*4.1. Broccoli and Cauliflower*

Broccoli (*Brassica oleracea* L., var. *italica* Plenk) and cauliflower (*B. oleracea* L., var. *botrytis*) are the two most popular vegetable crops belonging to the Brassicaceae family. Native of the Mediterranean Basin, both species are adapted to a wide range of environmental conditions and are cultivated in all five continents, with an annual production that reached about 26 million tons in 2017, from an estimated harvested area of over 1.39 million hectares worldwide [27]. Primarily known and appreciated for their typical organosulfur compounds [224–226], broccoli, cauliflower, and other *Brassica* species are also a rich source of anthocyanins which are responsible of the purple pigmentation of some varieties [18,227]. Typically green, some cultivars and populations of broccoli are characterized by a purple pigmentation of the sepals of the inflorescence [227,228]. Branca et al. [228] found high levels of anthocyanins in a Sicilian broccoli landrace called "Broccolo nero" (Black broccoli) grown around Mount Etna and characterized by a dark violet pigmentation of the inflorescence, stem, and leaf midribs. In a recent work, Yu et al. [227] identified and mapped a major locus and two minor loci associated with the purple sepal trait in broccoli, while the authors hypothesized that the development of purple color may be induced by cold temperatures. In another recent study, Rahim et al. [229] working on the hypocotyl of young green and purple broccoli seedlings identified seven putative candidate genes (BoPAL, BoDFR, BoMYB114, BoTT8, BoMYC1.1, BoMYC1.2, and BoTTG1) responsible for the biosynthesis of anthocyanins. Among those, BoTT8 was expressed considerably more in purple hypocotyl compared to the green ones. Testing the in vitro cytotoxic effect of Sicilian black broccoli stem and leaf extracts at different concentrations (0.05%, 0.1%, 0.5%, 1%, and 5%) against HT29 (colon cancer) and A2058 (melanoma cancer) cells after 24 h treatment in presence or not of the myrosinase enzyme (responsible for the hydrolyzation of glucosinolates), Terzo et al. [230] found that the juice was less toxic in presence of myrosinase especially at higher concentration of the extract (1%–5%), suggesting that factors other than the glucosinolate content, such as polyphenols (including anthocyanins) could be responsible for the cytotoxic effects against HT29 and A2058 cancer cells. Examining the anthocyanin profile of three cultivars of heat-tolerant purple sprouting broccoli, Rodríguez-Hernández et al. [231] found that cyanidin 3-*O*-diglucoside-5-*O*-glucoside derivatives were the major acylated anthocyanins, and each cultivar and plant portion (leaves, inflorescence) had a particular prominent acylated anthocyanin. The same study revealed that compared to green broccoli cv. "Marathon", purple sprouting broccoli was characterized also by higher levels of glucosinolates. Similarly, Verkerk et al. [232] observed exceptionally high glucoiberin content (396.5 μmol/100 g FW) in purple sprouting broccoli cv "Bordeaux" compared to other green broccoli genotypes, which suggests that there is some sort of interaction between purple pigmentation and glucosinolate profile of broccoli. In another study, analyzing the acylated anthocyanin profile of purple sprouting broccoli and three other green broccoli varieties at the sprouting stage, Moreno et al. [233] observed a significantly higher content of anthocyanins in the purple genotype compared to the green ones and observed that the quantity and quality of anthocyanin pigments were highly variable among the tested genotypes. Out of seventeen anthocyanins identified in the four genotypes only three isomers were predominant in all the genotypes examined: Cyanidin 3-*O*-(acyl)diglucoside-5-*O*-glucoside, cyanidin 3-*O*-(acyl1)(acyl2)diglucoside-5-*O*-glucoside, and cyanidin 3-*O*-(acyl1)(acyl2)diglucoside-5-*O*-(malonyl)glucoside. The purple sprouting genotype was characterized by a higher content of cyanidin 3-*O*-(sinapoyl)(sinapoyl)diglucoside-5-*O*-glucoside, cyanidin 3-*O*-(sinapoyl)diglucoside-5-*O*-glucoside, cyanidin 3-*O*-(feruloyl)diglucoside-5-*O*-glucoside, cyanidin 3-*O*-(sinapoyl)(feruloyl)diglucoside-5-*O*-(malonyl)glucoside, and cyanidin 3-*O*-(sinapoyl) (sinapoyl)diglucoside-5-*O*-(malonyl)glucoside (Table 3). In agreement with previous studies, Moreno et al. [233] concluded that broccoli sprouts could be an excellent source of bioactive compounds rich of flavonoids, including acylated anthocyanins, along with glucosinolates, vitamins, and minerals [234–236], and that future studies should evaluate the potential of further enhancing the content of bioactive compounds in broccoli sprouts.

In the case of cauliflower, while most of the cultivars have been traditionally selected for their white curds [237], many local landraces and commercial cultivars are characterized by colored heads with characteristic pigmentation ranging from green to dark violet. In Italy, green cauliflowers are traditionally grown in Lazio and Marche, while dark violet selections are typically grown in Sicily, Puglia, and other Southern regions characterized by high levels of solar radiation which make it more challenging to produce white curds as traditionally required by the European market [228,238]. Lately, the interest for colored cauliflower varieties substantially increased due to the potential health-beneficial properties of the phenolic compounds that provide the pigmentation of plant

tissues [239]. Anthocyanins are in fact responsible for the purple–violet pigmentation also in the case of cauliflower and are considered highly beneficial for human health [8]. The biosynthesis of anthocyanins in cauliflowers is regulated mainly at transcriptional level, and in a particular purple cauliflower mutant it has been demonstrated that the tissue-specific activation of the gene BoMYB2 up-regulated the expression of both BobHLH1 and BobHLH2, leading to the formation of a complex regulation network MYB–bHLH–WD40 (MBW), consisting of MYB, basic Helix-Loop-Helix (bHLH), and WD40 proteins, which in turn activates the structural genes responsible for the biosynthesis of anthocyanins [18,240]. β-carotene accumulation has been also observed in cauliflower curds due to a rare carotenoid gene (Or orange) mutation that activates the biosynthesis of carotenoids in tissues that otherwise would be white [241,242]. Nevertheless, such mutation received limited attention at commercial level and is more relevant to advance our understanding of the carotenoid biosynthesis regulation [243,244].

Analyzing by LC–MS/MS nine Sicilian landraces of violet cauliflower, Scalzo et al. [19] identified cyanidin-3-(6-p-coumaryl)-sophoroside-5-glucoside as the main anthocyanin along with p-coumaryl and feruloyl esterified forms of cyanidin-3-sophoroside-5-glucoside. Scalzo et al. [19] and Kapusta et al. [245] examined also the stability of anthocyanins after processing (blanching, microwave-heating, convection steaming and freezing, and conventional water cooking) and observed substantial changes with the formation of isomers from cyanidin-3-sophoroside-5-glucoside rather than the hydrolysis of anthocyanins, suggesting good stability especially after microwave-heating which could be interesting for food processing applications.
