**6. Anthocyanin extraction methods**

Anthocyanins may be extracted from different vegetable tissues, the most common method being the solid-liquid extraction. However, novel methods have been developed, such as the methodology developed by Japanese researchers where anthocyanins are extracted after fermenting the vegetable matrices that contain them [37].

Nowadays, anthocyanin extracts are usually applied without separating the individual components, since all the compounds have shown antioxidant activity, not only those with color [38].

On the other hand, the polar nature of the anthocyanin molecule permits solubility in a variety of solvents, such as alcohols, acetone, and water. However, their stability is easily affected by structural modifications by hydroxyl and methoxy groups, glycosides, and particularly acyl groups, as well as by environmental factors such as temperature and light [25].

Amongst those technologies, those currently available for anthocyanin extraction are the use of polar organic solvents, such as ethanol and methanol, and sometimes acidified media. In many cases, the solvents or chemical synthesis involved in the extraction are derived from petroleum, which leaves a strong carbon print on the environment [39].

Extraction of natural colorings by organic solvents has been the method of choice for decades. The toxicity of these solvents complicates the marketing of the final product due to their toxicity and environmental concerns. Many technologies for exploitation of agro-industrial residues have been developed out of the need of solving the problem of accumulation of solid organic residues. Green and clean technologies focus on lessening the environmental impact, while helping the processing and marketing of the final products [40].

Novel methods for the extraction of anthocyanins are ultrasound-assisted extraction [41] and extraction using supercritical fluid CO2 [42].

A viable method for anthocyanin extraction is the use of hydrolytic enzymes, which accelerate the reaction at which a substance is broken down into simpler components when reacting with water. This is the case of cellulase and pectinase that hydrolyze cellulose and pectin, respectively; both are found in the cell wall of fruits and vegetables [43].

A response surface methodology based on the Box-Behnken design may also be used to optimize an extraction method [44]. Identification and quantification of anthocyanins are based on the use of chromatographic methods, mainly the HPLC and UHPLC liquid analyses; mass spectrophotometry is also very helpful for the identification of individual compounds [44].

## **7. Antioxidant capacity highlights**

Antioxidants are molecules that inhibit or delay the oxidation in two ways: by trapping free radicals, in which case they are known as primary antioxidants (phenolic compounds) and are destroyed in the induction process, or by mechanisms such as chelation with heavy metals, capture of oxygen, conversion of hyperoxides into nonradical species, absorption of UV radiation, or inactivation of singlet oxygen; substances exhibiting these properties are known as secondary antioxidants [45].

Antioxidant activity is defined as the capacity of one or several compounds within a substance to inhibit oxidative degradation of another compound, acting mainly on free radicals [46].
