Soilless Cultivated Halophyte Plants: Volatile, Nutritional, Phytochemical, and Biological Differences

The use of halophyte plants appears as a potential solution for degraded soil, food safety, freshwater scarcity, and coastal area utilization. These plants have been considered an alternative crop soilless agriculture for sustainable use of natural resources. There are few studies carried out with cultivated halophytes using a soilless cultivation system (SCS) that report their nutraceutical value, as well as their benefits on human health. The objective of this study was to evaluate and correlate the nutritional composition, volatile profile, phytochemical content, and biological activities of seven halophyte species cultivated using a SCS (Disphyma crassifolium L., Crithmum maritimum L., Inula crithmoides L., Mesembryanthemum crystallinum L., Mesembryanthemum nodiflorum L., Salicornia ramosissima J. Woods, and Sarcocornia fruticosa (Mill.) A. J. Scott.). Among these species, results showed that S. fruticosa had a higher content in protein (4.44 g/100 g FW), ash (5.70 g/100 g FW), salt (2.80 g/100 g FW), chloride (4.84 g/100 g FW), minerals (Na, K, Fe, Mg, Mn, Zn, Cu), total phenolics (0.33 mg GAE/g FW), and antioxidant activity (8.17 µmol TEAC/g FW). Regarding the phenolic classes, S. fruticosa and M. nodiflorum were predominant in the flavonoids, while M. crystallinum, C. maritimum, and S. ramosissima were in the phenolic acids. Moreover, S. fruticosa, S. ramosissima, M. nodiflorum, M. crystallinum, and I. crithmoides showed ACE-inhibitory activity, an important target control for hypertension. Concerning the volatile profile, C. maritimum, I. crithmoides, and D. crassifolium were abundant in terpenes and esters, while M. nodiflorum, S. fruticosa, and M. crystallinum were richer in alcohols and aldehydes, and S. ramosissima was richer in aldehydes. Considering the environmental and sustainable roles of cultivated halophytes using a SCS, these results indicate that these species could be considered an alternative to conventional table salt, due to their added nutritional and phytochemical composition, with potential contribution for the antioxidant and anti-hypertensive effects.


Introduction
Salinity is a worldwide problem that affects a large percentage of the earth's surface, in which salts gradually accumulate in the soil, eventually transforming fertile soil to barren soil [1]. In this process, water-soluble salts are deposited in the soil to an extent that impacts crop productivity, agricultural economics, and the ecosystem [2]. Currently, salt-affected soils cover over 15 percent of the world's cultivated land [3]. Despite this critical factor that imposes severe constraints on the ecosystem, there is a type of plant able of growing in these salt-affected areas; they are called salt-tolerating plants or halophyte plants [4].
Halophyte plants can grow in extreme conditions of drought, temperature, and salinity (NaCl concentrations above 200 mM) since they respond to salt stress at three levels, e.g., cellular, tissue, and whole plant level [5]. In this context, halophyte plants are a potential solution for degraded soil, freshwater scarcity, and coastal area utilization [6]. Therefore, these plants have been considered alternative crops (biosaline and soilless agriculture) for sustainable use of natural resources.
In Portuguese territory, the halophytes grow along the country's coast salt marshes, and some companies are already producing some edible species in salt-affected areas using a soilless cultivation system (SCS) to improve their productivity and marketing, assuring their safety and quality. In this cultivation system, nutrients and salt are added to the halophytes through the cultivation media, and these plants are irrigated by flooding, simulating tides, under controlled temperature, and humidity [7]. Regarding food safety, salt marshes ecosystems are natural deposits of heavy metals near a polluted area. As the metals spread along with the tides and periodic floods, they interact with soil and the biotic community [8]. In this way, the halophytes may accumulate large amounts of these contaminants in their aerial and belowground organs, leading to potential impacts on human health and safety [9].
Portugal has become an important producer of edible halophytes. So far there are few studies carried out with cultivated halophytes using a SCS that report their nutraceutical value, as well as their benefits on human health when consumed in equilibrated diets. Therefore, studies involving halophyte species are crucial. In a previous study, the halophyte (Sarcocornia fruticosa L.) cultivated using a SCS showed higher nutrient contents (ash, proteins, and fat contents), antioxidant activity, and total phenolic compound content than wild plants [7].
Halophytes are used traditionally as herbs and vegetables and some species are appreciated in gourmet cuisine as a promising substitute for table salt because of their characteristic salty taste. These plants are attracting attention for being sources of phenolic acids, flavonoids, coumarins, vitamins, and carotenoids, which are reported to be health promoters due to their antioxidant properties [10][11][12]. These natural antioxidants are produced by halophytes to maintain their cellular functioning under oxidative stress conditions (e.g., high ionic content, waterlogging, and submersion) [13,14]. Studies show that the regular consumption of phenolic acids and flavonoids exerts cardiovascular protective effects and may reduce the onset or progression of cardiovascular diseases, particularly hypertension [15,16].
In this context, some studies have demonstrated that extracts from halophyte species could be used for the prevention and treatment of heart disease and hypertension (angiotensinconverting enzyme (ACE)-inhibitory activity) due to the presence of polyphenols and bioactive compounds [17][18][19]. For instance, Mesembryanthemum crystallinum extract displayed potent ACE-inhibitory activity (90.5% at a concentration of 1 mg/mL), presumably due to the polyphenols identified (three flavonoids (apigenin, diosmin, and luteolin), two phenolic acids (p-coumaric and 4-hydroxybenzoic acids), and a hydroxycinnamic acid derivative (2-O-(pcumaroyl)-l-malic acid)) [12]. A similar response in terms of the antihypertensive effect was obtained with Salicornia ramosissima extract, which can also be attributed to its composition in bioactive compounds, mainly p-coumaric, flavonoids (quercetin and apigenin glycosides), and hydroxycinnamic acids (chlorogenic, ferulic, and dicaffeoylquinic acids) [11]. In the control of blood pressure, the enzyme ACE (a dipeptidyl carboxypeptidase) catalyzes the for 60 min at 25 ± 3 • C. Afterward, the samples were centrifuged at 6000× g for 15 min (Sorvall ST16 centrifuge, Thermo Scientific, Osterode, Germany), and the supernatant was collected. The supernatant was evaporated to almost dryness at 40 ± 1 • C under reduced pressure (120 Bar) using a rotavapor (Büchi R-114, Flawil, Switzerland). The residue was dissolved in 2 mL of ethanol:water (50:50, v/v) solution and filtered through a 0.22 mm SFCA membrane (Branchia, Barcelona, Spain), and then the samples were stored at −18 • C until analysis. The extractions were performed in triplicates for each plant.

HPLC-DAD-ESI-MS/MS
Halophyte extracts were analyzed in a high-performance liquid chromatography (HPLC) system (Waters Alliance 2695, Milford, MA, USA), coupled to a diode array (DAD) detector (Detector Waters 2996, Milford, MA, USA), and outfitted with electrospray ionization source (ESI) and a triple quadrupole mass spectrometer (MS/MS) (Micromass, Waters) [11]. The HPLC system contained an autosampler, solvent degasser, quaternary pump, a pre-column (100RP-18, 5 mm), and a reversed-phase C18 column (LiCrospher 100 RP-18, 250 × 4 mm; 5 mm). Additionally, a thermostatic oven at 35 • C was used for the compounds' separation. The mobile phase consisted of water-formic acid (99.5%:0.5%) as eluent A and acetonitrile-formic acid (99.5%:0.5%) as eluent B at a flow rate of 0.30 mL/min. All solvents were filtered through a 0.22 mm PVDF membrane (Millipore, Billerica, MA, USA) before analyses. The gradient elution program was as follows: 0-10 min from 99 to 95% A; 10-30 min from 95 to 82% A; 30-44 min from 82 to 64% A; 44-64 min at 64% A; 64-90 min from 64 to 10% A; 90-100 min at 10% A; 100-101 min from 10 to 95% A; 101-120 min at 95% A; and finally returning to the initial conditions. The auto sampler's temperature was set at 7 • C, and the injection volume was 20 µL. DAD was used to scan the wavelength absorption from 200 to 650 nm. Tandem mass spectrometry (MS/MS) detection was carried out using an electrospray ionization source (ESI) at 120 • C and applying a capillary voltage of 2.5 kV and cone voltage of 30 V. The compounds were ionized in the negative mode and spectra were recorded in the range of m/z 60-1500. Analytical conditions were optimized to maximize the precursor ion signal ([M−H] − ). Ultra-high purity argon (Ar) was used as a collision gas. High-purity nitrogen (N 2 ) was used both as a drying gas and nebulizing gas. The data acquisition and processing were accomplished using MassLynx software (version 4.1, Waters, Milford, MA, EUA).

HPLC-DAD
The quantification of phenolic compounds was carried out using high-performance liquid chromatography (HPLC) (Vanquish system, Thermo Fisher Scientific, Waltham, MA,USA) equipped with a pump, auto-sampler, and coupled a diode array (DAD) detector [11]. An RP-18 pre-column was also used and thermostated at 35 • C, and the chromatographic separation was performed on a Luna C18 reversed phase (Luna 5 µm C18 (2) 100 Å, 250 × 4 mm; Phenomenex) column. DAD detector was programmed to scan wavelength absorption from 200 to 798 nm at a speed of 1 Hz with a bandwidth of 5 nm. The detection of compounds was monitored using the individual channels (280, 320, and 360 nm) at a speed of 10 Hz with a bandwidth of 11 nm. The autosampler's temperature was set at 7 • C, and the injection volume was set at 20 µL. The eluents used were eluent A (water-formic acid (99.5%:0.5%)) and eluent B (acetonitrile-formic acid (99.5%:0.5%)) at a flow rate of 0.30 mL/min. The solvents used in chromatography conditions were filtered through a 0.22 µm PVDF membrane (Millipore, Billerica, MA, USA) prior to analysis. The following gradient program was used: 0-10 min from 99 to 95% A; 10-30 min from 95 to 82% A; 30-44 min from 82 to 64% A; 44-64 min at 64% A; 64-90 min from 64 to 10% A; 90-100 min at 10% A; 100-101 min from 10 to 95% A; 101-120 min at 95% A; and finally returning to the initial conditions. Calibration curves (range from 0.78-100 ppm) for chlorogenic acid, gallic acid, and quercetin-3-hexoside were used for the quantification of the phenolic compounds. The DAD data acquisition was performed using the software Chromeleon version 7.0 (Waltham, MA, USA).

Total Phenolic Content
The extracts were quantified in terms of total phenolic content (TPC) according to Folin-Ciocalteu's colorimetric method [29,30]. Summarily, 230 µL of milli-Q water, 10 µL of the sample, and 15 µL (0.25 N) of Folin-Ciocalteu's reagent were added in the microplate and mixed at room temperature for 3 min. Afterward, 45 µL of sodium carbonate solution (solution 35%) was added, and the microplate was left to rest in the dark, protected from sunlight, at room temperature for 1 h. The absorbance of the samples was measured at 765 nm on a microplate spectrophotometer (Epoch2 Biotek, Winooski, VT, USA) with the Gen5 3.02 data analysis software spectrophotometer. The standard, gallic acid (1000 mg/L), was used for the calibration curve. The results were expressed as gallic acid equivalents per gram of fresh plant (mg GAE/g). The samples were analyzed in triplicates. The ORAC assay was performed using a microplate fluorescent reader (FL800 Bio-Tek Instruments, Winooski, VT, USA) according to Serra et al. [31,32]. This method evaluates the antioxidant capacity of the antioxidant species present in the samples to inhibit the oxidation of fluorescein (3 × 10 −4 mM) catalyzed by peroxyl radicals generated from AAPH. All samples were analyzed in triplicates. Trolox was used as a reference standard, and the results were expressed as Trolox equivalent antioxidant capacity per gram of fresh plant (µmol TEAC/g), Equation (2). Conc.: concentration.

Hydroxyl Radical Scavenging Capacity (HOSC) Assay
The HOSC assay was carried out based on the method of Moore et al. [33], using the microplate fluorescence reader (FL800 Bio-Tek Instruments, Winooski, VT, USA). This assay measured the hydroxyl radical scavenging capacity of a sample using fluorescein (9.96 × 10 −8 M) as a probe and a classic Fenton reaction with FeCl 3 (3.42 mM) and H 2 O 2 (0.20 M) as a source of hydroxyl radicals. Samples were analyzed in triplicates, and results were expressed as Trolox equivalent antioxidant capacity per gram of the fresh plant (µmol TEAC/g), see Equation (2).

Antihypertensive Activity Assay
The antihypertensive activity of the halophyte plants' extracts was evaluated using an angiotensin-converting enzyme (ACE) activity assay kit (Sigma-Aldrich, Saint Louis, MO, USA) [11]. This kit provides a direct and sensitive procedure for measuring ACE levels with the goal of screening for ACE inhibitors on samples, as it is based on the cleavage of a synthetic fluorogenic peptide. Briefly, 10 µL of extract and 40 µL of ACE were added to a 96-well black microplate and incubated at 37 • C for 5 min. The incubation allows contact between the enzyme and the inhibitor. Afterward, 50 µL of the substrate (ACE fluorogenic) was added, and the fluorescence was read every minute for 5 min. A standard curve (0.1 to 0.8 nmol) was used for the quantification of the fluorescent product formed, and the percentage of inhibition was calculated according to the manufacturer's protocol (CS0002, Sigma-Aldrich). A positive control, Lisinopril (Sigma-Aldrich, Darmstadt, Germany), was used during the assay. The IC 50 values that correspond to the needed amount of extract to inhibit 50% of ACE, were calculated. The range of extracts' concentrations tested was between 31.25 and 500 mg/mL.

Statistical Analysis
Descriptive statistical analysis, analysis of variance (ANOVA), and Tukey's test for multiple data comparisons at a significance level of 5% were performed using GraphPad Prism 9.4 software (GraphPad Software, Inc., La Jolla, CA, USA). The pairwise correlation between the analyzed parameters was established based on Pearson's correlation coefficient Antioxidants 2023, 12, 1161 7 of 36 test, at a significance level of 5%. The IC 50 values were determined using non-linear regression (dose-response inhibition) for the antihypertensive assay.
Multivariate analysis by partial least squares-discriminant analysis (PLS-DA) was performed in the Unscrambler X version 10.4, by CAMO software (Oslo, Norway) as a supervised classification technique that maximizes the differences between the samples and allows the correlation between the predictors, quantified parameters, and the response groups established based on the corresponding halophyte species. For data interpretation, the threshold of |0.5| was used to select the most relevant parameters associated with the sample discrimination [34].

Nutritional Characterization
Halophyte plant species are a category of marine vegetables and have a nutritional composition suitable for human consumption due to their content of micro and macronutrients, such as minerals, proteins, fibre, and polyunsaturated fatty acids. The nutritional content of the different studied halophyte species is shown in Table 1.
The increase of water content (succulence) in the tissues of halophytes is an adaptive strategy when exposed to salinity stress and extreme conditions [7,35]. These plants need to regulate their cellular Na + , Cl − , and K + concentrations as they adjust to the external water potential and in the accumulated solutes [36]. The content of salts in vegetative organs of halophytes increases with ageing and might reach toxic levels; therefore, these plants tend to lower such unfavourable salt concentrations by increasing the water content in their tissues (succulence) [37]. According to Table 1, D. crassifolium and M. crystallinum displayed significantly higher moisture content compared to other halophyte species: 96.20 g/100 g FW and 96.30 g/100 g FW, respectively.
The increase of succulence (water content per unit area of the leaf) observed for halophytes makes them more attractive to consumers [7,35]. In this context, the moisture values reported in this study were higher than the ones reported for wild halophytes growing in Portugal, such as S. ramosissima (88.20 g/100 g FW), M. nodiflorum (91.40 g/100 g FW), and Sarcocornia perennis (84.00 g/100 g FW) [7,10,11,38]. The results obtained in this study are according to Castañeda-Loaiza et al. (2020) [7], who reported that cultivated halophytes (S. maritima and S. fruticosa) showed higher values of moisture content than wild plants. This can be related to the water availability in SCS, since these plants are irrigated twice a day, in a process simulating diurnal tidal floods, contributing to the higher moisture content of cultivated plants [7].
Halophytes have a higher ash content than other edible plants due to their ability to retain minerals, which is highly correlated with the saline environment in which they grow and their mechanisms of tolerance to salt stress [39][40][41]. According to Table 1, halophyte species contain a higher ash content and show a higher NaCl content. Among the halophyte species, S. ramosissima (1.50 g/100 g FW; 2.39 g/100 g FW) and S. fruticosa (2.80 g/100 g FW; 4.84 g/100 g FW) displayed higher NaCl and chloride concentrations, respectively. Salicornia sp. and Sarcocornia sp. are highly appreciated in gastronomy due to their salty taste, being recognized as a promising natural ingredient and salt substitute. The significantly greater value of ashes in S. fruticosa (5.70 g/100 g FW) is justified by the significantly higher detected salt content, but also other salts that may be present due to the mineral composition. The opposite is also visible since M. crystallinum contains a significantly lower ash content (1.39 g/100 g FW) and also presents the lowest salt content.
Total ashes, proteins, and salt contents were significantly higher in S. fruticosa and lower in M. crystallinum, while C. maritimum displayed higher total fat, TDF (total dietary fibre), and energy. A higher concentration of proteins was found in S. fruticosa (4.44 g/100 g FW), C. maritimum (3.98 g/100 g FW), and I. crithmoides (3.13 g/100 g FW), while in D. crassifolium (1.28 g/100 g FW) and M. crystallinum (1.27 g/100 g FW), contents were significantly lower. Plant proteins contribute to the down-regulation of insulin and the up-regulation of glucagon, and their intake has been shown to provide protective effects against cardiovascular diseases and cancer [42]. Halophytes have been described as a good source of dietary fibre [7,10,43]. Dietary fibre provides many health benefits, reducing the risk of developing diabetes, obesity, hypertension, and others [44] [45], the claim "source of dietary fibre" may be used for these halophytes when their content in total dietary fibre (TDF) is higher than 3 g/100 g FW. According to this claim, C. maritimum (4.40 g/100 g FW) can be considered a source of dietary fibre. Table 2 shows the fatty acids profile of the halophytes; these plants are sources of essential fatty acids [46][47][48][49][50][51]. Results indicate that polyunsaturated fatty acids (PUFA) are predominant, making up more than 50% of the total fatty acids in all plants, ranging from 52.80% in C. maritimum to 63.10% in S. ramosissima. Palmitic, linoleic, and linolenic acids were the main fatty acids present in halophyte plants. Linoleic and linolenic acids present several physiological functions, such as intervention in blood coagulation and in inflammatory and immunological responses [52]. C. maritimum (28.7%) and I. crithmoides The mineral composition of halophyte plants is summarized in Table 3. Storage and distribution of minerals in halophytes are strongly influenced by environmental conditions (e.g., water and soil characteristics, pollutants, and climate) and can also be affected by some characteristics such as the plant maturation stage and part of the plant under study [5,41,53]. As previously described, S. fruticosa is the halophyte with the highest NaCl content and, consequently, has the highest sodium content (1120 mg/100 g FW) compared to other species. On the other quantified minerals, I. crithmoides and S. fruticosa showed the highest content in calcium and potassium, while S. ramosissima contained the highest content in magnesium, copper, and manganese.
Previous studies support the potential use of halophytes as natural salt substitutes due to their mineral composition, mainly as sources of potassium and magnesium, which contribute to vascular protection [54,55]. Hypertension and cardiovascular diseases in societies are associated with a high sodium content, as well as a low potassium and magnesium in human diets [56,57]. The interdependency of sodium and potassium in the pathogenesis of hypertension indicates that sodium restriction and increased potassium intake are important strategies for the primary prevention and treatment of hypertension and its cardiovascular consequences [58]. Furthermore, the risk factors for hypertension include, but are not limited to, age, race, family history, obesity, physical inactivity, and tobacco use [59]. In general, the nutritional parameters of halophyte species determined in this study are in accordance with data already reported in the literature [7,11,38,43,60,61].

Potentially Toxic Elements
Estuarine environments are extremely affected by anthropogenic-driven contamination, namely, potentially toxic metallic elements metal [53,62]. However, the use of the soilless cultivation system (SCS) allows for preventing the presence of contamination by toxic metals. Thus, the halophytes displayed lead and cadmium levels (values converted from dried weight to fresh weight according to the moisture content of each species) below 0.3 and 0.2 mg/kg FW, which is notably the maximum content allowed by Regulation (EU) number 1881/2006 for leaf vegetables [63]. Equally, arsenic levels (values were converted from dried weight to fresh weight according to the moisture content of each species) in plants are lower than the maximum levels allowed in certain foods by Commission Regulation (EU) number 2015/1006 (0.2 mg/kg FW) [64]. Hg was below the detection limit of the method of analysis in the plants analyzed (Table 4). Results are expressed as mean values ± standard deviation (n = 3). 1 Calculated: salt (g) = sodium (g) × 2.5. The letters (a-f) denote significant differences between the halophytes using Tukey's test (p < 0.05). For all halophytes the phenotypic stage was green. SFA-total saturated fatty acids; MUFA-total monounsaturated fatty acids; and PUFA-total polyunsaturated fatty acids. Results are expressed in percentages of total methyl esters ± standard deviation (n = 2).* LOQ < 0.05 g/100 g. The letters (a-g) denote the significant differences between the halophytes using Tukey's test (p < 0.05).  Results are expressed as means values ± standard deviation (n = 3). * LOD: limit of detection µg/g. The letters (a-e) correspond to the significant difference between halophytes using Tukey's test (p < 0.05).

Phytochemical Characterization and Bioactivity
As shown in Table 5, from all the halophyte plants, S. ramosissima and S. fruticosa stood out for their highest total phenolic content (TPC), 0.41 mg GAE/g FW and 0.33 mg GAE/g FW, respectively. Nevertheless, the value obtained in the studied S. ramosissima plants was clearly below the described value for the same species in previous works with plants cropped in 2019 (1.02 ± 0.04 mg GAE/g FW) [11]. The difference could be attributed to the impact of the cultivation system and environmental conditions on the plants' phenolic composition. Regarding the S. fruticosa plant, the TPC value of 0.33 mg GAE/g FW (2.47 mg GAE/g DW), was slightly lower than the values described for the Sarcocornia plants collected in the southwestern Iberian Peninsula (3.231-3.892 mg GAE/g DW) [65]. By opposition, M. nodiflorum, M. crystallinum, and D. crassifolium were the species with the lowest TPC values (0.098 to 0.11 mg GAE/g FW). For M. nodiflorum and M. crystallinum, the obtained TPC results (1.533 and 2.871 mg GAE/g DW, respectively) were higher than the ones described for the same species collected in south Tunisia (1.72 mg/g DW and 1.43 mg/g DW, respectively) [66]. Factors such as the variability in the halophyte species, the cultivation conditions, the cropping year, and the extraction conditions contributed to impairing the comparison of the results obtained and the ones described in the literature.
Following the same trend of the total phenolic content, the in vitro antioxidant activity, measured by ORAC and HOSC assays, expressed by fresh matter, reaches the highest values in S. ramosissima and S. fruticosa. However, the lowest values in M. nodiflorum, M. crystallinum, and D. crassifolium, suggesting the relevance of the halophytes' total phenolic content for the peroxyl and hydroxyl radicals' inhibition (R 2 = 0.829 and R 2 = 0.937; Figure S1-Supplementary Materials) and consequent prevention of the oxidative damage in cellular biomolecules.
Regarding the ACE-inhibitory activity, among the halophyte species evaluated, M. nodiflorum, M. crystallinum, I. crithmoides, S. fruticosa, and S. ramosissima showed the highest antihypertensive activity, requiring a lower extract concentration to inhibit an angiotensin-converting enzyme (ACE), and C. maritimum showed the lowest antihypertensive activity, showing a higher extract concentration to inhibit ACE. The weak correlation between the total phenolic content and the antihypertensive activity (R 2 = 0.0186) indicated that in the phenolic extracts obtained from the halophytes' specific phenolic compounds had a low impact on this inhibition [67]. However, the presence of other components in phenolic extracts, such as the ACE-inhibitory peptides (low molecular weight peptides), could contribute to the plant's antihypertensive activity [68,69].

Composition in Individual Phenolic Compounds
In the halophytes, the tentatively identified phenolic compounds were classified into different classes of phenolic acids (hydroxycinnamic and hydroxybenzoic acids), flavonoids (flavones, flavanals, flavonols, and flavanones), and hydrolysable tannins (gallotannins). These compounds exert potential antioxidant, anti-inflammatory, anti-carcinogenic [70,71], anti-microbial [72], and inhibitory activity in key enzymes associated with, neurodegenerative diseases, e.g., Alzheimer's disease (e.g., acetylcholinesterase, AChE and butyrylcolinesterase, and BuChE), diabetes mellitus type 2 (α-amylase and α-glucosidase), and skin hyperpigmentation/food oxidation (tyrosinase) [73]. The qualitative and quantitative abundance of phenolic compounds in the halophytes is dependent on several factors, such as the environmental growth conditions and the analyzed species. Despite the existent amount of collected information in the plant database for the halophyte and salt-tolerant plants [74], the data regarding their phenolic composition remains limited, which compromises qualitative and quantitative data comparison. The study herein aims to fill the gap of knowledge, describing the phenolic composition of different halophyte species grown under the same soilless cultivation conditions.
In the halophytes studied herein, 46 of the 94 tentatively identified compounds were hydroxycinnamic acids [75], mostly derivatives of caffeic acid ( Figure A1A Table A1-Appendix A. The presence of these hydroxycinnamic acid derivatives was previously reported in S. ramosisssima [76], in conventional and microwave S. ramosissima extracts [77], as well as in other halophyte plants such as Glaux maritima roots [78], and in C. maritimum [79], as a survival molecular adaptation to saline soil conditions, as described by Pungin et al. [78]. As shown in Table A1, there was high qualitative diversity in the phenolic composition of the different studied halophyte plants, with some compounds, such as the ferulic acid derivatives being mostly detected in the S. ramosissima, D. crassifolium, I. crithmoides, M. nodiflorum, and M. crystallinum, but not in the S. fruticosa and C. maritimum species. Considering the quantified amounts of hydroxycinnamic acids in the different halophyte species, Table 6 and Table S1-Supplementary Materials, S. ramosissima stood out as the species with the highest content of hydroxycinnamic acids, namely, caffeic acid derivatives, such as 3,4-, 3,5-, and 4,5-dicaffeoylquinic acids (compounds 61, 67, and 74) and caffeoylhydrocaffeoyl quinic acid (compound 80). These compounds were also described in S. europaea extracts [80], inhibiting the accumulation of cholesteryl ester hydroperoxide (CE-OOH) and therefore the blood plasma oxidation and the atherosclerotic plaques [81]. C. maritimum is highlighted from the remaining species as the one with the highest chlorogenic acid content (compound 15) [82], Table S1-Supplementary Materials. In the same way, S. fruticosa had the highest content of chlorogenic acid (compound 15) content, and a similar amount (13.661 µg/g FW) was reported for Sarcocornia perennis alpini (12.240 µg/g FW) [83]. In addition to the high chlorogenic acid content, C. maritimum plants were an important source of p-coumaroylquinic acid isomeric forms, compounds 38 and 41, 78.310 µg/g FW and 43.242 µg/g FW, respectively, which is in line with Siracusa et al. [84]. The compound p-coumaroylquinic acid was also present with a high content in S. fruticosa and S. ramosissima plants (11.764 µg/g FW and 3.644 µg/g FW, respectively). Diverse p-coumaric acid derivatives were found with considerably high contents among the analyzed halophytes (compounds 12 and 34 in C. maritimum; compounds 44 and 23 in S. fruticosa; compounds 88 in D. crassifolium; and compounds 54 and 56 in M. crystallinum). The non-conjugated form of p-coumaric acid (compound 13) was quantified in I. crithmoides and S. ramosissima plants in a relatively lower content than the p-coumaric acid conjugated forms. The presence of p-coumaric acid and its derivatives in halophyte plants, such as M. crystallinum, have been related to the in vitro and in vivo antioxidant metal-ion scavenging properties of these plants [12]. M. nodiflorum was the species with the lowest amount of hydroxycinnamic acids.  Results are expressed as mean ± standard deviation (n = 2), µg/g of fresh weight (FW). The hydroxycinnamic acids (HCA) and their derivatives were quantified as a 3-O-caffeoylquinic acid equivalent (CQAE), flavonoids were quantified as quercetin-3-glucoside equivalent (QGE), and hydroxybenzoic acids (HBA) were quantified as a gallic acid equivalent (GAE). The letters (a-g) indicate significant differences between the halophytes using Tukey's test (p < 0.05). ND: not detected; LOQ = 0.01 µg compound/g FW.
Regarding the hydroxybenzoic acids, based on the respective fragmentation mass pattern, in the studied halophyte plants, the main compounds were tentatively identified as derivatives of protocatechuic acid (m/z 153), gallic acid (m/z 169), and syringic acid (m/z 197), Figure A2-Appendix A. As described previously for the hydroxycinnamic acids, in the halophyte species, there was also a huge qualitative diversity in the hydroxybenzoic acids composition. Unlike most halophytes, in the D. crassifolium and M. crystallinum species, none of the studied hydroxybenzoic acids were detected. The lower content of D. crassifolium and M. crystallinum species in salt showed a positive association with the absence of hydroxybenzoic acids in these plants. As described by Pungin et al. [78] and Qasim et al. [85], despite the plant's adaptive variability in salt environmental conditions, the phenolic compounds, particularly the phenolic acids, accumulate in the halophytes as a protective response against the oxidate stress imposed by the soil salinization; therefore, in the plants with lower salt content a lower phenolic content is expected. From a quantitative point of view, S.ramosissima showed the highest content of hydroxybenzoic acids, which is particularly thanks to the protocatechuic acid arabinoside (compound 9) content, Table 6 and Table S1-Supplementary Materials. This compound was also detected in S. fruticosa in a lower amount. In C. maritimum, the protocatechuic acid esterified to a glycoside moiety was the most abundant hydroxybenzoic acid compound. The quantification of the aglycone form of protocatechuic acid was made previously by Pungin et al. [78] for other halophytes, namely, Spergularia marina (L.) and Glaux maritima.
In the studied halophyte plants, 35 compounds were classified as flavonoids. The most diverse flavonoids ( Figures A3 and A4-Appendix A) belong to the flavones and flavonols classes [86], which included 13 and 10 different compounds, respectively. The flavanol class included five tentatively identified compounds, and the remaining classes of flavanones and flavanonols comprised, respectively, six and one compounds, Table A1.  compounds 25 and 60), and in M. crystallinum, from the different flavones, the chrysin derivative (compound 50) stood out by its abundance. This last result contradicts the higher relative abundance of apigenin, diosmin, and luteolin described by Calvo et al. [12] for M. crystallinum plants collected from the northwest coast of Spain (Galicia). Contrarily to most of the halophyte species, in S. fruticosa and S. ramosissima no flavones were detected.
The flavanols, derived from catechin isomer, with trans configuration, and epicatechin isomer, [M−H] − at m/z 289, with cis configuration, were mostly found in their conjugated forms with galloyl, epigalloyl, and water residues. From the different studied halophytes, S. fruticosa was characterized by the highest abundance of the epicatechin derivative (compound 55), and M. nodiflorum was characterized by the highest abundance of epicatechin hydrate (compound 51) and gallocatechin (compound 10). The presence of epicatechin was previously reported in S. fruticosa and M. nodiflorum plants [7], as well as in other halophyte species, such as Spergularia marina L. and Glaux maritima L. [78]. Contrarywise, in C. maritimum, no flavanols were detected.
The The pinobanksin derivatives (compounds 16, 31, and 92) were identified for the first time in the studied halophyte plants. However, the presence of pinobanksin in halophytes was previously described in the propolis produced from Zuccagnia punctata, Larrea divaricata, and Larrea cuneifolia halophytic shrubs, present in the vegetation of the Monte region in Argentina [88]. The presence of eriodictyol hexoside and pinocembrin was previously reported by Kramberger et al. [89], in the halophyte Helichrysum italicum.
In the flavanonol class, only dihydroquercetin, also known as taxifolin (m/z 303), compound 36, was detected. S. fruticosa species was the only one with quantifiable amounts of this compound. This compound was recently associated with positive effects in nitric oxide production and endothelial dysfunction, contributing to a vasorelaxant effect and cardiovascular protection [90]. Compounds 28, 57, and 79, not classified in the previous classes, were less abundant in the plants and present in D. crassifolium, I. crithmoides, and M. nodiflorum, respectively. Nevertheless, the positive impact of these compounds on human health has been extensively described [91][92][93].

Integrating the Nutritional, Phytochemical, and Bioactivity Parameters
The diversity of the halophyte species, regarding the nutritional, phytochemical (total contents), and bioactivity parameters, can be summarized through multivariate analysis, namely, by partial least square-discriminant analysis (Figures 1 and 2). For data systematization, the phenolic compounds were classified into different families. The ones only quantified in one species were excluded from the multivariate analysis. Concerning the nutritional and the phytochemical composition, as shown in Figure 1, the most different species, occupying an external position on the score plot, were S. fruticosa, S. ramosissima, and M. crystallinum.
Based on the established correlations between the analyzed parameters and the defined groups (Figure 2), along with the first factor, the position of the S. fruticosa in the left-extreme corner of the plot was explained by its lowest moisture content and highest content in protein, fibre, ash, salt, chlorides, minerals (Na, K, Fe, Mg, Mn, Zn, Cu), TPC, flavanol, flavonol, and antioxidant activity parameters (ORAC and HOSC assays). These last in vitro antioxidant parameters, ORAC and HOSC, were highly correlated to the TPC value (Pearson's R of 0.945 and 0.968, respectively, Table S1 Table  S1-Supplementary Materials. S. ramosissima was characterized by a high abundance of the hydroxybenzoic acid C9; hydroxycinnamic acids C22, C40, C61, C67, C74, and C80; and the flavonol C82. In D. crassifolium, the hydroxycinnamic acids C13, C20, C47, C86, C88, C89, C90, and C93, the flavonoids C58, C64 (flavones), C73 (flavonol), and C14 (flavanol), and the coumarin C28 stood out as the most abundant compounds. Finally, in M. crystallinum, the compounds with higher abundance were the hydroxycinnamic acids C54, C56, and C62; flavones C46 and C50; flavanone C16; and the flavonols C35, C70, and C83. Figure 1. Partial least square-discriminant analysis (PLS-DA) score plot of the halophyte species distribution (IC, Inula crithmoides; SF, Sarcocornia fruticosa; SR, Salicornia ramosisssima; DC, Disphyma crassifolium; CM, Crithmum maritimum; MN, Mesembryanthemum nodiflorum; MC, Mesembryanthemum crystallinum) in a space defined by the two first factors. In the model, the average values for the nutritional and phytochemical parameters, determined for the analytical triplicates, were considered as predictor variables (accumulated variance of 67%) and the groups, defined by the halophyte's specie, as the response variable, with an accumulated variance of 34%. In the model, the inner circle indicates correlation loadings lower than |0.5| and the space between the inner and the outer circles the correlation loadings higher than |0.5| and lower than |1.0|.

Volatile Compounds Profile
The volatile profile of halophytes was studied by SPME-GC-MS, and the putative identification of their volatile compounds was carried out by comparing the mass spectra with the mass spectra bank from libraries and the Linear Retention Index (LRI). Compounds were classified by their chemical classes and odor descriptions according to the literature. In order to compare the volatile profile among species, the percentage of peak area was measured in relation to the total area of the chromatogram (Supplementary Materials, Table S2).

Conclusions
The halophyte species (I. crithmoides, S. fruticosa, S. ramosissima, D. crassifolium, C. maritimum, M. nodiflorum, and M. crystallinum) can be considered alternatives to the traditional table salt, with advantages due to their added nutritional and phytochemical values. When used as natural salt alternatives, these plants contribute with a salty taste due to their mineral composition, mainly as sources of sodium, potassium, and magnesium. This study showed that there are significant differences between species. Under the same soilless cultivation conditions, S. fruticosa contained a higher content of protein, ash, salt, chlorides, minerals (Na, K, Fe, Mg, Mn, Zn, Cu), and flavonoids. In contrast, S. ramosissima presented a higher content of phenolic acids and polyunsaturated fatty acids. Considering the phenolic profile of the plants, S. fruticosa and M. nodiflorum showed the most distinct profiles, with a relatively higher abundance of hydroxycinnamic acids in S. fruticosa, and a relatively higher abundance of flavonoids in M. nodiflorum and S. ramosisssima showed the highest content of hydroxycinnamic and hydroxybenzoic acids. The most abundant flavonoids class, especially in S. fruticosa, was the flavonols, namely, the compound tentatively identified, for the first time in this species, as rhamnetin hexosyl pentoside. The phytochemical diversity of the studied halophytes clearly indicates the advantages of consuming these species in the diet as a strategy to enhance the diet's nutraceutical value and, consequently, their antioxidant activity and antihypertensive effect. Future work is mandatory in order to increment the knowledge about these halophytes and the relationship between their bioactive composition and possible health effects.
In fact, there was a qualitative diversity of phenolic compounds  identified in halophytes, as shown in Table A1 (Appendix A). While, for the first time, the profile of vol-atile compounds  was studied (Table S2-Supplementary Materials).

Conclusions
The halophyte species (I. crithmoides, S. fruticosa, S. ramosissima, D. crassifolium, C. maritimum, M. nodiflorum, and M. crystallinum) can be considered alternatives to the traditional table salt, with advantages due to their added nutritional and phytochemical values. When used as natural salt alternatives, these plants contribute with a salty taste due to their mineral composition, mainly as sources of sodium, potassium, and magnesium. This study showed that there are significant differences between species. Under the same soilless cultivation conditions, S. fruticosa contained a higher content of protein, ash, salt, chlorides, minerals (Na, K, Fe, Mg, Mn, Zn, Cu), and flavonoids. In contrast, S. ramosissima presented a higher content of phenolic acids and polyunsaturated fatty acids. Considering the phenolic profile of the plants, S. fruticosa and M. nodiflorum showed the most distinct profiles, with a relatively higher abundance of hydroxycinnamic acids in S. fruticosa, and a relatively higher abundance of flavonoids in M. nodiflorum and S. ramosisssima showed the highest content of hydroxycinnamic and hydroxybenzoic acids. The most abundant flavonoids class, especially in S. fruticosa, was the flavonols, namely, the compound tentatively identified, for the first time in this species, as rhamnetin hexosyl pentoside. The phytochemical diversity of the studied halophytes clearly indicates the advantages of consuming these species in the diet as a strategy to enhance the diet's nutraceutical value and, consequently, their antioxidant activity and anti-hypertensive effect. Future work is mandatory in order to increment the knowledge about these halophytes and the relationship between their bioactive composition and possible health effects.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox12061161/s1, Figure S1: Correlation matrix (Pearson's R values from −1 to 1) between the phenolic composition and the bioactivity measured by in vitro antioxidant and anti-hypertensive activities. ACE inhibition expressed as inverse values for the antihypertensive activity. Table S1: Individual phenolic compounds quantified in the different halophyte species. Results are expressed as mean ± standard deviation (n = 2), µg/g of fresh weight (FW). The hydroxycinnamic acids (HCA) and their derivatives were quantified as a 3-O-caffeoylquinic acid equivalent (CQAE), flavonoids were quantified as quercetin-3-glucoside equivalent (QGE), and hydroxybenzoic acids (HBA) were quantified as a gallic acid equivalent (GAE). Table S2: References           Table A1. Figure A3. Molecular structure of some flavonoids derived from the flavonols quercetin, m/z 301; kaempferol, m/z 285; isorhamnetin-glucoside, m/z 477 and flavanone pinocembrin, m/z 255, identified in the halophyte species. The tentative identification of the remaining compound is detailed in Table A1.  Table A1.  Table A1.