The search for new anti-plasmodial agents: fate of mitragyna inermis, pseudocedrela kotschyi and moringa oleifera
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Date
2015-04-21
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Abstract
Malaria treatment keeps failing because of resistance. With recent speculations about genes likely to be coding for Artemisinin resistance, it has become pertinent to extend the search for new agents. This study was undertaken to evaluate the In-vivo antiplasmodial effects of traditionally used medicinal plants for malaria treatment. It also sought to evaluate them for possible future compound (s) isolation and drug development. Leaves and stem bark of Moringa oleifera (Moringaceae), twigs of Mitragyna inermis (Rubiaceae) and leaves of Pseudocedrela kotschyi (Meliaceae), were selected based on ethnopharmacological studies, collected and authenticated in the Department of Herbal Medicine, KNUST, Kumasi. The samples were dried at room temperature for a month and then pulverized. Aqueous and methanolic extracts were prepared and stored under refrigeration (-18 oC) until use. Samples of the powdered plant materials were also kept for investigation. Phytochemical screening was carried out on both powdered plant materials and the extracts. The extracts were also evaluated for their In-vivo antiplasmodial activity. Initial screening was carried out on the aqueous extracts of Moringa oleifera leaves (ML) and stem bark (MSB) at 500 and 750mg/kg respectively, and Pseudocedrela kotschyi leaves (PK) and Mitragyna inermis twigs (MT), both at 500 mg/kg for 7 days. Extracts were administered orally to Plasmodium berghei infected ICR mice (25-30 g). The most effective aqueous extract, ML, was then evaluated at 250, 500, 750 and 1000 mg/kg using Artemether-Lumefantrine (A/L) at 4 mg/kg as reference drug. Both Four-Day Suppressive Test and seven days of Curative Test were conducted. The weights of the animals, physical signs showing either clinical deterioration or wellness and survival of the animals were also monitored. The methanolic extracts of Moringa oleifera leaves (MOR), Pseudocedrela kotschyi leaves (PSD) and Mitragyna inermis twigs (MIT), each at 500 mg/kg, were also evaluated on P. berghei infected BALB/c mice (20-25 g) using A/L at 4 mg/kg as reference drug. The weights of the experimental animals were also monitored. Chromatographic fingerprints for the extracts were developed using reversed high performance liquid chromatography. The aqueous extracts obtained were MT (16.736 g; 1.67%), PK (16.6736 g; 3.33%), MSB (23.3355 g; 1.17%) and ML (24.1123 g; 2.41%). The methanolic extracts were MOR (16.4624 g; 11.65%), MIT (33.9513 g; 2.26%) and PSD (60.019 g; 5.91%). Phytochemical screening showed presence of alkaloids, tannins, coumarins, phytosterols, flavonoids and glycosides, with some variations in the compositions in the aqueous and methanolic extracts. The suppressions from the initial screening were as follows; ML (84% - 99.15%; AUC = 521.3), MSB (65% - 96.88%; AUC = 491.6), MT (47.67% – 84.49%; AUC = 340.8) and PK (22.47% - 82.91%; AUC = 275.0). ML extracts (250-1000 mg/kg) exhibited significant reduction in parasitemia in the four-day suppressive test (F6,49 = 4.309; p =0.0014). However, 250 mg/kg (69.31%; p < 0.001) and 500 mg/kg (77.26%; p < 0.001) extracts exhibited relatively higher activities compared to 750 mg/kg (25.28%; p < 0.001) and 1000 mg/kg (07.12%; p > 0.05). In the curative test, similar results were obtained with significant parasitemia reduction for 250 mg/kg (AUC = 52.52 ± 6.732; p < 0.01) and 500 mg/kg (AUC = 49.62 ± 3.804; p < 0.01) compared to the positive control group (AUC = 101.3 ± 14.32). In addition, physical signs such as piloerection, lethargy and decreased locomotor activity were observed to be progressive in all experimental groups except A/L. Finally, survival analysis showed that although 750 mg/kg and 1000 mg/kg groups recorded relatively higher mortalities, statistical analysis didn’t show any significant difference. In evaluating the methanolic extracts, percentage suppressions by day-3 post infection were as follow; MOR (67.08%), MIT (44.37%) and PSD (25.79%). Parasite reduction ratio (PRR), an indication of extract potency, was observed to be declining with day for the extracts whereas that of A/L kept increasing exponentially. The chromatograms developed gave indication of the complexity of the extracts and also showed feasibility of optimizing the experimental conditions for preparative HPLC fractionation of the extracts.
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A thesis submitted in partial fulfilment of the requirements for the degree of master of philosophy, 2014