*2.1. Lettuce*

Lettuce (*Lactuca sativa* L.) belongs to the Asteraceae (Compositae) family and is a very popular vegetable crop used for fresh consumption and as salad ingredient owing to its sensory and health-promoting properties [14,24–26]. Lettuce is widely cultivated throughout the globe and it is rightly considered the most important of leafy greens as it ranks highest in production (27 million tons in 2017; [27]). Lettuce comes in a wide variety of head formations, textures, sizes, leaf shapes, and colors and it is conventionally classified according to Mou [28] into six major groups (i.e., types): (i) Butterhead, (ii) Cos or Romaine, (iii) Crisphead, (iv) Leaf or Cutting, v) Stalk or Stem, and vi) Latin. Compared to several other leafy vegetables, lettuce is an excellent source of vitamin B9 and total flavonoids. Wang et al. [29] and Gan and Azrina [30] reported that total vitamin B9 and flavonoid contents in lettuce were higher by 16% and 220%, respectively, compared to spinach, which is another important and widely consumed leafy green. Quali-quantitative variation in lettuce vitamins and secondary metabolites (i.e., phytochemicals) depends on many pre-harvest factors such as genotype, environmental conditions, harvest maturity, and agricultural practices [31]. However, the genetic material is the predominant pre-harvest factor and the major determinant of

the biosynthesis and accumulation of lipophilic (i.e., carotenoids, chlorophylls, and vitamin E) and hydrophilic (i.e., phenolic compounds and vitamin C) antioxidant molecules [32]. Vitamins are essential micronutrients required for human metabolism and functionalities implicated in the reduction of cardiovascular and degenerative diseases [33]. Folate (vitamin B9) and vitamin C (as ascorbic and dehydroascorbic acids) are eminently present in lettuce [14]. In their review article Kim and co-workers reported that folate and vitamin C contents vary with leaf type and particularly leaf coloration, with red leaf, butterhead, and romaine lettuces being particularly good sources of folate [15,29,34], while leaf green lettuce had the highest vitamin C concentration [24,25,34,35].

Carotenoids, which constitute an important group of lipophilic pigments frequently present in yellow-orange vegetables and in dark green leafy vegetables, vary in concentration among lettuce types and colors. Mou [36] assessed the genetic variability in β-carotene and lutein content, the most abundant carotenoids in lettuce, across 52 genotypes (including butterhead, crisphead, Latin, leaf, primitive, romaine, stem lettuce and wild species) that were categorized by type in the following order: Romaine and green leaf > red leaf > butterhead > crisphead. The author also reported that the two target carotenoids were significantly and positively correlated with chlorophyll a and b as well as with total chlorophyll content. Contrarily to the findings of Mou [36], Baslam et al. [37], and Nicolle et al. [25] demonstrated that the content of β-carotene and lutein may not entirely correlate with leaf green pigmentation, since the carotenoids content appeared to be lower in green compared to red-pigmented lettuce plants. The contradiction between these results may indicate that the content in carotenoids may not be consistently related to leaf pigmentation [14]. Nevertheless, the frequent consumption of carotenoids-rich lettuce could be of high importance since several epidemiological studies demonstrated that the onset of chronic diseases such as heart disease, vision impairment, and certain types of cancer (lung, prostate, and colon) could be reduced [38–41].

Several authors reported that the contents of secondary metabolites in lettuce differed greatly among genotypes and depended particularly on leaf color (dark red, red, green/red, and green) [14,42]. According to Mulabagal et al. [43] red lettuce contains a single anthocyanin, namely cyanidin-3-*O*-(6"-malonyl-β-glucopyranoside) which is further converted in two cyanidin derivatives (cyanidin-3-*O*-(6"-malonyl-β-glucopyranoside methyl ester) and cyanidin-3-*O*-β-glucopyranoside), all presenting significant antioxidant activities against lipid peroxidation and cyclooxygenase activity. Kim et al. [42] explored the genetic material of 23 lettuce cultivars belonging to three major lettuce groups (crisphead, oak-leaf, and romaine) in respect to their phytochemical content and antioxidant potential. The authors reported that most phytochemicals varied significantly with genetic material and were associated mainly with leaf color. The red-leaf and to a lesser extent the green/red cultivars exhibited the highest concentration of the following antioxidant molecules: Cyanidin, carotenoids (lutein, violaxanthin and lactucaxanthin), fatty acids such as α-linolenic and linoleic acid, total polyphenols, and antioxidant potential [42]. In the same study, the authors were also able to demonstrate that the methanolic extract of red-pigmented lettuce contained potent scavengers of ABTS (scavenging assay of 2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt radical) and DPPH (scavenging assay of 2,2-diphenyl-1-picrylhydrazyl radical) radicals. The higher radical-quenching activity of red-pigmented lettuce cultivars irrespective of type renders them more bioactive thus their systematic inclusion in the human diet could be an efficient tool to minimize the impact of oxidative stress-related diseases [25]. Similarly, Hao et al. [44], reported the differences in nutritive quality (e.g., cellulose, protein, starch, sugar, and vitamin C contents) among 74 red/purple and green varieties of leaf lettuce using grey correlation analysis (a methodology of treating and analyzing qualitative data according to grey system procedure). The authors concluded that purple-leaf lettuce "P-S23" exhibited significantly higher grey comprehensive evaluation value (0.8) in comparison to the green counterparts (values < 0.5). In addition, leaf pigmentation significantly correlates to the constitution and concentration of phenolic compounds belonging mainly in the subgroups of phenolic acids, flavonoids, and anthocyanins [14]. Chicoric, caffeic, and chlorogenic acids and their derivatives are the most abundant phenolic acids present in lettuce, whereas the most outstanding flavonoids include

anthocyanins, quercetin, kaempferol, and flavone luteolin [35,45–47]. Several authors reported a higher total phenolic content in red butterhead, red leaf and red romaine lettuces compared to their green counterparts [16,35,48–51]. The red color of lettuce has been associated with a higher total phenolics content, known to impart a greater antioxidant activity than vitamins C and E [52], and has been attributed primarily to anthocyanins, an important group of flavonoids responsible for the red/purple coloration [53].

Regarding the health benefits attributed to lettuce, according to an in vivo study carried out by Lee et al. [54] on mice fed with a high-fat diet, supplementation of the diet with 8% red lettuce on a body weight (bw) basis decreased the total cholesterol and the low density lipoprotein (LDL) by 9% and 123%, respectively, thus highlighting the potential effects of red-pigmented lettuce consumption against cardiovascular disease. A putative mechanism behind the cholesterol reduction could be the synergistic effects of lipophilic and hydrophilic antioxidant molecules such as α-tocopherol, anthocyanins, β-carotene, and phenolic compounds. Similarly, Nicolle et al. [24] observed that feeding male rats with 20% of red oak-leaf lettuce decreased significantly the LDL (low density lipoproteins)/HDL (high-density lipoproteins) cholesterol ratio and the liver cholesterol content.

In addition to preclinical trials, clinical studies demonstrated that weekly consumption of lettuce was able to reduce the incidence of colorectal cancer [55]. The protective effect of frequent lettuce consumption against colorectal cancer has been attributed to the presence of β-carotene and vitamin C and not to Ca and vitamins B9 and E contents [55]. Recently, Qin [56] reported that the new cultivar B-2 of red-pigmented lettuce, characterized by high concentration of anthocyanins, flavones, and phenolic acids, can minimize oxidative stress-related diseases, leading to anti-tumor effects against human lung adenocarcinoma, hepatoma, and human cancer colorectal adenoma cell lines. Based on the above considerations, clinical and preclinical studies demonstrated that frequent consumption of fresh lettuce, in particular the red-pigmented varieties, carries potential anti-diabetic, cholesterol lowering, and anti-tumor properties.
