# Analytical Methods for Exploring Nutraceuticalticals Based on Phenolic Acids and Polypphenols
## 4 Analytical Methods for the Determination of Active Compounds in Nutraceutical Products
### Spectroscopic Methods  
#### 4.1.1 Antioxidant Indexes  
Colorimetric assays are broadly used to determine the antioxidant capacity (AC) in nutraceuticalals, which is mainly related to the presence of phenolic compounds. Although there is some controversy about the reliability of AC values obtained by these in vitro chemical assays, they are simple and require cheap equipment, which makes them an attractive approach to estimate AC. Among the most used, we can highlight the assays of Folin-Ciocalteu, ferric reducing antioxidant power (FRAP), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and 2,2\({}^{\prime}\)-azino-bis(3-ethylbenzothiazazoline-6-sulfonic acid) (ABTS) [6]. The assays are based on a redox reaction (e.g., Folin-Ciocalteu and FRAP), some of them involving a radical reagent (e.g., DPPH and ABTS), which leads to a change in the UV-visible spectrum of the solution. In all of the methods, the calibration is performed with an individual compound (e.g., gallic acid and Trolox) and AC is expressed in terms of the equivalent concentration of the compound used for calibration. The Folin-Ciocalteu (FC) method is, probably, the most applied to determine the total phenolic content, and it is considered a proper assay to estimate AC in samples of plant origin. The method was initially developed for the analysis of proteins and was later extended to the analysis of polyphenols [44] based on a redox reaction involving the reduction of Mo (VI) to blue Mo (V) under alkaline conditions. The FRAP test is based on the reduction of the Fe (III)-2,2,6-tripyridyl-s-triazine (TPTZ) complex (Fe (TPZT)\({}_{2}^{3+}\)) to the Fe (II)-TPTZ complex (Fe (TPZT)\({}_{2}^{2+}\)), which has an intense blue color [45; 46]. The FRAP assay is very simple and rugged, provided that the time elapsed between the start of the reaction and the measurements is kept under control. A large number of FRAP data of plant products can be found in the literature [47]. Other ligands (e.g., Fe (CN)\({}_{6}\)\({}^{3-}\)) have also been proposed to develop alternative colorimetric reactions. Regarding radical-based reactions, the DPPH assay is related to the scavenging activity of antioxidants against the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) [45]. DPPH is a stable radical, commercially available, and its solutions are violet in color, with an absorption maximum at 517 nm. The reduced form (DPPH-H) has a pale yellow color. Thus, when DPPH is reduced to DPPH-H by an antioxidant, the absorbance of the solution decreases. Although the method is widely applied to estimate AC, DPPH is not the best option for samples rich in anthocyanins, such as berries, cherries, and plums, because these compounds absorb in a similar range of wavelengths and interfere. The ABTS method is based on the scavenging activity of antioxidants against the ABTS\({}^{+}\) radical cation, whose solutions have a dark blue color. ABTS\({}^{+}\) is not commercially available and has to be generated by the oxidation of 2,2\({}^{\prime}\)-azino-bis(3-ethylbenzothazoline-6-sulfonic acid) (ABTS) with peroxodisulfate [45]. A lot of AC data from ABTS assays for a wide variety of plant samples have been reported [47]. The comparison of results from different assays is not a trivial issue, as the sensitivity of each particular reducing species in each method is characteristic of the compound [48]. Figure 2, adapted from Reference [48] by [PERSON] et al., compared various antioxidant indexes for the series of hydroxybenzoic acids. Despite the structural similarity of these molecules, the antioxidant powers differed significantly. The higher activities seemed to be associated with the presence of more than one hydroxyl group conveniently oriented in _ortho_ or _para_ positions, while those in _meta_ or monohydroxylated species were less active. Hence, the strongest reducing agents according to the FC assay were dihydroxy- and trihydroxybenzoic acids with _o_- and _p_-configurations, while those not following this pattern were less efficient.