[ES] La presente Tesis Doctoral se ha centrado en el aislamiento y caracterización de materiales celulósicos y extractos activos, procedentes de las cascarillas de arroz y café, y su incorporación a películas de almidón y mezclas compatibilizadas de almidón-PLA, para mejorar sus propiedades funcionales como materiales para el envasado de alimentos. Las fibras de celulosa (CF) se obtuvieron mediante tratamiento alcalino y de blanqueo, con un rendimiento de 41 y 53 g fibras/100 g cascarilla, respectivamente para cascarilla de arroz y café. Los nanocristales de celulosa (CNC) se aislaron de las fibras mediante hidrólisis ácida, con un rendimiento del 5% respecto a las fibras y con alta cristalinidad (90-92%), resistencia térmica y relación de aspecto (L/d: 20-40). Los compuestos activos se obtuvieron mediante extracción hidrotérmica (180 ºC; 9,5 bares), con un rendimiento de 17-18 g/ 100 g de cascarilla. Dichos extractos exhibieron capacidad antioxidante (EC50: 5,37-5,29 mg sólidos extraídos/ mg DPPH) y antimicrobiana (cuantificada en términos de concentración mínima inhibitoria: MIC) frente a L. innocua (MIC: 48-52 mg polvo/mL) y E. coli (MIC: 50-66 mg polvo/mL). Los materiales celulósicos procedentes de cascarilla de arroz y café se incorporaron a películas de almidón termoplástico (TPS), obtenidas mediante mezclado en fundido y moldeo por compresión. El módulo elástico aumentó un 186 y 121% cuando se incorporó a la matriz un 1% (p/p) de CNC de cascarilla de arroz y café, respectivamente. Del mismo modo, las CF se añadieron a las películas de TPS al 1, 5 y 10 pt%. Ambas CF aumentaron la rigidez y redujeron la extensibilidad de los films, aunque las CF de cascarilla de café mantuvieron mejor la ductilidad al 1 y 5% (p/p). La permeabilidad al vapor de agua de las películas de TPS no se redujo en los materiales compuestos, aunque la permeabilidad al oxígeno se redujo en aproximadamente un 17%. Al incorporar extractos activos a los films de almidón, mejoraron sus propiedades de tracción; el módulo elástico aumentó un 350%, a la vez que se hicieron menos extensibles. Las fibras de celulosa de ambos residuos fueron más efectivas como agentes de refuerzo en los films con extractos sólidos que en los de almidón solo. Se estudiaron también mezclas de almidón-PLA utilizando como compatibilizador policaprolactona funcionalizada con anhídrido maléico y/o glicidil metacrilato (PCLMG o PCLG). Se analizó el efecto de la proporción de PLA en la mezcla (20 y 40% respecto al almidón), y la de ambos compatibilizadores (2,5 y 5%), en las propiedades de los films. Los análisis de la microestructura, el comportamiento térmico y las propiedades funcionales (mecánicas, ópticas y de barrera) de los films, demostraron que sustituir el 20% del almidón por PLA e incorporar el 5% de PCLG podría ser una buena estrategia para obtener materiales adecuados para envasado de alimentos. Además, se estudió el efecto de la adición de rellenos celulósicos (CF y CNC) y del extracto antioxidante de cascarilla de café en la mezcla de almidón-PLA compatibilizada seleccionada. Las propiedades antioxidantes de los films se probaron a través de su eficacia para preservar al aceite de girasol de la oxidación. Se observaron diferencias significativas en las propiedades funcionales de los films cuando los CNC se incorporaron mediante dos métodos diferentes. El efecto de refuerzo de los materiales celulósicos en mezclas de S-PLA fue menos notable que en las películas de almidón, probablemente debido a la superposición del efecto de refuerzo de PLA. El extracto antioxidante no mejoró el comportamiento mecánico en la mezcla, pero le confirió capacidad antioxidante, adecuada para aplicaciones en el envasado de alimentos. / [CAT] La present Tesi Doctoral s'ha centrat en l'aïllament i caracteritzaciò de materials cel.lulòsics i extractes actius, procedents de pellorfa d'arròs i café, i la seua incorporació a pel·lícules de midó i mescles compatibilitzades de midò-PLA, per a millorar les seues propietats funcionals com materials per al envasat d'aliments. Les fibres de cel.lulosa (CF) s'obtingueren mitjançant tractament alcalí i de blanqueig, amb un rendiment de 41 i 53 g fibres/100g pellorfa, respectivament per a pellorfa d'arròs i cafè. Els nanocristalls de cel·lulosa (CNC) es van aïllar de les fibres de cel·lulosa per mig d'hidròlosi àcida, amb un rendiment del 5% respecte a les fibres; en tots dos casos, amb alta cristal·línitat (90-92%), resistència tèrmica i relaciò d'aspecte (L/d: 20-40). Els composts actius s'obtingueren mitjançant l'extracció hidrotèrmica (180 ºC; 9,5 bars), amb un rendiment del 17-18 g/100 g de pellorfa. Aquests composts exhibiren capacitat antioxidant (EC50: 5,37-5,29 mg extracte solit/ mg DPPH) i antimicrobiana, (quantificada en termes de concentració mínima inhibitòria: CMC) enfront a L. innocua (MIC: 48-52 mg pols/mL) i E. coli (MIC: 50-66 mg pols/ mL). Els materials cel·lulòsics procedents de pellorfa d'arròs i cafè es van incorporar a pel·lícules de midó termoplàstic (TPS), obtingudes mitjançant mesclat en fos i modelatge per compressió. El mòdul elàstic va augmentar un 186 i 121% quan es va incorporar a la matriu un 1 pt% CNC de pellorfa d'arròs i café, respectivament. De la mateixa manera, les CF es van afegir a les pel·lícules de TPS al 1, 5 i 10 pt%. Ambdues CF va augmentar la rigidesa de les pel·lícules i es va reduir la seua capacitat d'estirament. No obstant, les CF de pellorfa de cafè mantingueren millor la ductilitat al 1 i 5%. La permeabilitat al vapor d'aigua de les pel·lícules de TPS no es va reduir en els materials compostos, encara que la permeabilitat a l'oxigen es va reduir en aproximadament un 17%. A l'incorporar extractes actius a les pel·lícules de midó, milloraren les propietats de tracció de les pel·lícules ; el mòdul elàstic va augmentar un 350%, mentre que les pel·lícules es feren menys extensibles. Les CF dels dos residus foren més efectives com agents de reforç en pel·lícules que contenien extractes actius, que en pel·lícules de midó pur. També es van estudiar mescles de midò-PLA utilitzant com a compatibilitzador policaprolactona funcionalitzada amb anhídrid maleic i/o glicidil metacrilat (PCLMG o PCLG). Es va analitzar l'efecte de la proporció de PLA en la mescla (20 i 40% respecte al midó), i de la tots dues compatibilitzadors (2,5 i 5%), en les propietats de les pel·lícules. Els anàlisis de la microestructura, el comportament tèrmic i les propietats funcionals (mecàniques, óptiques i de barrera) de les pel·lícules, demostraren que substituir el 20% del midó per PLA i incorporar el 5% de PCLG podria ser una bona estratègia per a obtindré pel·lícules adequades per a l'envasat d'aliments. A demés, es va estudiar l'efecte de l'addició de reforçaments cel·lulòsics (CF i CNC) i extracte antioxidant de pellorfa de cafè, en mescles de midó-PLA compatibilitzades. Les propietats antioxidants de les pel·lícules s'analitzaren a través de la seua eficàcia per a preservar de l'oxidació l'oli de gira-sol. S'observaren diferències significatives en les propietats funcionals de les pel·lícules quan els CNC s'incorporaren mitjançant dos mètodes diferents. L'efecte de reforç dels materials cel·lulòsics en mescles de S-PLA va ser menys notable que en les pel·lícules de midó, provablement degut a la superposició de l'efecte de reforç del PLA. L'extracte antioxidant no va millorar el comportament mecànic en les mescles, però li va conferir la capacitat antioxidant adequada per a aplicacions a l'envasat d'aliments. / [EN] This Doctoral Thesis has focused on the isolation and characterisation of cellulosic materials and active extracts from coffee and rice husks, and their incorporation into starch films and starch-PLA compatibilised blend films in order to improve their functional properties as food packaging materials. Cellulose fibres were obtained through alkali and bleaching treatment with a final yield of 41 and 53 g fibres/100 g husk, respectively for rice and coffee husks. Cellulose nanocrystals were isolated from the bleached fibres by acid hydrolysis, with a yield of 5% with respect to bleached fibres, in both cases, with high crystallinity (90-92%), thermal resistance and aspect ratio (L/d: 20-40). The active compounds were obtained by hydrothermal extraction (180 ºC, 9.5 bar) with yields of 17 -18 g/100 g husks. They exhibited antioxidant properties (EC50: 5.37-5.29 mg extract solids/mg DPPH) and antibacterial activity against L. innocua (MIC: 48-52 mg powder/mL) and E. coli (MIC: 50-66 mg powder/mL), which were quantified in terms of the minimal inhibitory concentration. Cellulosic material from rice and coffee husks were incorporated into thermoplastic starch films (TPS) by melt blending and compression moulding. The elastic modulus increased by 186 and 121% when 1 wt% of cellulose nanocrystals (CNC) from rice and coffee husks, respectively, was incorporated into the matrix. Likewise, cellulose fibres (CF) were incorporated into TPS films at 1, 5 and 10 wt%. Both CF increased the film stiffness while reducing its stretchability. However, CF from coffee husk better maintained the film ductility at 1 and 5 wt%. The water vapour permeability of TPS films was not reduced in composites, although oxygen permeability was lowered by about 17%. When active extracts were incorporated into starch films, they improved the tensile properties; the elastic modulus increased by about 350%, while films became less stretchable. The cellulosic fibres from both residues were more effective as reinforcing agents in films containing extract solids than in net starch films. Starch-PLA blend films were also studied using grafted polycaprolactone with maleic anhydride and/or glycidyl methacrylate (PCLMG or PCLG) as compatibilisers. The effect of both the PLA ratio in the blend (20 and 40% with respect to starch) and the amount of both compatibilisers (2.5 and 5%) on the film properties was analysed. The analyses of microstructure, thermal behaviour and functional properties (mechanical, optical and barrier) of the films led to the conclusion that substituting 20% of the starch by PLA, and incorporating 5% of PCLG would be a good strategy to obtain films suitable for food packaging. The effect of the addition of cellulosic fillers (CF and CNC) and antioxidant aqueous extract from coffee husk to compatibilised starch-PLA blends was also studied. The antioxidant properties of the films were tested through their efficacy at preserving sunflower oil from oxidation. Significant differences were observed in the functional properties of the films when CNC was incorporated by two different methods. The reinforcing effect of cellulosic materials in S-PLA blends was less noticeable than in starch films, probably due to the overlapping of the PLA reinforcing effect. The antioxidant extract did not improve the mechanical performance in the blends, but conferred antioxidant capacity suitable for food packaging applications. / Collazo Bigliardi, S. (2019). Lignocellulosic fractions from rice and coffee husks to improve functionality of biodegradable films based on starch and poly-lactic acid [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/123055 / TESIS

This master´s thesis deals with production and characterization of fresh cheese with addition of herb extracts, specifically extracts from Plectranthus amboinicus and Echinacea purpurea. The purpose of this thesis was to prepare samples with higher sensory quality and nutritional value. Produced cheeses were characterized in terms of volatile (aroma) substances, fatty acid content, total phenolic substances, antioxidant activity and sensory quality. Cheese sample with added extract from E. purpurea reached the highest value of total phenolic compounds (0,172 ± 0,019 mgGAEg-1 of cheese) and antioxidant activity (91,2 ± 2,2 gTEACg-1 of cheese). Cheese sample with added extract from P. amboinicus, compared to standard cheese sample (without added extract) also reached higher values of total phenolic compounds and antioxidant activity. 43 volatile (aroma) compounds were identified in both samples with added extracts (P. amboinicus, E. purpurea), in standard cheese sample it was 33. In sample with E. purpurea camphor was present in high amount (15,23 %) responsible for the camphor aroma, camphor was also identified in small amount in sample with P. amboinicus. Highest volatile matter content was identified in sample with P. amboinicus. Standard cheese sample was rated as the best from the sensory perspective. Addition of extracts had negative effect on color, taste, and aroma perception of cheese. Strongest herbal taste was identified in sample with E. purpurea, brownish color of this sample manifested in lower pleasantness of color. Obtained results show, that extract addition increases the nutritional value of cheese (higher content of aromatic substances and phenolic compounds, higher antioxidant activity), but lowers sensory quality and overall consumer acceptability.

The aim of this diploma thesis was to prove the antimicrobial actvivity of the selected syntetic preservatives used in cosmetics, such as methylparaben, butylparaben, phenoxyethanol, bronopol, imidazolidinyl urea and methylisothaizolinone, in different concentrations (up to the highest permitted concentration and in increased concentrations), and of some essential oils, which are possible to use in cosmetics (essential oil from Eucalyptus globulus, Pimpinella anisum, Lavandula officinalis, Rosmarinus officinalis, Foeniculum vulgare, Mentha piperita, Juniperus communis and Pinus sylvestris). The effect was investigated in every preservative system alone, in order to evaluate the spectrum and the rate of each one. As indicator microorganism was chosen Bacillus subtilis and Penicillium chrysogenum. The used methods was disk difusion method and modify difusion method. From synthetic preservatives had the best effects bronopol, imidazolidinyl urea and methylisothiazolinone. From chosen natural antimicrobails had the effect against both indicator microorganisms the Mentha piperita, Foeniculum vulgare and Juniperus communis extracts.

The presented diploma thesis is focused on the preparation of new materials with antimicrobial effect. Liposomes and nanofibers from polyhydroxybutyrate containing clotrimazole and natural extracts with good antifungal and antioxidant effects were prepared. The theoretical part contains examples and short description of using nanoparticles and nanofibers in cosmetics and medicine and the description of plants which have positive and potential antimycotic effects. Moreover, methods for particles and fibers characterisation were shortly described. In the experimental part, natural water and lipid extracts were prepared and spectrophotometrically characterised for the content of polyphenols, flavonoids and the antioxidant activity. Liposomes and liposomes containtng PHB were prepared from selected extracts and the encapsulation effectivity, shortterm and longterm stability via determination of polyphenols were determined. Prepared particles were characterized with DLS method (size) and zeta- potential (stability). PHB nanofibers functionalised with selected lipid extracts and clotrimazole were prepared via electrospinning and forcespinning, and examined via FLIM and FTIR-ATR methods and spectrophotometry was used for antioxidant activity and release of active substances determination. Antifungal properties of prepared particles, extracts and fibers using the test system Candida glabrata were studied. Finally, cytotoxicity of selected samples was tested with MTT assay using human keratinocytes.

The use of plants for cosmeceutical applications is becoming more important since “safer” and more “natural” skin products are gaining popularity. The effect of different drying methods and storage conditions on metabolite changes and biological activity of two species with cosmeceutical application namely Greyia flanaganii and Leucosidea sericea were investigated using 1H-NMR metabolomics. The multivariate analysis (PCA and OPLS-DA), and 1H-NMR sample spectra were used to analyse the significant differences (P<0.05) resulting from the different treatments. The effect of these treatments on anti-tyrosinase and anti-bacterial (against Staphylococcus aureus) activity of G.flanaganii and L.sericea ethanol leaf extracts respectively, was further investigated to assess the quality. Four different drying methods adopted were freeze drying, oven drying at 50°C, air drying at room temperature and sun drying in a greenhouse. The dried leaf extracts were stored in three different conditions of fridge, freezer and shade conditions and samples from each storage condition taken for analysis at three and six months of storage. The chemical constituents of the leaf extracts of both species were not affected by the drying method and the storage condition, but the concentrations of the metabolites changed. The treatments did not show a significant difference (P<0.05) on the biological activity of the extracts. However, G.flanaganii plant material harvested from the University of Pretoria exhibited a higher anti-tyrosinase activity than material harvested from Mothong heritage site. In G. flanaganii freshly freeze and oven dried extracts exhibited a higher anti-tyrosinase activity with fifty percent inhibitory (IC50) activity of 16.8±0.69 μg/ml and 15.73±0.85 μg/ml respectively than the activity of sun and air dried with IC50 values of 33.08±0.78 μg/ml and 36.86±2.01 μg/ml respectively. The metabolite concentrations and anti-tyrosinase activity dropped significantly after storage. Leucosidea sericea oven and freeze dried extracts, exhibited good anti-bacterial activity with a minimum inhibitory concentration (MIC) value of 0.25 μg/ml and 0.125 μg/ml respectively as compared to sun and air dried extracts with the same MIC value of 0.5 μg/ml. Freeze dried samples showed the best anti-bacterial activity (MIC 0.125 μg/ml) compared to other drying methods. Fridge and freezer storage conditions enhanced the activity of stored sample. / College of Agriculture and Environmental Sciences / M. Sc. (Agriculture)

Diseases caused by bacteria, fungi and viruses pose a significant threat especially to poor rural communities. Viral infections are frequently complicated by secondary bacterial and fungal infections which remain a major challenge globally and in particular, in sub Sahara Africa amongst humans and animals alike. The main aim of this study was to develop a low toxicity plant extract or isolated compound active against viral, bacteria and fungal pathogens from selected plant species. Seven tree species that were investigated were Acokanthera schimperi, Carissa edulis, Ekebergia capensis, Podocarpus henkellii, Plumbago zeylanica, Annona senegalensis and Schrebera alata traditionally used in the treatments of various ailments were selected and extracted using solvents of varying polarity. Extracts of selected plants were tested for activity against two Gram positive and two Gram negative bacterial namely Enterococcus faecalis and Staphylococcus aureus and two Gram-negative species, Pseudomonas aeruginosa and Escherichia coli respectively, three fungal pathogens: Candida albicans, Cryptococcus neoformans and Aspergillus fumigates and four enveloped animal viruses: feline herpes virus–1 (FHV-1, dsDNA), canine distemper virus (CDV, ssRNA), canine parainfluenza virus-2 (CPIV-2, ssRNA) and lumpy skin disease virus strain V248/93 (LSDV, dsDNA). The presence of antioxidant constituents in the different extracts and cytotoxicity against three cell types CRFK, bovine dermis and Vero cells were determined. Bioautography and the serial microplate dilution methods were used to determine the number of antimicrobial compounds and antimicrobial activity of extracts against bacterial and fungal pathogens. Virucidal and attachments assays were used to determine the activity against viral pathogens. Qualitative antioxidant activities of extracts were tested using the DPPH reagent and cytotoxicity using the MTT assay. Biological activity was observed in all the extracts against one or more organisms on bioautography. The intermediately polar system (CEF) separated more active constituents. Some extracts had compounds with similar Rf values active against one or more organisms. In both the antibacterial and antifungal assays, acetone extracts had the highest activity followed by DCM against one or more pathogens. Hexanes extracts were the least active. P. henkellii extracts had more active compounds against the bacteria and Annona senegalensisagainst the fungi. In the micro-dilution assay, S. aureus was the most susceptible bacterial organism to extracts of the different plant, followed by P. aeruginosa andEscherichia coli, and E. faecalis the least. C. neoformans on the other hand was the most susceptible fungal pathogen. In the antiviral assay, although activity was observed with hexane extracts of some plants in the virucidal assay, the most potent inhibition was observed with the acetone and methanol extracts of Podocarpus henkelii against CDV and LSDV in the virucidal assay and acetone extracts in the attachment assay. In general the hexane was the least toxic while the intermediate polarity extracts were generally the most toxic indicating that highly polar compounds were possibly poorly or highly absorbed through membranes in the former and later respectively. Of the three cell types used CRFK was the most sensitive followed by bovine dermis and Vero cells the least. Cytotoxicity studies of extracts of the different plants revealed A. senegalensis and A. schimperi extracts were the most toxic plants in the cellular assay. These plants are toxic to animals and the cytoxicity is in line with the in vivo toxicity. The protective effects of antioxidant constituents in some extracts varied and appear to be influenced by the metabolism of the type of cell in culture. It also appears to suggest that metabolism in kidney derived cells can be influenced by species variation in the origin of cells. P. henkellii was selected for isolation of bioactive compound. Three compounds were isolated and their structure elucidated using 13C and 1H NMR and mass spectrometric data. The antibacterial, antifungal and antiviral activity of the isolated compounds 7’, 4’, 7’’, 4’’’, tetramethoxy amentoflavone (C1), isoginkgetin (C2) and Podocarpusflavone–A (C3) were determined. Compound C2 was the most active against E. coli and S. aureus (MIC = 60 ìg/mE) and a selectivity index (SI) value of 16.67. The compound was also active against A. fumigatus and C. neoformans (SI = 33.33) suggesting both antibacterial and antifungal activity with relative safety. Compound C3 had a broad spectrum of activity against E. faecalis and P. aeruginosa with SI values of 4. A less potent activity of the compounds was obtained in both the virucidal and attachment assays against test pathogens, indicating the lower activity of the compounds against tested viral pathogens. The studies further suggest structural activity relationship in the antimicrobial activity of biflavonoids. The compounds C1 and C2 had no toxic effect on the three cell types and mutagenicity studies indicated no activity of these compounds. Podocarpusflavone-A occurs in every species of Podocarpus so far investigated, except P. latifolius. These studies represent the first isolation of bioactive compounds from P. henkellii. Although a different extractant was used than that used by traditional healers, the presence of antiviral compounds in Podocarpus henkelii against two unrelated viruses may justify on a chemotaxonomic basis the traditional use of related species Podocarpus latifoliusand Podocarpus falcatus in the traditional treatment of canine distemper infection in dogs. / Thesis (PhD)--University of Pretoria, 2011. / Paraclinical Sciences / unrestricted

Colorectal cancer (CRC) is the third most prevalent and second leading causes of cancer-associated deaths globally. Over the last few decades, ent-kaurane diterpenes have been widely investigated for their anticancer potentials. Flexicaulin A (9) is a naturally occurring ent-kaurane. Previously, our lab modified the structure of 9 by replacing the C-11 hydroxyl group with carbonyl group and obtained a novel compound oxoflexicaulin A (11). However, anticancer activities and mechanistic pathway of these two compounds are yet to be explored. In the current thesis, we evaluated the cytotoxicity of compounds 9 and 11 in A549 lung, A375 melanoma, PANC1 pancreatic, HCT-116 and HT-29 colon cancer and 293T human embryonic kidney cells as well as compared the activity with a number of known natural ent-kauranes to describe their structure-activity relationship. Our study found that the presence of α, β-unsaturated ketone groups in ent-kaurane structure acted as the pharmacophore. The replacement of C-11 hydroxyl group by carbonyl in 9 (IC50: 3.68 µM) gives a novel potent anticancer compound 11 (IC50: 0.77 µM). Considering the novelty and superior activity of 11, its mechanistic pathway was studied in HCT-116 cells and compared with the natural scaffold 9. Flow cytometry analysis by Annexin V/PI staining along with fluorescent staining by DAPI showed that both compounds induced apoptosis in HCT-116 cells. Induction of apoptosis is mediated through up-regulation of tumor suppressor protein p53 and pro-apoptotic protein Bax, Bak and puma as well as promoting the cleavage of PARP while down-regulation of anti-apoptotic protein Bcl-2 and PARP. Apart from their effect on apoptosis, compounds 9 and 11 stimulated the event of senescence, a process of cellular aging, as confirmed by β-galactosidase assay. Induction of senescence is related with up-modulation of p21 and p27 while down-modulation of p16, Rb and its transcription factor E2F1. Moreover, immunofluorescence staining showed translocation of p21 and p27 from the cytoplasm to nucleus after treatment with 9 and 11. Further study found that the two ent-kauranes inhibited the protein level of two NF-κB sub-units p65 and p50 in the nucleus as well suppressed the cytoplasmic level of NF-κB inhibitor IkB-α. Both compounds also inhibited the expression and phosphorylation of STAT3 in the cytoplasm and nucleus, so as for the expression and phosphorylation of Src, an up-stream kinase of STAT3. In xenograft nude mice model, compound 11 remarkably inhibited tumor growths (volume and size) but the body weights of the mice were also reduced (p < 0. 05). Therefore, we designed to synthesis a series of prodrug analogs from 11 by adding acetal protecting group to reduce the toxicity. One of the prodrug (37) significantly attenuated the tumor volume and size (p < 0.05) at 50 mg/kg without any toxicity (p > 0.05). The prodrug 37 is actually released as compound 11 in the mice due to its cleavage in the acidic microenvironment of tumor. Taken together, antitumor effect of compound 11 in CRC model is supposed to be mediated through induction of apoptosis and senescence via modulation of NF-κB and STAT3 signaling pathway. Keywords: Colorectal cancer, ent-kaurane, flexicaulin A, oxoflexicaulin A, cytotoxicity, anticancer, apoptosis, pathway, NF-κB, STAT3.

Replacing the lost Phosphates from different ecosystems is not a question it’s an onus. This none replaceable and renewable resource is one of the vital nutrients where without it, cells, crops and human beings cannot function and live. Though available in environment, its natural cycle is disturbed as its need especially in agriculture sector increases significantly. Phosphorus recovery from waste water sorbents is one of the innovative and promising concepts. The core goal of this paper is to evaluate the phosphorus desorbing capacity of two reactive materials (waste water sorbents) using chemical extracts. The experimental work emphasized on investigation four extracts, HNO3, NaHCO3, P-AL and H2O on phosphate desorption capacity of two inorganic reactive materials namely Polonite® and Sorbulite® which are widely known for their high P-sorption capacity. The kinetics and desorption were examined in batch experiments and consequently the plant availability were investigated using Flow Injection Analyzing (FIA) spectroscopy (wet chemistry) method. The maximum average orthophosphate desorption at 100 rpp for 48 hrs was interpreted to be 33.12 mg g−1and 3.11 mg g−1 from Sorbulite® and Polonite® respectively using 4M HNO3 extract. The orthophosphate desorption characteristic using the above extracts tended to decrease in the following order HNO3 ≤ P-AL ≤ NaHCO3 ≤ H2O. Polonite® shows a higher recovery potential, where almost 6 times higher ammonium lactate (AL)-extractable P was observed when compared with Sorbulite®. Batch experiment was proved to be a tool for investigating and evaluating Phosphorus desorption capacity of mineral-based filter materials.

Doctor Educationis / The fungus Botrytis cinerea is an opportunistic pathogen on a wide variety of crops, causing a disease known as grey mould through infections via wounds or dead plant parts. Synthetic fungicides for controlling this disease are fast becoming ineffective due to the development of resistance. This, coupled with consumers' world wide becoming increasingly conscious of potential environmental and health problems associated with the build-up of toxic chemicals, (particularly in food products), have resulted in pressure to reduce the use of chemical pesticide volumes as well as its residues. An emerging alternative to random chemical synthesis is the study and exploitation of naturally occurring products with fungicidal properties. One group of compounds known as strobilurins produced by Strobilurus species, woodland basidiomycete fungi, is a good example of this phenomenon. Plants produce an enormous array of secondary metabolites, and it is commonly reasoned that a significant part of this chemical diversity serves to protect plants against plant pathogens. A problem with plant-produced compounds as potential fungicides is that in the natural state, they are generally only weakly active compared to synthetic fungicides. There have been reports on the uses of mixtures of synthetic fungicides for the control of plant pathogenic fungi. When utilized in two-way mixtures, such fungicides may maintain or enhance the level of control of a pathogen at reduced rates for both components utilized in combinations, or alone at normal rates. These studies provide an important precedent for the idea of synergism. For this study, we hypothesize that the addition of plant extracts may enhance the antifungal efficacy of the synthetic strobilurin fungicide, kresoxim-rnethyl against B. cinerea. We selected South African medicinal plant species such as Artemesia afra, Elyptropappus rhinocerotis, Galenia africana, Hypoxis hemerocallidea, Siphonochilus aetheopicus, Sutherlundia frutescence, Tulbaghia violacea and Tulbaghia alliaceae for this study. For the in vitro study, indigenous medicinal plant extracts were prepared at twofold dilution concentrations and combined with kresoxim-rnethyl at concentrations of 0.25 and 0.5% (w/v). The B. cinerea mycelial plug assays showed potent antifungal inhibitory effects with the plant extract and kresoxim-rnethyl mixtures. Further analyses of the mixtures indicate synergistic effects between the fungicide and plant extracts. I surmise that these in vitro effects are also achievable in vivo. Combinations of these agents represent an attractive avenue for the development of new management strategies for controlling B. cinerea in the future. A second study was conducted to analyse the final dose rates for synergistic reactions for combinations of kresoxim-methyl and medicinal plant extracts against B. cinerea in vivo. A series of two-fold concentrations of medicinal plant extracts were combined with kresoxim-methyl to conduct decay inhibition studies on Granny Smith apples. Synergistic effects were observed for many of the kresoxim-methyl and plant extract combinations. I, therefore, came to the conclusion that indigenous South African plant species produce modulators that potentiate the activity of fungicides. Whether these synergistic effects are due to the inhibition of fungal multi-drug resistant pumps require further studies at the molecular level. However, these inhibitory effects are likely to be advantageous for developing fungicide formulations and application strategies with low toxicity effects on the environment. This approach not only makes it possible to reduce fungicide concentrations while maintaining adequate decay control, but also ensures a reduction of the chemical residue on the fruit.

Thesis (M.Sc. (Biochemistry)) -- University of Limpopo, 2021 / Lung cancer is the most diagnosed cancer with an estimated 3 million deaths expected by 2035. Bioactive phytochemicals present in plants are preferred as anticancer therapeutic agents, due to their ability to differentiate between cancerous and normal cells. One such plant, Senna italica, is traditionally used to treat diabetes, malaria, constipation, jaundice, fever and sexually transmitted diseases. Several studies have reported on its anti-proliferative potential against different types of cancers. However, there is scanty information regarding its molecular mechanism of action against different types of cancers, more especially lung cancer. This study, therefore, aims to determine the differential expression profiles of apoptotic genes in lung A549 cancer cells induced by treatment with S. italica leaf and root extracts in an attempt to understand its purported anticancer molecular mechanism of action. The leaves and roots of S. italica were dried in the dark and extracted with ethyl acetate and methanol. Screening for the presence of secondary metabolites was performed using thin layer chromatography and various standard chemical-based tests. The total phenolic and flavonoid compounds were evaluated using gallic acid and quercetin equivalence assays. The antioxidant activity of S. italica extracts was determined using DPPH free radical scavenging and ferric ion reducing power assays. The cytotoxicity of both leaf and root extracts on lung A549 cancer cells was evaluated using 3-(4,5- dimethylthiazol-2-yl)-2-5-diphenyl tetrazolium bromide (MTT) and further confirmed by Muse cell count and cell viability assays. The proliferation of cells, after treatment with different concentrations of the extracts, was examined using the Ki67 proliferation assay. Genotoxicity was determined to assess the potential damage caused by the extracts on the DNA using a MUSETM multicolour DNA damage kit following manufacturer’s protocol. The morphological change of cells treated with different concentrations of S. italica ethyl acetate root extract was analysed using acridine orange/ ethidium bromide (AO/EB) dual staining assay and examined under fluorescent light. The total number of cells undergoing apoptosis was also determined using the Annexin V assay. The expression of 84 key genes, involved in programmed cell death or apoptosis, was determined using the Human Apoptosis RT² Profiler PCR array kit. Senna italica methanol extract had a high content of plant materials in both leaves and roots compared to the ethyl acetate extract. A higher phenolic content was observedxii mainly in the leaf extract and a higher flavonoid content was observed in the root extract. Phytochemicals, such as phenols, tannins, flavonoids, terpenoids and steroids, which are known to exhibit anti-cancer activity against cancerous cells were abundant in the ethyl acetate leaf and root extracts as compared to the methanol leaf and root extracts. Additionally, the ethyl acetate root extract exhibited more antioxidants and radical scavenging activity in comparison to the methanol root extract. The IC50 of ethyl acetate root extract was determined to be 200µg/ml. Both methanol and ethyl acetate root extracts had little to no effect on the viability of lung A549 lung cancer cells. The results were confirmed by cell count and viability assay results. The cytotoxicity of ethyl acetate root extract was also evaluated against the normal kidney HEK-293 cells, which displayed little cytotoxic effect. The proliferation results indicated that S. italica ethyl acetate root extract has the potential to reduce the proliferation of lung A549 cancer cells. The ethyl acetate root extract was found to induce late apoptosis in A549 cells, but the genotoxicity data indicated that the DNA double strand breaks (DSBs) were repairable. The results further showed an expression of different genes that inhibit apoptosis, such as XIAP in lung A549 cells, following treatment with S. italica ethyl acetate root extract. In conclusion, the ethyl acetate root extract displayed a promising anti-cancer therapeutic potential, and thus warrants further investigation to elucidate the identity of the inherent chemical components that are responsible for the observed biological activity. / University of Limpopo and SAMRC