Analytical Methods A physiologic approach to test the global antioxidant response of foods. The GAR method S. Pastoriza a , C. Delgado-Andrade b , A. Haro b , J.A. Rufián-Henares a,⇑ a Departamento de Nutrición y Bromatología, Facultad de Farmacia, Universidad de Granada, Granada, Spain b Instituto de Nutrición (IFNA), Estación Experimental del Zaidín, Spanish National Research Council, Granada, Spain article info Article history: Received 13 December 2010 Received in revised form 26 May 2011 Accepted 7 June 2011 Available online 15 June 2011 Keywords: Total antioxidant activity In vitro gastrointestinal digestion GAR QUENCHER ABTS FRAP abstract Several methods have been applied to measure antioxidants in foods. Extraction methods have previ- ously relied on chemical methods which are non-physiological or based on enzymatic hydrolysis. What- ever the method used, the insoluble fraction is systematically excluded. The global antioxidant response (GAR) method use an in vitro approach with enzymatic digestion, designed to mimic digestion through the gastrointestinal tract, aimed to release antioxidants from foods. A total of 27 samples were analysed using the ABTS and FRAP assays applied to the soluble and insoluble fractions. The GAR method showed a higher antioxidant activity compared with the usual chemical extraction and the Quencher (direct) method. The soluble fraction was more antioxidant than the chemical extracts due to the release of com- pounds from the hydrolysis of carbohydrates and proteins. In addition, the GAR method allowed the mea- surement of the antioxidant activity in the insoluble fraction, which was important in fibre-rich samples. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction In the last decades there is a growing interest in the study of the antioxidant activity of foods and diets due to the known implica- tions of oxygen free radicals in the progress and development of cardiovascular and neurodegenerative disease, aging, cancer, etc. (García-Parrilla, 2008). On this purpose, many different procedures have been developed to test the total antioxidant capacity (TAC) of foodstuffs (Pellegrini et al., 2003; Pérez-Jiménez et al., 2008). The different studies show that there are two key issues: (i) the antiox- idant method applied and (ii) the extraction procedure of the antioxidant compounds. Regarding the first question, it is well- established that in the TAC determination it is mandatory the use of measurement procedures that include different mechanisms of action of antioxidant compounds, whether radical scavenging or metal-reducing activities. In relation to the second issue, the importance of the extraction procedure of the antioxidants (chem- ical aqueous–organic solvents or enzymatic hydrolysis) in the final results is also known. Thus, the solubility of the compounds responsible for the antioxidant activity in the reaction media uses to be a limiting step (Pérez-Jiménez et al., 2008), since whatever the extraction procedure, there is always an insoluble fraction of antioxidant material present in the food that is systematically dis- carded, among which insoluble proteins, tannins and melanoidins (final products of the Maillard reaction) must be considered (Gokmen, Serpen, & Fogliano, 2009). To overcome this problem, Gokmen et al. (2009) have recently developed a direct procedure called QUENCHER to evaluate the TAC of foods without the extrac- tion step, and thus, work with the whole antioxidant material pres- ent in solid stage. From a physiologic point of view, the biological properties of antioxidants will depend on their release from the food matrix dur- ing the digestion process. Moreover, the non-extracted antioxi- dants after digestion will follow the intestinal transit, being at some extent released and metabolised by the microflora action, and then producing significant biological effects (Delgado-And- rade, Conde-Aguilera, Haro, de la Cueva, & Rufián-Henares, 2010). Thus, the best approach to the physiologic conditions is the application of a gastrointestinal in vitro digestion to the foods be- fore determining the TAC by any antioxidant assay. Previous stud- ies of our research group have shown the increased antioxidant activity detected in the bioaccessible fraction after in vitro diges- tion of breakfast cereals when compared with the solvent extrac- tion procedure (Rufián-Henares & Delgado-Andrade, 2009). The new QUENCHER procedure provides the possibility of evaluating the antioxidant activity corresponding to the inaccessible fraction after digestion, as it has been recently applied in pre-baked bread (Delgado-Andrade et al., 2010). The previous work establishes that the TAC of bread can be estimated by adding the antioxidant 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.06.009 ⇑ Corresponding author. Address: Dpto. Nutrición y Bromatología, Facultad de Farmacia, Universidad de Granada, Campus de Cartuja, 18012 Granada, Spain. Tel.: +34 958 24 1000x20463; fax: +34 958 24 95 77. E-mail address: jarufian@ugr.es (J.A. Rufián-Henares). Food Chemistry 129 (2011) 1926–1932 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem