Made available in DSpace on 2014-08-20T14:37:54Z (GMT). No. of bitstreams: 1 tese_maria_cecilia_florisbal_dame.pdf: 53923 bytes, checksum: e82c085a8b324064158221bf46a754ba (MD5) Previous issue date: 2013-12-12 / This thesis is a continuation of a research project started with a diagnosis of dermatosis mechano-bullosa in a herd of buffaloes from a farm in southern Rio Grande do Sul. After this diagnostic it was created an experimental herd where several congenital defects and / or hereditary disorders have been diagnosed during more than two decades. These diseases were studied by a research group which the author of this thesis is a member. Thus, three papers are presented: the first one is a literature review about what has been diagnosed in Brazil on congenital defects and hereditary diseases in buffalo. It was concluded that undesirable genes are widespread in the population of buffaloes in the country; the second was a study conducted in partnership with UNESP / Jaboticabal, SP and UFCG / CSTR-Patos, PB to identify the mutation in the gene that determines the oculo-cutaneous albinism in Murrah buffalo. Another paper was accomplished to describe the occurrence of malignant melanoma in two albino buffalo. Although it is not a congenital or hereditary disease it was observed only in the buffalo with oculocutaneous albinism and probably the condition is associated with a predisposition of these animals to develop tumors in the skin. / Esta tese dá continuidade a um projeto de pesquisa iniciado com o diagnóstico de dermatose mecanobolhosa em 1985 em um rebanho de búfalos pertencente a uma propriedade da zona sul do Rio Grande do Sul. A partir desse diagnóstico foi criado um rebanho experimental no qual por mais de duas décadas foram diagnosticados diversos defeitos congênitos e/ou hereditários que foram estudados por um grupo de pesquisa do qual faz parte a autora desta tese. Assim sendo, são apresentados três artigos científicos: o primeiro trata-se de uma revisão bibliográfica sobre o que foi diagnosticado no Brasil em relação a defeitos congênitos/doenças hereditárias em bubalinos concluindo-se que alguns genes indesejáveis estão disseminados na população de búfalos no País; o segundo foi um trabalho realizado em parceria com a UNESP/Jaboticabal, SP e com a UFCG/CSTR-Patos, PB que identificou a alteração no gene que determina o albinismo óculo-cutâneo em búfalos da raça Murrah. O terceiro artigo trata-se da descrição de melanoma maligno observado em dois búfalos albinos. Embora não seja um defeito congênito ou hereditário este neoplasma ocorreu somente nos búfalos com albinismo óculo-cutâneo do rebanho e acredita-se que a condição esteja associada à predisposição desses animais a desenvolverem tumores de pele.

Introduction: Dark spots appearing on the skin caused by hyperpigmentation results from the action of tyrosinase, an enzyme whose activity leads to the production of the skin pigment melanin. Extracts of the plant Glycyrrhiza glabra, also known as liquorice, are commonly used to treat a range of conditions including skin hyperpigmentation. This study aimed at isolating and identifying compounds in extracts from South African liquorice root and assaying these compounds as to their antioxidant activity, their ability to inhibit the tyrosinase enzyme and their level of cytotoxicity. Methods: The ability of plant extracts to scavenge free radicals was tested using the 2,2-diphenyl-1-picrylhydrazyl (DPPH), [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonicacid)] (ABTS) and the ferric ion reducing power (FRAP) tests. The polyphenolic content of extract fractions was determined and extract compounds were identified using UHPLC-QToF-20 MS. In vitro anti-tyrosinase activity was also investigated as well as cytotoxicity in HepG2 liver and SK-MEL-1 melanoma cells using the MTT cell viability assay. Results: Of the four fractions prepared from the 70% methanolic extract of liquorice root, fraction 3 (F3) showed increased polyphenolic content and antioxidant properties with IC50 of 56.1 ± 6.32, 39.14 ± 1.1 and 66.34 ± 1.4 μg/ml against DPPH, ABTS and FRAP, respectively. The anti-tyrosinase activity of this fraction showed an IC50 of 358.54 μg/ml compared to Kojic acid (0.75 mM) used as the control. In addition, this fraction showed reduced liver toxicity as a higher percentage cell viability was noted in the HepG2 cells compared to the SK-MEL-1 skin melanoma cells. However, both cell types showed higher percentage viability compared to acetaminophen that was used as cytotoxic control. The LC-MS analysis revealed the presence of a wide variety of compounds including 4-azido-3-benzyl-coumarin, ferulic acid, glycyrrhizin, quercitrin, cirsilineol, gentioflavine and 4'',6,7-trihydroxyisoflavone. The literature indicates the use of these compounds regarding antioxidant and anti-tyrosinase activity. Significantly, cularidine was identified in this study, a compound not previously reported in studies involving liquorice root. Conclusion: The results from this study concur with previous reports as to the anti-tyrosinase and antioxidant activities associated with liquorice roots, activities perhaps due to the relatively high polyphenolic content in extracts from South African liquorice root. / Life and Consumer Sciences / M. Sc. (Life Sciences)

Greyia radlkoferi is a South African indigenous tree, which has recently been discovered to be a source of extracts that have a potential in the development of cosmeceutical herbal products having the ability to treat hyperpigmentation disorders. For product development however, G. radlkoferi would need to be available in a commercial scale. Greyia radlkoferi grows naturally in the wild and is often available for cultivation as an ornamental plant. In order to establish this plant into cultivation, suitable propagation techniques must be established for rapid multiplication of trees and thus a sustainable leaf production. For consistent and improved leaf supply to the market, agronomic practices that will enhance leaf production were investigated in the current study. Furthermore, in order to meet market demand in terms of good quality extracts with guaranteed therapeutic efficiency, pre-harvest and post-harvest factors that affect the chemical composition of the extracts were investigated. Recently developed biotechnology techniques such as metabolomics using 1H-NMR and multivariate data analysis offered a platform to study the chemical variation of extracts. Therefore, the current study was aimed at understanding the requirements for propagation and optimum leaf production as well as conditions that favour optimum production of secondary metabolite of G. radlkoferi plant material (at pre and post-harvest) and thus assess its commercial viability. To understand the effects of temperature on seed germination of G. radlkoferi, seeds were exposed to five temperatures (10°C, 15°C, 20°C, 25°C and 30°C) in the incubators in the laboratory. Germination of G. radlkoferi by seeds was discovered to be temperature dependent. The optimum germination temperature of 81% was obtained at 25°C. In the case of vegetative propagation by stem cuttings, the effect of cutting position (basal or apical), exogenous rooting hormone (Seradix1, Seradix 2, 0.1% IBA, 0.3% IBA and 0.8% IBA) and cutting position were investigated in the glasshouse. The cutting position had a significant effect on rooting of G. radlkoferi cuttings with basal cuttings exhibiting 35% rooting as compared to 6% rooting attained for the apical cuttings. A clear trend in rooting response to application of rooting hormones was observed, with 0.1% Indole butyric acid (IBA) showing the highest rooting percentage of 63%. Considering the outcomes of the propagation studies as well as the limited material for vegetative propagation, seed propagation appears to be the most suitable technique for large-scale multiplication of G. radlkoferi. The effect of different pruning techniques as well as harvesting frequencies on fresh and dry weights of G. radlkoferi leaves were evaluated. Factors considered were four pruning treatments (‘pruned but not tipped’, ‘tipped but not pruned’, ‘not pruned nor tipped’ as well as ‘pruned and tipped’) and three harvesting periods (monthly, bimonthly and once–off). Bimonthly harvests highly increased leaf production compared to trees that were harvested monthly and once-off with higher leaf fresh weight yield of 238 g per tree or 2.38 tons/per hectare as well as dry weight yield of 83 g per tree or 0.830 tons/hectare. This outcomes of this study further suggested that a suitable pruning practice for G. radlkoferi would be to either ‘prune only’ or ‘cut back the main stem’ rather than a combination of the two treatments. The influence of seasons (summer, autumn, winter and spring) on the anti-tyrosinase activity and metabolomics profile of G. radlkoferi leaf extracts were investigated. Seasons significantly influenced the chemical composition and the efficacy of the plant extracts. Tyrosinase enzyme inhibition was investigated against monophenolase (tyrosine) with kojic acid as positive control. The highest tyrosinase inhibition concentration with IC50 (50% tyrosinase inhibition concentration) value of 30.3±1.8 μg/ml were obtained in winter harvested leaves compared to the other seasons. The lowest IC50 values were obtained in spring. Metabolomics analysis using orthogonal partial least square discriminant analysis (OPLS-DA) provided a clear class separation according to the harvest season. Extracts from winter harvested leaves contained sucrose, acetamide, alanine and a compound of the catechin group (gallocatechin-(4 alpha->8)-epigallocatechin) as revealed by 1H-NMR metabolomics with assistance of LC-MS. Since compounds of the catechin group are well-known tyrosinase inhibitors, the high tyrosinase activity exhibited in extracts of winter harvested G. radlkoferi leaves could be ascribed to the presence of gallocatechin-(4 alpha->8)-epigallocatechin. Based on the outcomes of the seasonal study, we suggest that in order to obtain extracts with high bioactivity, the best suitable time for harvesting leaf samples is in late autumn-early winter. Processing leaf material using three different drying methods (sun, oven and air drying) significantly influenced chemical composition and the efficacy of the plant extracts. Extracts prepared from air-dried leaf material showed the highest tyrosinase inhibition with IC50 value of 17.80 μg/ml compared to extracts of the other drying methods. Extracts of leaves processed with air drying preserved most metabolites during processing while extracts of sun-dried and oven-dried leaves clearly depleted some metabolites especially amino acids and some aromatic compounds. 1H-NMR metabolomics approach with the assistance of LC-MS data successfully determined a positive association of alanine, acetamide, sucrose and gallocatechin-(4 alpha->8)-epigallocatechin as the chemical constituents contributing to the variation in the air-dried leaves compared to the oven-dried leaves. A positive association of valine, alanine, leucine, isoleucine, gallocatechin-(4 alpha->8)-epigallocatechin and glucose contributed to the variation in air-dried group, compared to the sun-dried group. The highest tyrosinase inhibitory activity exhibited in air-dried samples compared to the other drying methods was associated with the presence of gallocatechin-(4 alpha->8)-epigallocatechin. Because air drying preserved most leaf metabolites compared to sun and oven drying, it was regarded as the most suitable method for processing G. radlkoferi leaf material. This study is the first scientific account that provides guidelines and recommendations to (1) establish G. radlkoferi as a cultivated plant for commercialization, (2) optimize leaf production for sustainable supply to the commercial markets and (3) optimize medicinal content of G. radlkoferi related to harvesting time and post-harvest processing (drying), for enhanced quality of extracts and its products / Agriculture, Animal Health and Human Ecology / Ph. D. (Agriculture)