Thesis (M.Sc.) - University of KwaZulu-Natal, Pietermaritzburg, 2009. / Full text also available online. Scroll down for electronic link.

The rhizomes of Alepidea amatymbica and Alepidea natalensis are used for medicinal purposes. Because of the increase in demand for these plants the species is becoming scarce. As the seed biology of neither species is well defined, conditions as well as treatments required for optimum germination and vigour were studied. Seeds were exposed to various physical factors such as varying light and temperature conditions and cold stratification, sowing depth and seed storage. The effects of smoke-water, butenolide (3-methyl-2H-furo [2, 3-c] pyran-2-one) a novel smoke compound and chemical substances (gibberellins, kinetin and KNO3) were also tested in order to improve seed germination. Alepidea amatymbica and A. natalensis achieved the highest seed germination (72.5% and 80%, respectively) at 25 °C under a 16 h photoperiod with a mean germination time (MGT) of 18 and 12 days, respectively. Phytochrome studies showed that A. natalensis requires light for germination. Cold stratification (5 °C) for 14-28 days significantly improved the percentage germination of both species (> 90%) compared to non-stratified seeds (control) at 25 °C under a 16 h photoperiod. Sowing A. amatymbica and A. natalensis seeds at a depth of 0.5 cm resulted in higher percentage germination compared to 2.5 cm. The highest emergence rate for A. amatymbica was 40% at a sowing depth of 0.5 cm and the lowest emergence rate was 3% at 2.5 cm. Six months storage of A. natalensis seeds at room temperature (25 ± 2 °C) showed maximum germination (99%) with a MGT of 9 days. Smoke-water treatment of A. amatymbica seeds significantly enhanced germination from 72% to 91%. Smoke and butenolide at 10 °C and 25 °C promoted germination of A. natalensis seeds in a 16 h photoperiod. Smokewater application significantly improved both germination and seedling vigour of A. natalensis. GA3 (10-8 M) was the best treatment for achieving maximum percentage germination of A. natalensis seeds. Antibacterial (two Gram-positive bacteria: Bacillus subtilis, Staphylococcus aureus and two Gram-negative bacteria: Escherichia coli, Klebsiella pneumoniae), antifungal (Candida albicans), anti-inflammatory (COX-1 and -2) and genotoxicity tests (Ames test) were carried out on petroleum ether (PE), dichloromethane (DCM), 80% ethanol (EtOH) and water extracts of the two Alepidea species. Water extracts of A. natalensis rhizomes exhibited high activity (MIC values of 0.78 mg/ml) against the four bacterial strains. High activity was also observed in the PE and DCM leaf extracts of the same plant against the Gram-positive bacteria. The PE and DCM extracts of A. amatymbica rhizomes exhibited the best activity (MIC values of 0.39 mg/ml) against Bacillus subtilis. The rest of the extracts showed low activity (MIC values >1 mg/ml). All the extracts showed activity against Candida albicans, with A. natalensis leaf extracts exhibiting the highest antifungal activity with MIC values of 0.88, 0.20 and 0.78 mg/ml for PE, DCM and EtOH, respectively. EtOH extracts had inhibition less than 40% for both A. natalensis and A. amatymbica. All the PE extracts showed higher inhibitory activity for COX-2 than for COX-1. PE and DCM extracts had percentage inhibitions above 70% in both COX-1 and COX-2 assays. The Ames test for genotoxicity revealed that none of the plant extracts were genotoxic to the Salmonella TA98 tester strain. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

D. Tech. (Department of Chemistry, Faculty of Applied and Computer Sciences)|, Vaal University of Technology. / Medicinal plants provide valuable alternative sources of drugs and drug discovery because many have been used in traditional practices for centuries to manage or treat various forms of ailments. The aim of this study was to evaluate the biological activities of nine medicinal plants used by Zulus in Mabandla village, KwaZulu-Natal province, South Africa to treat inflammation and to isolate selected active compounds against studied pathogens from Alepidea amatymbica and Pomaria sandersonii. The plants were selected on the basis of an ethnobotanical survey based on questionnaire response and verbal interviews that were conducted in Mabandla village with the local traditional healers and herbalists. The isolation of compounds from Alepidea amatymbica and Pomaria sandersonii was based on the bioassay based study which was carried out in this study. Bioassay guided study involving in vitro anti-inflammatory measurement using soya bean derived 15 Lipoxygenase, free radical scavenging capacity against the ABTS●+ radical cation and DPPH● radicals; antimicrobial and bioautography assays against Staphylococcus aureus, ATCC 29213, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, Escherichia coli, ATCC25922, Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus were carried out using the plants extracts, fractions and pure compounds. Isolation of compounds displaying biological activity was carried out by using open column chromatography and preparative thin layer chromatography (PTLC). The compounds were characterised by use of Nuclear Magnetic resonance, (NMR) and Mass Spectrometry (MS). The DPPH sprayed TLC showed that all the nine plants contained antioxidants. Most of which were contained in polar fractions of acetone and methanol. Results of the assays displayed a range of biological activities comparable to the positive controls used for each assay. DPPH● scavenging displayed EC50 values ranging between 1.008 and 467 Kg/ml. The highest activity was observed with the methanol fraction of Berkheya setifera with an EC50 value of 1.008 Kg/ml followed by the crude extract of Gunnera perpensa with EC50 value of 1.069 Kg/ml. Carissa bispinosa hexane fraction had the lowest activity of 467.7 Kg/ml. The Pomaria sandersonii DCM extract had the highest ABTS●+ radical scavenging activity by Pomaria sandersonii DCM extract, (1.273 Kg/ml) for the ethyl acetate, (5.973 Kg/ml) while the hexane fraction from Eucomis autumnalis had the lowest activity (929.4 Kg/ml). The activity of Pomaria sandersonii extracts and fractions demonstrated that the plant contains antioxidants that react with both DPPH and ABTS radicals although higher activities were shown by ABTS as displayed by the lower EC50 values. All the crude fractions and extracts had high to moderate antibacterial activities (20-625 Kg/ml) and anti-fungal activities (20-2500 Kg /ml). Pomaria sandersonii crude and fractions had the highest antimicrobial activity compared to other plants. Some MIC values for P. sandersonii dichloromethane and ethyl acetate fractions (80 Kg/ml in each case) compared well with gentamycin (4 Kg/ml) since they showed same values against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudonomus aeruginosa. The dichloromethane, acetone and methanol fractions were also active (20 Kg/ml) against both Candida albicans and Aspergillus fumigatus. Inhibition of pathogen growth demonstrated by the polar fractions of the studied plants suggested that some of the active compounds would be soluble in water. A total of seven compounds were isolated from Alepidea amatymbica and Pomaria sandersonii. We propose three were new compounds after considering literature search involving closely related research to this investigation. These were two diterpenes from Alepidea amatymbica, namely, 14-acetoxo-12-oxokaur-16-en-19-oic acid labelled as 0657 and 16-hydroxy-kaur-6-en-19-oic acid given the label 06-2 in this study. The third suspected new compound is the chalcone dimer, which is referred to as EM86 in this study from Pomaria sandersonii. EM80-2 was obtained as a mixture of the cis and trans of 2’, 4, 4,’-trihydroxychalcone or 1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)-2-propen-1-one, from Pomaria sandersonii. The three diterpenes, 14-acetoxokaur-16-en-19-oic acid (0652), 13-hydroxy-16-kauren-19-oic acid (06B) and 14-oxokaur-16-en-19-oic acid (06431) were isolated from Alepidea amatymbica for the first time. Isolated compounds were further tested as individual compounds and results showed that 16-hydroxy-kaur-6-en-19-oic acid (06-2) had weak activity against tested bacteria and fungi with the MIC: Staphylococcus aureus (320 Kg/ml) and Candida albicans, (320 Kg/ml). On the other hand 13-hydroxy-kaur-16-en-19-oic acid (06B) was more active against, Staphylococcus aureus (160 Kg/ml) and Aspergillus fumigatus (40 Kg/ml). The yellow compound that was isolated from Pomaria sandersonii, 1-(2, 4-ihydroxyphenyl)-3-(4-hydroxyphenyl)-2-propen-1-one was antimicrobial with the following MICs: Candida albicans: 80 Kg/ml; Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus: 160 Kg/ml and Aspergillus fumigatus: 625 Kg/ml. There were two mixtures referred to as EM 49 and EM 77 from Pomaria sandersonii which were difficult to purify but had anti-microbial inhibitory activities worth reporting. EM49 had MIC against Candida albicans of: 160μg/ml; Pseudomonas aeruginosa: 320 Kg/ml, Escherichia coli: 80μg/ml, Enterococcus faecalis 80μg/ml, and Staphylococcus aureus: 80μg/ml and Aspergillus fumigatus: 320μg/ml. EM 77 had MIC against Escherichia coli: 80 Kg/ml and Cryptococcus neoformans: 80μg/ml. Further work on their purification need to be done since in this research we are just reporting on their high MIC activities. The medicinal plants used to treat inflammation under different disease conditions in the Zulu community of Mabandla village, Kwa-Zulu Natal, South Africa have some relevant biological activities. The various antimicrobial, antioxidant and anti-inflammatory activities support the validity of their healing capacities that the traditional healers of the community claim to possess. Although there is evidence of good antimicrobial, antioxidant and anti-inflammatory activities by the crude extracts, the high levels of sucrose in P. prunelloides and glucose in G. perpensa should be borne in mind when using their decoctions in traditional medicine particularly by diabetic patients. In vitro results for the antioxidant, antinflammtory and antimicrobial activities carried out in this investigation illustrate that the plants can be a source of treatment and management for inflammation related conditions. These therefore justify their use in Zulu traditional medicine. However, in vivo assays should be carried out in order to completely validate claims by the traditional healers that they treat inflammation related conditions. / Vaal University of Technology