J.W. Gathirwa

Anti-parasitic activity and cytotoxicity of selected medicinal plants from Kenya.

Indigenous rural communities in the tropics manage parasitic diseases, like malaria and leishmaniasis, using herbal drugs. The efficacy, dosage, safety and active principles of most of the herbal preparations are not known. Extracts from 6 selected plant species, used as medicinal plants by indigenous local communities in Kenya, were screened for in vitro anti-plasmodial and anti-leishmanial activity, against 2 laboratory-adapted Plasmodium falciparum isolates (D6, CQ-sensitive and W2, CQ-resistant) and Leishmania major (IDU/KE/83=NLB-144 strain), respectively. The methanol extract of Suregada zanzibariensis leaves exhibited good anti-plasmodial activity (IC(50) 4.66+/-0.22 and 1.82+/-0.07 microg/ml for D6 and W2, respectively). Similarly, the methanol extracts of Albizia coriaria (IC(50) 37.83+/-2.11 microg/ml for D6) and Aspergillus racemosus (32.63+/-2.68 and 33.95+/-2.05 microg/ml for D6 and W2, respectively) had moderate anti-plasmodial activity. Acacia tortilis (IC(50) 85.73+/-3.36 microg/ml for W2) and Albizia coriaria (IC(50) 71.17+/-3.58 microg/ml for W2) methanol extracts and Aloe nyeriensis var kedongensis (IC(50) 87.70+/-2.98 and 67.84+/-2.12 microg/ml for D6 and W2, respectively) water extract exhibited mild anti-plasmodial activity. The rest of the extracts did not exhibit any anti-plasmodial activity. Although the leishmanicidal activity of extracts were lower than for pentosam (80%), reasonable activity was observed for Aloe nyeriensis methanol (68.4+/-6.3%), Albizia coriara water (66.7+/-5.0%), Maytenus putterlickoides methanol (60.0+/-6.23%), Asparagus racemosus methanol and water (58.3+/-8.22 and 56.8+/-6.58%, respectively), Aloe nyeriensis water (53.3+/-5.1%) and Acacia tortilis water (52.9+/-6.55%) extracts at 1000 microg/ml. Leishmania major infected macrophages treated with methanol extracts of Suregada zanzibariensis and Aloe nyeriensis var kedongensis and pentostam had infection rates of 28+/-2.11, 30+/-1.22 and 40+/-3.69%, respectively at 1000 microg/ml, indicating better anti-leishmanial activity for the extracts. The methanol extract of Albizia coriara (44.0+/-3.69%) and aqueous extracts of Asparagus racemosus (42+/-3.84%) and Acacia tortilis (44+/-5.59%) had similar activity to pentosam. Multiplication indices for Leishmania major amastigotes treated with methanol extracts of Albizia coriaria, Suregada zanzibariensis and Aloe nyeriensis var kedongensis, aqueous extract of Acacia tortilis and pentosam were 28.5+/-1.43, 29.4+/-2.15, 31.1+/-2.22, 35.9+/-3.49 and 44.0+/-3.27%, respectively, at 1000 microg/ml, confirming better anti-leishmanial activity for the extracts. Aqueous extracts of Aloe nyeriensis (46.7+/-3.28%) and Albizia coriaria (47.5+/-3.21%) had similar activity level to pentosam. The plant extracts have better inhibitory activity while pentosam has better leishmanicidal activity. All extracts exhibited very low cytotoxicity (CC(50) > 500 microg/ml) against human embryonic lung fibroblast (HELF) cells. The investigations demonstrated the efficacy and safety of some extracts of plants that are used by rural indigenous communities for the treatment of parasitic diseases.

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.

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 Peters 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.

Preparation, characterization, and optimization of primaquine-loaded solid lipid nanoparticles

Primaquine (PQ) is one of the most widely used antimalarial drugs and is the only available drug that combats the relapsing form of malaria. PQ use in higher doses is limited by severe tissue toxicity including hematological- and gastrointestinal-related side effects. Nanoformulation of drugs in an appropriate drug carrier system has been extensively studied and shown to have the potential to improve bioavailability, thereby enhancing activity, reducing dose frequency, and subsequently reducing toxicity. The aim of this work was to design, synthesize, and characterize PQ-loaded solid lipid nanoparticles (SLNs) (PQ-SLNs) as a potential drug-delivery system. SLNs were prepared by a modified solvent emulsification evaporation method based on a water-in-oil-in-water (w/o/w) double emulsion. The mean particle size, zeta potential, drug loading, and encapsulation efficiency of the PQ-SLNs were 236 nm, +23 mV, 14%, and 75%, respectively. The zeta potential of the SLNs changed dramatically, from −6.54 mV to +23.0 mV, by binding positively charged chitosan as surface modifier. A spherical morphology of PQ-SLNs was seen by scanning electron microscope. In vitro, release profile depicted a steady drug release over 72 hours. Differential scanning calorimeter thermograms demonstrated presence of drug in drug-loaded nanoparticles along with disappearance of decomposition exotherms, suggesting increased physical stability of drug in prepared formulations. Negligible changes in characteristic peaks of drug in Fourier transform infrared spectra indicated absence of any interaction among the various components entrapped in the nanoparticle formulation. The nanoformulated PQ was 20% more effective as compared with conventional oral dose when tested in Plasmodium berghei-infected Swiss albino mice. This study demonstrated an efficient method of forming a nanomedicine delivery system for antimalarial drugs.

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.

Development, characterization and antimalarial efficacy of dihydroartemisinin loaded solid lipid nanoparticles

UNLABELLED Effective use of dihydroartemisinin (DHA) is limited by poor water-solubility, poor pharmacokinetic profile and unsatisfactory clinical outcome especially in monotherapy. To reduce such limitations, we reformulated DHA into solid lipid nanoparticles (SLNs) as a nanomedicine drug delivery system. DHA-SLNs were characterized for physical parameters and evaluated for in vitro and in vivo antimalarial efficacy. DHA-SLNs showed desirable particle characteristics including particle size (240.7 nm), particle surface charge (+17.0 mV), drug loadings (13.9 wt %), encapsulation efficacy (62.3%), polydispersity index (0.16) and a spherical appearance. Storage stability up to 90 days and sustained release of drug over 20 h was achieved. Enhanced in vitro (IC50 0.25 ng/ml) and in vivo (97.24% chemosuppression at 2mg/kg/day) antimalarial activity was observed. Enhancement in efficacy was 24% when compared to free DHA. These encouraging results show potential of using the described formulation for DHA drug delivery for clinical application. FROM THE CLINICAL EDITOR Malaria still poses a significant problem worldwide. One of the current drugs, artemisinin has been shown to be effective, but has poor water-solubility. The authors here described their formulation of making dihydroartemisinin (DHA) into solid lipid nanoparticles, with subsequent enhancement in efficacy. These results would have massive potential in the clinical setting.

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.

Bisbenzylisoquinoline and hasubanane alkaloids from Stephania abyssinica (Dillon & A. Rich) (Menispermeceae).

Two bisbenzylisoquinoline and one hasubanane alkaloids: (-)-pseudocurine (1), (-)-pseudoisocurine (2) and (-)-10-oxoaknadinine (3), were isolated from leaf extract of Stephania abyssinica, a plant used in traditional medicine in South Nyanza region of Kenya. They were characterized using 1D ((1)H, (13)C and DEPT) and 2D (COSY, NOESY, HMQC and HMBC) NMR techniques. (-)-Pseudocurine (1) and (-)-pseudoisocurine (2) exhibited strong to moderate anti-plasmodial activity while (-)-10-oxoaknadinine (3) showed moderate to mild activity.

Evaluation of Ethnomedical Claims II: Antimalarial Activities of Gongronema latifolium Root and Stem

Methanolic extract and chromatographic fractions of Gongronema latifolium were tested against clinical isolates of Plasmodium falciparum-, P. yoelii nigeriensis-infected mice, chloroquine-sensitive (D6) and chloroquine-resistant (W2) P. falciparum clones. The isolates, characterized as a 1:1 mixture of α-amyrin and β-amyrin cinnamates (1a/1b), lupenyl cinnamate (2) and lupenyl acetate (3), were assayed using the clones. Extract, most active vacuum liquid and column chromatographic fractions had respective ED50 values of 120.85, 32.03, 25.62 mg.kg-1 and IC50 of 36.27, 9.45, 7.05 μg.mL-1, against W2 clones. Lupenyl acetate had 18.96 μg.mL-1, indicating synergistic action of the constituents. Results justified its ethnomedical use for treating malaria.