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Atividade antioxidante da casca da pêra abacate (Persea americana) e identificação dos seus compostos
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A fruta e os legumes são essenciais na alimentação humana, uma vez que contêm muitos dos nutrientes essenciais à nutrição humana. A pêra abacate (Persea americana) é muito consumida devido ao seu elevado valor nutricional e também por apresentar benefícios para a saúde, nomeadamente, a atividade anticancerígena, antioxidante in vivo e inibição da oxidação de lípidos e proteínas, o que aumenta a interesse científico na determinação da sua composição. Devido às propriedades demostradas por este fruto e, sendo a casca da pêra abacate um importante resíduo das indústrias de processamento deste fruto, o principal objetivo deste trabalho foi a valorização destes resíduos, com vista à recuperação dos compostos de elevado valor acrescentado, nomeadamente, compostos fenólicos presentes nos extratos aquosos da sua casca. Tendo em conta que a pêra abacate foi descrita como uma mistura complexa de proteínas, açúcares, vitaminas, taninos, ácidos gordos, aminoácidos, compostos fenólicos, entre outros, as várias espécies apresentam entre si diferenças ao nível nutricional e na composição. Neste trabalho fez-se uma decocção da casca da pêra abacate do tipo Hass e testaram-se vários métodos de purificação dos compostos fenólicos presentes no extrato. Os processos de purificação foram a diálise a pH ácido, a digestão com pepsina seguida de diálise e o método de precipitação em etanol. Este último foi o que revelou melhores resultados, permitindo obter no sobrenadante recuperado 69,28± 0,71 𝜇𝑔 𝑑𝑒 𝑓𝑒𝑛ó𝑖𝑠/ 𝑚𝑔 𝑑𝑒 𝑒𝑥𝑡𝑟𝑎𝑡𝑜 e 36,96 ± 2,79×10−3𝜇𝑔 𝑑𝑒 𝑡𝑎𝑛𝑖𝑛𝑜𝑠 /𝑚𝑔 𝑑𝑒 𝑒𝑥𝑡𝑟𝑎𝑡𝑜, valores significativamente superiores a resultados já descritos por outros processos mais complicados de extração de compostos fenólicos e com maior impacto ambiental. Para o sobrenadante recuperado por este processo de purificação foi analisada a sua atividades antioxidante na capacidade de extinção do radical DPPH 𝐸𝑐50 =51,45 ± 1,09×10−3 𝜇𝑔/𝑚𝐿, na capacidade de inibição da peroxidação lipídica 𝑇𝐵𝐴𝑅𝑆 = 451,3 ± 0,041 𝜇𝑔/𝑚𝐿, na capacidade de eliminação do óxido nítrico 𝑁𝑂 =110,49 ± 0,01 𝜇𝑔/𝑚𝐿 e na atividade inibitória da acetilcolinesterase 𝐼𝐶 50= 2602,01 ± 2,62×10−4 𝜇𝑔/𝑚𝐿. Foi também feito o estudo da citotoxicidade, em linhas celulares humanas do cancro do fígado, HepG-2, do extrato, do sobrenadante e do precipitado, tendo-se considerado que estes não eram tóxicos. A identificação dos compostos bioativos presentes no extrato da casca da pêra abacate foi feita com recurso a LC-MS e HPLC-DAD. Os compostos fenólicos identificados foram: o ácido quínico, a epicatequina, o ácido glucónico, a procianidina B, a procianidina C, o dímero da etil-epicatequina, a quercetina di-hexose e a quercetina – O-hexose-O-pentose, o 7-Glucosilo-11-metil-oleosido e o ácido 3,4,5-Tri-O- galoilquínico, sendo os maioritários presentes o ácido glucónico, o ácido quinico, a procianidina B e a epicatequina. A quantificação de ácido glucónico e epicatequina presentes no sobrenadante foi efetuado sendo de 673,7 μ𝑔 𝑑𝑒 á𝑐𝑖𝑑𝑜 𝑔𝑙𝑢𝑐ó𝑛𝑖𝑐𝑜/𝑚𝑔 𝑑𝑒 𝑒𝑥𝑡𝑟𝑎𝑐𝑡𝑜 e 6,50 μ𝑔 𝑑𝑒 𝑒𝑝𝑖𝑐𝑎𝑡𝑒𝑞𝑢𝑖𝑛𝑎/𝑚𝑔 𝑑𝑒 𝑒𝑥𝑡𝑟𝑎𝑐𝑡𝑜, respetivamente. Foi ainda analisada a contribuição dos compostos maioritários presentes no sobrenadante para as atividades biológicas demonstradas. O ácido glucónico contribui para a capacidade de extinção do radical DPPH do sobrenadante em 0,6 %, 22,1 % para a inibição da peroxidação lipídica pelo método dos TBARS e 7,8% para a eliminação do NO. Quanto à epicatequina a percentagem de contribuição para a capacidade de extinção do radical DPPH é de 5 %, 0,21 % para a inibição da peroxidação lipídica pelo método dos TBARS e 0,4 % para a eliminação do NO. É possível concluir que foi com este trabalho foi conseguido implementar com sucesso um processo de recuperação dos compostos naturais bioativos da casca da pêra abacate para a valorização de um resíduo. Os compostos fenólicos recuperados revelaram ser poderosos antioxidantes e apresentar uma baixa toxicidade e a sua identificação e quantificação torna-se extremamente importante no sentido de caracterizar quimicamente a mistura de compostos recuperados e perspetivar outras potencialidades de aplicação destes compostos de elevado valor acrescentado.
Fruit and vegetables are essential in human nutrition, since it contains numerous essencial nutrients for human nutrition. The avocado (Persea americana) is much consumed due to its nutritional value and also because it has health benefits, including anticancer activity, in vivo antioxidant and inhibition of lipid and protein oxidation, which increases the scientific interest in determining its composition. Due to the properties evidenced by the avocado and because its peel is an important waste of the processing fruit industry, the main objective of this work was the valorization of such waste for the recovery of compounds of high added value, in particular phenolic compounds present in the aqueous extracts of the peel. Although the avocado has been described as a complex mixture of proteins, sugars, vitamins, tannins, fatty acids, amino acids, phenolic compounds, among others. Different species of avocado have differences in nutritional value and in chemical composition. In this work an infusion of type Hass avocados peel was prepared and several methods for purification were applied for the recovery of the phenolic compounds present in the extract. The purification procedures tested were dialysis at acid pH, the digestion with pepsin followed by dialysis and ethanol precipitation method. This latter has showed better results allowing to obtain in the recovered supernatant 69.28± 0.71 μg of phenols/ mg of extract and 36.96 ± 2.79×10−3 μg of tannins /mg of extract, these were significantly higher values than the results previously described in the literature using more complicated processes of extraction of phenolic compounds and with more environmental impact. The supernatant recovered by this purification process was analyzed and the antioxidant activities determined were the capacity of radical DPPH extinction 𝐸𝑐50 =51.45 ± 1.09×10−3 μg/mL, the capacity of lipid peroxidation inhibition 𝑇𝐵𝐴𝑅𝑆 = 451.13 ± 0.041 μg/mL, the capacity of nitric oxide elimination𝑁𝑂 =110.49 ± 0.01 μg/mL and the acetylcholinesterase inhibitory activity 𝐼𝐶 50= 2602.01 ± 2.62×10−4 μg/mL. It was also studied the cytotoxicity against human liver cancer cell line of HepG-2 of the extract, the supernatant and the precipitate obtained in the purification, which revealed not to be toxic. The identification of bioactive compounds present in the extract of avocado peel was accomplished by LC-MS and HPLC-DAD. The phenolic compounds identified were: quinic acid, epicatechin, gluconic acid, procyanidin B, procianidin C, epicatechin-ethyl dimer, quercetin di- hexose, quercetin-O-hexose-O-pentose, 7-glucosyl-11-methyl-oleosid and 3,4,5-tri-O-galoilquínic acid, the major compounds presents were gluconic acid, quinic acid, procianidin B and epicatechin. Quantification of gluconic acid and epicatechin in the supernatant was performed and 673.7 μg of gluconic acid/mg of extract and 6.50 μg of epicatechin/mg of extract were determined, respectively. It was also analyzed the contribution of the major compounds present in the supernatant to the exhibited biological activities. The gluconic acid contributes to the capacity of radical DPPH extinction in 0.6%, 22.1% to lipid peroxidation inhibition by the TBARS method and 7.8 % for NO elimination. Concerning epicatechin the percentage of contribution to the DPPH radical extinction capacity was 5 %, 0.21 % for lipid peroxidation inhibition by TBARS method and 0.4% for NO elimination. In conclusion, in this work it was successfully implemented a process for the recovery of bioactive natural compounds from avocado peel for this waste valorization. The recovered phenolic compounds revealed to be powerful antioxidants with low toxicity therefore its identification and quantification was extremely important in order to chemically characterize the recovered mixture of compounds and to predict potential applications of these high added value compounds.
Fruit and vegetables are essential in human nutrition, since it contains numerous essencial nutrients for human nutrition. The avocado (Persea americana) is much consumed due to its nutritional value and also because it has health benefits, including anticancer activity, in vivo antioxidant and inhibition of lipid and protein oxidation, which increases the scientific interest in determining its composition. Due to the properties evidenced by the avocado and because its peel is an important waste of the processing fruit industry, the main objective of this work was the valorization of such waste for the recovery of compounds of high added value, in particular phenolic compounds present in the aqueous extracts of the peel. Although the avocado has been described as a complex mixture of proteins, sugars, vitamins, tannins, fatty acids, amino acids, phenolic compounds, among others. Different species of avocado have differences in nutritional value and in chemical composition. In this work an infusion of type Hass avocados peel was prepared and several methods for purification were applied for the recovery of the phenolic compounds present in the extract. The purification procedures tested were dialysis at acid pH, the digestion with pepsin followed by dialysis and ethanol precipitation method. This latter has showed better results allowing to obtain in the recovered supernatant 69.28± 0.71 μg of phenols/ mg of extract and 36.96 ± 2.79×10−3 μg of tannins /mg of extract, these were significantly higher values than the results previously described in the literature using more complicated processes of extraction of phenolic compounds and with more environmental impact. The supernatant recovered by this purification process was analyzed and the antioxidant activities determined were the capacity of radical DPPH extinction 𝐸𝑐50 =51.45 ± 1.09×10−3 μg/mL, the capacity of lipid peroxidation inhibition 𝑇𝐵𝐴𝑅𝑆 = 451.13 ± 0.041 μg/mL, the capacity of nitric oxide elimination𝑁𝑂 =110.49 ± 0.01 μg/mL and the acetylcholinesterase inhibitory activity 𝐼𝐶 50= 2602.01 ± 2.62×10−4 μg/mL. It was also studied the cytotoxicity against human liver cancer cell line of HepG-2 of the extract, the supernatant and the precipitate obtained in the purification, which revealed not to be toxic. The identification of bioactive compounds present in the extract of avocado peel was accomplished by LC-MS and HPLC-DAD. The phenolic compounds identified were: quinic acid, epicatechin, gluconic acid, procyanidin B, procianidin C, epicatechin-ethyl dimer, quercetin di- hexose, quercetin-O-hexose-O-pentose, 7-glucosyl-11-methyl-oleosid and 3,4,5-tri-O-galoilquínic acid, the major compounds presents were gluconic acid, quinic acid, procianidin B and epicatechin. Quantification of gluconic acid and epicatechin in the supernatant was performed and 673.7 μg of gluconic acid/mg of extract and 6.50 μg of epicatechin/mg of extract were determined, respectively. It was also analyzed the contribution of the major compounds present in the supernatant to the exhibited biological activities. The gluconic acid contributes to the capacity of radical DPPH extinction in 0.6%, 22.1% to lipid peroxidation inhibition by the TBARS method and 7.8 % for NO elimination. Concerning epicatechin the percentage of contribution to the DPPH radical extinction capacity was 5 %, 0.21 % for lipid peroxidation inhibition by TBARS method and 0.4% for NO elimination. In conclusion, in this work it was successfully implemented a process for the recovery of bioactive natural compounds from avocado peel for this waste valorization. The recovered phenolic compounds revealed to be powerful antioxidants with low toxicity therefore its identification and quantification was extremely important in order to chemically characterize the recovered mixture of compounds and to predict potential applications of these high added value compounds.
Descrição
Tese de mestrado, Química, Universidade de Lisboa, Faculdade de Ciências, 2016
Palavras-chave
Pêra abacate LC-MS Compostos fenólicos Atividade antioxidante Valorização de resíduos Teses de mestrado - 2014