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Quantitative and qualitative optimization of antimicrobial bioactive constituents of Helichrysum cymosum using hydroponics 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.