*4.4. Asparagus*

Used since ancient time for the diuretic and medicinal properties of its spears [284], cultivated asparagus (*Asparagus o*ffi*cinalis* L.) is now a popular vegetable grown worldwide on over 1.5 million hectares producing over 8.9 million tons of spears [27]. Native of Eastern Europe, cultivated asparagus is grown at commercial level mainly in China, followed far behind by Perú, Mexico, Germany, Spain, USA, Italy, Japan, France, and the Netherlands. The edible portion of this herbaceous perennial is constituted by its spears or young stems produced by the underground crown, which may be green, green-purple, or purple when harvested above ground or white when purposely harvested before exposed to sunlight. Asparagus spears are considered a rich source of minerals [285], amino acids and dietary fibers [286], saponins [287,288], vitamins and volatile sulfur organic compounds [289], and especially of phenolic

compounds and flavonoids [290], including anthocyanins that are responsible for the purple color of the bracts of green spears or of the whole spears in purple asparagus genotypes [23,154,291]. Being rich of all these bioactive compounds it is difficult to isolate the effect of anthocyanins and specific pigments, nevertheless several studies have demonstrated the antioxidant properties of anthocyanins in the species [284]. Comparing green, white, and purple spears, Maeda et al. [291] found that purple spears had significantly higher levels of rutin compared to green spears, while rutin was not detected in white asparagus spears. The same authors observed a positive correlation between spears total polyphenol content, rutin, and DPPH radical scavenging activities, suggesting that purple asparagus may provide higher levels of antioxidants compared to green and white asparagus [291]. In this perspective, while most of the cultivars have been selected to produce green spears and have a relatively small content of anthocyanins, and even less pigments when etiolated to produce white spears, more recently, as for other crops, specific functional breeding programs have developed purple cultivars characterized by high levels of anthocyanins to satisfy the need of consumers attracted by new colors and seeking richer sources of natural antioxidants [291]. Most of the modern commercial cultivars of green and white asparagus are diploid (2*n* = 20) and were developed from an old population called "Purple Dutch" from which French growers selected two stocks called "Precoce d'Argenteuil" and "Tardive d'Argenteuil" which were subsequently used to develop modern cultivars and hybrids in The Netherlands, France, Germany, United Kingdom, and in the USA [292,293]. Although any green asparagus genotype may be used to produce etiolated white spears, Dutch, French, and German breeding programs have selected cultivars suitable specifically for white spears production to satisfy the European market requirements by reducing as much possible the content of anthocyanins that may develop and accumulate in the tips of white-harvested spears even during storage [292,294]. On the other hand, using "Violetto d'Albenga" a tetraploid local population traditionally grown in Northern Italy, likely derived from interspecific crossing between *A. o*ffi*cinalis* and *A. maritimus* [295] and characterized by the production of larger, sweeter, and less fibrous spears ranging in color from green to dark purple, breeding programs in the US and New Zealand have developed in the early 1990s the first cultivars producing purple spears such as "Purple Passion" [296] and "Purple Pacific" and "Stewarts Purple" [297].

Using HPLC and NMR to analyze the fresh peel of "Purple Passion" asparagus, Sakaguchi et al. [23] isolated two major anthocyanins identified as: cyanidin 3-[3"-(*O*-β-d-glucopyranosyl)-6"-(*O*-αl-rhamnopyranosyl)-*O*-β-d-glucopyranoside], and cyanidin 3-rutinoside characterized by high antioxidant activity as determined through ORAC (Oxygen Radical Absorbance Capacity) assay. Recently, Dong et al. [298] comparing the anthocyanin profile of three purple asparagus cultivars ("Jing Zi-2", "Purple Passion", and "Pacific Purple") with a green control ("Jing Lv-1"), identified sixteen anthocyanins, with peonidin, cyanidin and their glycoside derivatives being the predominant compounds. In the same study, through transcriptomics and quantitative real-time polymerase chain reaction (qRT-PCR) analysis, several anthocyanin synthesizing genes (PAL, C4h, 4CL, CHS, CHI, F3H, F3'H, DFR, ANS, and 3GT) and transcription factors genes (bHLH137-like, TT2-like, WD40-like, bZIP61-like, and MADS18-like) that regulates the biosynthesis of anthocyanins like reported for other species were identified and showed to be differentially expressed between purple and green cultivars [298]. The expression of the same genes and the content of anthocyanins were examined also comparing spears grown in the presence of light and in the dark. In accordance with previous studies [299,300], results demonstrated that anthocyanin biosynthetic and regulatory genes are considerably down-regulated in absence of light and pigments are not synthesized in dark conditions, suggesting that light conditions are a key factor for anthocyanin biosynthesis and accumulation. This is further corroborated by the findings of Huyskens-Keil et al. [301] who showed the effects of light quality (white, red, blue, UV-C) on the activity of phenylalanine ammonia-lyase (PAL) and peroxidase (POD) and the synthesis and accumulation of anthocyanins in basal and apical segments of white asparagus spears during postharvest storage. Postharvest conditions and handling may in fact considerably affect the stability of anthocyanins. In another recent study, Barberis et al. [302] reported that dipping the spears of "Purple Passion" for 5 min in a 3 mM solution of oxalic acid (pH 2.9) reduced

the lightness of the spears after 12 days only by 13% compared to spears treated with water (control pH 8) or with 1 mM oxalic acid (pH 6) solution, suggesting that the low pH treatment enhanced the stability of the anthocyanin pigments. Analyzing the phenolic profile of green and purple asparagus at harvest and during storage, in accordance with Sakaguchi et al. [23] the same authors [302] found that cyanidin glucosyl rutinoside, cyanidin rutinoside and peonidin rutinoside (at harvest 774.2, 125.5, and 84.8 mg/kg, respectively) were the main anthocyanins identified in "Purple Passion" spears and determining the typical purple color. During storage, the first anthocyanin decreased overtime and was negatively affected by oxalic acid treatment, cyanidin rutinoside increased over time, while peonidin rutinoside remained stable.
