Thesis submitted in fulfilment of the requirements for the degree Master of Technology: Horticulture in the Faculty of Applied Sciences at the Cape Peninsula University of Technology / The high demand for medicinal plants has favoured over-exploitation of wild plants. The search for alternative and sustainable methods of medicinal plant cultivation is imperative and desirable. Biotechnological approaches particularly hydroponic technology has the potential for large scale plant cultivation and production of secondary metabolites. The current study aims at optimizing the production of antimicrobial secondary metabolites by an indigenous South African medicinal plant species (Helichrysum cymosum) through hydroponics N and K fertilization. In Chapter 1, the conceptual framework and justifications of the study are presented. In Chapter 2 the research objective was to discern the optimal potassium (K) supplement level for H. cymosum by evaluating the effects of different hydroponic K levels on growth, K-leaf content, and anti-Fusarium oxysporum f.sp.glycines (Ascomycota: Hypocreales) and total activities. Six weeks old seedlings of H. cymosum were treated with varied concentrations of K in the form of potassium chloride, potassium nitrate and monopotassium phosphate (58.75, 117.5, 235 and 470 ppm). These concentrations were based on a modification of Hoagland’s hydroponic nutrient formula. Plants were maintained under greenhouse conditions and growth parameters (plant height and number of leaves) were recorded weekly. At 8 weeks post treatment, plants were harvested and fresh weights were recorded and tissue nutrient content analysed. Sub-samples of the aerial parts of plants grown in the different treatments were air dried, extracted with acetone and tested against F. oxysporum. Plants exposed to 235 ppm K showed a marked increase in leaf number, plant height and fresh weight. Overall there was no significant difference (P > 0.05) among the treatments with respect to tissue nutrient content; K ranged from 3.56 ± 0.198 to 4.67 ± 0.29 %. The acetone extraction yield increased with increasing K fertilization: 58.75 ppm (16.67 ± 2.35 mg), 117.5 ppm (22.5 ± 4.79 mg), 235 ppm (210 ± 38.5 mg) but dropped to 40 ± 4.08 mg at 470 ppm K. Results from the anti-F. oxysporum bioassay showed that 58.75 and 235 ppm K treatments produced the most bioactive acetone extracts; MIC values of 0.49 and 0.645 mg/l, respectively. Acetone extracts obtained from plants exposed to 235 ppm K yielded the highest total activity, comparatively (P < 0.05). In conclusion, the optimum nutrient K level for growing H. cymosum hydroponically was 235 ppm. Chapter 3 focused on another important macro nutrient N and the objective was to determine the optimum nutrient requirements for growing the medicinal plant, Helichrysum cymosum (L.) (Asteraceae), hydroponically. Experiments were conducted to assess the effects of varied nitrogen (N) concentrations supplied as nitrate and ammonium on growth, tissue nutrient content, antimicrobial and total activities of acetone extracts of aerial parts. Treatments were based on a modified Hoagland’s nutrient formula. Six week old rooted cuttings were treated with 52.5 ppm, 105 ppm, 210 ppm and 420 ppm of N. Leaf number and stem height (cm) were recorded at weekly intervals and leaf analysis conducted. The effects of N treatments on plant growth parameters varied significantly among treatments; 52.5 ppm of N yielded the tallest plants (height) [19.4 ± 0.7 cm], while 105 ppm N yielded the maximum leaf number (68.1 ± 6.2) as well as maximum fresh weight of aerial parts was obtained with 105 ppm (15.12 ± 1.68 g). Nitrogen content of plant tissue ranged between 0.53 ± 0.03 and 4.74 ± 0.29% (d, f, 3, 12; f=14; P ≤ 0.002) depending on treatments. Powdered aerial parts (5 g) of H. cymosum obtained from the different N treatments were extracted with 100 ml of acetone. N treatment significantly affected the yield of crude extracts, which ranged from 87.5 ± 15.5 (52.5 ppm) to 230 ± 23.5 mg (105 ppm). Acetone extracts of plants that were exposed to varied N treatments were screened for anti-Fusarium oxysporum activity using minimum inhibitory concentration (MIC) method. The MIC value (0.073 ± 0.014 mg/ml) obtained with acetone extracts of plants exposed to 52.5 ppm N was significantly lower compared to the MICs of the other N treatments (105 [0.47 ± 0 and 0.705 ± 0.135 mg/ml], 210 [0.234 and 0.47 mg/ml] and 420 ppm [0.29 ± 0.101 mg/ml]) at 24 and 48 hours respectively. However, the total activities of extracts obtained among the four N treatments, which ranged from 0.062 ± 0.02 to 0.26 ± 0.06 ml/g was not statistically different at 24 or 48 hours (P > 0.05). LC-MS analysis of acetone extracts of H. cymosum plants obtained from the four treatments hinted that known anti-microbial agents such as apigenin, quercetin, kaempferol, helihumulone and quinic acids were present in the extracts and the quantity of helihumulone increased with increased nutrient N level. These results suggest that H. cymosum may be cultivated hydroponically and that the antimicrobial activity and/or the phytochemical profile of the crude acetone extracts is affected by nutrient nitrogen levels. Hydroponic cultivation of plants may be able to alleviate to an extent the pressure on wild medicinal plants.

Helichrysum longifolium and H. pedunculatum belong to the Astereceae family and are used extensively in folkloric medicine in South Africa to manage stress-related ailments and as dressings for wounds normally encountered in circumcision rites, bruises, cuts and sores. The in vitro antibacterial time-kill studies, the synergistic potentials, the phytochemical screenings and antioxidant potentials as well as the isolation of the bioactive compounds from the extracts of these two plants were carried out in this study. The in vitro antibacterial activities and time kill regimes of crude extracts of H. pedunculatum was assessed. The extracts was active against both Gram positive and Gram negative bacteria tested at a concentration of 10 mg/ml. Minimum Inhibitory Concentration (MIC) values for all the susceptible bacteria ranged between 0.1 – 35 mg/ml. The average log reduction in viable cell count in time kill assay ranged between 0.17 Log10 to 6.37 Log10 cfu/ml after 6 h of interaction, and between 0.14 Log10 and 6.99 Log10 cfu/ml after 12 h interaction in 1 × MIC and 2 × MIC of the extract. The effect of the aqueous extract was only bacteriostatic on both reference and environmental strains and the clinical isolates were outrightly resistant to aqueous extract. This is worrisome and this could be one reason why, there is an incidence of high death rate resulting from circumcision wounds infection even after treating such wounds with H. pedunculatum leaf. In vitro antibacterial time kill studies of extracts of H. longifolium was assessed. All test bacteria were susceptible to the methanol extract, while none was susceptible to the aqueous extract. Two of the test bacteria were susceptible to the ethyl acetate extract, while ten and seven were susceptible to the acetone and chloroform extracts respectively at the test concentration of 5 mg/ml. The minimum inhibitory concentrations (MICs) ranged between 0.1 and 5.0 mg/ml, while minimum bactericidal concentrations (MBCs) ranged between 1.0 and >5 mg/ml for all the extracts. Average log reductions in viable cell counts for all the extracts ranged between 0.1 Log10 and 7.5 Log10 cfu/ml after 12 h interaction at 1 × MIC and 2 × MIC. Most of the extracts were rapidly bactericidal at 2 × MIC achieving a complete elimination of most of the test organisms within 12 h exposure time. The effect of combinations of the crude extracts of H. pedunculatum leaves and eight antibiotics was investigated by means of checkerboard and time-kill methods. In the checkerboard method, synergies of between 45.83-56.81 percent were observed and this is independent of Gram reaction, with combinations in the aqueous extract yielding largely antagonistic interactions (18.75 percent). The time kill assay also detected synergy that is independent of Gram reaction with a ≥ 3Log10 potentiation of the bactericidal activity of the test antibiotics. We conclude that the crude leaf extracts of H. pedunculatum could be potential source of broad spectrum antibiotics resistance modulating compounds. The interactions between crude extracts of H. longifolium in combination with six first-line antibiotics using both the time-kill and the checkerboard methods were carried out. The time-kill method revealed the highest bactericidal activity exemplified by a 6.7 Log10 reduction in cell density against Salmonella sp. when the extract and Penicillin G are combined at ½ × MIC. Synergistic response constituted about 65 percent, while indifference and antagonism constituted about 28.33 percent and 6.67 percent in the time kill assay, respectively. The checkerboard method also revealed that the extracts improved bactericidal effects of the antibiotics. About 61.67 percent of all the interactions were synergistic, while indifference interactions constituted about 26.67 percent and antagonistic interactions was observed in approximately 11.66 percent. The in vitro antioxidant property and phytochemical constituents of the aqueous crude leaf extracts of H. longifolium and H. pedunculatum was investigated. The scavenging activity on superoxide anions, DPPH, H2O2, NO and ABTS; and the reducing power were determined, as well as the flavonoid, proanthocyanidin and phenolic contents of the extracts. The extracts exhibited scavenging activity in all radicals tested due to the presence of relatively high total phenol and flavonoids contents in the extracts. Our findings suggest that H. longifolium and H. pedunculatum are endowed with antioxidant phytochemicals and could serve as a base for future drugs. Bioactivity-guided fractionation of the leaves of H. longifolium and H. pedunculatum yielded two known compounds. From the n-hexane fraction of H. longifolium a compound was isolated (Stigmasterol) and from the ethyl acetate fraction of H. pedunculatum another compound (β-sitosterol) was isolated. The compounds were isolated and identified using various techniques. The antimicrobial, anti-inflammatory, antioxidant, analgesic and anti-pyretic activities of these compounds have been reported in literatures. In general, the experiments and tests conducted in this study appear to have justified the folkloric medicinal uses of H. longifolium and H. pedunculatum for the treatment of stress related ailments and wound infections and make a substantial contribution to the knowledge base of the use of herbal medicine for the treatment of the microbial infections.