Implementation and continued validation of the malaria Plasmodium falciparum lactate dehydrogenase-based colorimetric assay for use in antiplasmodial drug screening.

Antimalarial drug discovery has been facilitated by the development of various in vitro drug susceptibility testing methods suitable for medium-throughput or high-throughput campaigns. Among many, the Plasmodium falciparum lactate dehydrogenase (PfLDH) assay has acceptable demand on equipment, labour, technical skills and affordability and offers a good opportunity for scientists in low- and middle-income countries to participate in the global effort of discovering future antimalarial drugs. Hence, to enable our search for novel antimalarial drugs, we implemented and examined assay conditions and validated the PfLDH-based method in our laboratory using a reference set of standard antimalarial drugs with known activity against Plasmodium falciparum strains. The PfLDH assay revealed acceptable linearity profiles of R2 = 0.97 and 0.92 for Pf3D7 and PfDd2, respectively, achieved at 2% parasitaemia and 1% haematocrit. The detection and quantitation limits (DL and QL) of the PfLDH-based assay were 0.09% and 0.4% parasitemia, respectively. The assay showed an acceptable average Z-factor between 0.76 and 0.79 and was considerably robust. The average interassay reproducibility via percent coefficient of variation (%CV) was 5.47 between independent experiments. Overall, the PfLDH-based method produced a reliable and reproducible drug screening profile for in vitro assays in our setting. There were no significant interassay variability or hazards of other screening assays.

Extracts from Annona Muricata L. and Annona Reticulata L. (Annonaceae) Potently and Selectively Inhibit Plasmodium Falciparum.

The aim of this work was to screen extracts from Annona muricata and Annona reticulata in vitro against Plasmodium falciparum. Crude ethanolic extracts, methylene chloride fractions, aqueous fractions, subfractions and isolated compounds (stigmasterol-3-O-ß-d-glucopyranoside, lichexanthone, gallic acid and ß-sitosterol-3-O-ß-d-glucopyranoside) were tested for cytotoxicity on erythrocytes and Human Foreskin Fibroblasts cells and against the W2 strain of P. falciparum in culture. Results indicated that none of the extracts was cytotoxic at concentrations up to 10 µg/mL. Most of the extracts, fractions and subfractions inhibited the growth of P. falciparum with IC50 values ranging from 0.07 to 3.46 µg/mL. The most potent was the subfraction 30 from A. muricata stem bark (IC50 = 0.07 µg/mL) with a selectivity index of ˃ 142. Subfraction 3 from A. muricata root also exhibited very good activity (IC50 = 0.09 µg/mL) with a high selectivity index (SI ˃ 111). Amongst the isolated compounds, only gallic acid showed activity with IC50 of 3.32 µg/mL and SI > 10. These results support traditional claims for A. muricata and A. reticulata in the treatment of malaria. Given their limited cytotoxicity profile, their extracts qualify as promising starting points for antimalarial drug discovery.

Potent and Synergistic Extract Combinations from Terminalia Catappa, Terminalia Mantaly and Monodora tenuifolia Against Pathogenic Yeasts.

Mycoses caused by Candida and Cryptococcus species, associated with the advent of antifungal drug resistance have emerged as major health problems. Improved control measures and innovative therapies are needed. This paper describes results from the screening of bio-guided fractionated extracts alone and combinations of Terminalia catappa, Terminalia mantaly and Monodora tenuifolia harvested in Cameroon. Crude ethanolic, hydro-ethanolic and aqueous extracts and bio-guided fractions were screened for antifungal activity against isolates of C. albicans, C. glabrata, C. parapsilosis and Cr. neoformans and the reference strain C. albicans NR-29450. Minimal inhibitory concentrations (MIC) were determined using a broth micro dilution method according to the Clinical & Laboratory Standards Institute (CLSI). Time kill kinetics of extracts alone and in combination were also evaluated. Extracts from T. mantaly stem bark were the most active with the best MIC values ranging from 0.04 mg/mL to 0.16 mg/mL. Synergistic interactions were observed with combinations of sub-fractions from M. tenuifolia, T. mantaly and T. catappa. Combination of sub-fractions from M. tenuifolia and T. mantaly (C36/C12) showed synergistic interaction and fungicidal effect against four out of five tested yeasts. These results support further investigation of medicinal plant extracts alone and in combination as starting points for the development of alternative antifungal therapy.