Neglected and Underutilized Plant Species (NUS) from the Apulia Region Worthy of Being Rescued and Re-Included in Daily Diet

: Neglected and underutilized species (NUS) are cultivated, semi-domesticated, or wild plant species, not included in the group of the major staple crops, since, in most cases, they do not meet the global market requirements. As they often represent resilient species and valuable sources of vitamins, micronutrients, and other phytochemicals, a wider use of NUS would enhance sustainability of agro-systems and a choice of nutritious foods with a strategic role for addressing the nutritional security challenge across Europe. In this review, we focused on some examples of NUS from the Apulia Region (Southern Italy), either cultivated or spontaneously growing species, showing interesting adaptative, nutritional, and economical potential that can be exploited and properly enhanced in future programs.


Introduction
Often considered a central argument in the scientific debates at a local or global scale, the biodiversity loss issue is becoming a critical challenge that needs to be carefully considered in future years. Following this debate, the newly launched EU Biodiversity strategy has put forward measures to address the biodiversity loss across the European Union [1]. Within this issue, a lively interest has been addressed towards the agro-biodiversity, which includes cultivated species and landraces, wild flora, soil microorganisms, pollinators, and the relative interconnections between plant and environment or genetic resources and agricultural management/practices [2]. Furthermore, local knowledge and culture also have an important role and should be considered part of agro-biodiversity [2].
Localized in the central part of the Mediterranean area, Italy offers a wide variety of ecological, pedoclimatic, and orographic conditions. The Italian flora is characterized by rare and endemic plants, with many domesticated crops and vegetables showing high genetic and phenotypic variability [3]. In the Italian territory, particularly South Italy, small family-owned farms and rural areas are rich in vegetable germplasm, represented by wild flora, different landraces, and plant species closely linked to the local historical memory [3].
The neglected and underused plant species (NUS) are cultivated varieties, semidomesticated, or wild plant species that tend to be underutilized locally or globally, due to their relatively low value for the global production and marketplace, since they most often do not meet the modern standards of uniformity [4] as major cultivated varieties [4][5][6][7]. Indeed, starting from the Green Revolution, we assisted the decline of many local/traditional species and varieties, which were less competitive compared with commercial cultivars, and, therefore, they have been replaced by high-yielding and uniform cultivars developed by modern breeding programs [2,7]. This genetic erosion has been also amplified by urban their nutritional significance can be also extended to other molecular properties, since a body of evidence has associated polyphenols dietary administration to anti-inflammatory, anti-aging, and anti-tumoral effects, thus providing a preventive effect against chronic and inflammatory human diseases [23,24]. Based on these nutraceutical features, multicolored carrots represent important horticultural species that can be valorized in breeding programs aimed at biodiversity preservation and sustainable agriculture. lturae 2021, 7, x FOR PEER REVIEW 3 of 14 polyphenols, mainly chlorogenic acid and anthocyanins, maintaining, at the same time, a carotenoids content similar to orange carrots (Table 1) [16,18,[22][23][24]. Due to the presence of high levels of phenolic compounds, the extracts from yellow-purple carrots have shown to be high in vitro antioxidant capacities compared to the orange carrots, but their nutritional significance can be also extended to other molecular properties, since a body of evidence has associated polyphenols dietary administration to anti-inflammatory, anti-aging, and anti-tumoral effects, thus providing a preventive effect against chronic and inflammatory human diseases [23,24]. Based on these nutraceutical features, multicolored carrots represent important horticultural species that can be valorized in breeding programs aimed at biodiversity preservation and sustainable agriculture.

Roquette
Roquette (Figure 2), also known as arugula, belongs to the Brassicaceae family and is an important leafy salad worldwide. In the Mediterranean region, the main cultivated roquette species are: Eruca vesicaria L. Cav. (formerly E. sativa Mill.) [25], which is prevalently cultivated in rich soils, or alternatively can be found mixed with ruderal flora in marginal areas; Diplotaxis tenuifolia L., which has succulent leaves and is well adapted to harsh and poor soils and is mostly collected as a wild species [26]. In Apulia, Eruca vesicaria is currently suffering a strong genetic erosion, due to the growing attention focused on the wild Diplotaxis tenuifolia, which is preferred for culinary preparations. However, E.

Roquette
Roquette (Figure 2), also known as arugula, belongs to the Brassicaceae family and is an important leafy salad worldwide. In the Mediterranean region, the main cultivated roquette species are: Eruca vesicaria L. Cav. (formerly E. sativa Mill.) [25], which is prevalently cultivated in rich soils, or alternatively can be found mixed with ruderal flora in marginal areas; Diplotaxis tenuifolia L., which has succulent leaves and is well adapted to harsh and poor soils and is mostly collected as a wild species [26]. In Apulia, Eruca vesicaria is currently suffering a strong genetic erosion, due to the growing attention focused on the wild Diplotaxis tenuifolia, which is preferred for culinary preparations. However, E. vesicaria is still cultivated in small gardens in the area of Bari but with rare cases of global marketing placement.
Roquette is characterized by a pungent and bitter taste, provided by a range of beneficial compounds (Vitamin C, carotenoids, phenolics, and glucosinolates) ( Table 2) that contribute to its antioxidant capacity. Conversely, it can also accumulate anti-nutrients (e.g., nitrates) and heavy metals [27][28][29][30][31]. Besides the culinary uses, roquette has interesting medicinal properties, such as diuretic and depurative effects [26], and its extracts have antimicrobial properties [32], antigenotoxic properties in D. melanogaster [33], and cytotoxic effects in tumoral cell lines [34]. vesicaria is still cultivated in small gardens in the area of Bari but with rare cases of global marketing placement. Roquette is characterized by a pungent and bitter taste, provided by a range of beneficial compounds (Vitamin C, carotenoids, phenolics, and glucosinolates) ( Table 2) that contribute to its antioxidant capacity. Conversely, it can also accumulate anti-nutrients (e.g., nitrates) and heavy metals [27][28][29][30][31]. Besides the culinary uses, roquette has interesting medicinal properties, such as diuretic and depurative effects [26], and its extracts have antimicrobial properties [32], antigenotoxic properties in D. melanogaster [33], and cytotoxic effects in tumoral cell lines [34].

Salicornia spp.
Salicornia spp. (Figure 3) is a group of edible halophytes able to grow in high salt soil conditions, commonly named glasswort, pickle-weed, or sea asparagus [43]. Salicornia spp. can be found as a wild species in transition zones between permanently flooded muds and perennial vegetation, characterized by a winter flooding period and dry summer. The geographical distribution of the wild species is very wide, since it can be found in USA, Mexico, Canada, Europe (e.g., Britain, Ireland, France, Spain, Italy), India, Iran, Korea, and some Africa regions [43].
Apart from the historic usage as a source of sodium carbonate for glass making and an additive for soap production [43], some Salicornia spp. are utilized for culinary purposes.

Purslane
Purslane (Portulaca oleracea L.) ( Figure 4) is a very common spontaneous plant in gardens, lawns, vineyards, cultivated fields, eroded slopes, and bluffs, where it is considered one of the most common weeds. It is a very common plant in the temperate and subtropical regions, but it also grows in the tropics and at higher latitudes [55]. P. oleracea is a synanthropic species that can tolerate mechanical disturbance and can be derived from anthropic activities. It has fleshy, succulent, and very branched leaves and stems. The origin of P. oleracea is uncertain, but it has been suggested that it comes from India, even though it was also found in America in pre-Columbian times [56]. Purslane has a broad physiological adaptability and high morphological variability (highly polymorphic); therefore, the taxonomy of P. oleracea is still under debate [56]. This is quite important because the Italian peninsula and adjacent islands provide fragmentary information on the infraspecific diversity of P. oleracea. However, a recent elucidation about the distribution of various P. oleracea morphotypes has been provided [56,57]. Thus, in the P. oleracea Apart from the historic usage as a source of sodium carbonate for glass making and an additive for soap production [43], some Salicornia spp. are utilized for culinary purposes.
The natural adaptation to saline environments, as well as the salt tolerant traits and the contextual content of bioactive compounds, makes Salicornia spp. interesting for many landscapes due to its cultivation in adverse, harsh environments and its contribution to human nutrition. In fact, Salicornia cultivation could represent a valid option in the context of global warming, in which edible plants with high salt tolerance are needed. Salicornia spp. can be also good candidates for reclamation of barren lands, salt flats, and seashores [43]. Its use has also been proposed in heavy metal removal and phytoremediation, but these applications are not compatible with nutritional purposes, as they can be a source of toxic metal ions and antinutrients. In fact, it is important to keep in mind that some species can accumulate high contents of oxalic acid and iatrogenic iodine and excessive content of salt, heavy metals, and saponines (as in the case of S. bigelovii) [44].
In the Apulia region, wild Salicornia spp. gathering is quite common, linked to ancient culinary uses, even though some cultivation practices (as in the case of Salicornia patula, belonging to the S. europea group) are also consolidated, especially in the northern area of Gargano, close to the areas of the Lesina and Varano salt lakes [44,45]. The first attempts of Salicornia cultivation have been reported along the Lesina lagoon, which occupies an area of about 51 km 2 , with a length of 22 km, an average width of 2.4 km, and a depth of about 0.7 m [45]. Other scattered coastal sites suitable for Salicornia spp. growth and gathering are present in the southern parts of the Apulia region, such as "Torre Guaceto" coastal lagoon (province of Brindisi), "Le Cesine" (province of Lecce), and "Salina dei monaci" (province of Taranto).
S. patula can be generally cultivated from February-March to August-September in a soil that is typically black, sandy, acidic, and very rich in organic matter. The harvest of fresh and tender parts can be repeated depending on the level of development of the plant, with a final yield that can reach 10-15 tons per hectar [44]. The propagation can be carried out by gamic or agamic techniques, and in the case of gamic techniques, seeds need strategies for dormancy under hypersaline conditions and germination at low salt levels. Furthermore, germination is affected either by the type of salt or its concentration [45].
Phytochemical analyses on S. patula have focused on fatty acids content, with a percentage of saturated fatty acids reaching 80% and phenolic content ranging from 2.989 to 4.209 mg GAE/g DW, with the major components represented by salicylic and transcinnamic acids [51] (Table 3).

Purslane
Purslane (Portulaca oleracea L.) ( Figure 4) is a very common spontaneous plant in gardens, lawns, vineyards, cultivated fields, eroded slopes, and bluffs, where it is considered one of the most common weeds. It is a very common plant in the temperate and subtropical regions, but it also grows in the tropics and at higher latitudes [55]. P. oleracea is a synanthropic species that can tolerate mechanical disturbance and can be derived from anthropic activities. It has fleshy, succulent, and very branched leaves and stems. The origin of P. oleracea is uncertain, but it has been suggested that it comes from India, even though it was also found in America in pre-Columbian times [56]. Purslane has a broad physiological adaptability and high morphological variability (highly polymorphic); therefore, the taxonomy of P. oleracea is still under debate [56]. This is quite important because the Italian peninsula and adjacent islands provide fragmentary information on the infraspecific diversity of P. oleracea. However, a recent elucidation about the distribution of various P. oleracea morphotypes has been provided [56,57]. Thus, in the P. oleracea complex, the P. trituberculata morphotype has been identified in the Apulia region. This morphotype is one of the most common in continental Italy since the Roman period [57].
been extracted from purslane leaves, particularly with oleracein A and C, found as major components in leaves, reaching 8.2-103.0 mg and 21.2-143 mg/100 g dried weight [59]. Concerning the biological activities, purslane has shown antioxidant and lipid oxidation inhibiting capacities [60][61][62][63] and provides protection against DNA damage in in vitro studies [61]. Di Cagno et al. [64] have also tested purslane juice obtained by lactic acid bacteria fermentation, finding that the fermented juice strongly decreased the levels of pro-inflammatory mediators and reactive oxygen species in the CaCo2-cell line.   In the Apulia region, purslane has always been traditionally harvested, and recently, it has been officially recognized as a traditional food product [58]. Due to its sour and salty taste, similar to fresh spinach, purslane is generally served raw in salads to give flavor and freshness or cooked to prepare soups. In the past, it was used as a medicinal herb due to its purifying, diuretic, and anti-diabetic properties. Purslane is a good source of omega-3 fatty acids, tocopherols, and vitamin C (Table 4) and contains minerals, such as magnesium, manganese, potassium, iron, and calcium. Flavonoids and polyphenols have also been extracted from purslane leaves, particularly with oleracein A and C, found as major components in leaves, reaching 8.2-103.0 mg and 21.2-143 mg/100 g dried weight [59]. Concerning the biological activities, purslane has shown antioxidant and lipid oxidation inhibiting capacities [60][61][62][63] and provides protection against DNA damage in in vitro studies [61]. Di Cagno et al. [64] have also tested purslane juice obtained by lactic acid bacteria fermentation, finding that the fermented juice strongly decreased the levels of pro-inflammatory mediators and reactive oxygen species in the CaCo 2 -cell line.

Leopoldia comosa L.
Leopoldia comosa (L.) Parl., (Figure 5), previously named Muscari comosum (L.) Mill, is a perennial bulb, belonging to the Hyacinthaceae family and originating from South-East Europe, Turkey, and Iran, naturalized elsewhere and eaten in some Mediterranean countries. It is called the tassel of hyacinth or tassel grape hyacinth. It is a wild species, but it can also be properly cultivated. The wild specimens can be found in rocky ground or cultivated lands, cornfields, or vineyards. The cut bulbs transude mucilages, sugars, latex, tannins, salts, triterpenes, homoisoflavones, and muscarosides [66].

Milk Thistle
Milk thistle (Sylibum marianum L.) ( Figure 6) is a member of the Asteraceae family and is native to the Mediterranean basin, although it is widespread in Northern Africa, Asia, North and South America, and South Australia [73,74]. It can be cultivated as an ornamental plant, but it often grows widely as a proper weed in yields and roadsides, in warm environments and dry soils. Flowering season is between July and August. It is also considered a heavy metals tolerant species [73]. Milk thistle fruits, sometimes confused as seeds, have been used for medical purposes since ancient Greek civilization, especially for the treating of liver diseases for its hepatoprotective activities. A recent study has evidenced that wild accessions of S. marianum in Italy can be identified in three different stable chemotypes, based on the biochemical profile of these accessions. Two of these chemotypes have been reported from different Italian regions, including Apulia, with no clear correlation between the chemical profile and geographic features [75].
Apulian traditional culinary usages included the leaves and the tender stems of the milk thistle, together with other well-known and appreciated species, Cynara cardunculus L. and Scolymus hispanicus L. (golden thistle). However, the main problem that has greatly limited its uses in recent years is represented by the first cleaning phase, which consists of eliminating leaf blade, which is exceedingly spiny. However, once cooked the milk thistles can be used to prepare very tasty dishes rich in beneficial compounds. The bulbs of L. comosa are characterized by a typical strong sour and bitter taste and, in the culinary uses of the Apulia region, are traditionally boiled and consumed with olive oil, vinegar, and salt, or they can be fried. Additionally, they can be part of the preparation of other traditional local dishes [67]. Other popular usages of L. comosa bulbs include the cure of toothache and skin spots [68].
L. comosa bulbs are rich in several classes of phytochemicals, including flavonoids, phenolic acids, and fatty acids [69] (Table 4). Among the fatty acid fraction, palmitic acid has been reported as the major component, followed by linoleic, linolenic, and stearic acids [69] (Table 5).
Phytochemicals in Leopoldia comosa bulbs have shown metal chelating, antioxidant properties, pancreatic lipase inhibitory activity, and hypoglycemic activity via the inhibition of carbohydrate digestive enzymes, such as α-amilase and α-glucosidase [70]. Furthermore, enzyme-inhibitory effects and in vitro antitumoral activities in breast adenocarcinoma cells have also been reported [71].
In a comparative study of extracts deriving from wild and cultivated bulbs of L. comosa, Marrelli et al. [69] have shown higher radical scavenging activity and good in vitro pancreatic lipase inhibitory activity from the wild bulb extracts compared to the cultivated bulb extracts. In light of these data, the extracts from wild L. comosa bulbs have been suggested to be considered for subsequent in vivo studies and the activity could be attributed to phenolic compounds [69]. Accordingly, Casacchia et al added L. comosa extracts (20 or 60 mg/die) to a high-fat diet in rats fed for 2 weeks. Following these conditions, L. comosa extracts inhibited lipase and pancreatic amylase activities, counteracting abdominal obesity, dyslipidemia, liver steatosis, and improving glucose tolerance, suggesting an important effect of prevention of obesity-dependent metabolic disorders [72]. In another study, Casacchia et al. [71] used raw bulbs or bulbs cooked with two different methods (boiled or steam-cooked), confirming higher antioxidant activities and inhibition of pancreatic lipase and α-amylase, especially in the raw bulbs, relating these in vitro activities mainly to the phenolic compounds and suggesting that the traditional cooking methods can partially deplete the observed biological activities.

Milk Thistle
Milk thistle (Sylibum marianum L.) ( Figure 6) is a member of the Asteraceae family and is native to the Mediterranean basin, although it is widespread in Northern Africa, Asia, North and South America, and South Australia [73,74]. It can be cultivated as an ornamental plant, but it often grows widely as a proper weed in yields and roadsides, in warm environments and dry soils. Flowering season is between July and August. It is also considered a heavy metals tolerant species [73]. Milk thistle fruits, sometimes confused as seeds, have been used for medical purposes since ancient Greek civilization, especially for the treating of liver diseases for its hepatoprotective activities. A recent study has evidenced that wild accessions of S. marianum in Italy can be identified in three different stable chemotypes, based on the biochemical profile of these accessions. Two of these chemotypes have been reported from different Italian regions, including Apulia, with no clear correlation between the chemical profile and geographic features [75]. The most important biological activities of milk thistle are related to silymarin, a mixture of flavonoid complexes and flavolignans. In silymarin, many compounds have been reported, among which are silybin, isosilybin, silychristin, isosilychristin, sylidianin, and silimonin [76][77][78]. Apart these compounds, some flavonoids (quercetin, kaempferol, apigenin, naringenin, eriodyctiol, and taxifolin), tocopherol, sterols, sugars, and proteins have been reported [76], even though silybin is the most abundant compound in the extracts [74] (Table 6).
Milk thistle extracts, from the heads, leaves, and stems, have shown several biological properties [79], including strong antioxidant and anti-inflammatory properties and antitumoral activities [79][80][81]. In an experimental model of nonalcoholic steatohepatitis, the administration of S. marianum extract reduced the severity of steatohepatitis and the levels of alanine amino transferase and aspartate amino transferase and improved the levels of glutathione [82]. The hepatoprotective activities were also observed in human hepatocytes and human liver microsomes by inhibiting cytochrome-P450 isoenzymatic activities [83].   Apulian traditional culinary usages included the leaves and the tender stems of the milk thistle, together with other well-known and appreciated species, Cynara cardunculus L. and Scolymus hispanicus L. (golden thistle). However, the main problem that has greatly limited its uses in recent years is represented by the first cleaning phase, which consists of eliminating leaf blade, which is exceedingly spiny. However, once cooked the milk thistles can be used to prepare very tasty dishes rich in beneficial compounds.

Conclusions
In this review, we focused on some examples of NUS from the Apulia region that are worthy of being enhanced, with the intent to preserve the living heritage and biodiversity. The major staple crops, intensively cultivated because they ensure the standards of global market requirements, are preferred to NUS, thus hiding their great potentials to contribute to the process of adaptation to changing climates. Indeed, most of NUS are characterized by a high resilience to harsh and adverse environments and a rich source of nutrients. Further efforts need to address: 1.
The molecular basis and genetic traits linked to the adaptation to harsh environmental conditions (with a special look at tolerance to heat/salt/heavy metals stresses); 2.
The characterization of main nutrient classes and their biosynthesis pathways; 3.
The quantification and characterization of the main antimetabolic factors/antinutrients; 4.
A better knowledge of the biological activities in the prevention of human diseases.
Expanding our knowledge on these issues will increase awareness of the importance of NUS and the activities related to their recovery and enhancement. Investing in research on NUS, an inter/multi-disciplinary approach and shared scientific and traditional knowledge will help to fully realize the benefits of these crops.