Efeito da biotransformação do resíduo de citrus na proteção vascular

Orientador: Gabriela Alves MacedoTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: O Brasil é considerado o maior produtor mundial de suco de laranja, resultando na produção de toneladas de resíduos industriais de citrus. Esses resíduos são considerados uma fonte importante de compostos fenólicos, especialmente de flavononas. Os compostos fenólicos são conhecidos devido aos seus efeitos bioativos, que envolvem suas propriedades antioxidantes e de proteção cardiovascular. Estudos recentes têm mostrado que os fenólicos na forma aglicona apresentam maior bioatividade de que na forma glicosídica. Entretanto, esses estudos utilizam padrões analíticos de alto custo, já que não existem fontes comerciais dessas agliconas. Uma das estratégias empregada para produzir extratos ricos em agliconas é a biotransformação enzimática. Baseado nisso, esse estudo teve como objetivo avaliar os efeitos da biotransformação enzimática no potencial antioxidante e anti-hipertensivo de extratos cítricos preparados a partir de resíduos industriais de citrus. Dois resíduos cítricos foram estudados, um proveniente da extração de suco de laranja e outro resíduo proveniente da extração de pectina. Diferentes processos enzimáticos foram aplicados empregando as enzimas celulase, pectinase, tanase e ?-glicosidase de forma isolada ou combinada. O perfil fenólico dos extratos cítricos foi analisado por HPLC-DAD. O potencial antioxidante dos extratos foi avaliado pelos métodos de Folin-Cicalteou, ORAC, DPPH e FRAP. O potencial anti-hipertensivo in vitro dos extratos foi avaliado pela inibição da enzima conversora de angiotensina (ECA) e pelo efeito vasorelaxante em anéis de artéria ilíaca de rato. Testes paralelos foram conduzidos utilizando padrões analíticos e drogas comerciais para elucidar os mecanismos envolvidos no efeito anti-hipertensivo dos extratos de cítricos. Os resultados mostraram que os extratos dos resíduos da extração de pectina apresentaram maior concentração de hesperidina em relação aos extratos dos resíduos de suco. A análise por HPLC mostrou claramente a mudança do perfil fenólico dos extratos de cítricos após o tratamento enzimático com tanase e ?-glicosidase, levando a produção de 3,044 e 5,338 ?g/ g de hesperetina a partir dos resíduos da extração de suco e de pectina, respectivamente. Entre os extratos estudados, o extrato dos resíduos de pectina biotransformado apresentou maior atividade antioxidante e foi o mais eficaz em inibir a atividade da ECA (Emáx = 100% e IC50 =0,21 mg/ ml). Esse extrato também foi o mais eficiente em induzir o relaxamento de vasos (Emáx = 100% e EC50 =0,47 mg/ ml). Nossos resultados confirmaram o maior potencial anti-ACE (100% e 76%) e vasorelaxante (EC50 = 20 µM e 1,3 mM) da aglicona hesperetina em relação ao glicosídeo hesperidina, justificando os melhores resultados dos extratos biotransformados ricos em flavononas agliconas. O vasorelaxamento induzido pelos extratos dos resíduos da extração de pectina foi independente do endotélio. Ainda, o relaxamento foi potencializado quando a enzima GCs estava oxidada devido ao estresse oxidativo, sendo comparável ao BAY58-2667. Assim, esse trabalho mostrou que a biotransformação enzimática pode ser utilizada para a redução e reutilização de resíduos da maior indústria processadora de citrus do mundo, podendo ser utilizados na indústria farmacêutica e alimentícia como ingredientes para o desenvolvimento de produtos para a prevenção e para auxiliar no tratamento de doenças vascularesAbstract: Brazil is the world's largest orange juice producer, resulting in the production of tons of industrial citrus waste. These residues are considered an important source of phenolic compounds, especially flavanones. Phenolic compounds are known due to their bioactive effects, which involve their antioxidant properties and cardiovascular protection. Recent studies have shown that phenolics in the aglycone form exhibited greater bioactivity than in the glycosidic form. However, these studies use high cost analytical standards, since there are no commercial sources of these aglycones. One of the strategies to produce extracts rich in aglycones is the enzymatic biotransformation. Based on this, this study aimed to evaluate the effects of enzymatic biotransformation on the antioxidant and antihypertensive potential of citrus extracts from industrial citrus wastes. Two citrus residues were studied, one from the extraction of orange juice and another from pectin extraction. Different enzymatic processes were applied using the enzymes cellulase, pectinase, tannase and ?-glucosidase alone or in combination. The phenolic profile of the citrus extracts was analyzed by HPLC-DAD. The antioxidant potential of the extracts was evaluated by Folin-Cicalteou, ORAC, DPPH and FRAP assays. The in vitro antihypertensive potential of the extracts was evaluated by the inhibition of the angiotensin converting enzyme (ACE) and by the vasorelaxant effect in rat iliac artery rings. Parallel tests were conducted using analytical standards and commercial drugs to elucidate the pathways involved in the antihypertensive effect of citrus extracts. The results showed that extracts of pectin extraction residues showed higher concentration of hesperidin in relation to extracts of juice residues. HPLC analysis clearly showed the changes in the phenolic profile of citrus extracts after the enzymatic treatment with tannase and ?-glycosidase, leading to the production of 3044 and 5338 ?g/ g hesperetin from extracts of juice and pectin residues, respectively. Among the extracts studied, the extract of pectin residues biotransformed presented higher antioxidant activity and was the most effective in inhibiting ACE activity (Emax = 100% and IC50 = 0.21 mg/ ml). This extract was also the most efficient in inducing vasorelaxation (Emax = 100% and EC50 = 0.47 mg/ ml). Our results confirmed the higher anti-ACE (100% and 76%) and vasorelaxant (EC50 = 20 ?M and 1.3 mM) effects of the hesperetin aglycone in relation to the hesperidin glycoside, justifying the best results of biotransformed extracts rich in flavanones aglycones. The vasorelaxation induced by extracts from pectin extraction residues was independent of the endothelium. Furthermore, relaxation was potentiated when the GCs enzyme was oxidized due to oxidative stress, being comparable to BAY58-2667. Thus, this work showed that enzymatic biotransformation can be used to reduce and reuse residues from the world's largest citrus processing industry, since they could be used in the pharmaceutical and food industry as ingredients for the development of products for the prevention and in the treatment of vascular diseasesDoutoradoNutrição Experimental e Aplicada à Tecnologia de AlimentosDoutora em Alimentos e Nutrição140537/2015-52015/04555-2CNPQFAPES

Conditions of enzyme-assisted extraction to increase the recovery of flavanone aglycones from pectin waste

The citrus pectin by-product (CPB), generated from pectin industry, is a rich-source of flavanones, but not explored until now. As most of these compounds are inside vacuoles or bound to cell wall matrix, enzymatic hydrolysis was applied on their recovery, followed by hydroalcoholic and ultrasound extraction. Different parameters were studied: enzymes (β-glucosidase, tannase, and cellulase), their concentration (5, 10, and 20 U g−1 CPB), and reaction time (6, 12, and 24 h). Extracts were characterized in total phenolic content (TPC), antioxidant capacity (ORAC and DPPH assays), and polyphenolic profile (HPLC–DAD). All enzymatic treatments significantly improved CPB antioxidant capacity and TPC, compared with hydroalcoholic and ultrasound extraction. β-glucosidase (5 U) for 24 h was the most effective in polyphenol extraction and bioconversion, followed by β-glucosidase (5 U) for 12 h and tannase (5 U) for 24 h. Thus, the concentration of these enzymes was increased (10 and 20 U) to improve flavanones extraction. β-glucosidase at 20 U offered the highest amount of naringenin (77.63 mg 100 g−1 of CPB) and hesperetin (766.44 mg 100 g−1) obtained so far by biological processes. According to Person’s correlation analysis, TPC and antioxidant activity were highly correlated with CPB contents of hesperetin and naringenin. The aglycone flavanones are rarely found in natural sources and have higher biological potential than their glycosylated forms. Our results indicated enzyme-assisted extraction as a good choice for recovering aglycone flavanones from CPB, and increased knowledge on the biological activity of this agroindustrial waste, amplifying their application in food and pharmaceutical fieldCONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPnão tem2015/04555-

INFUSÃO DE Aloysia triphylla: EFEITOS OPOSTOS EM UM TESTE DE ATIVIDADE ANTIOXIDANTE IN VITRO E NA ESTABILIDADE OXIDATIVA DE PATÊS DE PESCADO REFRIGERADOS

O objetivo deste trabalho foi avaliar o efeito da infusão de Aloysia triphylla (IAT) sobre os valores de pH e a estabilidade oxidativa de patês de carne de jundiá (Rhamdia quelen) armazenados refrigerados. A IAT apresentou 1,8 mg de fenólicos totais/mL e teve boa capacidade antioxidante no ensaio de remoção do radical 2,2-difenil-1-picrilhidrazila (DPPH) (equivalente a uma solução 3,2 mM de trolox). As formulações de patês, sendo uma controle (água) e três formulações com quantidades crescentes de IAT (18 mg, 45 mg e 90 mg de fenólicos totais/kg de patê) foram armazenadas a 5°C e avaliadas ao longo de 28 dias quanto ao pH, oxidação lipídica (substâncias reativas ao ácido tiobarbitúrico, TBARS) e proteica (carbonilação de proteínas) e cor instrumental (CIELab). Os valores de pH reduziram ao longo da armazenagem, enquanto os de TBARS aumentaram até o 9º dia e depois reduziram (p<0.05), mas nenhuma dessas medidas foi influenciada pela IAT (p<0,05). A oxidação proteica aumentou ao longo do armazenamento (p<0.05) e as formulações com 45 mg e 90 mg de fenólicos/kg tiveram maior carbonilação proteica que o controle nos dias 21 e 28 de armazenagem, respectivamente (p<0,05). Comparadas ao controle, as formulações com IAT tiveram maiores valores de L* no início da armazenagem e menores valores de a* e croma a partir do 15º dia de armazenagem (p<0,05). A IAT não foi efetiva como antioxidante em patês de jundiá, ainda que tenha apresentado boa atividade antioxidante no ensaio de remoção do radical DPPH in vitro.

Oxidative stability of refrigerated fish pates containing loquat seed extract

This study investigated the effects of hydroethanolic E. japonica seed extracts (EJSE) as inhibitors of lipid and protein oxidation on fish pates subjected to refrigerated storage. Five fish pate formulations were developed. These formulations included two control pates (water-control and ascorbic acid-control) and three pates with added EJSE (0.1, 0.2 and 0.4g of seed 100g-1 product, equivalent to 3.4, 6.8 or 13.6mg phenolic compounds kg-1 product), which were then stored under refrigeration for 35 days. Conjugated dienes (CD) and peroxide (PV) values increased along with the storage time; however, these values decreased and were similar among all samples at the end of 35 days of analysis (P<0.05). However, the thiobarbituric acid reactive substances levels (TBARS) did not change along the storage and were not affected by the EJSE. Additionally, there was a linear increase in the protein carbonyl content of fish pates over the storage period (P<0.05), but no effect of EJSE on protein oxidation. The results show that, at the concentrations evaluated, hydroethanolic E. japonica seed extract was unable to inhibit or reduce lipid and protein oxidation in fish pates, but the observed phenolic content emphasizes the need for further studies on the wastes of this fruit

Antioxidant potential of barley extract in rats subjected to a high-fat diet

Antioxidants have the ability to neutralize free radicals produced in the body during lipid oxidation. The objective in this article was to study the effect of the barley extract on lipid oxidation in rats subjected to a high-fat diet. The experiment lasted 67 days. The animals were separated into three experimental groups: standard (P), high-fat diet group (L), and group with high-fat diet supplemented with barley extract (C). The feed intake of L and C groups was the lowest (p < 0.05). The treatments did not influence weight gain, organ weight, and the blood parameters measured. However, the levels of malondialdehyde present in the liver tissue were higher in the L group and lower in the P and C groups. Therefore, the results indicated an increased level of lipid peroxidation in the liver of rats subjected to high-fat diet, which was reduced by the consumption of barley

Antioxidant potential of barley extract in rats subjected to a high-fat diet Potencial antioxidante de extrato de cevada em ratos submetidos à dieta hiperlipídica

Antioxidants have the ability to neutralize free radicals produced in the body during lipid oxidation. The objective in this article was to study the effect of the barley extract on lipid oxidation in rats subjected to a high-fat diet. The experiment lasted 67 days. The animals were separated into three experimental groups: standard (P), high-fat diet group (L), and group with high-fat diet supplemented with barley extract (C). The feed intake of L and C groups was the lowest (p < 0.05). The treatments did not influence weight gain, organ weight, and the blood parameters measured. However, the levels of malondialdehyde present in the liver tissue were higher in the L group and lower in the P and C groups. Therefore, the results indicated an increased level of lipid peroxidation in the liver of rats subjected to high-fat diet, which was reduced by the consumption of barley.<br>Os antioxidantes têm a capacidade de neutralizar os radicais livres gerados no organismo durante a oxidação lipídica. O objetivo deste artigo foi estudar o efeito do extrato de cevada na oxidação lipídica em ratos submetidos a uma dieta rica em gordura. O experimento durou 67 dias. Os animais foram separados em três grupos experimentais: grupo padrão (P), grupo de dieta rica em gordura (L) e grupo com dieta rica em gordura suplementada com extrato de cevada (C). O consumo de ração dos grupos L e C foi mais baixa (p < 0,05). Os tratamentos não influenciaram no ganho de peso, no peso dos órgãos e nos parâmetros sanguíneos avaliados. No entanto, os níveis de malondialdeído presentes no tecido hepático foram maiores no grupo L e menores nos grupos P e C. Portanto, os resultados indicaram um aumento do nível de peroxidação lipídica no fígado de ratos submetidos à dieta rica em gordura, a qual foi reduzida com o consumo de cevada

Aglycone-rich extracts from citrus by-products induced endothelium-independent relaxation in isolated arteries

Citrus by-products are rich in flavanone aglycons which have lower bioactivity than aglycone forms. Based on this, the study evaluated the effects of enzyme biotransformation on antioxidant and vasorelaxant capacity of polyphenolic rich-extracts from citrus waste. Enzymatic treatment produced extracts with high hesperetin concentrations and higher antioxidant capacity than non-treated extracts. At the highest concentration tested (3 mg mL−1), citrus juice by-products exhibited only a partial vasorelaxant effect. After the enzymatic treatment, the extract had a maximal effect of 100% relaxation and enhanced their pEC50. The relaxation promoted by citrus pectin by-products was potentiated in the presence of ODQ, suggesting that vasorelaxant effects of these extracts may occur even when sGC is oxidized because of vascular oxidative stress. Thus, the enzyme-assisted extraction appears to enhance antioxidant and vasorelaxant properties of citrus extracts, which could be used to develop new products with antihypertensive properties23CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP140537/2015-52015/04555-

Recovery of phenolic compounds from citrus by-products using pressurized liquids — an application to orange peel

Citrus industry residues are sources of phenolic compounds, which may be extracted by pressurized liquid extraction (PLE) and volatile compounds, extractable by supercritical fluid extraction (SFE). PLE and SFE are fast and allow using non-toxic solvents and moderate temperatures. Therefore, the objective of this work was to extract volatile and phenolic compounds from orange peel by SFE and PLE. The raw material was orange peel without (Lot 1) and with (Lot 2) previous supercritical CO2 extraction performed at 40 °C and 35 MPa. The volatile profile was evaluated in the SFE extract by HS-SPME-CG-MS. PLE solvents were absolute ethanol and mixtures of ethanol and water (75% and 50% ethanol, v/v) at the temperatures 45, 55, and 65 °C and pressure of 10 MPa. Global yield, total phenolic content (TPC), antioxidant capacity by DPPH and FRAP methods, total reducing sugars and the concentration of the major phenolic compounds by HPLC were evaluated in the extracts. Temperature and ethanol concentration had significant effects on all responses. The main volatile compound found in the SFE extract was α-Terpineol, followed by d-Limonene. The major phenolic compound was hesperidin, which highest recovery (19.3 ± 0.9 mg/g dry peel) was achieved with 75% ethanol at 65 °C from Lot 1. At the same condition, high TPC and antioxidant capacity were also achieved. The three-line spline and two-site kinetic models provided good adjustments to the PLE curves, being able to describe their behavior. PLE using water and ethanol can be applied to recover phenolics from a large variety of fruit by-products112921CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP140883/2015-0Sem informação2015/11932-