Bioassay-guided phytochemical study of indigenous medicinal plants of Ethiopia
Gutu, Ketema Tolossa
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In many developing countries, farmers and pastoralists still rely on their indigenous knowledge, practices and locally available plants to control nematode parasitic infections, both in livestock and humans. The overall aim of my thesis was to undertake bioassay-guided phyto-chemical study of extracts and their constituents from Ethiopian anti-parasitic plants used by healers to control gastrointestinal nematode parasites in livestock to validate their ethno-medicinal use and to characterise and identify their active ingredients. As a first experiment (Chapter Three), four types of crude extracts (water, 70% methyl-alcohol, absolute methanol and acetone) of four indigenous Ethiopian medicinal plants (Adenia species, Cissus ruspolii, Ipomoea eriocarpa and Euphorbia thymifolia) were screened against Teladorsagia circumcincta egg hatching in vitro, not only as a first step to validate the traditional healers claim but also to choose the most promising plant extract(s) for further phyto-chemical studies. The egg hatching inhibition (EHI) test results revealed that the anti-parasitic properties of these plants depended on plant species, dose, and solvent polarity. The water extracts of both C. ruspolii and Adenia sp. exhibited largest, up to 100% EHI but also larval migration inhibition activities, and were selected for further studies. The second experiment (Chapter Four) assessed the nature of active constituents in these extracts by physico-chemical methods. It was observed that the major constituents of both plant extracts responsible for the EHI activities are likely highly polar, water-soluble, small and moderately heat-labile molecules. The third and fourth experiments (Chapters Five and Six) consisted of separating Cissus ruspolii and Adenia sp. water extracts into discrete fractions by gel-permeation chromatography, EHI tests of Bio-Gel P-2 fractions followed by thin layer chromatography (TLC) profiling of these fractions to detect separated spots (in day light, under UV-light or after staining with various staining reagents) and also to see how elution patterns of separated spots affected by column parameters. The EHI tests on the fractions obtained revealed that the active constituents of C. ruspolii and Adenia sp. water crude extracts were eluted into few fractions based on their molecular sizes. The TLC profilings of these fractions identified spot patterns of active and inactive fractions, which allowed pooling of active constituents based on their EHI and TLC profiling into three pools for each plant. The fifth experiment (Chapter Seven) was to isolate and purify compounds from these pools using various preparative planar and column chromatographic methods. Sequential applications of column chromatography followed by preparative thin layer chromatography isolated and purified five active compounds from C. ruspolii and two active compounds from Adenia sp. The sixth experiment (Chapter Eight) was to characterize and propose/elucidate structures of compounds from the active fractions using chromatographic, analytical and spectroscopic methods. In this regard, the structures of two oleanane type triterpenoid saponins isolated from one of active fractions of Adenia sp. were proposed based on their mass spectrometry (MS) and nuclear magnetic resonance (NMR) data with support of compounds property, TLC and literature. Similar outcomes for C. ruspolii were not achieved due to lack of sufficient sample to run 13C-nuclear magnetic resonance spectroscopy and distortionless enhancement by polarization transfer (DEPT), contamination of some purified compounds with ill-characterised substance from the preparative TLC matrix and in some cases mass spectrometry (MS) and nuclear magnetic resonance (NMR) data did not support each other. The last experiment (Chapter Nine) was to assess anthelmintic efficacy and safety of C. ruspolii and Adenia sp. crude water extracts in Heligmosomoides bakeri infected mice. This in vivo test revealed that both plant extracts exhibited significant reduction in worm burdens and worm egg excretion, with moderate effects on haematology and organ weights at tolerated dosages. In conclusion, both in vitro and in vivo data revealed that Adenia sp. and C. ruspolii have anthelmintic properties, thus validating traditional healer claims and supporting ethno-medicinal use. The bioassay-guided phytochemical study resulted in the isolation of a number of active compounds from these plants, for some of which a structure has been proposed.