Compostos bioactivos em plantas Brassicacea e o potencial efeito na Saúde Humana
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2009
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Em Portugal, entre a grande diversidade de espécies hortícolas produzidas e
consumidas, destacam-se as pertencentes à família Brassicaceae, em particular o género
Brassica, normalmente designadas por “Brássicas” ou simplesmente “couves”. Estas plantas, para além de constituírem uma fonte de proteína e fibra, hidratos de carbono, vitaminas e minerais, contêm também outro tipo de compostos que desempenham um papel biológico importante, na medida em que podem intervir em mecanismos de eliminação de radicais livres resultantes do metabolismo celular e na prevenção de doenças de diversa etiologia. São
os chamados “compostos bioactivos”. Entre estes, destacam-se os glucosinolatos e seus
hidrolisados e os polifenóis. A investigação que se apresenta nesta dissertação centra-se no estudo dos glucosinolatos, compostos fenólicos totais e flavonóides totais, vitamina C, aminoácidos-livres e minerais, tendo por objectivo avaliar as suas variações e alguns dos seus efeitos biológicos. Com este propósito foram estruturados os seguintes capítulos:
Capítulo 1 - “Introdução Geral”. Neste capítulo, faz-se uma introdução geral ao tema, com a respectiva justificação e apresentam-se os principais objectivos.
Capítulo 2 - “Revisão Bibliográfica”. Compreende a informação relativa à importância sócio-económica das Brássicas em Portugal e no Mundo e da respectiva composição e valor alimentar, bem como dos seus potenciais efeitos biológicos e factores que os influenciam.
Capítulo 3 - “Material e Métodos”. Em síntese, neste capítulo descreve-se o material vegetal que foi produzido em Vila Real, ao longo de 2 anos consecutivos e em duas épocas de produção: entre Setembro e Janeiro e Abril e Julho, períodos de maior consumo de couves e nabo. São descritas igualmente todas as metodologias analíticas, ensaios efectuados e análise
estatística.
Capítulo 4 - “Componente Experimental ”. Neste capítulo apresentam-se separadamente
os trabalhos já publicados em revistas do JCR e outros em fase de preparação ou já
submetidos, que passamos a discriminar:
4.1. -“Estudo da variação dos teores em compostos bioactivos”. Neste trabalho (em
preparação) faz-se a identificação e quantificação, bem como a avaliação do potencial qualitativo de glucosinolatos, compostos fenólicos totais e flavonóides totais, vitamina C, aminoácidos-livres e minerais em couve--brócolo, couve-galega, couve-lombarda, couve-repolho, couve-tronchuda, nabiça e nabo, produzidas em duas épocas climáticas distintas
(Primavera-Verão e Verão-Inverno) em dois anos consecutivos (2005-2006 a 2006-2007).
Pelos resultados obtidos, constatámos que genótipo, época e ano de produção condicionam
significativamente os perfis e os teores médios dos diferentes compostos. Verificámos ainda que as condições de stress climático durante o período em que decorre a produção, tendem a induzir alterações nos teores dos diferentes compostos.
4.2. - “Estudo da actividade antioxidante das diferentes Brássicas em função da época
de produção.” Neste trabalho (submetido), avalia-se o potencial antioxidante das couves
produzidas, com base nos métodos espectrofotométricos do DPPH (2,2'-difenil-1-
-picrilhidrazil) e capacidade redutora. Correlaciona-se ainda esta capacidade com o teor em compostos fenólicos totais, flavonóides totais e vitamina C, determinados no trabalho 4.1.
Verificou-se que com a aplicação dos dois métodos, a capacidade antioxidante das diferentes Brássicas variou com genótipo, a época e o ano de produção. As inflorescências de couve-brócolo e as folhas de couve-galega e couve-tronchuda, foram aquelas que apresentaram os
valores mais elevados de actividade antioxidante e globalmente foi nas épocas de produção de Verão-Inverno que se obteve a maior actividade antioxidante média. Foram detectadas correlações entre o teor em fenólicos totais, flavonóides totais, vitamina C e a actividade
antioxidante, pelo que podemos afirmar que existe uma forte associação entre a capacidade e/ou actividade antioxidante dos extractos aquosos das Brássicas e a concentração destes compostos.
4.3. - “Estudo da actividade da enzima mirosinase e do efeito da conservação e
tratamento culinário no teor em glucosinolatos em quatro Brássicas”. Neste trabalho (submetido) avalia-se a actividade da enzima mirosinase, e o efeito das condições de conservação (temperatura ambiente e 4 ºC) e o tipo de cozedura (tradicional, vapor, e microondas) no perfil e teor dos glucosinolatos presentes. Os resultados obtidos permitiram
observar a existência de diferenças não significativas na actividade da mirosinase. Contudo,foi possível agrupar os genótipos em três grupos diferentes: um grupo com valores iguais e superiores a 5,5 Unidades.g-1 de peso seco, constituído pelas couve-brócolo, nabiça e couve-tronchuda; um outro grupo com valores entre 4,5 e 5,5 Unidades.g-1 de peso seco no qual se inclui o nabo; e por último um grupo com valores inferiores a 4,5 Unidades.g-1 de peso seco, onde se incluiu a couve-galega, couve-lombarda e couve-repolho. Relativamente ao efeito da
conservação verificou-se que a 4 ºC se registaram menores perdas de glucosinolatos; também o método de cozedura a vapor induziu a menor degradação de glucosinolatos. Neste trabalho, a couve-repolho e a couve-tronchuda Portuguesa revelaram ser menos susceptíveis a perdas do que o nabo e a couve-brócolo.
4.4. - “Avaliação do efeito dos glucosinolatos e seus produtos de hidrólise, em bactérias aeróbias facultativas do segmento gastrointestinal Humano.” Este trabalho (já publicado), reporta o efeito antibacteriano de diferentes glucosinolatos e respectivos hidrolisados, em condições controladas de crescimento (in vitro) em 18 bactérias aeróbias facultativas
(Gram-positivo e Gram-negativo) do segmento gastrointestinal humano. Foram testados 19
compostos, a 5 concentrações (0,015, 0,15, 0,75, 1,5 e 3,0 μmoles) utilizando o método de difusão em disco em meio sólido. Verificou-se que os glucosinolatos intactos não apresentaram qualquer efeito antibacteriano, ao contrário dos seus hidrolisados que se mostraram activos na inibição do crescimento in vitro daquelas bactérias, ainda que a sua eficácia tenha sido variável. Dos hidrolisados, os isotiocianatos foram os mais eficientes.
Verificou-se ainda que a ocorrência do efeito de inibição foi proporcional à concentração aplicada, embora este efeito dependa do tipo de bactérias.
4.5. - “Avaliação do efeito dos hidrolisados de glucosinolatos em Xanthomonas
campestris pv. campestris.” Neste trabalho (já publicado) avaliaram-se 11 importantes
hidrolisados dos glucosinolatos, a 5 concentrações diferentes (0,015, 0,15, 0,75, 1,5 e 3 μmoles) no crescimento in vitro da bactéria aeróbia fitopatogénica Xanthomonas campestris pv. campestris, uma das mais patogénicas bactérias das Brássicas, disseminada mundialmente. Para avaliar a actividade antibacteriana foi utilizado o método de difusão em disco em meio sólido. Os dados obtidos permitiram demonstrar que entre 11 hidrolisados analisados, 10 apresentaram actividade antibacteriana, e que o efeito antibacteriano foi sempre
proporcional à concentração aplicada. No entanto o efeito antibacteriano dependeu
significativamente da estrutura química dos hidrolisados, sendo os isotiocianatos os mais
eficazes na inibição do crescimento in vitro.
Capítulo 5 - “Conclusões e Considerações finais”. Neste capítulo, incluem-se as
principais conclusões obtidas com os diferentes trabalhos realizados e apontam-se
perspectivas futuras.
In Portugal, within the great diversity of vegetables produced and consumed are the plants belonging to Brassicaceae family, namely the genus Brassica, normally designated by “Brassica” or simply “cabbage”. These plants are a good dietary source of essential amino acids, proteins, dietary fibre, various carbohydrates, vitamins and minerals, but they also contain another class of compounds generally defined as “secondary metabolites” and more recently as “phytochemicals”. Although phytochemicals are not essential for the diet they can have important biological effects, and specifically in relation to reducing the risks of certain diseases such as cancer and coronary heart disease. Different biochemical and gene-induction effects have been reported for different classes of phytochemicals e.g. they can function as direct antioxidants preventing the formation of free radicals resulting from cellular metabolism, they can modulate the expression of genes associated with anticancer and antioxidant defences, and they can modulate the gut microflora. Phytochemicals have are described as “bioactive compounds”. Within the Brassica vegetables the most prominent are the glucosinolates and their enzymatic derivatives namely the isothiocyanates and indoles; depending on the Brassica species. In addition many Brassica foods are also rich in polyphenols (specifically flavonols and various hydroxycinnamic acid derivatives) and carotenoids. The current research, selected Brassica species common to Portugal, focuses on i. the composition of specific bioactives and nutrients (glucosinolates, total phenolics, total flavonoids, vitamin C, free amino acids and minerals), ii. the effect of growth conditions and climate on the composition of these bioactives and nutrients, and iii. the effects of crude Brassica extracts and specific Brassica phytochemicals in different bioassays. With this focus in mind the research is presented in the following logical sequence of chapters: Chapter 1 - “General Introduction”. In this chapter a general introduction on the subject matter is presented. The justifications for the research foci and the main objectives are also defined in this chapter. Chapter 2 - “Review”. Within this chapter information about the social and economic importance of Brassica plants in Portugal and globally are presented. Details on the respective composition and food values, as well the potential biological effects and factors which can influence them are also reviewed. Chapter 3 - “Material and Methods”. This chapter describes the production and utilization of the plant material produced in Vila Real, throughout two consecutive years in the two different production seasons: between September and January and April and July. The protocols and methods are clearly described for the sample preparations, the analytical methodologies, the various activity assays and the statistical analysis performed. Chapter 4 - “Experimental component”. Within the sub-chapters of this main chapter the major research results are presented - in the form of published, in press, or submitted papers to refereed journals: 4.1. - “Study of the variation of bioactive compounds”. In these studies the identification and quantification of individual and total glucosinolates, total phenolics and total flavonoids, vitamin C, free amino acids and minerals in broccoli, kale, savoy cabbage, white cabbage, tronchuda cabbage and turnip (green leaves and roots) were done. The materials evaluated were from two different climatic seasons (Spring-Summer and Summer-Winter) in two consecutive years (2005-2006 and 2006-2007). The results obtained, showed a significant effect of genotype, season and production year on the profile and average levels of the different bioactive compounds. It was noted that climatic stress during the production season increased the levels of several compounds, although each genotype presented specific reactions to the variations climatic conditions. 4.2. - “Study of the antioxidant activity of different Brassicas produced in two different production seasons”. In these studies the antioxidant potentials of the selected Brassica were evaluated, using the DPPH (2,2’-difenil-1-picrilhidrazil) and reducing capacity spetrophotometric methods. This capacity is correlated with total phenolics, total flavonoids and vitamin C contents, determined in 4.1. The results, with two methods, showed a significant variation in the antioxidant activity depending on the genotype, climate seasons and production year. The inflorescences of broccoli and the leaves of kale and tronchuda cabbage had the highest values of antioxidant activity. In general, the highest levels of antioxidant activity were achieved in the Summer-Winter season. Statistical correlations between total phenolics, total flavonoids and vitamin C were performed and show a strong association between the antioxidant activity of Brassica and the levels of the specific antioxidant compounds present in these vegetables. 4.3. - “Study of the myrosinase activity and the effect of storage and cooking treatment on glucosinolates content of four Brassicas”. In these studies two compositional parameters were measured (total myrosinase activities and profiles/contents of glucosinolates) in relation to two processing parameters (the effect of storage at room temperature and 4 ºC, and the type of cooking method used – either traditional-boiling, steam cooking or microwave cooking). The results showed that there were no significant differences in myrosinase activities between Brassicas. However, it was possible to aggregate the genotypes into three different groups – i.broccoli, turnip green leaves and Portuguese tronchuda cabbage, with myrosinase activities that > 5,5 Units g-1 dry weight (DW); ii. turnip with a myrosinase activity between 4,5 and 5,5 Units g-1 DW; iii. kale, savoy cabbage and white cabbage, with myrosinase activities < 4,5 Units g-1 DW. The post-harvest studies showed that the 4 ºC temperature lead to a higher level of glucosinolates and the steam method lead to a lower degradation of glucosinolate levels. The tronchuda cabbage and white cabbage were less affected during the processing method and the turnip and the broccoli were the most affected. 4.4. – “Evaluation of the effect of glucosinolates and their hydrolysis products on facultative aerobic bacteria from human gastrointestinal segments.” These studies were focused on studying the antibacterial effects of several glucosinolates and their respective hydrolysis products in controlled conditions (in vitro), on 18 aerobic bacterial strains(Gram-positive and Gram-negative) isolated from human gastrointestinal segments. Nineteen compounds at 5 different concentrations (0.015, 0.15, 0.75, 1.5 and 3 μmoles) were evaluatedusing a disc diffusion method. The results showed that the antibacterial effects were restricted to the glucosinolate hydrolysis products, and the isothiocyanates were the most effective. There was a clear proportional relationship between the antibacterial activities and the concentrations applied, although the effect was variable according the class of bacteria. 4.5 – “Evaluation of the effect of glucosinolates hydrolysis products on Xanthomonas campestris pv. campestris.” In these studies the antibacterial effects of 11 glucosinolate hydrolysis products at 5 concentrations (0.015, 0.15, 0.75, 1.5 and 3 μmoles) were evaluated on the in vitro growth of the aerobic phytopathogenic bacteria Xanthomonas campestris pv. campestris, globally one of the most pathogenic bacteria of Brassica. The antibacterial activities were determined using a disc diffusion method. The results showed inhibition effects were restricted to 10 of the 11 compounds tested, and the antibacterial effect wasalways proportional to the concentration applied. Nevertheless, the antibacterial effect was strongly dependent on the structure of the glucosinolate hydrolysis product – the isothiocyanates were the most effective inhibitors of in vitro bacterial growth. Chapter 5 – “Conclusions and Final considerations”. In this chapter the main conclusions of the research are presented and discussed.
In Portugal, within the great diversity of vegetables produced and consumed are the plants belonging to Brassicaceae family, namely the genus Brassica, normally designated by “Brassica” or simply “cabbage”. These plants are a good dietary source of essential amino acids, proteins, dietary fibre, various carbohydrates, vitamins and minerals, but they also contain another class of compounds generally defined as “secondary metabolites” and more recently as “phytochemicals”. Although phytochemicals are not essential for the diet they can have important biological effects, and specifically in relation to reducing the risks of certain diseases such as cancer and coronary heart disease. Different biochemical and gene-induction effects have been reported for different classes of phytochemicals e.g. they can function as direct antioxidants preventing the formation of free radicals resulting from cellular metabolism, they can modulate the expression of genes associated with anticancer and antioxidant defences, and they can modulate the gut microflora. Phytochemicals have are described as “bioactive compounds”. Within the Brassica vegetables the most prominent are the glucosinolates and their enzymatic derivatives namely the isothiocyanates and indoles; depending on the Brassica species. In addition many Brassica foods are also rich in polyphenols (specifically flavonols and various hydroxycinnamic acid derivatives) and carotenoids. The current research, selected Brassica species common to Portugal, focuses on i. the composition of specific bioactives and nutrients (glucosinolates, total phenolics, total flavonoids, vitamin C, free amino acids and minerals), ii. the effect of growth conditions and climate on the composition of these bioactives and nutrients, and iii. the effects of crude Brassica extracts and specific Brassica phytochemicals in different bioassays. With this focus in mind the research is presented in the following logical sequence of chapters: Chapter 1 - “General Introduction”. In this chapter a general introduction on the subject matter is presented. The justifications for the research foci and the main objectives are also defined in this chapter. Chapter 2 - “Review”. Within this chapter information about the social and economic importance of Brassica plants in Portugal and globally are presented. Details on the respective composition and food values, as well the potential biological effects and factors which can influence them are also reviewed. Chapter 3 - “Material and Methods”. This chapter describes the production and utilization of the plant material produced in Vila Real, throughout two consecutive years in the two different production seasons: between September and January and April and July. The protocols and methods are clearly described for the sample preparations, the analytical methodologies, the various activity assays and the statistical analysis performed. Chapter 4 - “Experimental component”. Within the sub-chapters of this main chapter the major research results are presented - in the form of published, in press, or submitted papers to refereed journals: 4.1. - “Study of the variation of bioactive compounds”. In these studies the identification and quantification of individual and total glucosinolates, total phenolics and total flavonoids, vitamin C, free amino acids and minerals in broccoli, kale, savoy cabbage, white cabbage, tronchuda cabbage and turnip (green leaves and roots) were done. The materials evaluated were from two different climatic seasons (Spring-Summer and Summer-Winter) in two consecutive years (2005-2006 and 2006-2007). The results obtained, showed a significant effect of genotype, season and production year on the profile and average levels of the different bioactive compounds. It was noted that climatic stress during the production season increased the levels of several compounds, although each genotype presented specific reactions to the variations climatic conditions. 4.2. - “Study of the antioxidant activity of different Brassicas produced in two different production seasons”. In these studies the antioxidant potentials of the selected Brassica were evaluated, using the DPPH (2,2’-difenil-1-picrilhidrazil) and reducing capacity spetrophotometric methods. This capacity is correlated with total phenolics, total flavonoids and vitamin C contents, determined in 4.1. The results, with two methods, showed a significant variation in the antioxidant activity depending on the genotype, climate seasons and production year. The inflorescences of broccoli and the leaves of kale and tronchuda cabbage had the highest values of antioxidant activity. In general, the highest levels of antioxidant activity were achieved in the Summer-Winter season. Statistical correlations between total phenolics, total flavonoids and vitamin C were performed and show a strong association between the antioxidant activity of Brassica and the levels of the specific antioxidant compounds present in these vegetables. 4.3. - “Study of the myrosinase activity and the effect of storage and cooking treatment on glucosinolates content of four Brassicas”. In these studies two compositional parameters were measured (total myrosinase activities and profiles/contents of glucosinolates) in relation to two processing parameters (the effect of storage at room temperature and 4 ºC, and the type of cooking method used – either traditional-boiling, steam cooking or microwave cooking). The results showed that there were no significant differences in myrosinase activities between Brassicas. However, it was possible to aggregate the genotypes into three different groups – i.broccoli, turnip green leaves and Portuguese tronchuda cabbage, with myrosinase activities that > 5,5 Units g-1 dry weight (DW); ii. turnip with a myrosinase activity between 4,5 and 5,5 Units g-1 DW; iii. kale, savoy cabbage and white cabbage, with myrosinase activities < 4,5 Units g-1 DW. The post-harvest studies showed that the 4 ºC temperature lead to a higher level of glucosinolates and the steam method lead to a lower degradation of glucosinolate levels. The tronchuda cabbage and white cabbage were less affected during the processing method and the turnip and the broccoli were the most affected. 4.4. – “Evaluation of the effect of glucosinolates and their hydrolysis products on facultative aerobic bacteria from human gastrointestinal segments.” These studies were focused on studying the antibacterial effects of several glucosinolates and their respective hydrolysis products in controlled conditions (in vitro), on 18 aerobic bacterial strains(Gram-positive and Gram-negative) isolated from human gastrointestinal segments. Nineteen compounds at 5 different concentrations (0.015, 0.15, 0.75, 1.5 and 3 μmoles) were evaluatedusing a disc diffusion method. The results showed that the antibacterial effects were restricted to the glucosinolate hydrolysis products, and the isothiocyanates were the most effective. There was a clear proportional relationship between the antibacterial activities and the concentrations applied, although the effect was variable according the class of bacteria. 4.5 – “Evaluation of the effect of glucosinolates hydrolysis products on Xanthomonas campestris pv. campestris.” In these studies the antibacterial effects of 11 glucosinolate hydrolysis products at 5 concentrations (0.015, 0.15, 0.75, 1.5 and 3 μmoles) were evaluated on the in vitro growth of the aerobic phytopathogenic bacteria Xanthomonas campestris pv. campestris, globally one of the most pathogenic bacteria of Brassica. The antibacterial activities were determined using a disc diffusion method. The results showed inhibition effects were restricted to 10 of the 11 compounds tested, and the antibacterial effect wasalways proportional to the concentration applied. Nevertheless, the antibacterial effect was strongly dependent on the structure of the glucosinolate hydrolysis product – the isothiocyanates were the most effective inhibitors of in vitro bacterial growth. Chapter 5 – “Conclusions and Final considerations”. In this chapter the main conclusions of the research are presented and discussed.
Descrição
Tese de Doutoramento em Ciências Agronómicas,