## 2 Discussion

Table 1: Some bioactive compounds from plants, their properties, and the methodologies used for their identification and quantification. Figure 1: The chemical structures for some glycosides. During the last decade, our research group has been working in the extraction, purification, and characterization of anthocyanins (Figure 3), which are flavonoids that have demonstrated ample possibilities for their application as natural pigments, nontoxic food additives, nutraceuticals, and antioxidants. For the above, a detailed description of this research, and references from others related, are described. \begin{table}
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Figure 2: The general structures for flavonoids and proanthocyanidins. Anthocyanin identification and separation from foods are widely performed activities. Individual components must be separated from foods and beverages to evaluate their biological activities. For the specific case of anthocyanins, using HPLC-UV-vis has become the most important approach for identifying and separating these compounds [47; 48]. The ultraviolet absorption spectra of these phenolic compounds can be taken advantage of to follow and separate different anthocyanins from foods and beverages using the UV-vis interval (200-750 nm). Chromatographic column techniques are useful in separating anthocyanins from extracts and different anthocyanins based on their retention properties. A study performed by our research group on the extracts of roselle calves (_Hibiscus sabdariffa_) afforded a chromatogram with 25 different compounds having maximum absorbances in the range of 270-520 nm. From these peaks, we identified 23 compounds employing MS\({}^{\text{n}}\). The most abundant compounds in the extract comprise chlorogenic acid, 5-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, and the anthocyanins delphinidin-3-O-sambubioside (D3S), delphinidin-3-O-glucose (D3G), cyanidin-3-O-sambubioside (C3S), cyanidin-3-O-glucose (C3G), quercetin-3-O-sambubioside (Q3S), quercetin-3-O-glucoside (Q3G), quercetin-3-O-rutinoside (Q3R), myricetin-3-O-sambubioside (M3 YS), quercetin-3-O-rutinosil-7-O-xyloside (Q3R7X), kaempfer-3-O-rutinoside (K3R), quercetin-3-O-(2\"-cournosyl)-sambubioside (Q3(2C)S), quercetin-3-O-(2\"-cournosyl)-rutinoside (Q3(2C)R), and quercetin (unpublished results). In the same way, [PERSON] et al. [50] obtained the phenolic compound profile by liquid chromatography (LC)-MS, including chlorogenic acid, quercetin, myricetin, caffeic acid, syringic acid, kaempfer, gallic acid, sinapic acid, unsolic acid, ferulic acid, p-coumaric acid, protocatechuic acid, and epicatechin. Differences exist in the composition and abundance of phenolics caused by the differences in the sample origin [51; 52]. Spectrophotometric measures with multidiode array detectors must be performed individually or in association with HPLC equipment. This technology allows the analysis of a sample on a broad range of UV-vis wavelengths. Using combined techniques for the separation and identification of phenolic compounds determines the association of the active properties observed on a natural product to a specific substance present on food. The phenolic compound characterization is an affordable process applying specific techniques. For example, characterization of anthocyanins from roselle calves (_Hibiscus sabdariffa_), blue maize (_Zea mays_), coffee fruit (_Coffea arabica_), blueberry (_Vaccinium ashei_),  
Figure 3: The general structure of anthocyanins and most representative molecules with their substituents. Adapted from [49]. and from beverages prepared from these products can be carried out using spectroscopic and spectrometric techniques. The characterization of extracts from vegetal products with anthocyanins implies the measurement of the total phenolic compounds, total monomeric anthocyanins, antioxidant activity, and color assessment. The Folin-Ciocalteu method is used to determine the total phenolic compounds. It quantifies the antioxidant or reductive capacity of the phenolic compounds present in a sample.