Bioactive Compounds and Aroma Profile of Some Lamiaceae Edible Flowers

Edible flowers are consumed for their appearance, colours, nutritional and healthy properties, but the use is limited by the actual number of the species. Seven edible flowers of the Lamiaceae family (Ocimeae and Mentheae tribes) were investigated: Monarda didyma ‘Fireball’, Nepeta × faassenii ‘Six Hills Giant’, Ocimum basilicum ‘Blue Spice’, O. basilicum ‘Cinnamon’, Ocimum × citriodorum, Salvia discolor, and Salvia microphylla ‘Hot Lips’. Total soluble sugars, proteins, polyphenols, carotenoids, ascorbic acid and antioxidant activity were detected. The species of the Mentheae tribe contained higher sugar content than Ocimeae flowers, the opposite with regard to protein content. Ocimeae tribe flowers showed high polyphenols and carotenoids content. The Ocimeae tribe together with two specie of the Mentheae tribe showed an aroma profile dominated by sesquiterpene hydrocarbons (58.0% in S. discolor to 77.9% in Ocimum × citriodorum). Oxygenated monoterpenes prevailed in Nepeta and Monarda, also present in the essential oil of this latter species (84.5%). By contrast, Nepeta and S. discolor evidenced non-terpenes as the principal class (41.2% and 77.5%, respectively), while the oxygenated sesquiterpene was the main one in S. microphylla. The two varieties of Ocimum spp. showed oxygenated monoterpenes as the main class of volatiles.


Introduction
Lamiaceae (order Lamiales) is a family of flowering species, also known as the mint family [1]. The taxonomy rank is composed of 236 genera and 6900-7200 species, distributed all over the world [2]. Lamiaceae is divided in 12 subfamilies [3] of which Nepetoidae is one of the most clearly defined [4] and has strongly aromatic species with volatile terpenoids [1].
Lamiaceae are usually herbs, subshrubs, or shrubs. Their leaves and flowers are generally scented, and this is a distinctive feature of this family. Many Lamiaceae species produce a wide spectrum of bioactive compounds (flavonoids, terpenoids, phenolics and alkaloids), that are characterized by numerous biological activities (e.g., antioxidant, anti-inflammatory, and antibacterial properties) [5][6][7][8][9]. Therefore, several species were listed in the official Pharmacopoeias [10] and currently used in pharmaceutical, cosmetic, food and pesticides industries [11]. Furthermore, many members of the Lamiaceae family are widely cultivated as culinary herbs, such as basil, oregano, rosemary, thyme,     Table 2 reported the contents of total crude proteins and soluble sugars (glucose, fructose, and sucrose) in the different flowers.

Bioactive Compounds
Sugars are an important component of flowers, since the flavor is often related to that content. The Mentheae tribe members resulted in higher sugars content than Ocimeae ones. The two sage species, Salvia microphylla Kunth (S. micro) and Salvia discolor Kunth (S. disc), characterized by a fruity taste, showed the highest content of sucrose (7.91 and 9.6 mg/g FW) and of hexoses (glucose and fructose) in comparison to Monarda didyma L. (M. did) and Nepeta × faassenii Bergmans ex Stearn (N. × faas). Within the Ocimum flowers, Ocimum × citriodorum Vis (Ob-Ct) showed the lowest content of soluble sugars ( Table 2).
The total crude proteins were higher in the Ocimeae tribe than in the Mentheae members. The three different Ocimum spp. showed a proteins percentage in the range of 9.62-16.16%. In the Mentheae tribe only N. × faas evidenced similar proteins percentage (12.69%), while low content was observed in the sage flowers and M. did (3.19-6.29% and 6.79%, respectively).
The carotenoids and anthocyanins amounts were determined and reported in Table 2. The higher contents of carotenoids were detected in the Ocimum genotypes, 51.59 µg/g FW in the "Blue spice" (Ob-BS), 68.33 µg/g FW in the "cinnamon" (Ob-Cn) and the highest amount in Ob-Ct (Thai lemon basil) with 81.86 µg/g FW. Within the Mentheae tribe, S. disc had the highest content of carotenoids (61.34 µg/g FW), due to the dark color, while the lowest amount was detected in M. did (1.91 µg/g FW).
In relation to the color of flowers, S. disc and M. did showed the highest content of anthocyanins, while the flowers with pale color had lower content, especially Ob-Ct and the Ob-Cn measured 0.03 and 0.06 mg/g FW, respectively. Anthocyanins were abundant in the following sequence: M. did = S. disc > S. micro > Ocimum species (0.98, 0.2, 0.16, 0.06 and 0.03 mg/g FW). The higher polyphenols content was detected in the Ocimum species, in the range between 7.42-8.06 mg/g FW, and the lowest amount in the S. micro (Andean sage, 2.41 mg/g FW). The ascorbic acid content (ASA TOT , vitamin C), an important nutritional value, was of highest measured in S. micro (2.57 mg/g FW), M. did and N. × faas (2.42 and 2.34 mg/g FW respectively. Lower amounts of total ASA were detected in the S. disc and in the flowers of Ocimeae tribe. The radical scavenger activity by DPPH assay was monitored as the IC 50 value: the highest activity was revealed in Ob-Ct (Thai lemon basil, 0.43 mg/mL), followed by the other two Ocimum and the Andean sage (S. disc). The lower antioxidant activity was measured in M. did and S. micro plants. Related to the higher antioxidant activity observed in the Ocimum flowers a negative correlation was observed with the highest content of total polyphenols, and is underlined in Figure 2.   Table 2. Determination of primary and secondary metabolites in the seven studied flowers of Lamiaceae family. Data are presented as means ± standard error (SE, n = 3). Abbreviations: FW = fresh weight; DW = dry weight; GAE-gallic acid equivalents; CE-± catechin equivalents; ME-malvin equivalents; sig.= significant post hoc test at p < 0.05.

Phytochemical Analyses
Overall, 118 chemical constituents were identified in the volatiles from Lamiaceae spp. samples (Table 3) with the number of peaks detected varying between 21 (N. × faas and S. micro) and 51 (O. basilicum 'Cinnamon', 'Ob-Cn). Sesquiterpene hydrocarbons represented the main class in all O. basilicum varieties as well as in S. disc (58.0% in S. disc to 77.9% in Ob-Ct), nevertheless they did not have the same characteristic compounds. β-caryophyllene, which was the only compound in common among all the studied species, represented the highest amount in both S. disc (36.2%) and Ob-Ct (23.7%). Ob-BS evidenced β-bisabolene (26.2%) as main constituent, while germacrene D (17.3%) and β-elemene (16.8%) prevailed in Ob-Cn. The presence of these latter constituents is conspicuous in all the previous species even though with different amounts, except for β-bisabolene, which was almost the exclusive compound of Ob-BS, present with lesser amount in S. disc (4.0%).
The second species of the Salvia genus (S. microphylla) showed a heterogeneous profile because all the classes were present. In fact, the EO composition evidenced the presence of MH (36.8%), OM (25.6%), OS (21.2%) and SH (14.7%) in this decreasing order. This species was characterized by limonene (25.8%) followed by isobornyl acetate (14.3%) and guaiol (11.5%) as principal components.

Multivariate Explorer Analyses
Principal component analysis (PCA) was performed with the spontaneous emission compounds present in a percentage greater than 3% in addition to the nutritional values of flowers. The result of this multivariate analysis ( Figure 3) where the first two axes account for 54.9% for a correlation matrix, showed two first macro groups, one with positive loading on PC1 and the other one with a negative loading in the same axis. All Ocimum varieties, scored negatively along PC1, were located in the upper left quadrant. This loading was generated mostly by the content of both distinguished compounds such β-citral in Ob-Ct (5.5%), (E)-β-ocimene (19.8%), eugenol (6.9%) and β-bisabonene (26.2%) in Ob-BS, and common compounds such as trans-α-bergamotene (11.6% and 6.4%, respectively), trans-α-bisabolene (15.7% and 17.3%, respectively), in addition to their nutritional value as regards proteins (13.8 in Ob-Ct and 16.16 in Ob-BS, respectively) and carotenoids (81.86 in Ob-Ct and 51.59 in Ob-BS respectively). Ob-Cn, even though it had a negative loading along PC1 and plotted in the same quadrant as the other two varieties, was slightly separated from them. In fact, this basil was distinguished by the presence of α-bulnesene (9.5%) and α-guaiene (9.0%) together with the high value of polyphenols (8.06 mg/g FW) and fructose (FRU) (6.85 mg/g FW). S. disc, with its negative loading along both axes, was positioned deep down in the left quadrant by dint of characteristic compounds: elemol acetate (9.0%) and methyl neoabietate (6.3%), together with their amount in saccharose (SACC) and anthocyanins. In the opposite quadrant relative to the Y-axis two out of three remaining species were present: M. did and N. × faas. These species were scored positively along PC1 and negatively along PC2. This position is mainly due to the main compounds as for Monarda and Nepeta. S. micro was the only sample with a positive loading on both PC1 and PC2, and it was located in the upper right quadrant because both its main constituents were previously cited as well as other specific compounds such as camphor (6.5%), α-copaene (6.3%), δ-cadinene (5.3%), eucalyptol (4.8%) and cubebol (3.0%), with the addition of glucose (GLU) and ASA TOT content.  These results were confirmed by the heat map of the two-way HCA analysis ( Figure 4) which differentiated S. microphylla (A) from other studied species gathered together in the group (B). This latter was further divided into two clusters. The cluster B1 was composed by S. disc and M. did, which in spite they showed a VOC with different compositions, the two species pointed out the highest amount in anthocyanins (0.98 in both spices). The cluster B2 included the remaining plant species. Including Nepeta with basil varieties was not strange, owing to fact that this species shared with basil its high percentage of germacrene D as well as proteins and carotenoids.         -

Essential Oil (EO) Analysis
The different constituents of the EOs from the seven Lamiaceae species studied herein, identified by gas chromatography-mass spectrometry (GC-MS) analysis, are reported in Table 4. Ninety-five compounds were present accounting for 92.7% in Nepeta to 100% of the total identification in the oil composition of Ob-BS. The striking thing was the drastic decrease of the number of identified peaks in all the Ocimum varieties. This decrease was about 58% in Ob-BS to more than 85% in Ob-Cn.
Another important thing to note was how the fragile and thermosensitive constituents decomposed into artefacts due to the heating during the hydrodistillation. All the O. basilicum volatiles were dominated by sesquiterpene compounds which were biosynthesized by the mevalonic acid (MVA) pathway, while the EO distillation originated the volatile monoterpenes (C10) [44]. This is because the two varieties of basil, Ob-Cn and Ob-Ct, showed OM as the main class of compounds in their EOs (72.3% and 52.4%, respectively) except for the Ob-BS that seemed not to be affected by heating since the EO profile evidenced SH (77.4%) as in VOCs. Linalool (48.6%) and terpinene-4-ol (23.7%) were the main monoterpenes in Ob-Cn; αand β-citral in Ob-Ct (32.2% and 18.8%, respectively). This latter species evidenced also a good percentage of SH (32.6%), represented by trans-α-bisabolene (29.3%). This compound (38.7%), together with β-bisabolene (34.4%), were peculiar in Ob-BS.
M. didyma showed a trend not very different from its spontaneous emission because it conserved the predominance of the same class of compounds: OM (84.5%). Thymol (68.6%) became the chief compound, while thymol methyl ether completely disappears. By contrast, Nepeta evidenced aliphatic hydrocarbons as the most abundant class (NT, 41.2%) together with a good amount of OS (35.2%). In detail of composition, caryophyllene oxide (17.2%) and tetracosane (14.7%) were most abundant constituents.
S. disc had a radically different profile, and its EO was distinguished by its high rate of NTs (77.5%). More than the 63% of this fraction was represented only by three compounds: tetracosane (24.3%), pentacosane (14.6%) and docosyl-isopropyl ether (10.2%). Important was also the amount of apocarotenoids exclusively represented by hexahydrofarnesyl acetone (15.7%). This constituent was also present in the second species of the Salvia genus in a notable amount (11.9%). The membership class of this compound was one of the main class in S. micro, even though it was not the prevalent one. In fact, OS (46.1%) and OM (27.0%) were mainstream. Davana ether (16.3%) and carvacrol (10.9%) showed to be the most representative compounds.

Multivariate Explorer Analyses
The PCA analysis performed with compounds of EOs > 3% was reported in Figure 5. The first two axes account for more than the half (52.6%) of a correlation matrix. Here PC2 plays a key role in the agglomeration of the species rather than PC1. In fact, two macro groups were present: S. micro, S. disc and N × faas were of positive loading on PC2 while the remaining ones were of negative loading. It is interesting to note that only S. micro was positioned in the upper right quadrant (load positively in both axes) and this was due to the exclusive compound (E,E)-farnesyl acetone and guaiol as well as its high amount of carvacrol. The species with the highest percentage of NTs, S. disc and N. × faas, were positioned on the opposite quadrant. All the basil species together with Monarda were located in the borderline along Y-axis, except for Ob-BS which shifted a little to the left, this was because of its content in β-bisabonene.
The heat map of the two-way HCA analysis ( Figure 6) confirmed what observed in PCA analysis and distinguish S. micro from the others (group I). The second group II was further divided into two subgroups: II.1 homogeneous constituted only by S. disc; II.2 which gathered M. did with all the basil species.

Bioactive Compounds
Carbohydrates are the most abundant macronutrient in edible flowers, reaching even 90% of Rosa micrantha's dry weight [13,45]. Nectar is a relevant source of soluble sugars [14], and it is composed of water, sucrose, glucose, fructose, and traces of 10 minor sugars [46]. Most Lamiaceae flowers are known to produce nectar in significant amounts and several species are cultivated as melliferous plants [47,48]. In our study, Salvia spp. and M. did flowers contained the highest quantities of glucose and sucrose, while Ob-Cn was characterised by the highest amount of fructose (Table 2). Soluble sugars were poorly represented in Ob-Ct, compared to the other six flowers under evaluation ( Table 2). Sucrose amount in M. did was fully comparable with the results obtained by Stefaniak and Grzeszczuk [49] who analysed the same species. However, some discrepancy in the amount of total reducing sugars was evidenced. This could be due to other components of reducing sugars and/or to the genetic difference between M. did and plant material, the origin of the outset plant material and the cultivation methods used in other reports. Very few studies were performed on the detection of soluble sugars in O. basilicum edible flowers. Shanaida et al. [50] quantified total soluble sugars and reducing sugars in O. americanum, with similar range of contents as those presented here.
Usually, in edible flowers carbohydrates are followed by proteins, ranging between 2.0 and 52.3 g/100 g DW (reviewed in [13]). In our work, these primary metabolites were of the highest amount in Ocimum spp. and N. × faas, exceeding 10% of the flowers' dry weight. Similar results were obtained analysing other well-known edible flowers, such as Allium schoenoprasum [51] and Cucurbita pepo [52], although these flowers belong to different families. However, previous work on flowers of M. didyma showed higher percentage of proteins, due to the cultivation systems [49].
Secondary metabolites are classified in phenolics, terpenes and steroids, and alkaloids [53]. They are usually involved in the adaptation of plants to their environment, playing a role in plant defense against biotic and abiotic stresses [53], ultraviolet radiation and oxidants [54]. Flowers assigned on these molecules the role to attract pollinators as well as the fragrance and brightness [53][54][55]. Flowers' colours are determined by flavonoids (mostly anthocyanins), betalains and carotenoids [53,56], that often contribute in mixture to the final flowers' hue [56]. Carotenoids are involved in yellow, orange and red flowers' pigmentation [57], while anthocyanins are mainly responsible for the bluish to purple and reddish colors [54]. Betalains are the yellow and violet pigments that replace anthocyanins in plants belonging to the order Caryophyllales [58] and, for this reason, they were not evaluated in this work. In our study, carotenoids were higher in S. disc and Ocimum spp. flowers (Table 2) than in the other three varieties under evaluation and some species reported in literature, such as the pale colored Telosma minor (Andrews) W. G. Craib and Piper retrofractum Vahl (12.9 each µg/g DW) [59], as well as other 11 species (0.020-0.992 µg/g FW), including Lavandula angustifolia Mill. and Salvia spendens Sellow ex Roem. et Shult. [60].
On the other hand, the examined flowers contained less carotenoids than Hemerocallis × hybrida Hort., Mimulus × hybridus 'Magic Yellow', and black Dianthus chinensis L. 'Chianti' [49]. In fact, all of them, with the only exception for D. chinensis 'Chianti', were decribed as flowers with intense yellow, orange or red colourations. This feature makes these flowers very different from those described herein, which are characterised by softer tones. Anyway, regarding M. did, there was a strong discrepancy between our carotenoid quantification and the one obtained by Stefaniak et al. [49] (1.91 vs. 167.20 µg/g FW). This could be due to different genetic background and cultivation systems.
S. disc and M. did were rich in anthocyanins, as their colour suggested. This class of metabolites is higher in these two flowers than in Begonia semperflorens Link and Otto (0.05 mg/g FW), Fuchsia hybrida Hort. Ex Siebert and Voss (0.08 mg/g FW) and Pelargonium peltatum (L) L'Hér. (0.14 mg/g FW), which are characterised by red petals [61]. Nevertheless, D. chinensis 'Chianti' and M. didyma contain more than 2 mg/g FW of anthocyanins [49]. These last results were obtained with different methods, therefore the comparison may be not similar.
Between the species studied herein, S. disc and Ocimum spp. showed the highest content in total polyphenols (TPC). The same metabolites are comparable between S. micro (2.41 mg/g FW) and S. splendens (2.16 mg/g FW) [60]. To the best of our knowledge, the TPC of O. basilicum flowers was reported in only one paper [62], with the quantification of three different basil cultivars ('Subja', 'Holy green' and 'Red rubin') in freeze-dried samples, making difficult the comparison with the fresh flowers used in this work. No studies were published on the TPC in N. × faas flowers, but some data are available for other species belonging to the same genus, such as N. cataria L. [63] and N. nepetella L. [64]. Dried flowers of these two species are characterised by 2-and 4-fold more TPC than N. × faas fresh flowers [63,64].
Ascorbic acid (vitamin C, ASA) is known to take part in essential human biochemical and physiological processes. This molecule plays a relevant role in the development and maintenance of connective tissues, in bone formation and wound healing [65]. ASA is also involved in several metabolic pathways, in the proper functioning of the immune system and it protects the human body from free radicals' damages [65]. However, human organisms are not able to synthetize this vitamin, since humans lack the terminal enzyme of its biosynthetic pathway [66]. For this reason, ASA must be present in a well-balanced diet, and the consumption of edible flowers can help to supply the EU daily requirements intakes (80 mg per days) [67]. In fact, Tagetes tenuifolia Cav. and Viola tricolor L. are considered good sources of vitamin C, containing 241.20 and 182.16.20 mg/100g FW respectively [60].
In this work, M. did, S. micro and N. × faas were characterised by higher levels of total (ASA TOT ) and the reduced form (ASA) than the other species under evaluation (Table 2). Nevertheless, the amounts of vitamin C were very low, and due to their small size, thousands of flowers would be needed to reach the EU recommended intakes. However, compared to other Lamiaceae flowers, these two varieties and N. × faas were characterized by ASA TOT content similar to some Agastache spp. [17]. On the other hand, M. did and S. micro contained around −18 and −15 fold less vitamin C than S. splendens and L. angustifolia [60].

Spountaneous Emissions
In this study, we evaluated the aroma profile spontaneously emitted from the seven selected Lamiaceae flowers. The analyses of the volatile organic compounds increased our knowledge concerning their ecological role. In spite of this, the species that have been investigated for their spontaneous emission were too negligible compared to the high number of plants present in nature. The spontaneous emission of Ocimum basilicum (Ob) was already widely studied [75][76][77][78][79], but only a few papers investigated its varieties and none of these works reported the varieties studied herein. In 2008, Klimánková et al., [80] evaluated five cultivars of basil green cultivar I (Prava zelena), green cultivar II (Trpaslici), red cultivar III (Cinamonette), red cultivar IV (Purple Opaal), and red cultivar V (Rot), and their volatile composition was characterized by linalool, methyl chavicol, eugenol, bergamotene, and methyl cinnamate. A more recent study reported the SPME components of two basil varieties (Violetto and Genovese) where linalool (18.94% and 22.57% respectively), eugenol (3.95% and 15.02%, respectively) and methyl eugenol (39.17% and 19.39%, respectively) were identified as the main constituents [77]. On the contrary, the whole spontaneous emission of Malaysian O. basilicum flower was represented by estragole (88.18%) [78]. In the three Ocimum varieties studied herein only linalool and eugenol were detected even though their presence was not detected in all varieties (Ob-Cn and Ob-Ct for linalool and Ob-Cn and Ob-BS for eugenol) obviously with lesser percentage. Bergamoptene was observed in Ob-BS studied here. Methyl eugenol and methyl cinnamate were not present while chavicol (= eustragol) was of lesser amount and only present in Ob-BS.
To the best of our knowledge, no works considered the aroma profile of M. didyma or any other species belonging to the same genus.
Concerning the studied Nepeta × faassenii no work was found related to its VOC composition. Moreover, this genus seemed to be not attractive seeing the scant reports in this context. The first work found dates back to 2010 when the authors used the Proton-Transfer-Reaction Mass Spectrometry (PTR-MS) method to evaluate the VOCs of three Nepata species cultivated in vitro. High concentration of nepetalactone was evidenced in N. sibirica L. and especially in N. rtanjensis Diklic and Milojevic shoot cultures, even though this constituent was detected in traces in N. nervosa Royle ex Benth. [81]. Recently Yayali and collaborators [82] investigated the Turkish Nepeta conferta Hedge and Lamond and reported that p-cymene (25.5%), eucalyptol (9.8%), limonene (5.0%), sabinene (4.8%), carvacrol (3.7%), (E)-linalool oxide (3.3%), (Z)-linalool oxide (3.0%) [82]. Moreover, Barhoumi et al. [83] studied the VOCs of two wild Nepeta curviflora Boiss originating in two Jordan regions (Salt, Northwest of Amman capital and Irbid, in the Northern of Jordan). Fully expanded flowers from Salt were characterized by a high SH content (75.94%) especially represented by trans-caryophyllene (26.50%) and OMs (18.53%) represented mainly by 4aα,7α,7aα-nepetalactone (12.74%). The main sesquiterpene hydrocarbons detected in the emission profiles of the flowers from the northern species, collected during the full blossoming stage, included β-bourbonene (19.45%), α-copaene (13.37%) and bicyclogermacrene (7.09%) [83]. All these compounds were completely absent in the VOCs of the species studied herein, except for nepetalactone and trans-caryophyllene.
No work was also found in both the studied species of Salvia genus, notwithstanding numerous published works on this subject [84][85][86][87][88][89][90][91][92][93][94]. The two studied species S. disc and S. micro, originating from South America, were grown under uniform conditions in CREA-Sanremo (Italy). As reported by Ascrizzi et al. [93], with the exception of only 3 species, all the South American studied plants were rich in SHs with a percentage ranging from 54.4% and 96.5% and showed β-caryophyllene and germacrene D as the most abundant ones. This is in a total agreement with the class of compounds in S. disc also for the presence of these two compounds because the first one showed a similar amount likewise what reported by the cited work while the second one had a very low amount. The same work underlined the presence of other sesquiterpene constituents in South American species such valencene, α-copaene, cis-muurola-3,5-diene, β-bisabolene and γ-muurolene. Except for β-bisabolene, all the other compounds were lacking. S. micro profile followed the same trend of one of the exceptions of South American species: S. dorisiana [93]. In this latter the whole volatile emission profile was mainly composed by MH (77.9%), with limonene of the most abundant compound (11.65%).

Essential Oils
Numerous reports are present in the literature on the EO composition of O. basilicum taxa which are very complex and show wide compositional variability according to the presence of several chemotypes within the species and according to the varied climatic/geographical conditions and agronomic practices [95]. Regardless of these factors, monoterpenes were commonly distributed in basil EOs and the linalool percentage was very high (ranging from 29.2% to 75.9%) as in the 'Cinnamon' variety, reported by many studies [96][97][98][99][100][101][102][103][104][105][106]. Only few papers reported the EO composition of O. basilicum varieties and among these we can find the study of Sajjadi [107], who investigated two Iranian basil varieties (O. basilicum L. cv. purple and O. basilicum L. cv. Green). Methyl chavicol was the characteristic compounds of both (52.4% and 40.5%, respectively). Although the oil of green basil was characterized by a high content of citral (both neral and geranial, 46.1%), citral was not detected in purple basil oil [107]. The same varieties from Yemen evidenced a completely different behaviour where linalool prevailed in both varieties (44.3% in O. basilicum var. purpurascens (purple) vs. 46.2% in O. basilicum var. basilicum (green) [108]. In the same year, another work was published by Tsasi and co-workers [109], where the effect of harvesting was studied in five O. basilicum varieties. The Ob-Cn EO were in agreement with O. basilicum var. latifolia and O. basilicum var. minimum, cultivated in the field, concerning their linalool content (49.5% and 52.0%, respectively) and with var. violetto (11.9) and var. latifolia (10.1%) cultivated in the greenhouse regarding the eugenol amount. Among the studied varieties in this work, Ob-Ct was the only one reported by the literature. The first work was done by Turkish scientists, who actually did not study directly this variety, but compared some investigated EOs with a high amount of citral compared to what is found in lemon balm basil, known as O. × citriodorum or O. basilicum var citriodorum (a hybrid of O. basilicum × O. americanum) [110]. Further on, in 2000, another Turkish research team succeeded in the cultivation of O. × citriodorum and confirmed the domination of neral (43.3%) and geranial (43.4%) in the flower EO [111]. A quite recent work confirmed a good percentage of citral (20.7%) in O. × citriodorum even though it was not the main compound which was represented on the contrary by nerol (23.0%) [112]. Asian O. × citriodorum showed the presence of two chemotypes: the first one was rich in geranial/neral, which is the same as this study, and another one with methyl chavicol [113]. The behaviour of the investigated Ob-BS followed the O. basilicum var. 'Blue Spice' [29].
The studied species of M. didyma showed an EO almost exclusively formed by thymol. The richness in this compound was confirmed by many scientific publications. In fact, Fraternale et al. [114], showed the prevalence of thymol (51.7%) and γ-terpinene (14.3%) in the flower EO of M. didyma. Also, a Monarda species grown in Canada underlined thymol (41.17%), γ-terpinene (15.88%), carvacrol (15.20%), and p-myrcene (12.58%) as main constituents [115]. Two other studies published in 2017 reported the EO composition from this plant species cultivated in central Italy: the EO from the flowering aerial parts pointed to thymol (59.3%) and p-cymene (10.3%) as major compounds [116], while the second work evidenced thymol 62% [43]. Other Monarda species, always cultivated in Italy, were very rich in monoterpenes, but with o-cymene (13.42), γ-terpinene (22.15), and carvacrol (13.80%) as the main constituents, and thymol with a lesser amount (5.87%) [42]. The chemical characterisation of the EO from Nepeta can be traced back to 1967 when Regnier [117] studied three species and each one showed a different main compound: nepetalactone in N. cataria L. (77%), epi nepetalactone in N. mussini Spreng. Ex Henckel (70%) and citronellol in N. citriodora Dumort. Since then several species were studied. The bulk of investigated plants were distinguished by the presence of a good amount of at least one of the nepetalactone isomers (ranging between 16% to 72%) such as N. cataria [118], N. rtanjensis [119], N. cataria var. citriodora and N. nuda L. [120]. All these works disagree with what was found in the analysed Nepeta × faassenii where these compounds were completely absent. The presence of non-terpene compounds was observed in the Lebanese Nepeta species such as N. cilicica Boiss. ex Benth [121], N. nuda ssp. Pubescens and N. curviflora Boiss [122]. These results did not agree with the data found herein since, despite this class was the main one, the constituents were completely different. Caryophyllene oxide, one of the most important compounds in our Nepeta × faassenii (17.2%), was evidenced in the higher amount in N. melissifolia Lam. and N. sibirica (22.06 and 20.35%, respectively) [120]. The only work which analysed the studied Nepeta hybrid was that of Ali and his co-workers [123], who found an EO rich in 1,8-cineole. This compound was present in very fewer amount in our study.

Plant Material and Cultivation
Monarda didyma "Fireball" and Nepeta × faassenii "Six Hills Giant" plants were bought at L'Erbaio della Gorra (Str. Gianardo, 11 Casalborgone, To, Italy,) plant nursery, and then grown in open field for two years in private garden. Cutting were used for plant propagation in greenhouse. Seeds of Ocimum basilicum 'Blue Spice', Ocimum basilicum 'Cinnamon' and Ocimum × citriodorum were provided to the Conservatoire National des Plantes à Parfum, Medicinales et Aromatiques (Milly-la-Forêt, France). Salvia discolor and S. microphylla "Hot Lips" are currently part of the plants collection at CREA-Research Centre for Vegetable and Ornamental Crops (CREA, Sanremo, IM, Italy, GPS: 43.816887, 7.758900) where they were propagated by cuttings. All the plants used in this work, both deriving from seed or cutting, were cultivated in pots kept in an unheated greenhouse covered with an anti-insect net at CREA, as reported by Najar et al. [17]. Briefly, the plants were cultured in substrate (Hochmoor-Terflor, Capriolo, BS, Italy) with slow release fertilizer (Nitrophoska, Eurochem Agro, Cesano Maderno, MB, Italy) and irrigated with nutrient solution (Ferti 3, Planta-D Plants 2020, 9, x

.2. Biochemical Analyses
Fresh flowers were picked e replica, and stored at −80 °C. Froz total polyphenolic content (TPC) ( content [17]. Radical scavenging a as IC50 (mg/mL). Soluble sugars ( reduced ascorbate (ASA) were qu performed with an ultraviolet (UV nitrogen content determination w in Jones et al., [132]. Data were multiplying the percentage of nitr

Phytochemical Analysis
A fresh flower of each plant conical flask (20 mL) and sealed w time). The evaluation of VOC emis (PDMS) fibre manufactured by Su conditioned according to the man injector of a gas chromatograph. E place for 15 min at a temperature o and immediately transferred to th the analytes were thermally desor desorbed from SPME fibre was ex Essential oil (EO) was extra material was barely sufficient to separately hydrodistilled for 2 h European Pharmacopeia [133]. Th high-performance liquid chromato MS.
ngemittel, Regenstauf, Germany) every week. Supplemental irrigations with water were carried out according to the needs of the plants and the season in order to avoid water stress to the plants. The plants were grown applying the organic cultivation method (without pesticides), using antagonist insects (Koppert Italia Srl., Bussolengo, VR, Italy) and microorganisms [17]. Full-bloom flowers were picked during their flowering time (see Table 1).

Biochemical Analyses
Fresh flowers were picked early in the morning, divided into three homogeneous biological replica, and stored at −80 • C. Frozen samples (200 mg) were used to quantify total carotenoid [130], total polyphenolic content (TPC) (Folin-Ciocalteu method, according to [17]), and total anthocyanins content [17]. Radical scavenging activity was determined by DPPH assay [131], reporting the results as IC 50 (mg/mL). Soluble sugars (D-glucose, D-fructose and sucrose), total ascorbate (ASA TOT ) and reduced ascorbate (ASA) were quantified as described in Najar et al. [17]. All measurements were performed with an ultraviolet (UV)-1800 spectrophotometer (Shimadzu Corp., Kyoto, Japan). Total nitrogen content determination was performed by Kjeldhal method following the protocol described in Jones et al., [132]. Data were reported as percentage of crude protein content, obtained by multiplying the percentage of nitrogen by 6.25 as conversion factor (%N × 6.25).

Phytochemical Analysis
A fresh flower of each plant was picked (an average of 0.5 to 1 g), placed separately in a glass conical flask (20 mL) and sealed with a cap provided with aluminium foil for 30 min (equilibration time). The evaluation of VOC emission was performed with the use of 100 µm polydimethylsiloxanes (PDMS) fibre manufactured by Supelco Ltd (St. Louis, MO, USA). Prior to the analyses, the fibre was conditioned according to the manufacturer's instruction, at 250 • C for a duration of 30 min in the injector of a gas chromatograph. Exposition of the fibre in the headspace phase of the samples took place for 15 min at a temperature of 23 • C. Subsequently the fibre was reinserted back into the needle and immediately transferred to the injector of the gas chromatograph (temperature 250 • C), where the analytes were thermally desorbed for a duration of 30 min. The composition of the compounds desorbed from SPME fibre was examined using GC-MS.
Essential oil (EO) was extracted from fresh flowers even though the weight of these plant material was barely sufficient to undertake a microdistillation. Therefore, the fresh flowers were separately hydrodistilled for 2 h using a micro-Clevenger like apparatus as recommended by the European Pharmacopeia [133]. The yield of the EOs were very low and were collected directly in high-performance liquid chromatography (HPLC)-grade n-hexane and immediately analysed by GC-MS.
GC-MS analyses were performed with an Agilent 7890B gas chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with an Agilent HP-5MS (Agilent Technologies Inc., Santa Clara, CA, USA) capillary column (30 m × 0.25 mm; coating thickness 0.25 µm) and an Agilent 5977B single quadrupole mass detector (Agilent Technologies Inc., Santa Clara, CA, USA). Analytical conditions were as follows: injector and transfer line temperatures 220 and 240 • C, respectively; oven temperature programmed to raise from 60 to 240 • C at 3 • C/min; carrier gas helium at 1 mL/min; injection of 1 µL (0.5% HPLC grade n-hexane solution); split ratio 1:25. The acquisition parameters were as follows: full scan; scan range: 30-300 m/z; scan time: 1.0 sec. Identification of the constituents was based on a comparison of the retention times with those of the authentic samples, comparing their linear retention indices relative to the series of n-hydrocarbons. Computer matching was also used against commercial [134,135] and laboratory-developed mass spectra library built up from pure substances and components of known oils and MS literature data [135][136][137][138][139][140].

Statistical Analysis
Biochemical results were statistically analysed using either Tukey's honest significant difference (HDS) or the Games-Howell test according to the homogeneity of variance (Levene's test) [141]. The analyses were performed using IBM SPSS software (IBM Corp. Released 2017. IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp).
Linear correlation between polyphenols and radical scavenging activity were determined using Microsoft Excel ® 2013 (Microsoft Corporation, Redmond, WA, USA).
Multivariate explorer, principal component and hierarchical clustering analyses were carried out which allowed the co-evaluation of all variables [142]. For each treatment, the chemical compounds and their proportions (%) were plotted in Excel spreadsheets. Compounds present with amounts less than 5% were omitted from the analysis. The data were transformed by orthogonal rotation into latent variables named as the principal components. These are linear combinations of original variables created from the eigenvalues of the data correlation matrix. The Euclidean distance was used as a dissimilarity metric to represent the straight-line distance between the centroids of each cluster of chemical compounds identified in EO analysis. The unweighted pair group method with arithmetic averages (UPGMA) was used to cluster the compounds. The results were presented in a dendrogram that characterized the clusters. Both analyses were run in the JMP software package 13.0.0 (SAS Institute, Cary, NC, USA).