Eucomis (Family Hyacinthaceae) are deciduous geophytes with long, narrow leaves and erect, densely packed flower spikes. The bulbs are greatly valued in traditional medicine for the treatment a variety of ailments, and are thus heavily harvested for trade in South Africa's "muthi" markets. Eucomis species propagate relatively slowly from offsets and seed, and this, together with their over-utilization ethnopharmacologically, has led to their threatened status. This investigation focussed mainly on the study of the anti-inflammatory activity of plant extracts prepared from the leaves, bulbs and roots, and the development of suitable tissue culture protocols for the bulk propagation of the species under study. Common underlying symptoms in the majority of ailments treated with traditional remedies prepared from Eucomis species are pain and inflammation. Prostaglandins are the primary mediators of the body's response to pain and inflammation, and are formed from essential fatty acids found in cell membranes. This reaction is catalysed by cyclooxygenase, a membrane-associated enzyme occurring in two isoforms, COX-1 and COX-2. Non-steroidal anti-inflammatory drugs (NSAIDs) act by inhibiting the activity of COX. The use of commercially available COX-1 inhibitors is associated with side-effects, including gastric and renal damage. Selective COX-2 inhibitors do not have these undesired effects, and are thus potentially very valuable to the pharmaceutical industry. The relative inhibitory effects of different extracts of Eucomis species on the activities of purified cyclooxygenase enzyme preparations (COX-1 in sheep seminal vesicles, COX-2 in sheep placenta) were assessed. The COX-1 assay was used to screen extracts from 10 species of Eucomis and one hybrid species at a concentration of 250 μg mℓ ¯¹ in the assay. High levels of anti-inflammatory activity were exhibited by the ethanolic extracts prepared from the dried leaves, bulbs and roots. Aqueous extracts (screened at 500 μgmℓ ¯¹) showed much lower levels of activity. In general, the highest levels of anti-inflammatory activity were observed for the ethanol bulb and root extracts. Comparison of the activity of the bulb extracts from bulbs harvested in summer and winter revealed very little difference in COX-1 inhibitory activity. Eucomis extracts were separated using thin layer chromatography. The plates were developed in a solvent system of benzene : 1,4-dioxan : acetic acid, 90:25:4 and stained with anisaldehyde-sulphuric acid. The TLC fingerprints prepared from these extracts showed different chemical profiles for the leaf, bulb and root extracts, but many similarities between the different species. The position of the active R[f] fractions was determined and correlated with the TLC-fingerprints. The most widely utilized species medicinally, E. autumnalis subspecies autumnalis, was chosen for further investigation. The fluctuation of anti-inflammatory activity with season and physiological age was determined. Young plants were found to have high levels of COX-1 inhibitory activity, particularly in the leaves. As the plant matured, higher levels of activity were associated with the bulb and root extracts. The antiinflammatory activity of the leaf, bulb and root extracts varied slightly throughout the year, with the highest levels detected towards the end of the growing season, shortly before the onset of dormancy. This study of E. autumnalis autumnalis was extended to investigate the effects of environmental conditions on the levels of COX-1 inhibitory activity. The extent to which high temperature and light intensity, fertilization of the plants in summer with Kelpak preparations, and cold storage of the dry bulbs during winter, affected the levels of active compounds accumulated, was determined. Kelpak application decreased the anti-inflammatory activity of the leaf, bulb and root extracts, while high temperature / high light intensity had no significant effect on the COX-1 inhibitory activity of the leaf or bulb extracts. The root extract did show a significant increase in anti-inflammatory activity. Bulbs that were removed from the soil and stored at 10°C exhibited significantly higher COX-1 inhibitory activity than the control bulbs maintained in the soil. Higher COX-1 inhibition was observed in the leaf extracts from these plants when harvested half-way through the growing season. No significant difference was observed at this stage between the bulb and root extracts from the different treatments. Bioassay-guided fractionation (using the COX-1 assay) was used to isolate the active principle(s) in the bulb extract. The bulb material was subjected to serial extraction using a Soxhlet apparatus. The ethyl acetate fraction showed the highest levels of COX-1 inhibition, and this was further fractionated using a Sephadex LH-20 column and a solvent system of cyclohexane : dichloromethane : methanol (7:4:1). The most active fraction from this separation was then purified using semi-preparative TLC and HPLC. The primary compound eluting in this fraction had an IC₅₀ value of 14.4 μgmℓ ¯¹ in the COX-1 assay, and 30.5 μgmℓ ¯¹ in the COX-2 assay. This compound was tentatively characterized as a phenol ring attached to a conjugated hydrocarbon chain (with a molecular weight of 390), and was a potent COX-1 inhibitor. The COX-2 / COX-1 inhibitory ratio was calculated to be 2.1. A second, highly active compound, with IC₅₀ values of 25.7 μgmℓ ¯¹ and 21.8 μgmℓ ¯¹ in the COX-1 and COX-2 assays respectively, crystalized from one of the Sephadex LH-20 column fractions. This compound was identified as a spirostane-type triterpenoid, eucosterol, previously isolated from Eucomis species but not specifically linked to the pharmacological activity of the extracts. This compound showed COX-2 / COX-1 inhibitory ratio of 0.8, indicating that it was a selective COX-2 inhibitor. Two further compounds were identified from this extract, after crystallization from different fractions obtained from Sephadex LH-20 chromatography. These were both homoisoflavanones, 5,7-dihydroxy-6-methoxy-3-(4-methoxy benzyl)-chroman-4-one, and 5,7-dihydroxy-3-(4-methoxy benzyl)-chroman-4-one [eucomin], the latter having been isolated previously. The first compound exhibited very low levels of both COX-1 and COX-2 inhibition, and the second compound (eucomin) exhibited high COX-1, but low COX-2 inhibitory activity. The in vitro propagation of the genus Eucomis was undertaken primarily to provide a source of material for experimentation, and also to optimize this technique for the bulk production of plants for commercial and conservation purposes. Multiple shoot production was initiated from leaf explants, in all species studied. A Murashige and Skoog (MS) medium, supplemented with 100 mg ℓ ¯¹ myo-inositol, 20 g ℓ ¯¹ sucrose, and solidified with 2 g ℓ ¯¹ Gelrite® was used. The optimal hormone combination for shoot initiation in the majority of species was determined to be 1 mg ℓ ¯¹ NAA and 1 mg ℓ ¯¹ BA. Optimal root initiation was demonstrated on media supplemented with 1 mg ℓ ¯¹ IAA, IBA or NAA, depending on species. A continuous culture system using this protocol produced 25-30 plantlets per culture bottle, with 10-25 specimens per bottle available for acclimatization. To maximize plantlet survival, different support media used during the acclimatization process were necessary. Certain species responded best on a vermiculite medium, while perlite (which holds less water) was necessary for the optimal survival rate of other species. Acclimatized plantlets were repotted in a sand : soil mix (1:1). Further experimental work aimed to determine the factors affecting the accumulation of anti-inflammatory compounds in in vitro plantlets. Extracts prepared from in vitro plantlets showed high levels of COX-1 and COX-2 inhibitory activity, with a C0X-2/C0X-1 ratio of 1.1. High levels of sucrose (40 g ℓ ¯¹) significantly increased the number of shoots initiated, but had no effect on the anti-inflammatory activity. Low levels of sucrose (10 g ℓ ¯¹) led to a significant decrease in COX-1 inhibition. Changing the levels of nitrogen in the medium (but not the ratio of nitrate to ammonium ions) had no significant effect on the COX-1 inhibitory activity of the extracts. Callus was initiated from leaf explants and experiments were conducted to maximize callus proliferation. Optimal callus growth occurred on an MS medium supplemented with 100 mg ℓ ¯¹ myo-inositol, 30 g ℓ ¯¹ sucrose, 2 g ℓ ¯¹ Gelrite® , and a hormone combination of 10 mg ℓ ¯¹ 2,4-D and 2 mg ℓ ¯¹ kinetin. Callus cultures maintained in the dark grew best. Callus extracts tested in the COX assays (250 μgmℓ ¯¹) showed a higher level of COX-2 inhibition (69%) than COX-1 inhibition (46%). Lastly, the conclusive identification of the species under study was attempted, using DNA fingerprinting. Protocols were developed for the extraction of DNA from the leaves of Eucomis plants, and the optimization of the AP-PCR technique. Random sequence (10-base) oligonucleotide primers were screened, each primer used singly. Primers were selected on the basis that more than five distinct bands were detected. Differences were detected in the amplification products visualized using nondenaturing agarose gel electrophoresis stained with ethidium bromide. This work provides the basis for further studies into the phylogenetic relationships between the various species (and hybrids) of Eucomis. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1999.

Eucomis L’ Hér. is a genus of 10 species that fall within the Hyacinthaceae family. Eucomis zambesiaca Baker is a summer-blooming bulbous geophyte occurring from northern South Africa to Malawi. Eucomis species are used in southern African traditional medicine for the treatment of various ailments, in particular, pain and inflammation. As a result, the bulbs are heavily harvested for trade in South Africa’s traditional ‘muthi’ markets. Over-collection of Eucomis species has seriously depleted natural populations and now Eucomis plants are among the 15 scarcest medicinal species to be traded. Micropropagation is a useful technique for rapid clonal multiplication of plant material which could potentially yield useful secondary metabolites as well as alleviate the pressure on the wild plant populations. The in vitro induction of storage organs is especially beneficial as it can limit the loss of plants during acclimatization as bulblets are hardier than shoots or plantlets. The aim of this research was to determine optimal growth conditions for bulblet induction of Eucomis zambesiaca. The effect of environmental and physiological parameters on the initiation and growth of bulblets was investigated. These included the effect of temperature, photoperiod, various carbohydrates at different concentrations and combinations as well as various plant growth regulators. Maximum number of bulblets per explant was obtained at 20 °C, with an average of three bulbs p er leaf explant. The average bulblet mass was 57 mg, which was significantly higher than bulblets formed at other tested temperatures. An 8 h light regime was the optimum photoperiod. The highest mean number of bulblets (1.4 per leaf explant) developed under the 8 h photoperiod and the bulblets that formed were large in size. They had a mean bulb diameter of 3.4 mm and a mean bulb weight of 42 mg. Different carbohydrates such as fructose, sucrose and glucose were tested at concentrations of; 1, 3, 6, 9 and 12%. Fructose at a concentration of 3% was found to produce the best results. An average of 1.2 bulbs formed per explant. The mean bulb diameter was 3.4 mm and mean bulb weight was 56.6 mg. Plant growth regulators (GA3, IAA, IBA, NAA, BA, zeatin, iP and others) were tested at concentrations of 1, 2 and 5 mg/L. 1 mg/L IBA was found to be the optimum hormone treatment for bulblet induction. Bulblets were large, had good leaves and well established roots. Medium supplemented with 1 mg/L IBA produced bulblets that had an average bulb diameter of 4.36 mm and a mean bulblet weight of 79.1 mg. Bulblets grown in vitro were transferred to vermiculite and placed in a misthouse to acclimatize. After 2 months the plantlets were transferred to pots containing a sand:soil mixture of 1:1 and placed in a greenhouse. There was a 80 to 90% survival rate. / 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