*2.3. LC–PDA/UV–ESI–MS Profiles*

#### 2.3.1. Lignan Content of Flaxseed Meal

Flaxseed is known as a major source of lignan SDG, which is present in the form of high molecular oligomers due to ester bonds with 3-hydroxy-3-methylglutaric acid (HMGA) and glycosidic linkages with phenolic compounds, such as hydroxycinnamic acid derivatives and herbacetin diglucoside [14]. Both alkaline and acid hydrolysis of SDG oligomers is commonly used to analyze the lignan content of flaxseed [15]. In the present study, the chemical characterization of flaxseed lignans from the four analyzed extracts (defatted seed meal of flaxseed Sideral and Buenos Aires in the two cultivation sites Pisa and Bologna) was performed on the alkaline hydrolysates of SDG oligomers by the means of HPLC coupled to a PDA/UV detector and an electrospray ionization mass spectrometer (ESI–MS). The PDA/UV chromatograms (Figure 3) were acquired at 280 nm, which is the maximum absorption of SDG. All the extracts showed very similar profiles, with the presence of phenolic compounds due to the breaking of oligomer ester linkages. Indeed, the alkaline hydrolysis led to the formation of phenolic acid glucosides, such as *p*-coumaric acid glucoside (two isomeric forms, peaks **1** and **2**) and ferulic acid glucoside (two isomeric forms, peaks **3** and **4**), the flavonoid herbacetin diglucoside (peak **5**), the lignan SDG (two isomeric forms, peaks **6** and **7**), and ferulic acid (peak **8**), according to previous studies [15]. The tentative identification of all compounds was carried out comparing their elution order, ESI–MS/MS and PDA/UV data (Table 3) with those previously reported [16].

Masses of identified phenolics were detected in negative ion mode, originating deprotonated [M − H]<sup>−</sup> molecules and except for compound **6**, formiate [M + HCOO]<sup>−</sup> and acetate [M + CH3COO]− adducts, leading to establish the molecular weight of detected substances. MS/MS of [M + CH3COO]− ions for compounds **1**/**2** (*m/z* 385) and **3**/**4** (*m/z* 415) showed losses of a hexosyl moiety ([M−162]−) due to the cleavage of the *O*-sugar bond, generating aglycon portions attributable to *p*-coumaric acid and ferulic acid, respectively. Thus, compounds **1**/**2** and **3**/**4** were identified as two isomeric forms of *p*-coumaric acid glucoside and ferulic acid glucoside, respectively, that cannot be distinguished on the basis of UV and MS data. The alkaline hydrolysates showed also the presence of ferulic acid (**8**), with λmax 237 and 323 nm and diagnostic product ions (*m/z* 178, 149, and 134) generated in the MS/MS of deprotonated molecule [M − H]<sup>−</sup> at *m/z* 193. The full mass spectrum of compound **5** showed a deprotonated molecule [M − H]<sup>−</sup> at *m/z* 625, while MS/MS displayed two diagnostic fragment ions at *m/z* 463 and 301 generated by the subsequent losses of two hexosyl moieties. Thus, compound **5** was identified as herbacetin diglucoside, a flavonol considered part of the lignan macromolecule [17]. Compounds **6** and **7** were identified as two isomeric forms of SDG, the most abundant lignan present in linseed. The MS/MS experiment for the acetate adduct [M + CH3COO]− at *m/z* 745 provided a product ion at *m/z* 583, due to the loss of a hexosyl moiety, according to the presence of a glucosyl residue. A few minor peaks remained unidentified.

The percentage composition of SDG oligomers in terms of detected phenols **1**–**8** after alkaline hydrolysis, calculated by integrating the peak areas at 280 nm in all camelina extracts, is shown in Figure 4. The most representative compound was *p*-coumaric acid glucoside (isomers **1** and **2**) with a percentage amount ranging from 48.8 to 60.0%, followed by SDG (isomers **6** and **7**; 20.2–26.4%), ferulic acid glucoside (isomers **3** and **4**, 7.6–16.0%), ferulic acid (**8**. 4.2–9.4%), and herbacetin diglucoside (**5**. 1.0–2.1%). Based on these results, meal extract from flaxseed Sideral cultivated in Pisa was the richest in *p*-coumaric acid glucoside, while meal extract from flaxseed Buenos Aires cultivated in Bologna was the richest in SDG and ferulic acid glucoside.

**Figure 3.** HPLC–PDA/UV profiles (detected at 280 nm) of secoisolariciresinol diglucoside (SDG) oligomers alkaline hydrolysates from the defatted seed meal extracts of flaxseed Sideral cultivated in Bologna (**A**) and Pisa (**B**), and Buenos Aires cultivated in Bologna (**C**) and Pisa (**D**). For peak data, see Table 3.




 Compound numbers correspond to peak numbers in Figure 3. b For peaks **1**–**4**, **6**, and **7** MS/MS data were obtained by the fragmentation of the [M + CH3COO]− precursor ions, whereas for peaks **5** and **8,** data were obtained by the fragmentation of the deprotonated molecule [M − H]−.

#### 2.3.2. Glucosinolate and Phenol Contents of Camelina Meal

The LC–ESI–MS analyses of camelina meal extracts (Figure 5), registered in negative ion mode, showed the presence of two major classes of compounds, represented by glucosinolates (peaks **9**, **10**, and **15**) and flavonoids (peaks **11**–**13**). The compounds were tentatively identified on the basis of spectral data. The MS/MS of all glucosinolates ([M − H]<sup>−</sup> at *m/z* 506, 520, and 534) showed diagnostic fragments due to the losses of a SO<sup>2</sup> molecule and were identified as glucoarabin (**9**), glucocamelinin (**10**), and 11-(methylsulfinyl)undecylglucosinolate (**15**), in agreement with previous studies [18,19]. The three detected flavonoids, characterized by two strong UV absorptions at 256–258 and 350–356 nm were all in the form of glycosides characterized by the presence of the same aglycone identified as quercetin due to the diagnostic fragment at *m/z* 301 in the MS/MS. Compound **11** ([M − H]<sup>−</sup> at *m/z* 741) was a triglycoside as deduced by its fragmentation pathway ([M − H − 132 − 146 − 162]−) and was identified as quercetin 2"-*O*-apiosyl-3-*O*-rutinoside, previously isolated from camelina by Quéro et al. [18]. Both compounds **12** ([M − H]<sup>−</sup> at *m/z* 595) and **13** ([M − H]<sup>−</sup> at *m/z* 609) contain a disaccharide chain constituted by pentose–hexose and deoxyhexose–hexose, respectively. Thus, compound **13** was identified as quercetin 3-*O*-rutinoside (rutin) [19], while compound **12** is probably a quercetin apiosyl-glucoside in which the exact position of sugars cannot be determined only on the basis of MS/MS data; based on similar components previously found in camelina seeds, it can be assumed that compound **12** is quercetin 2"-*O*-apiosyl-3-*O*-glucoside. Finally, compound **14** ([M − H]<sup>−</sup> at *m/z* 623) remained not completely identified, but it could be a synapoil derivative as can be deduced by the presence of fragment [M − H − 206]<sup>−</sup> at *m/z* 417.

The LC–MS quantitative analyses (Table 5) showed that among glucosinolates glucocamelinin was the most representative in both camelina meal extracts, while rutin was the most abundant among flavonoids. Furthermore, meal extract from camelina cultivated in Bologna showed the highest content in term of glucosinolates [18.6 ± 0.3 vs. 14.5 ± 0.5 mg/g dry weight (DW)], whereas meal extract from camelina cultivated in Pisa showed the highest content in terms of flavonol glycosides (4.8 ± 0.08 vs. 4.5 ± 0.06 mg/g DW).

**Figure 5.** HPLC–ESI–MS profiles (registered in negative ion mode) of camelina meal extracts cultivated in Pisa (**A**) and Bologna (**B**). For peak data, see Table 4.

**Table 4.** Spectral (UV and ESI–MS/MS) and chromatographic data (*t*R, retention time) of compounds **9**–**15**, detected in camelina meal extracts.


<sup>a</sup> Compound numbers correspond with peak numbers in Figure 5.


**Table 5.** Quantitative amount (mg/g ± SD DW) of constituents found in meal extracts from *Camelina sativa* Italia in two cultivation sites, Pisa and Bologna.

DW: dry weight; SD: standard deviation.

#### **3. Discussion**

The obtained results showed that both flaxseed and camelina were able to reach satisfactory yields in both environments, even if clear effects of environment and variety were observed, with the highest productive performance reached by Sideral and Italia grown in Pisa. The obtained results are consistent with those reported in the literature for different environments and cultivars [4,20–22]. The analysis of the chemical composition of flax seeds showed that oil contents were not significantly different under the influence of variety or environmental conditions, with average values of 46%. This content was higher in comparison with those reported in previous studies [23,24] in which flaxseed oil content ranged between 34 and 45%, depending on the geographical area, genotype and environmental conditions. Flaxseeds were characterized by a very stable proportion of polyunsaturated fatty acids, in both varieties and environments, with α-linolenic acid as the most abundant fatty acid. It is known that this fatty acid is characterized by beneficial effects on the prevention of several diseases, such as cardiovascular diseases, hypercholesterolemia, chronic kidney diseases, atherosclerosis, and neurological disorders [25,26]. In the present study, the content of α-linolenic acid in both flaxseed varieties, ranging from 57 to 65%, consistent and sometimes higher than that reported in literature (from 50 to 59%, depending on genotype and environment) [27,28].

In camelina, oil content and fatty acid profiles were dependent on the environment, with higher oil and oleic acid contents in Pisa samples, compared to the Bologna ones. On the contrary, the seed oil of camelina grown in Bologna showed an increased content of eicosenoic acid and α-linolenic one. These differences could be due to the highest temperatures experienced during flowering and seed filling by the crop cultivated in Pisa, for which a spring sowing was performed. It is known that elevated temperatures during flowering and seed ripening determine a rise in oleic acid since temperature can interfere with the activity of the enzymes involved in the biosynthesis of fatty acids [29]. Interestingly, eicosenoic acid can be a valuable source of medium chain fatty acids for the bio-based industry, which nowadays are not produced in Europe as they are totally derived from palm and coconut oils [30]. Nonetheless, the oil content fell in the range typically reported for this oilseed crop [21,30]. Finally, as a general observation, the crude protein content of both types of oilseeds was negatively correlated with the oil content confirming previous findings [5,31].

The evaluation of the total phenols and flavonoids and their related antioxidant activities showed that both types of meal were characterized by interesting levels of these beneficial substances, regardless of the variety and growing environment, except for that given for total phenolic content and the anti-radical activity of camelina meals. In this case, in fact, higher phenols and stronger anti-radical activity, measured by DPPH, were observed for defatted meal deriving from camelina grown in Pisa, in comparison with camelina meal obtained from Bologna. In the literature, regarding total phenols and flavonoids in flaxseed and camelina cakes and/or meals, lower and higher values are reported, in comparison with our findings, depending on the variety, extraction method, and growing conditions [32–35]. For example, Teh and Birch [32] found, in defatted flaxseed cake, levels of total phenols

and total flavonoids in the range of 474–807 mg GAE/100 g FW (FW = fresh weight) and 5.6–15.6 mg luteolin equivalents (LUE)/100 g FW, respectively, depending on the extraction method (ultrasonic and conventional method), solvent volume and extraction temperature. In the defatted camelina meal, Rahman et al. [34] observed a mean total phenolic content of 11.69 ± 0.44 mg GAE/g DW with a total flavonoid content equal to 6.81 ± 0.68 mg CAE/g DW.

Phenolic compounds are recognized as important food metabolites able to prevent several pathologies, such as cardiovascular and neurodegenerative diseases and cancer [36,37]. Polyphenols exhibit, in fact, interesting antioxidant activity thanks to their ability to transfer a hydrogen atom or an electron, acting as a reducing agent, as well as by the possible chelation of metal ions and the inhibition of the activity of oxidases [38]. The antioxidant activity of the hydroalcoholic extracts of the two kinds of meals tested in the present study was in line with the results on phenol and flavonoid concentration. In fact, a lower value of EC<sup>50</sup> (for both DPPH and ABTS assays) were revealed for camelina meal, suggesting a positive correlation with the higher phenols and flavonoids detected in this kind of meal. The lower the EC<sup>50</sup> value is, in fact, the higher the ability of the extract to scavenge radicals is. Previous reports underlined that camelina and flaxseed meals, after solvent oil extraction, contain good amounts not only of phytochemicals but also crude proteins (32–45%, with the presence of important essential amino acids, such as lysine, methionine and cysteine), insoluble fiber, carbohydrates and minerals [34,39–41], which make these meals good candidates for food and feed applications.

As expected, all flaxseed meal extracts were found to be a good source of SDG, herbacetin diglucoside, and hydroxycinnamic acid glucosides (ferulic and *p*-coumaric acid glucosides), herein characterized by HPLC–UV–MS analyses of alkaline hydrolysate, since they are accumulated in flaxseed seeds in form of oligomers [15]. Although the chemical compositions of the four extracts showed the same profiles in terms of constituents and their relative abundance, Buenos Aires flaxseed's meal cultivated in Bologna resulted, even if with small distances from others, the major source of SDG, a lignan whose potential health benefits are under investigation in many recent studies [8]. SDG, one of the most representative monomeric constituents of the lignan macromolecule, is reported to have many biological activities such as antioxidant and anti-inflammatory properties, playing a role in the prevention against chronic diseases, such as cardiovascular events and metabolic syndrome [8].

In agreement with previous investigations [18], three major glucosinolates were found in camelina meals, showing glucocamelinin as the most representative (about 64 and 66% of the total glucosinolate composition in the varieties growing in Pisa and Bologna, respectively). Long-chain glucosinolates predominate in camelina compared with short-chain glucosinolates that comprise the majority of glucosinolates in canola meal. Previous studies evidenced that glucosinolate content in camelina seeds is dependent on the geographic origin of seeds, as well as climatic factors and soil conditions [9]. A large variation was also observed between different cultivars [42]. In a recent work, Russo and Reggiani [43] reported an amount of total glucosinolates ranging from 19.6 to 40.3 mmol/kg DW in camelina meal from 47 accessions, with an average of 30.3 mmol/kg DW. Compared to these data, the amount of total glucosinolates in camelina meal obtained in the present work from the two Italian cultivation sites were moderately high, with camelina from Bologna richer (35.7 mmol/kg DW) than camelina from Pisa (27.9 mmol/kg DW), probably due to the different environmental conditions. In addition to glucosinolates, both camelina extracts were shown to contain phenolic constituents in quite a similar amount, with camelina from Pisa slightly above camelina from Bologna. Flavonoids, mainly kaempferol and quercetin derivatives, were previously reported in camelina seeds [19] and their profile compared to that of camelina meal [34,35]. The LC–MS analyses of camelina meal extracts from Pisa and Bologna showed that both samples contained three major quercetin glycosides, with quercetin 3-*O*-rutinoside the most representative, followed by quercetin 2"-*O*-apiosyl-3-*O*rutinoside, herein more expressed than previous studies. The presence of glucosinolates and

flavonoids is important for defining the nutraceutical value of camelina meals. Glucosinolates, secondary metabolites typical of the Brassicaceae family, have received great attention for their potential benefits in the prevention of carcinogenesis as well as cardiovascular and neurological diseases [44–46]. In a recent preliminary study, glucocamelinin and glucoarabin from defatted seed meal showed an ability to upregulate the phase II detoxification enzyme quinone reductase (NQO1) [18]. Similarly, rutin, a common flavonol glycoside found in a large number of plant species, is known for its antioxidant activity and its role in the treatment and prevention of various diseases [47].

## **4. Materials and Methods**
