Phytochemical and pharmacological investigation of four Tanzanian medicinal plants for anti-protozoal activity

Abstract

Protozoal diseases like malaria, trypanosomiasis and leishmaniasis, caused by Plasmodium, Trypanosoma and Leishmania, respectively, have overwhelming impact on public health in developing regions. Due to their prevalence, virulence and drug resistance, they are the most serious and widespread parasitic diseases encountered by mankind The inadequate armory of drugs, high cost and lack of new drugs are the major limiting factors in the fight against the three diseases. Consequently, there is continuing need of research on the discovery and development of new effective and safe anti-protozoal drugs. In this work, solvent extracts from four Tanzanian medicinal plants: Annickia kummeriae, Pselalospondias microcarpa, Drypetes natalensis and Acridocarpus chloropterus, were tested for anti-protozoal activity. Anti-protozoal bioassays revealed 14 solvent extracts with strong i ) moderate in vitro antiplasmodial activity (IC5o 0.12_+0.01-5.50_+0.27 gg/ml), 6 with moderate anti-trypanosomal activity (IC5o 2.3±0.43-5.40_+0.64 pg/ml) and only 3 with mild inti-leishmanial activity (IC5o 9.25+0.54-9.79+2.5 gg/ml). Annickia kummeriae, P. micro arpa and D. natalensis solvent extracts exhibited good anti-plasmodial activity and favourable selectivity (SI 29.22,250). The strong anti -plasmodial, moderate anti-trypanosomal aid mild anti -lei shmanial activity of the methanolic extract of A. kummeriae leaves encoura~ ed the isolation of antiprotozoal compounds. Bioassay-guided chromatographic fraction ion led to isolation and identification of fractions with stronger anti-plasmodial activi and more favourable selectivity than the solvent extracts. Seven anti-protozoal al Voids: palmatine (82), jatrorrhizine (83), lysicamine (127), trivalvone (128), annickine ( 129), columbamine (130) and (-)-tetrahydropalmatine (131), were identified by spectroscopic methods and subjected to in vitro anti-protozoal assays. The isolated alkaloids showed good selectivity and strong to moderate anti-plasmodial activity (IC5o 0.08_+0.001-2.3+0.44 [t-'ml), mild to weak antitrypanosomal (IC5o 3.2++0.01-14.30_+0.1 pg/ml) and anti-leishmaniA activity (IC5o 2.7++0.120.4+_0.1 gg/ml). Chromatography of A. chloropterus extracted to the isolation and identification of 5 triterpenes: (3-sitosterol (132), stigmasterol (133), friedelin (134), oleanolic acid (124), ursolic acid (123); and 5 flavonoids: apigenin (135), luteolin (136), vitexin (137), kaempferol (138) and quercetin (139). Quercetin (139) exhibited moderate in vitro anti-plasmodial activity (IC5o 2.6++0.05 pg/ml) while the rest of compounds were inactive. Mild to weak in vitro anti -trypanosomal activity was observed in quercetin (139) (IC5o 3.60±0.1 Frg/ml), ursolic acid (123) (IC5o 7.80±0.1 pg/ml) and apigenin (135) (IC5o 9.0+0.1ug/ml). Good in vitro anti-leishmanial activity (IC50 0.80_+0.001, 2.10_+0.1, 2.20+0.1, 5.90_+ 0.1 and 3.5+0.2 ug/ml) and favourable selectivity were observed with ursolic acid (123), quercetin (139), kaempferol (138), apigenin (135) and oleanolic acid (124), respectively. Chemical structure-biological activity relationship comparisons confirmed that, the methoxyl groups at C-2, C-3 (ring A) and C-9, C-10 (ring D) together with the quaternary nitrogen atom in position 7 are the structural moieties required for strong anti-plasmodial activity in protoberberine alkaloids. Structure-activity-relationship (SAR) comparisons in the isolated triterpenoids confirmed that, the hydroxyl group at C-3 with methyl groups at C-23, C-25, C-26 and C-30, C-12/C-13 double h:md, the carboxylic acid group at C-28, and hydrogen at C-20 in the molecular framework )f ursolic acid (123) are the structural moeties responsible for the strong anti-leishmania! activity whereas in the isolated flavonoids, the hydroxyl groups at C-3 and C-3' in the apigenin (135) molecular framework are the structural moeties responsible for the strong anti-protozoal activity observed. The alkaloids with strong anti-plasmodial and me gate anti-trypanosomal activity may provide lead compounds for the development o drugs for malaria and trypanosomiasis. Similarly, the strong to moderate anti-leishmani I activity of the isolated triterpenes and flavonoids render them good candidates as molecular templates for new drug development.