The use of Toddalia asiatica (L) Lam. (Rutaceae) in traditional medicine practice in East Africa.

Toddalia asiatica (L) Lam. (Rutaceae) has been used by traditional health practitioners in East Africa for management of diseases, however, the extent of its usefulness has not been established to date. Fieldwork for this study was carried out in the Lake Victoria Basin between March and September 2006. The purpose was to collect ethnomedical information that will serve as a basis for further studies to establish current and potential medicinal uses. The ethnomedical information was obtained through interviews using semi-structured questionnaires. Consultative meetings were also conducted with traditional health practitioners and other members of the communities in Kenya, Uganda and Tanzania. Results of this study show that Toddalia asiatica is collected in the wild, prepared mostly as decoctions or concoctions and administered orally. It is used for the management of a number of disease conditions. The most frequently cited diseases were stomach problems (78%) followed by malaria (25%). Cough (22%), chest pain (13%), food poisoning (8%), sore throat (7%), were also mentioned among other disease conditions treated. Validation studies of therapeutic claims will be carried out at a later date.

Traditional healers and the management of malaria in Kisumu district, Kenya.

OBJECTIVE: To document the ethnobotanical information on malaria treatment with the goal of eventually testing the medicinal plant extracts for antiplasmodial activity. DESIGN: A prospective study. SETTING: Informants from Kisumu City and its environs were gathered at the Kenya Medical Research Institute, Centre for Vector Biology Control Research, Kisian, Kisumu. INTERVENTIONS: Semi-structured Questionnaires were administered to 16 traditional health practitioners (THPs) to evaluate the THPs' perceptions and practice relating to causation and treatment of malaria. MAIN OUTCOME MEASURES: The THPs described the signs, symptoms and cause of malaria. Details of the preparation and use of plants for management of malaria were recorded. RESULTS: Of the 16 respondents 12 (75%) knew that malaria is transmitted by mosquito bite and 12 (75%) recognised the main symptoms as fever. Of the 36 medicinal plants, claimed to treat malaria in Kisumu, 19 plants were identified at the East African Herbarium, National Museums of Kenya. CONCLUSION: The ethnomedical and ethnobotanical data generated form the basis for pharmacological evaluation of the medicinal plants collected to establish their potential in the treatment of malaria.

Liquid chromatography of polymyxin B sulphate.

A reversed-phase liquid chromatography method for analysis of polymyxin B sulphate is described. The method uses a YMC-Pack Pro, C18, 5 microm, 250x4.6 mm I.D. column maintained at 30 degrees C. The mobile phase comprises acetonitrile-sodium sulphate (0.7%, m/v)-phosphoric acid (6.8%, v/v dilution of 85%, m/m phosphoric acid)-water (22.25:50:5:22.75) at a flow-rate of 1.0 ml/min. Detection was by UV at 215 nm. The method is able to resolve polymyxin B1, the major component, from more than thirty other components present in the complex. Robustness was evaluated by performing a full-factorial design experiment. The method showed good selectivity, repeatability, linearity and sensitivity.

Isolation and structural characterization of polymyxin B components.

Polymyxin B is a peptide antibiotic complex present as sulphate. The components were separated preparatively on a poly(styrene-divinylbenzene) (PLRP-S), 1000 A, 8 microm, 250 x 12.5 mm I.D. stationary phase maintained at 60 degrees C and using 215 nm detection. Elution was carried out with acetonitrile-sodium sulphate solution (0.7%, m/v; pH adjusted to 2.5 with trifluoroacetic acid)-water (18:50:32, v/v) at a flow-rate of 4.0 ml/min. Seven polymyxin B components were isolated and characterized using 1H and 13C NMR. The molecular masses were confirmed by mass spectrometry. The structures of two components were determined for the first time. Polymyxins B5 and B6 were identified as having the same composition as polymyxin B1 except that the fatty acid moiety was nonanoic acid and 3-hydroxy-6-methyloctanoic acid, respectively.

Analysis of polymyxin B sulfate by capillary zone electrophoresis with cyclodextrin as additive. Method development and validation.

A capillary zone electrophoresis method for analysis of polymyxin B sulfate is described. In this method, triethanolamine (TEA)-phosphate buffer at pH 2.5 was employed to reduce the adsorption of analyte onto the capillary wall. Methyl-beta-cyclodextrin (M-beta-CD) and 2-propanol (IPA) were found to be necessary for selectivity enhancement. In order to optimize the method and to control its robustness, a central composite design was performed with four parameters, i.e. concentration of M-beta-CD, TEA, IPA and buffer pH. The optimal separation conditions were as follows: capillary, 55 cm (50 microm I.D., 47 cm effective length); 130 mM TEA-phosphate buffer (pH 2.5) containing 5 mM M-beta-CD and 5% IPA; 24 kV (51 microA) applied voltage; column temperature, 20 degrees C. Further, linearity and limits of detection quantification were examined. Three commercial samples were analyzed quantitatively.

Isolation and structural characterization of colistin components.

Preparative-scale separation of colistin sulphate bulk sample was carried out on a preparative poly(styrene-divinylbenzene) stationary phase. Isocratic elution with acetonitrile-sodium sulphate solution (0.7% m/v; pH adjusted to 2.5 with TFA) - water (16:50:34, % v/v/v) was carried out at a flow rate of 4.0 ml min(-1). Six colistin components were isolated and characterized using 1H and 13C NMR. The molecular weights were confirmed by mass spectrometry. The structures of 2 components were determined for the first time. Polymyxin E7 was identified as having the same composition as polymyxin E1, except that the fatty acid moiety was 7-methyloctanoic acid. Isoleucine polymyxin E8 was characterized as having the same composition as isoleucine polymyxin E1 with 7-methylnonanoic acid as the fatty acid moiety.

Liquid chromatography method for separation of clindamycin from related substances.

A reversed-phase liquid chromatography method has been developed for the separation of clindamycin from 7-epiclindamycin, clindamycin B, lincomycin, lincomycin B, 7-epilincomycin and other impurities of unknown identity. The method uses a Hypersil ODS, 5 microm, 250 x 4.6 mm i.d. column maintained at 45 degrees C. The mobile phase comprises acetonitrile phosphate buffer (1.35% v/v phosphoric acid, adjusted to pH 6.0 with ammonium hydroxide)-water (35:40:25, v/v) at a flow rate of 1.0 ml/min. UV detection is performed at 210 nm. The method was tested on several C-18 columns and showed good robustness. Robustness was further evaluated by performing a full-fraction factorial design experiment. The method showed good selectivity, linearity, and repeatability. It is also suitable for analysis of clindamycin formulations.

Study of the stability of polymyxins B(1), E(1) and E(2) in aqueous solution using liquid chromatography and mass spectrometry.

Polymyxins B(1), E(1) (colistin A) and E(2) (colistin B) were subjected to degradation in aqueous solutions of different pH values (1.4, 3.4, 5.4 and 7.4) and at different temperatures (37, 50 and 60 degrees C) in order to investigate the characteristics of decomposition. The progress of decomposition was followed by reversed-phase liquid chromatography on YMC-Pack Pro, C-18 stationary phase. The degradation curves showed (pseudo) first order kinetics. The pH-rate profiles indicate that colistin is more susceptible to degradation in solutions of pH above 5 and is more stable in acidic media. The degradation of polymyxin B(1) was most rapid at pH 7.4. Qualitative analysis of the degradation products by LC/MS reveals that racemization is the major mechanism of degradation in both acidic and neutral media.

Influence of manufacturing practices on quality of pharmaceutical products manufactured in Kenya.

OBJECTIVE: To establish the quality of pharmaceutical products manufactured by the respective industries in Kenya and determine the effect of manufacturing practices on the quality of these products. DESIGN: Cross-sectional study. SETTING: Industries examined are in Nairobi, Kenya. Laboratory analysis was carried out using available facilities at Kenya Medical Research Institute and University of Nairobi, Faculty of Pharmacy. INTERVENTIONS: Structured Questionnaires were administered to examine how the code of good manufacturing practices has been used in the production of each pharmaceutical product by respective companies. Questionnaires designed to evaluate the distribution and carry out limited post-market surveillance study were administered to community pharmacy outlets. Drugs were sampled and analyzed for their quality according to the respective monographs. MAIN OUTCOME MEASURES: The questionnaires administered to the industry included the source of raw materials, quarantine procedure before and after manufacture, manufacturing procedure, quality audit, quality assurance procedure, equipment, and staff. That administered to the pharmacy outlet included availability, affordability and acceptability of locally manufactured pharmaceutical products. Quality analysis of products involved the establishment of the chemical content, dissolution profile, friability, uniformity of weight and identity. For antibiotic suspensions the stability after reconstitution was also determined. RESULTS: There were 15 respondents and two non-respondents from the industry and six out of nine respondents from the pharmacy outlets. The ratio of qualified staff to product range produced seemed to influence product quality. Industries producing several products with only limited number of pharmaceutical staff had more products failing to comply with pharmacopoeia specifications compared to those producing only few products. Nevertheless, all companies are well equipped with quality control equipment, in accordance with type of product manufactured. Private pharmacies stocked few of the locally manufactured products. The reason, they said, was due to low doctor and/or patient acceptance. Compliance with quality specifications as set out in respective monographs was overall 76%. CONCLUSION: Although the local pharmaceutical industries have adopted good manufacturing practices leading to many good quality products currently in commerce, these manufacturing practices are not comprehensive and measures need to be taken to continue improving them.

Quality of intravenous infusion fluids manufactured in Kenya.

The incidence and nature of microbial contamination of intravenous fluids prepared by four manufacturing establishments in Kenya was evaluated using the European Pharmacopoeia membrane filtration method for sterility testing. The percentage failures were 28.6% for source D, 18.8% for source A, 12.5% for source B and 10.5% for source C. The major contaminant was aspergillus which was isolated from samples from three sources. Candida and Staphylococcus accounted for the contamination of samples from two sources. Failure rates due to the chemical composition of the products was 66.7% for Source A, 60.0% for D, 41.7% for C and 13.3% for B. The experience of the manufacturing sites appeared to correlate with the quality of the products, with the older manufacturing establishments showing lower percentage failures.

Antimalarial and safety evaluation of extracts from Toddalia asiatica (L) Lam. (Rutaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Toddalia asiatica (L) Lam. (Rutaceae) is a medicinal plant traditionally used in Kenya by many communities for the treatment of malaria and other ailments. All parts of the plant are claimed to have medicinal value, but the root bark in particular is believed to be more potent. Decoctions or infusions of the roots are taken orally to treat malaria, fever and stomach ache. AIM OF THE STUDY: To evaluate antimalarial activity of aqueous and organic extracts prepared from Toddalia asiatica and determine in vitro and in vivo safety of the extracts. MATERIALS AND METHODS: Aqueous, ethyl acetate, hexane and methanol extracts were obtained from Toddalia asiatica root bark, fruits and leaves. In vitro antiplasmodial activity was done using chloroquine-sensitive (D6) and chloroquine-resistant (W2) Plasmodium falciparum strains and the concentration causing 50% inhibition of radioisotope incorporation (IC(50)) was determined. In vivo assay was done by administering mice infected with Plasmodium berghei four consecutive daily doses of the extracts through oral route following Peters 4-Day suppressive test. The percentage suppression of parasitaemia was calculated for each dose level by comparing the parasitaemia in untreated control with those of treated mice. Quinine hydrochloride was used as positive control while double distilled water or 20% Tween-80 was used as a negative control. In vivo acute toxicity was determined in mice using standard procedures. In vitro cytotoxicity assay was carried out using actively dividing sub-confluent Vero cells. RESULTS: Inhibitory concentrations of ethyl acetate extract of Toddalia asiatica fruits showed high activity against chloroquine resistant (W2) strains of Plasmodium falciparum (IC(50)=1.87 µg/ml), followed by root bark aqueous extract (IC(50)=2.43 µg/ml). Tested in vivo against Plasmodium berghei, the fruit ethyl acetate extract (500 mg/kg) and root bark aqueous extract (250 mg/kg) reduced malaria parasitaemia by 81.34% and 56.8% respectively. Higher doses were found to be less effective in vivo. Acute toxicity and cytotoxictiy of the tested extracts, with the exception of hexane extract from the roots, showed LD(50)>1000 mg/kg and CC(50)>100 µg/ml respectively. CONCLUSIONS: The results obtained contribute to the validation of traditional use of Toddalia asiatica and provides in vivo and safety data of the plant extracts tested for the first time. Ethyl acetate extract of the fruits was active against chloroquine resistant Plasmodium falciparum as well as against Plasmodium berghei. These findings confirm the suitability of Toddalia asiatica as a good candidate for further tests to obtain a prototype for antimalarial medicine.