ALESSANDRA BRAGA RIBEIRO

Título da Tese: AVALIAÇÃO DA ATIVIDADE ANTIOXIDANTE POR MÉTODOS QUÍMICOS E CELULARES E ANÁLISES DE COMPONENTES BIOATIVOS EM VÁRIAS PLANTAS

Orientador: Prof. Dr. Jesuí Vergílio Visentainer

Data da Defesa: 22/04/2014

RESUMO GERAL: Brazil has a great biodiversity, considered one of the largest in the world; the diversity is primarily due to large territorial extension, variety of soil and favorable geographical and climate conditions. There are three important biomes: Brazilian Cerrado, Atlantic Forest and Amazon rainforest, where it can be found an enormous diverse flora, including a large number of native and exotic fruit species and an uncountable number of medicinal plants. Considering these facts, in recent years the whole world laid eyes on Brazil and its immense range of possibilities, such as the discovery new plants with biological activity, mainly based in the massive disclosure that plants (fruits, vegetables, flowers) can help prevent degenerative and inflammatory diseases, aging and many kinds of cancer. However, from the beggining of the discovery of a new plant, going through all the chemical, toxicological characterization and the determination of biological activity until their rational use directed to improve human health, there is a long way. Thus, the scientific research on each fruit/plant could support the rational dissemination and the development of higher added value products with latent prospects in food and pharmaceutical industry. In this context, one of the most desirable biological activities in a plant is the antioxidant capacity, since there is compelling evidence that oxidative stress is involved in the damage of cellular constituents, such as DNA, cell membrane lipids or proteins, consequently the most frequent chronicdegenerative diseases, as cardiovascular disease, cancer, neurological disorders, diabetes, and ischemia/reperfusion are highly associated to oxidative stress. Despite of it is well known that production of reactive oxygen ROS and RNS exert beneficial physiological effects in human body, the excess of free radicals and oxidants that generate oxidative stress, needs to be neutralized. In the most cases, the endogenous antioxidants are not enough, thus, it is necessary to supply antioxidants from an exogenous source. The bioactive compounds, such as phenolic compounds and carotenoids, have been positively associated to a reduction of oxidative related diseases. To initiate an antioxidant investigation of a new plant, scientists commonly perform total phenolic analysis, since they constitute the major group of antioxidant compounds; but it is necessary to emphasize that there are other compounds that can contribute to their antioxidant capacity. Furthermore, conducting a review in scientific literature, it could be found a lot of methodologies to evaluate antioxidant compounds and capacity, but the most common are in vitro analysis by spectrophotometry, highlighting DPPH , FRAP and ABTS+ . These methods are used worldwide in the quantification of free radical scavenging activity and may be considered as an important tool to procced screening research, since they are simple and fast technique carried out with relatively affordable solvents and equipment. Despite the fact of the most common antioxidant assays are based on exogenous radicals (DPPH and ABTS+ ), assays based on physiological reactive species has been considered a valuable tool to evaluate the antioxidant capacity. Moreover, the antioxidant capacity assays based in cellular models is appropriate to evaluate the protective effects of antioxidants in different steps of the pathway of stress oxidative, to assess the intake of the antioxidants into the cells and, cellular models help to perceive the underlying mechanisms of oxidative stress. Considering the lack of knowledge on the chemical profile and antioxidant capacity of some native and exotic fruits and a promising Amazonian plant, this study aimed to identify and quantify phenolic compounds and carotenoids, fatty acids composition, to verify antioxidant capacity using different methodologies and determinate their correlation. As well as, to verify in the most promising tested plants of this study, the protection against the oxidative burst of human neutrophils and the potential to inhibit the oxidative damage in human erythrocytes, including lipid peroxidation, oxidation of 12 hemoglobin and the depletion of glutathione. The main resuts obtained by DDPH, ABTS, ORAC, FRAP and total phenolic compounds (TPC) revelead that fruit P. cattleianum possess the highest antioxidant capacity and also, it was possible verify the best correlation between FRAP and other tested methods and FRAP and TPC. In this account, P. cattleainum was forwarded to more complete analysis, including identification and quantification of phenolic compounds and carotenoids by LC-MS/MS and in vitro scavenging capacity screening assays of skin and pulp extracts from P. cattleianum fruit against reactive oxygen species (ROS) and reactive nitrogen species (RNS). The major phenolic compounds identified in skin and pulp extracts of P. cattleianum were ellagic acid; while all-trans-lutein, all-trans-antheraxanthin and alltrans-β-carotene were the major carotenoids identified in both extracts. P. cattleianum pulp extract showed higher scavenging capacity than skin extract for all tested ROS and RNS, namely superoxide radical (O2 ●- ), hydrogen peroxide (H2O2), hypochlorous acid (HOCl), singlet oxygen (1O2), nitric oxide (●NO) and peroxynitrite (ONOO- ). Related to fatty acid composition, the oleic acid was predominant in the tested fruits (included P. cattleianum), this fact is very interesting, it is well known that a diet rich in monounsaturated fatty acids, represented mainly by oleic acid, is closely related to the prevention of cardiovascular diseases, since it decreases the serum concentrations of triacylglycerol, cholesterol and low density lipoprotein (LDL), and are precursors of fatty acids polyunsaturated long-chain, including docosahexaenoic acid (DHA) and arachidonic acid (ARA). In parallel of the research concerning to the fruits, analyzes to evaluate the scavenging capacity of five extracts from different parts of V. cauliflora (leaf, branch, stem bark, flower and whole fruit) were performed against reactive oxygen (ROS) and nitrogen species (RNS), namely, superoxide radical (O2 ●- ), hydrogen peroxide (H2O2), hypochlorous acid (HOCl), singlet oxygen (1O2), nitric oxide (●NO) and peroxynitrite (ONOO- ). All parts of V. cauliflora showed high efficiency against all tested ROS and RSN, although flower and stem bark extracts exhibited the most remarkable scavenging capacity. Furthermore, the scavenging capacity of the most extracts of V. caulifora seemed to be closely dependent on the phenolic compound contents. Considering the paramount antioxidant potential of this plant, the profile of phenolic compounds and carotenoids by LC-MS/MS were also determined. Epicatechin and proanthocyanidin dimers and trimer were the major phenolic compounds tentatively identified in leaf, branch, stem bark and flower extracts, while dihydroxybenzoic acids were the major compounds in whole fruit extracts. All-trans-zeinoxanthin and all-transβ-carotene were the major carotenoids tentatively identified in leaf extracts. As stem bark and flower presented better results than the other parts of the plant, they were directed to avaliation related to their capacity to neutralize neutrophils’ oxidative burst using specific probes (luminol, MLCA, amplex red and APF). Furthermore, the capacity of both parts of the plant to protect erythrocyte from oxidative damage was undertaken by hemoglobin oxidation, lipid peroxidation and the depletion of Gluthatione (GSH) assays. The results demonstrate that oxidative burst in activated human neutrophils were effectively neutralized by both extracts in a very low concentration range as well as the great capacity from both extracts to protect erythrocyte from oxidative damage. Considering the potential beneficial effects to human health, P. cattleianum and V. cauliflora may be considered as a promising source of bioactive compounds with high antioxidant properties and may be directed to research targeted effectively to develop products in food and/or phytopharmaceutical field. Keywords: Phenolic compounds, carotenoids, fatty acids, fruits, Vismia cauliflora, reactive oxygen species.

Artigos Publicados Vinculados a Tese:

https://www.sciencedirect.com/science/article/pii/S030881461400795X?via%3Dihub

https://www.tandfonline.com/doi/full/10.3109/13880209.2014.1001407

https://doi.org/10.3109/13880209.2014.974063

http://dx.doi.org/10.5935/0103-5053.20130105