PROTIC IONIC LIQUIDS AS EXTRACTIVE TOOL FOR ANALYSING ANTIOXIDANT POTENTIAL OF ACEROLA (Malpighia emarginata)

ISBN 978-85-85905-19-4

Área

Química Verde

Autores

Garcia-martinez, E. (UNIVERSIDAD DE VALENCIA) ; Passos, F. (UNIVERSIDADE FEDERAL DA BAHIA) ; Yuste, A. (UNIVERSIDAD DE VALENCIA) ; Martínez-navarrete, N. (UNIVERSIDAD DE VALENCIA) ; Iglesias, M. (UNIVERSIDADE FEDERAL DA BAHIA)

Resumo

Acerola fruit (Malpighia emarginata) is well known as an excellent food source of vitamin C, and it also contains phytochemicals such as carotenoids and polyphenols which significantly contribute to its total antioxidant capacity. The extractions and analyses of the bioactive substances present in fruits involve techniques that use organic solvents that are highly toxic to human health and environment. To improve the safety and environmental friendliness of conventional separation techniques, Protic Ionic Liquids (PIL’s) were used as ideal substitutes because of their stability, nonvolatility and adjustable miscibility and polarity. The protic ionic liquid 2HDEAPr gathered the best results as additive solvent in the extraction of bioactive compounds.

Palavras chaves

Protics Ionics Liquids; Antioxidants; Acerola

Introdução

Fruits, in particular the tropical ones, are aliments of great interest for human nutrition due to their high nutritious and therapeutic properties. (Schieber, 2001) Studies provide evidences that the ingestion of foods that are rich in natural compounds promotes the stabilization of free radicals (antioxidant capacity) in the human body and reduces infirmities which are of great concern, such as cancer and cardiovascular diseases. (Halliwell, 2007, Food Ingredients Bazil, 2015) The acerola, a tropical fruit found in Brazil roughly 60 years ago, is cultivated especially in the country’s northeastern region, markedly in the states of Pernambuco, Paraíba, Bahia and Ceará. It poses itself as a fruit richly laden with vitamins, e.g. ascorbic acid (Vitamin C), carotene (Vitamin A), thiamine (Vitamin B1), riboflavin (Vitamin B2), niacin and mineral salts (iron, calcium, phosphorus) and sugars, besides also being rich in bioactive substances (carotenoids and phenolic compounds) that grant it great antioxidant power. The high concentrations of ascorbic acid confer the acerola the ability to prevent fatigue and appetite loss, diminishing one’s odds of contracting infectious diseases and suffering from muscular and joint pains. However, despite its nutritious relevance, the acerola is yet to be amply studied (Yamashita, 2003). The productivity and quality of the acerola fruits have been perfected throughout the last few years, having the processing industries of Brazil absorbed most of the national production in the forms of pulps, frozen fruits and pasteurized juices. Both the internal and external markets have shown an ever-increasing demand, not only with respect to the quantities of the products but also to their diversification. Hitherto there haven’t been developed nutraceutical initiatives that are able to bring forth all of the potential that the acerola fruit bears, a fact that makes the investigation of new extraction techniques and possible applications of the obtained bioactives relevant. (Assis et al., 2001) In this context blossoms the so-called Green Chemistry, often defined as the application of the science and production of chemicals in a sustainable, safe and non-pollutant manner, consuming the least possible quantities of raw materials and energy while emitting no residues (Erias Rey and Alvarez-Campana Gallo, 2007). The usage of the protic ionic liquids (PILs) in extraction processes poses itself as an excellent alternative, since those new compounds confer a less potentially toxic load while promoting a highly efficient extraction, bearing greater selectivity and being biodegradable substances. The use of protic ionic liquids also promotes lower energy consumption: they are easily separable, can be reused as of the end of the process, have low costs and are of particularly easy synthesis. In this fashion, the processes become less toxic, safer and thus more sustainable with respect to the management of edibles (Reis et al., 2012). These substances are characterized as non-volatile organic salts, and this trait prevents their dispersion through evaporation. They have recently been the object of studies that demonstrate their myriad of social and industrial application (Adams et al., 1998, Cull et al., 2000, Baston et al., 2002, Zhao et al., 2002, Lei et al., 2003, Welton, 2004, Zhou, 2005, Liu et al., 2005, Zhao et al., 2005, Li et al., 2007, Sun et al., 2007, Borra et al., 2007, Hagiwara and Lee, 2007, Nakamoto and Watanabe, 2007, Cieniecka-Roslonkiewicz et al., 2007, Hough et al., 2007). An important aspect that is yet to be studied in sufficient depth is the application of the protic ionic liquids as selective extractors of biocompounds. In the field of food technology, some studies support the application of ionic liquids in the extraction of antioxidants stage as well as its use as the stationary phase in the chromatographic analysis (Torrecilla, 2012). This work aims to study and assess the effects of the addition of protic ionic liquids to the solvents employed in the extraction (water and methanol) of the acerola’s antioxidant compounds through the DPPH method. (Brand-Williams et al., 1995)

Material e métodos

Synthesis and Preparation of Ionic Liquids The protic ionic liquids were synthesized through simple Bronsted-Lowry neutralization reactions, in which the acid acts as proton donor and the base as its acceptor. Two bases were used in this work (monoethanolamine and diethanolamine) which were combined with a collection of organic acids (acetic acid, propionic acid and salicylic acid), resulting in six ionic liquids of distinct natures and properties. The chemical reactions are of exothermic character, thus demanding a strict temperature control to avoid thermal degradation. The purification was conducted under constant stirring and slight heating over 48 hours, assuring the thorough elimination of the residual reactants and of the absorbed water. Sample Preparation The acerola pulp samples were provided from local provider. 2 g of the pulp that was diluted in different solvents were used in order to assess its antioxidant capacity. The utilized solvents were: pure methanol, pure water, a mixture of methanol with 10% of each protic ionic liquids and a mixture of water with 10% of protic ionic liquids. The mixture of pulp and solvents underwent agitation for 20 minutes, being centrifuged short after at the conditions of 800 rpm throughout 10 minutes and at a temperature of 20° C. After the centrifugation, the obtained samples were filtered at 0.45 µm in order to finally be submitted to the analyses of their antioxidant capacities through the DPPH method.

Resultado e discussão

Determination of the Antioxidant activity through the use of the DPPH method. The method is based on the methodology described by Brand-Williams et al (1995), in which the DPPH radical undergoes reduction and the color change is proportional to the antioxidant capacity of the studied compound. 3 mL of the fruit sample were added to 1 mL of methanolic solution of DPPH at (6x10–5M). For the sake of solution control ethanol was used as the white. As the standard reference solution the Trolox solution was employed (6-Hydroxy-2,5,7,8- tetramethylchromane-2-carboxylic acid) at 97%. The mixture was homogenized and past 30 minutes of rest it underwent a reading effectuated through the use of a UV spectrophotometer at the wavelength of 517 nm. Further readings were conducted at intervals of five minutes until the results reached a stable value. From the absorbance data gathered the calculations of DPPH concentration were effectuated stemming from a calibration curve obtained through linear regression. The results were expressed in an activity equivalent to the Trolox (µM/g of fruit). In rows, different letters denote significant differences (p <0.05) Results demonstrate that in all cases, the addition of PIL’s significant increased (p <0,05) the values of antioxidant activity of acerola. Methanol fractions provided significant (p <0,05) better antioxidant activity results than water. In all cases, 2HDEASa extractions exhibited the highest (p <0,05) antioxidant activity, thus reducing the amount of methanol employed in the extraction. It seems feasible to use PIL’s to reduce the use of toxic and harmful organic reagents for the environment and to improve the extraction step phytochemicals.

Acid-Base pairs arranged in order to produce the protic ionic liquids that were utilized/. Acerola antioxidant activity analyzed by the methods of DPP

Conclusões

Attending to previously results, as expected, protic ionic liquids gathered an amazing capability as selective solvent for antioxidant compounds from acerola. In all tested samples, the protic ionic liquid 2HDEASa showed the best results, reducing the use of traditional organic solvents, improving a promising alternative procedure for nature phytochemicals extraction processes.

Agradecimentos

Special thanks are owed to the FAPESB, due to the scholarship it provided, and to the PROPCI, for its invaluable financial support.

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