoclada and Uvaria acuminata were the most active (IC(50)<10µg/ml) against both the chloroquine (CQ) sensitive (D6) and the CQ resistant (W2) Plasmodium falciparum clones. When tested in vivo in a mouse model, Azadirachta indica, Rhus natalensis and Grewia plagiophylla depicted the highest percent parasite clearance and chemo suppression of 89%, 82% and 78%, respectively. Evaluating effect of combining some of these extracts with one another against a multi-drug resistant Plasmodium falciparum (W2) clone revealed synergism among some combinations. The highest synergy was between Uvaria acuminata and Premna chrysoclada. The interaction between Grewia plagiophylla and Combretum illairii was largely antagonistic. Impressive cytotoxicity results were obtained with most of the plants tested revealing high selectivity indices an indication of enabling achievement of therapeutic doses at safe concentrations. Uvaria acuminata was, however, toxic to the cultured cells. Mild cytotoxicity was also observed in Hoslundia opposita and Lannea schweinfurthii (CC(50) 37 and 76 µg/ml, respectively). CONCLUSIONS: This study identified plants with low IC(50) values, high percent chemo suppression and low cytotoxicity thus potential sources for novel antiplasmodial agents. The findings remotely justify use of combined medicinal plants in traditional medicine practices as synergy among some plant species was demonstrated.

Anti-plasmodial activity of the extracts of some Kenyan medicinal plants.

ETHNOPHARMACOLOGICAL RELEVANCE: The spread of drug resistant Plasmodium falciparum strains necessitates search for alternative newer drugs for use against malaria. Medicinal plants used traditionally in preparation of herbal medicines for malaria are potential source of new anti-malarial drugs. AIM OF THE STUDY: To identify the anti-plasmodial potential of twelve plants used in preparing herbal remedies for malaria in Kilifi and Tharaka districts of Kenya. MATERIALS AND METHODS: Twelve plants used traditionally for anti-malarial therapy in Kilifi and Tharaka districts were extracted with water/methanol yielding twenty-three extracts. The extracts were tested against chloroquine sensitive (NF54) and resistant (ENT30) P. falciparum strains in vitro using (3)Hypoxanthine assay. RESULTS: Seven (30%) extracts showed activity against P. falciparum with IC(50) values below 20 microg/ml. The remaining 16 extracts showed low or no activity. The most active extracts were from Zanthoxylum chalybeum (Rutaceae) with an IC(50) value of 3.65 microg/ml, Cyperus articulatus (Cyperaceae) with 4.84mug/ml, and Cissampelos pareira (Menispermaceae) with 5.85 microg/ml. CONCLUSIONS: This study revealed plants, that are potential sources of anti-malarial compounds. Anti-plasmodial activities of extracts of T. simplicifolia, C. pareira, and C. articulatus are reported for the first time.

Anti-plasmodial activity of the extracts and two sesquiterpenes from Cyperus articulatus.

Two sesquiterpenes, corymbolone and mustakone, isolated from the chloroform extract of the rhizomes of Cyperus articulatus, exhibited significant anti-plasmodial properties. Mustakone was approximately ten times more active than corymbolone against the sensitive strains of the Plasmodium falciparum.

The in vitro anti-plasmodial and in vivo anti-malarial efficacy of combinations of some medicinal plants used traditionally for treatment of malaria by the Meru community in Kenya.

The use of herbal drugs as combinations has existed for centuries in several cultural systems. However, the safety and efficacy of such combinations have not been validated. In this study, the toxicity, anti-plasmodial and antimalarial efficacy of several herbal drug combinations were investigated. Lannea schweinfurthii, Turraea robusta and Sclerocarya birrea, used by traditional health practitioners in Meru community, were tested for in vitro anti-plasmodial and in vivo anti-malarial activity singly against Plasmodium falciparum and Plasmodium berghei, respectively. Methanolic extract of Turraea robusta was the most active against Plasmodium falciparum D6 strain. Aqueous extracts of Lannea schweinfurthii had the highest anti-plamodial activity followed by Turraea robusta and Sclerocarya birrea. D6 was more sensitive to the plant extracts than W2 strain. Lannea schweinfurthii extracts had the highest anti-malarial activity in mice followed by Turraea robusta and Sclerocarya birrea with the methanol extracts being more active than aqueous ones. Combinations of aqueous extracts of the three plants and two others (Boscia salicifolia and Rhus natalensis) previously shown to exhibit anti-plasmodial and anti-malarial activity singly were tested in mice. Marked synergy and additive interactions were observed when combinations of the drugs were assayed in vitro. Different combinations of Turraea robusta and Lannea schweinfurthii exhibited good in vitro synergistic interactions. Combinations of Boscia salicifolia and Sclerocarya birrea; Rhus natalensis and Turraea robusta; Rhus natalensis and Boscia salicifolia; Turraea robusta and Sclerocarya birrea; and Lannea schweinfurthii and Boscia salicifolia exhibited high malaria parasite suppression (chemo-suppression >90%) in vivo when tested in mice. The findings are a preliminary demonstration of the usefulness of combining several plants in herbal drugs, as a normal practice of traditional health practitioners.

Antimalarial activity of some plants traditionally used in treatment of malaria in Kwale district of Kenya.

Methanolic and water extracts of five medicinal plant species used for treatment of malaria in traditional/cultural health systems of Kwale people in Kenya were tested for antimalarial activity against Plasmodium falciparum and Plasmodium berghei, respectively and for their cytotoxic effects. The most active extracts (IC(50)<10 microg/ml) screened against chloroquine (CQ) sensitive (D6) and resistant (W2) P. falciparum clones, were the water and methanol extracts of Maytenus undata (Thunb.) Blakelock (Celasteraceae), methanol extracts of Flueggea virosa (Willd.) Voigt (Euphorbiaceae), Maytenus putterlickioides (Loes.) Excell and Mendoca (Celastraceae), and Warburgia stuhlmannii Engl. (Canellaceae). These extracts showed various cytotoxic levels on Vero E6 cells with the water extract of M. undata exhibiting least cytotoxicity. At least one of the extracts of the plant species exhibited a high chemo suppression of parasitaemia >70% in a murine model of P. berghei infected mice. These results indicate that there is potential for isolation of a lead compound from the extracts of the five plants. W. stuhlmannii and M. putterlickioides have not been reported before for antiplasmodial activity.

Antimalarial activity of some plants traditionally used in Meru district of Kenya.

Ten plant extracts commonly used by the Meru community of Kenya were evaluated for the in vitro antiplasmodial, in vivo antimalarial, cytotoxicity and animal toxicity activities. The water and methanol extracts of Ludwigia erecta and the methanol extracts of Fuerstia africana and Schkuhria pinnata exhibited high antiplasmodial activity (IC(50) < 5 microg/mL) against chloroquine sensitive (D6) and resistant (W2) Plasmodium falciparum clones. The cytotoxicity of these highly active extracts on Vero E6 cells were in the range 161.5-4650.0 microg/mL with a selectivity index (SI) of 124.2-3530.7. In vivo studies of these extracts showed less activity with chemosuppression of parasitaemia in Plasmodium berghei infected mice of 49.64-65.28%. The methanol extract of Clerodendrum eriophyllum with a lower in vitro activity (IC(50) 9.51-10.56 microg/mL) exhibited the highest chemosuppression of 90.13%. The methanol and water extracts of Pittosporum viridiflorum were toxic to mice but at a lower dose prolonged survival of P. berghei infected mice (p < 0.05) with no overt signs of toxicity. However, the extracts were cytotoxic (SI, 0.96-2.51) on Vero E6 cells. These results suggest that there is potential to isolate active non-toxic antimalarial principles from these plants.

Anti-plasmodial activity and toxicity of extracts of plants used in traditional malaria therapy in Meru and Kilifi Districts of Kenya.

The methanol and aqueous extracts of 10 plant species (Acacia nilotica, Azadirachta indica, Carissa edulis, Fagaropsis angolensis, Harrissonia abyssinica, Myrica salicifolia, Neoboutonia macrocalyx, Strychnos heningsii, Withania somnifera and Zanthoxylum usambarensis) used to treat malaria in Meru and Kilifi Districts, Kenya, were tested for brine shrimp lethality and in vitro anti-plasmodial activity against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum (NF54 and ENT30). Of the plants tested, 40% of the methanol extracts were toxic to the brine shrimp (LD(50)<100micro/ml), while 50% showed in vitro anti-plasmodial activity (IC(50)<100microg/ml). The methanol extract of the stem bark of N. macrocalyx had the highest toxicity to brine shrimp nauplii (LD(50) 21.04+/-1.8microg/ml). Methanol extracts of the rest of the plants exhibited mild or no brine shrimp toxicity (LD(50)>50microg/ml). The aqueous extracts of N. macrocalyx had mild brine shrimp toxicity (LD(50) 41.69+/-0.9microg/ml), while the rest were lower (LD(50)>100microg/ml). The methanol extracts of F. angolensis and Zanthoxylum usambarense had IC(50) values <6microg/ml while the aqueous ones had values between 6 and 15microg/ml, against both chloroquine-sensitive and resistant P. falciparum strains. The results support the use of traditional herbs for anti-malarial therapy and demonstrate their potential as sources of drugs.