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.

The antiplasmodial activity of spermine alkaloids isolated from Albizia gummifera.

In the present study the methanolic extract of Albizia gummifera was fractionated into various fractions. These fractions were tested against choroquine sensitive (NF54) and resistant (ENT30) strains of Plasmodium falciparum. All other fractions apart from the alkaloidal fraction showed low activity with IC 50 above 3 microg/ml. The alkaloidal fraction exhibited strong activity against NF54 and ENT30 with IC 50 of 0.16+/-0.05 and 0.99+/-0.06 microg/ml, respectively. Five known spermine alkaloids were isolated from the alkaloidal fraction. These alkaloids exhibited activities against NF54 and ENT30 with IC 50 ranging from 0.09+/-0.02 to 0.91+/-0.10 microg/ml. Four of the alkaloids were further evaluated for in vivo activity against rodent malaria parasite Plasmodium berghei. The alkaloids showed percentage chemosuppression of parasitaemia in mice ranging from 43 to 72%. The use of the extracts A. gummifera for treatment of malaria in traditional medicine seems to have a scientific basis.

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.

Anti-plasmodial activity of some Kenyan medicinal plant extracts singly and in combination with chloroquine.

Sixty organic and aqueous extracts of eleven plants used for the control of malaria by local communities in Kisii District, Kenya were screened for in vitro anti-plasmodial activity. The plants selection was based on existing ethnobotanical information and interviews with local communities. The extracts were tested against chloroquine sensitive and resistant laboratory adapted strains of Plasmodium falciparum. The study revealed that 63.6% of the plants were active (IC50 < or = 100 microg/mL). Extracts of four plants, Ekebergia capensis, Stephania abyssinica, Ajuga remota and Clerodendrum myricoides gave IC50 values below 30 microg/mL against both chloroquine sensitive and resistant P. falciparum strains. Combination of extracts of E. capensis and C. myricoides with chloroquine against the multi-drug resistant P. falciparum isolate (V1/S) revealed synergistic effect. The plants which showed activity may be useful as sources for novel anti-plasmodial compounds.

Brine shrimp toxicity and antiplasmodial activity of five Kenyan medicinal plants.

The organic extracts of leaves and roots of five plants used for treating malaria in Central, Nairobi and Rift Valley Provinces, Kenya were tested for brine shrimp lethality and in vitro antiplasmodial activity against chloroquine sensitive and resistant strains of Plasmodium falciparum. Of the plants tested, 60% were toxic to the brine shrimp (LC(50)<30 microg/ml) and eight out of ten plant parts (80%) showed in vitro antiplasmodial activity (IC(50)<50 microg/ml). Among the extracts screened, the leaves of Cyathula polcephala had the highest toxicity to the brine shrimp (LC(50)=2.9 microg/ml) while the leaves of Pentas longiflora had the best antiplasmodial activity (IC(50)=11. /ml). The plant extracts with low IC(50) values are potential sources for novel antiplasmodial compounds.

In vitro antiplasmodial activity of some plants used in Kisii, Kenya against malaria and their chloroquine potentiation effects.

Fifty-five organic and aqueous extracts of 11 plants used in malaria therapy in Kisii District, Kenya were tested in vitro against chloroquine (CQ)-sensitive and resistant strains of Plasmodium falciparum. Of the plants tested, 73% were active (IC(50) < 100 microg/ml). Three plants, Vernonia lasiopus, Rhamnus prinoides and Ficus sur afforded extracts with IC(50) values ranging less than 30 microg/ml against both CQ-sensitive and resistant strains. Combination of some extracts with CQ against the multi-drug resistant P. falciparum isolate V1/S revealed some synergistic effect. The plant extracts with low IC(50) values may be used as sources for novel antimalarial compounds to be used alone or in combination with CQ.

Physiological aspects of fungi isolated from root nodules of faba bean (Vicia faba L.).

The present study was made to isolate and assess some physiological characteristics of root nodule-colonizing fungi. During this study, 17 fungal species were isolated from root nodule samples taken from faba bean plants (Vicia faba L.) collected from different sites at Assiut area (Egypt). The growth of faba bean plants in pots was significantly promoted by soil inoculation with most fungi. Growth was checked in pots with inocula of Cladosporium cladosporioides, Fusarium moniliforme, F: oxysporium, F solani, Macrophominia phaseolina and Rhizoctonia solani which were added separately. All growth-promoting fungi were capable of producing cellulase, pectin lyase, polygalacturonase, protease, urease, amidase, acid phosphatase, alkaline phosphatase and arylsulfatase in growth medium supplemented with the corresponding substrates. Four fungal species, Aspergillus awamori, A. flavus, Penicillium chrysogenum and Trichoderma koningii showed the highest rates of enzyme formation. The effect of the addition of six trace elements to the growth media at 30 micromol/ml on enzyme production revealed some dependency on species, enzyme and metal ion. Cd2+, Hg2+ and Zn2+ generally inhibited enzyme activity. Cu(1+), Fe3+ and Al3+ showed a stimulatory effect. Fungicides (afugan and tilt) and herbicides (brominal and fusilade) at 50 ppm generally promoted enzyme activity, but insecticides (kelthane and fenvalerate) caused some inhibition to enzyme activities. Salinization of the growth media with NaCl strongly inhibited the enzymatic activity of all fungi at concentrations between 0.5 and 1.5%.

Effects of prenatal steroids on water and sodium homeostasis in extremely low birth weight neonates.

OBJECTIVE: We sought to determine if prenatal steroid (PNS) treatment affects water and sodium (Na) balance in extremely low birth weight infants (<1000 g). METHODS: PNS treatment enhances lung maturation in preterm infants and induces maturation of renal tubular function and adenylate cyclase activity in animals. We compared water and Na homeostasis for the first week of life in those infants whose mothers received steroids before delivery (PNS: n = 16) to those who did not (nonsteroid group [NSG]: n = 14). The data were collected prospectively, but PNS treatment was not given in a randomized manner. Fluids were initiated at 100 to 125 mL/kg/d and adjusted every 8 to 12 hours to allow a daily weight loss of 150 mmol/L compared with 36% of the NSG infants. PNS infants had a higher cumulative Na excretion at day 2 of life (10 +/- 2 mmol/kg vs 6 +/- 1 mmol/kg) but a less negative cumulative Na balance at 1 week (-10 mmol/kg vs -14 mmol/kg). CONCLUSION: PNS treatment was associated with lower estimated insensible water loss, a decreased incidence of hypernatremia, and an earlier diuresis and natriuresis in extremely low birth weight neonates. We speculate that PNS effects these changes through enhancement of epithelial cell maturation improving skin barrier function. PNS treatment may also enhance lung Na, K-ATPase activity leading to an earlier postnatal reabsorption of fetal lung fluid increasing extracellular volume expansion to help prevent hypernatremia.

Availability of phosphorus and sulfur of insecticide origin by fungi.

Thirteen fungal species isolated from soil treated with pesticides were tested for their ability to mineralize and degrade three organophosphate insecticides currently used in Egypt (Cyolan, Malathion and Dursban) in liquid media free from phosphorus (P) and sulfur (S). All fungal species grew successfully on the culture media treated with the three used doses of insecticides (10, 50 and 100 ppm active ingredient) but the growth rate varied with the species, the insecticide and the doses. At 10 ppm level, insecticide degradation expressed in term of organic P mineralization (calculated as % of applied P) was the highest with all fungi tested. Organic P mineralization from pesticides was decreased by increasing the dose used to 50 and 100 ppm. The highest amount of P mineralized was observed with Cyolan followed by Malathion whilst P mineralization from Dursban proceeded very slowly. Aspergillus terreus showed the greatest potential to mineralize organic P followed by A. tamarii, A. niger, Trichoderma harzianum and Penicillium brevicompactum whilst the remaining fungi only moderately mineralized the organic P component of the insecticides tested. Organic sulfur mineralization by the used fungal species paralleled, to some extent, organic P mineralization. The extracellular protein content of culture filtrates in the presence of various doses of insecticides was also decreased by increasing insecticide concentrations. The extracellular protein was significantly correlated with P and S mineralization (r = 0.89** and 0.64**, respectively) whilst correlation with cell dry mass was not significant (r = 0.03 and 0.003) suggesting a direct relationship between pesticide degradation and microbial protein production. The addition of P or S to the growth media enhanced extracellular protein excretion, and increased organic P and S mineralization by the most potent species tested (A. niger, A. tamarii, A. terreus and T. harzianum). This increment was significant in most cases, especially at the higher application rates. The relationship between extracellular protein excretion and organic P and S mineralization from insecticides was highly significant with the addition of inorganic phosphorus (r = 0.96** and 0.83**, respectively) or sulfur (r = 0.85** and 0.89**, respectively) to the growth media.

A new approach to encapsulating non-steroidal anti-inflammatory drugs. V. Biopharmaceutical study of microcapsules of azapropazone coated with pectin and rutin.

Azapropazone was encapsulated with pectin-rutin mixture using the fluidized bed technique. The encapsulated particles showed higher dissolution rate and bioavailability but lower ulcerogenic activity as compared with the drug alone.