Memoria para optar al título profesional de Ingeniero Agrónomo / La minería genera una gran cantidad de residuos, llamados relaves mineros, que pueden presentar altos contenidos de metales, siendo un potencial riesgo para el medioambiente. Una de las estrategias que se pueden utilizar para disminuir o mitigar la alta concentración de metales, es la fitorremediación, la cual utiliza plantas tolerantes a metales, que a través de su crecimiento y absorción de estos elementos, los retienen en sus tejidos, evitando la dispersión por el viento o lixiviación. Dentro de la estrategia de fitorremediación, se distinguen dos tipos, la fitoestabilización, donde la planta acumula metales preferentemente en la raíz y la fitoextracción, donde la planta acumula metales preferentemente en la parte aérea. Además, la fitorremediación puede ser complementada con la adición de enmiendas orgánicas, para favorecer el desarrollo de las plantas y la absorción de metales. El objetivo general del trabajo es evaluar la especie Carpobrotus aequilaterus como cubierta vegetal en el relave minero y el efecto de la aplicación de ácidos fúlvicos como estrategia de fitorremediación. El estudio se llevó a cabo en la Facultad de Ciencias Agronómicas de la U. de Chile, mediante un ensayo en macetas de 2,5 litros, en condiciones de exterior, entre los meses de enero a mayo 2015. Se aplicó como enmienda los ácidos fúlvicos en dosis total 1: 7 mg maceta-1 y dosis total 2: 14 mg maceta-1. Las dosis fueron aplicadas en forma parcializada y disueltas en el agua de riego. Los tratamientos fueron los siguientes, T0: relave minero + dosis 1; T1: relave minero + dosis 2; T2: relave minero + doca; T3: relave minero + doca + dosis 1; T4: relave minero + doca + dosis 2. Al final del ensayo, se determinaron las concentraciones de metales (Cd, Cr, Cu, Fe, Mn, Ni, Pb y Zn) en las plantas y la biomasa de parte aérea y raíz. En los relaves mineros, no se observaron concentraciones totales particularmente altas de metales, excepto para Cu, que también presentó mayor disponibilidad. Estos relaves carecen de fertilidad en relación a bajos niveles de N, P, K disponibles y materia orgánica. El pH fue ligeramente ácido y la conductividad eléctrica indica que el relave minero es no salino. Los metales Cd, Cr, Ni y Pb se encontraron bajo los límites de detección del instrumento de medición, por lo cual no fueron evaluados. La adición de ácidos fúlvicos, no mostró ningún efecto en la absorción de Cu y Zn en planta, a los 60 y 120 días de ensayo. Sólo en raíz, los ácidos fúlvicos con dosis 1, aumentaron los niveles de Fe y Mn. Respecto a la generación de biomasa, la adición de ácidos fúlvicos no contribuyó a un mayor crecimiento. Se observó un índice de transporte de metales a la parte aérea menor a 1 para Cu y Fe, a los 60 y 120 días de ensayo, esto indica que la planta presentó una mayor concentración en las raíces, mostrando una capacidad de fitoestabilización de estos elementos. En contraste, para Mn y Zn, los valores del índice de transporte fueron mayores a 1, lo que indica una capacidad de fitoextracción de estos elementos. Respecto al factor de bioconcentración los resultados obtenidos fueron menores a 1 para todos los elementos, excepto para el Mn, lo que permite declarar a Carpobrotus aequilaterus como una planta potencialmente acumuladora de este element

The use of medicinal plants in the treatment of infectious diseases is an acceptable and popular phenomenon in South Africa and worldwide. The potential of extracts from these plants as antimicrobial agents necessitates their scientific evaluation. Therefore, this study evaluated the antimicrobial activity of Carpobrotus edulis; Cotyledon orbiculata; Datura stramonium; Dodonaea angustifolia; and Zanthoxylum capense against Porphyromonas gingivalis; Tannerella forsythensis and Actinobacillus actinomycetemcomitan. Given that most currently used drugs are cytotoxic, the possible cytotoxic effect of these medicinal plants on human periodontal ligaments fibroblasts and human gingival fibroblasts was also determined. The modified broth micro dilution method incorporating resazurin as an indicator of cell growth in 96-well microtitre plates was used to determine the antibacterial activity of the test plants extracts. The extracts showed some significant antibacterialactivity against Porphyromonas gingivalis, Tannerella forsythensis and Actinobacillus actinomycetemcomitans. The activity varied with respect to individual test bacteria. Their minimum inhibitory concentration (MIC) values ranged from 10 to 0.01mg.mr-l. All bacteria tested were inhibited by the highest concentration of the selected plant extracts ( 1 Omg.ml-1). The MTT [3-( 4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] method was used to determine the cytotoxic effect of test extracts. All extracts tested with the exception of Carpobrotus edulis, inhibited the growth of both human periodontal ligament fibroblasts and human gingival fibroblasts at the tested dilutions, with the cytotoxicity levels being directly related to the concentration of the extracts. The extract of Carpobrotus edulis, inhibited the tested cells at 10-1 for human periodontal ligaments fibroblasts and ≥ 2: 10-2 for human gingival fibroblasts. All other tested concentrations of Carpobrotus edulis extracts enhanced the growth of both human periodontal ligaments and human gingival fibroblasts. The study provided a scientific evidence of the important role that medicinal plants play as antibacterial agents in the treatment of oral infections. / Dissertation (MSc)--University of Pretoria, 2009. / Community Dentistry / unrestricted

Philosophiae Doctor - PhD / South African Carpobrotus species have been found to contain hydrolysable tannins,various flavonoids e.g. rutin and hyperoside, phytosterols and aromatic acids which have a diverse range of pharmacological properties including antimicrobial and, antioxidant activities. The main aim of the thesis was to determine the natural products in C. mellei and C. quadrifidus using chromatographic techniques and electrochemical analysis. The antioxidant activity of both Carpobrotus species was determined by using a superoxide dismutase (SOD) biosensor. ESI-LC-MS was used to separate and determine flavonoids in C. mellei and C. quadrifidus. 8 flavonoid compounds: catechin, epicatechin, epicatechin-epicatechin, coumarylquinic acid, isorhamnetin, quercetin-hexose (hyperoside), rutin and myricetin-deoxyhexose were identified. Cyclic and square wave voltammetry were used to detect flavonoids from C. mellei and C. quadrifidus. Catechin was detected in the ethyl acetate extract of C. mellei and C. quadrifidus. The oxidation potential of the plant extracts were observed at +150.6 mV to +1072.6 mV. The oxidation mechanism proceeds in sequential steps, related to the catechol moiety, -OH groups in C ring and the resorcinol group. The oxidation process of the catechol moiety involves a two electron - two proton reversible reaction and forms o-quinone. This occurs first at low potential and is a reversible reaction. The hydroxyl group in the C ring and resorcinol group oxidise there after and undergo an irreversible reaction. UV-vis and FTIR spectroscopy were used to confirm the presence of catechin in the ethyl acetate extract of both plants.UV-visible spectroelectrochemistry confirmed the oxidation process of catechin at constant potential. Since C. mellei and C. quadrifidus were confirmed to contain flavonoids by ESI-LC-MS and electrochemical analysis, the antioxidant activity was further investigated using a SOD biosensor. The superoxide dismutase (SOD) enzyme was immobilised with 1% Nafion on a platinum electrode. Detection limit and sensitivity of the SOD biosensor were found to be 0.03918 μmol L-1 and 1.44 μA(μmol L-1)-1, respectively. The results showed that C. mellei and C. quadrifidus have antioxidant activity, with relative antioxidant capacity (RAC) of 24% and 42%, respectively. May 2009

The human immunodeficiency virus (HIV) is one of the most common and dreaded diseases of the 21th century. Today, the disease is still spreading with increasing incidence. “Since the beginning of the epidemic, almost 75 million people have been infected with the HIV virus and about 36 million people have died of HIV. Globally, 35.3 million [32.2–38.8 million] people were living with HIV at the end of 2012” (http://www.who.int/gho/hiv/en/). Several studies have been conducted on herbs under a multitude of ethnobotanical grounds. The use of medicinal plants for the management of HIV has become a common practice especially, in the Eastern Cape Province of South Africa (Wilfred Otang Mbeng, 2013 PhD thesis, UFH). At the beginning of this programme, an ethnomedicinal survey of plants used for the management of HIV infection was carried out in targeted areas of the Province and information on the names of plants, the parts and the methods of preparation were collected. The survey revealed that 18 species representing 12 families were found to be commonly used for the management of HIV, as well as other opportunistic diseases such as tuberculosis, diabetes mellitus, sores, high blood pressure, etc. Carpobrotus edulis was selected for this research because it was the most frequently used in the Province. The foliar micro morphological contents of the plant, its phytochemical and antioxidant activity, in vitro antimicrobial activity, inhibitory effect against HIV-1 protease and reverse transcriptase, mechanisms of action and cytotoxicity were investigated. In terms of the foliar micro morphological contents in plants, an electron microscopy scanning (SEM) was completed. Investigation revealed that both glandular tricomes and calcium oxalate crystals (CaOX) were observed. Consequently, it is hypothesized that the bioactive therapeutic compounds secreted by C. edulis may be produced in the glandular trichomes. An investigation of phytochemical content of the plant extracts (C. edulis) was completed using four solvent extracts (hexane, acetone, ethanol and water). Results of the phytochemical analysis showed that proanthocyanidins (86.9 ± 0.005%) where highest in the aqueous extract with phenolics at 55.7 ± 0.404% in acetone extract, tannin at 48.9 ± 0.28% in ethanol extract, while the hexane extract had the highest levels of flavonoids (0.12 ± 0.05%) and flavonols (0.12 ± 0.05%). Antioxidant studies of the various extracts revealed that aqueous and ethanol extracts were found to be the best solvents for antioxidant activity in C. edulis leaves. GC-MS analysis of the essential oil from C. edulis leaves revealed that the essential oil contained at least 28 compounds. These included, in order of abundance: Oxygenated monoterpenes (36.61%); fatty acids esters (19.25%); oxygenated diterpenes (19.24%); monoterpenes (10.6%); sesquiterpenes (3.58%); and diterpenes (1.43%). Similarly, a GC-MS analysis of the crude hexane, acetone and ethanol extracts from C. edulis leaves identified a total of 59 compounds. Of the 59 compounds, 12 major phyto-metabolites that are active against infectious diseases were identified. To comfirm the potential use of C. edulis to treat infectious disease, antifungal activity of the crude essential oil extract and the four solvent extracts were tested against Candida albicans, Candida krusei, Candida glabrata, Candida rugosa and Cryptococcus neoformans strains. The essential oil extract was found to be the most active against all the fungal strains tested and performed better than the four extracts used various solvents used to extract (hexane, acetone, ethanol and water) the C. edulis leaves were tested for antibacterial and anti HIV-1 reverse transcriptase (RT) activity. The results indicated that both gram-positive and gram-negative isolates were inhibited by the extracts (hexane, acetone and ethanol) but antimicrobial activity was observed for the water extract. The lowest minimum inhibitory concentration values were obtained for the ethanol extract, followed by acetone and hexane extracts. No inhibition of HIV-1 reverse transcriptase was observed for any of the leaf extracts, even up to concentrations of 16 mg/ml. The potential inhibitory activities of the various solvent extracts against HIV-1 protease were evaluated at four different concentrations (16, 1.6, 0.16 and 0.016 mg/ml). Results indicated that the water extract showed almost 100% inhibition of HIV-1 protease activity, with an IC50 of 0.86 mg/ml leaf extract. Other solvent extracts (hexane, acetone and ethanol) however, did not show any inhibition activity above that observed for the DMSO control. The metabolic components in the water extract were subjected to LC-MS/MS analysis, which identified at least 91 compounds present in the water extract. Further studies involving the molecular modelling need to be carried out to confirm the inhibitory potential of these compounds. The cytotoxicity of the water extract of C. edulis leaves was also screened using human Chang liver cells at concentrations ranging bewteen 0.005 mg/ml and 1 mg/ml. Results indicated that the water extracts were not toxic. In conclusion the results from this study support the use of water extracts of C. edulis leaves by traditional healers to treat HIV infections and have identified possible mechanisms of action of the water extracts of C. edulis.

Philosophiae Doctor - PhD / South African Carpobrotus species have been found to contain hydrolysable tannins, various tlavonoids e.g. rutin and hyperoside, phytosterols and aromatic acids which have a diverse range of pharmacological properties including antimicrobial and, antioxidant activities. The main aim of the thesis was to determine the natural products in C. mellei and C. quadrifidus using chromatographic techniques and electrochemical analysis. The antioxidant activity of both Carpobrotus species was determined by using a superoxide dismutase (SOD) biosensor. ESI-LC-MS was used to separate and determine tlavonoids in C. mellei and C. quadrifidus. 8 tlavonoid compounds: catechin, epicatechin, epicatechin-epicatechin, coumarylquinic acid, isorhamnetin, quercetin-hexose (hyperoside), rutin and myricetin-deoxyhexose were identified. Cyclic and square wave voltammetry were used to detect tlavonoids from C. mellei and C. quadrifidus. Catechin was detected in the ethyl acetate extract of C. mellei and C. quadrifidus. The oxidation potential of the plant extracts were observed at +150.6 mV to +1072.6 mV. The oxidation mechanism proceeds in sequential steps, related to the catechol moiety, -OH groups in C ring and the resorcinol group. The oxidation process of the catechol moiety involves a two electron - two proton reversible reaction and forms o-quinone. This occurs first at low potential and is a reversible reaction. The hydroxyl group in the C ring and resorcinol group oxidise there after and undergo an irreversible reaction. UV-vis and FTIR spectroscopy were used to confirm the presence of catechin in the ethyl acetate extract of both plants. UV -visible spectroelectrochemistry confirmed the oxidation process of catechin at constant potential. Since C. mellei and C. quadrifidus were confirmed to contain flavonoids by ESI-LC-MS and electrochemical analysis, the antioxidant activity was further investigated using a SOD biosensor. The superoxide dismutase (SOD) enzyme was immobilised with 1% Nafion on a platinum electrode. Detection limit and sensitivity of the SOD biosensor were found to be 0.03918 umol L-' and 1.44 !lA (umol i.'):', respectively. The results showed that C. mellei and C. quadrifidus have antioxidant activity, with relative antioxidant capacity (RAC) of 24% and 42%, respectively.

La llegada de plantas exóticas fuera de su rango de distribución original y su posterior asentamiento en ecosistemas naturales no es una excepción en las Islas Baleares. En la actualidad tenemos 308 especies no nativas naturalizadas y subespontáneas que representan el 16% del total de la flora Balear. En esta tesis se ha evaluado el estado de la cuestión a nivel global y local. Se ha confeccionado el primer catálogo de flora introducida y se ha analizado cuales de ellas son más abundantes y peligrosas; así como también qué ecosistemas son más vulnerables a la introducción de éstas. También se ha profundizado en el conocimiento de dos de las especies exóticas más peligrosas en el litoral Balear: Carpobrotus edulis y C. affine acinaciformis. Se ha evaluado su influencia sobre la polinización, su tasa de crecimiento bajo diferentes escenarios ambientales, se ha confeccionado un modelo de crecimiento y se ha evaluado su impacto sobre la composición y estructura de las comunidades nativas. / L'arribada de plantes exòtiques fora del seu rang de distribució original i el seu posterior assentament a ecosistemes naturals no és una excepció a les Illes Balears. A l'actualitat tenim 308 espècies no natives naturalitzades i subespontànies que representen el 16% del total de la flora Balear. En aquesta tesi s'ha avaluat l'estat de la qüestió a nivell global i local. S'ha confeccionat el primer catàleg de flora introduïda i s'han analitzat quines d'elles són més abundants i perilloses; així com també quins ecosistemes són més vulnerables a la introducció d'aquestes. També s'ha profunditzat en el coneixement de dues de les espècies exòtiques més perilloses del litoral Balear: Carpobrotus edulis y C. affine acinaciformis. S'ha avaluat la seva influència damunt els vectors de pol·linització, la taxa de creixement a diferents escenaris ambiental, s'ha confeccionat un model de creixement, i s'ha avaluat el seu impacte damunt la composició i estructura de les comunitats natives. / The constant arrival of exotic plants into areas outside their original distribution range and further settlement in natural ecosystems is not an exception in the Spanish Balearic archipelago. At the moment we have 308 non native species (naturalized and casual), the 16% from the total Balearic flora. This thesis has evaluated the exotic plant problematic at global and local level. We have elaborated the first exotic plants list, its distribution and abundance, as well as its degree of impact on the more vulnerable environments. We went deeply into the knowledge of two very invasive plants along the coast of the Balearic islands: Carpobrotus edulis y C. affine acinaciformis. We have evaluated its influence over pollen transmission, its growth rate at different environmental conditions, we have too elaborated a non liner growth model and its impact on composition and structure in natural communities