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Structures and Evaluation of Biologically Active Constituents of "Cussonia Zimmermannii" Harms

Senn, M. W. (2006) Structures and Evaluation of Biologically Active Constituents of "Cussonia Zimmermannii" Harms. Doctoral thesis, University of Basel.

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Abstract

For the present thesis 22 samples of seven African medicinal plants, the extracts of which had previously proved to possess an in vitro activity against T. b. rhodesiense and/or P. falciparum and 13 samples of six randomly selected plant species based on availability were collected in Tanzania. Of these 35 samples, 140 crude extracts were produced with four solvents of different polarity, and the extracts were tested for in vitro antitrypanosomal and antiplasmodial activity and for cytotoxicity. In addition the extracts were tested for the affinity to the GABAA receptor performed by radioligand binding experiments. Based on the considerable antitrypanosomal and antiplasmodial activity and the affinity to the GABAA receptor, two extracts were selected for bioassay-guided fractionation : the petroleum ether extract of the stembark of Commiphora fulvotomentosa Engl. because of its promising IC50 value of 2.1 mg/ml against T. b. rhodesiense and the high selectivity index of 21.4, and the petroleum ether extract of the rootbark of Cussonia zimmermannii Harms because of its IC50 value of 4.8 mg/ml against T. b. rhodesiense, its IC50 value of 3.3 mg/ml against P. falciparum, and the potent modulatory effect at the GABAA receptor of 151 %. After three fractionation steps the fractions of the petroleum ether extract of the stembark of Commiphora fulvotomentosa Engl. did not show any inhibitory activity against T. b. rhodesiense and were therefore not investigated further. Bioassay-guided fractionation of the petroleum ether extract of the rootbark of Cussonia zimmermannii Harms lead to the isolation of four polyacetylenes. By the application of MS, HR-MS, UV/VIS and IR methods and NMR experiments (1H-NMR, 13C-NMR, DEPT135, 1H-1H COSY, HMQC and HMBC) the structures of the four novel diynes were established : 8-Hydroxyheptadeca-4,6-diyn-3-yl acetate (MS-1 (25)), 8-Hydroxyheptadeca-1-ene-4,6-diyn-3-yl acetate (MS-2 (26)), 16-Acetoxy-11- hydroxyoctadeca-17-ene-12,14-diynyl acetate (MS-4 (27)), and 11,16-Diacetoxyoctadeca- 17-ene-12,14-diynyl acetate (MS-5 (28)). Additionally, stigmasterol (42) was isolated and identified by comparison of its spectroscopic data with those of an authentic sample. For the determination of the absolute configuration of MS-4 (27) at C(11) the Mosher method was used. The negative d values for the neighbouring protons suggested the S-configuration. But this result could not be confirmed by the 13C-NMR data since both positive and negative d values were obtained for the neighbouring carbons. Due to the fact, that the results for the protons and the carbons were not consistent the S-configuration for C(11) of MS-4 (27) could not be assigned with certainty. The isolated compounds MS-1 (25), MS-2 (26), and MS-4 (27) were tested for in vitro inhibitory activity against additional parasites like T. cruzi and L. donovani (axenic and in infected macrophages). 42 was not tested because it was a well known phytosterol which is ubiquitous in plants. Neither was MS-5 (28) tested, since the isolated amount was not sufficient. It was found that MS-1 (25) showed in all antiparasitic in vitro assays no higher inhibitory activity than the crude extract. MS-2 (26) showed promising activities in the T. cruzi and L. donovani (axenic and in infected macrophages) assay with IC50 values of 0.2, 0.039, and 0.098 mg/ml, respectively. The respective IC50 values of the standard drugs were 0.62, 0.18, and 0.29. The cytotoxicity was relatively high; therefore the selectivity with SI values of 18.0 and 36.7, respectively (T. cruzi, L. donovani in inf. mac.), was in a moderate range. MS-4 (27) showed also interesting activities in the T. cruzi and L. donovani (axenic) assays with IC50 values of 0.15 and 0.054 mg/ml. The SI value of 145.3 (T. cruzi) was high. Compared with the standard drugs the activities of MS-2 (26) and MS-4 (27) against T. b. rhodesiense and P. falciparum were in a moderate range. MS-1 (25), MS-2 (26), and MS-4 (27) were also tested in the GABAA receptor binding assay. Here MS-4 (27) showed the highest relative specific binding of 158 % at a concentration of 20 mg/ml, followed by MS-2 (26) with 152 % and MS-1 (25) with 138 %. In order to investigate whether the modulatory effect at the GABAA receptor leads to a chanel opening, the isolated compounds were investigated electrophysiologically. It was found that MS-1 (25), MS-2 (26), and MS-4 (27) act as potent positive allosteric modulators at GABAA receptors with a half maximal stimulation at a concentration of 0.6-3.5 mM and a maximal stimulation of 110-450 %. The compounds also showed unique subunit selectivity profiles, the stimulation was independend from the presence of the g subunit and resistant to the benzodiazepine antagonist Ro15-1788. These in vitro data suggested that MS-1 (25), MS-2 (26), and MS-4 (27) may be used to treat diseases of the central nervous system, specifically they may be used to treat states of anxiety, as sedatives/hypnotics, as muscle relaxants and as anticonvulsives (e.g. in epilepsy), and in the treatment of drug addiction. Therefore it was decided to patent the compounds for these indications [174]. All data are on the in vitro level. For a pharmaceutical application in vivo data are needed. Therefore MS-1 (25), MS-2 (26), and MS-4 (27) need to be synthesized in order to get more material for subsequent in vivo experiments.

Item Type: Thesis (Doctoral)
Keywords: Antimalarial Drug, Ecosystem, Medicinal Plants, Chemotaxonomic, Ecological, Ethnopharmacologica, Tanzania
Subjects: Malaria > Diagnosis & treatment
Divisions: Other
Depositing User: Mr Joseph Madata
Date Deposited: 19 Feb 2013 08:51
Last Modified: 19 Feb 2013 08:51
URI: http://ihi.eprints.org/id/eprint/1146

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