Bioactive Compounds in Sarcocornia and Arthrocnemum, Two Wild Halophilic Genera from the Iberian Peninsula

(1) Background: this study describes bioactive compounds in the following halophytes: Sarcocornia (S. alpini, S. pruinosa, and S. perennis) and Arthrocnemum (A. macrostachyum). The material comes from: coastal marshes in Tinto River, Guadiana River, and some interior provinces from the Iberian Peninsula. (2) Methods: the techniques used were Folin–Ciocalteu, GC-MS, and ESI-MS/MS. (3) Results: Five phenolic acids were found in Sarcocornia: trans-cinnamic, salicylic, veratric, coumaric, and caffeic acids. In addition, in Arthronemum, ferulic acid was also detected. The obtained flavonoids were cyanidin-3-O-arabinoside, luteolin-7-glucoside, dihydroquercetin, and p-coumaroyl-glucoside. They also presented fatty acids, such as palmitic, linoleic, and oleic acids in Sarcocornia, while palmitic, linolenic, and stearic acids were the main fatty acids in A. macrostachyum. (4) Conclusions: the high diversity of the compounds identified confirms the relation between nutritional interest and salt tolerance in halophytes.

The Chenopodiaceae species are generally characterized by a high content of minerals, polyphenols, and fatty acids, among other compounds of interest. The abundance of inorganic elements (Na + , K + , Mg 2+ , and Ca 2+ , among others) in the tissues of these plants, together with the wide diversity of bioactive compounds, have been related to their capacity to survive and grow in extreme environments with high salinity and long periods of intense drought [7][8][9][10].
Arthrocnemum macrostachyum has recently been used in soil desalination programs [11] due to its capacity to accumulate high concentrations of sodium chloride in its tissues and hence to reduce it in the cultivation medium. El-Naker et al. [12] recorded the presence of a wide range of phytochemical compounds in this genus and identified sixteen that were potentially bioactive, some of which have antioxidant (quercetin, 4-hydroxybenzoic, and caffeic acids), antiviral, antibacterial, and/or anti-tumoral properties (hesperidin, salicylic, chlorogenic, and coumaric acids), including compounds for the treatment of diabetes (rhamnetin).
Several species of Sarcocornia have been evaluated as edible plants due to their different nutritional properties, particularly including their antioxidant capacity and lipid composition. Riquelme et al. [13] characterized different phenolic compounds in Sarcocornia

Total Phenolic Compounds (TPC) and Phenolic Acids
Our values for the total phenolic compounds in the genus Sarcocornia expressed as gallic acid equivalent (G.A.E.) were between 3.892 mg G.A.E./g plant dw (dry weight) and 3.231 mg G.A.E./g plant dw ( Table 1). The phenolic acids found in the species of Sarcocornia and Arthrocnemum were benzoic acids (salicylic and veratric) and hydroxycinnamic acids (trans-cinnamic, caffeic, coumaric, and ferulic). All the material from the genus Sarcocornia presented trans-cinnamic acid, which is the most frequent and abundant compound (Table S1, Figures 1-3). Table 1. Data on the sample weight and total phenolic compounds (TPC ± SD (mg G.A.E./g plant dw)) for dry material and humidity. Note: gallic acid equivalent (G.A.E.) and standard deviation (SD). n = 3.

Flavonoids and Hydroxycinnamic Acids
All the samples of Sarcocornia and Arthrocnemum studied contained luteolin and this was the only flavonoid present in S. perennis. Cyanidin-3-O-arabinoside and luteolin-7-       Sample 1 of S. alpini presented veratric acid and trans-cinnamic acid with 39% of relative content, while coumaric and caffeic acid accounted for around 10%. For S. alpini material corresponding to samples 2, 3, and 4, only trans-cinnamic acid was detected between 96% and 98%.
All the material of S. perennis presented trans-cinnamic acid between 64% and 67%, and salicylic acid between 30% and 32%.
The total phenolic compounds in A. macrostachyum were between 4.891 mg G.A.E./g plant dw and 4.220 mg G.A.E./g plant dw.

Flavonoids and Hydroxycinnamic Acids
All the samples of Sarcocornia and Arthrocnemum studied contained luteolin and this was the only flavonoid present in S. perennis. Cyanidin-3-O-arabinoside and luteolin-7-glucoside ( Table 2 and Table S3) were found in S. alpini, while S. pruinosa contained dihydroquercetin and p-Coumaroyl tyrosine. A. macrostachyum contained dihydroquercetin and p-Coumaroyl-glucoside. The chemical structures of these compounds are shown in Figure S1.

Fatty Acids
Our results show that the total proportion of saturated fatty acids in the genus Sarcocornia represented a mean of 61.5% relative percentage, with lower proportions of monounsaturated fatty acids at 2.7% (Table S2, Figures 2-4). Polyunsaturated fatty acids were at 19.20% relative percentage.
In the genus Arthrocnemum, saturated fatty acids represented a mean of 65.2% relative percentage, monounsaturated fatty acids accounted for 7.8% relative percentage, and polyunsaturated fatty acids were 24.1% relative percentage.
Among the saturated fatty acids, the palmitic acid was notable, which was present between 30% and 20% relative percentage in all samples of both Sarcocornia and Arthrocnemum; this was also the case for stearic acid but with lower percentages between 20% and 10% relative percentage.
Lauric and myristic acids were found only in material from the genus Sarcocornia. Lauric acid was notable in S. pruinosa, S. alpini, and S. perennis, with a relative content of between 15% and 8% relative percentage. Myristic acid was detected in S. alpini and S. perennis with a content of over 10% relative percentage.

Total Phenolic Compounds (TPC)
Our data were within a range of 3.231 to 4.803 mg G.A.E./g plant dw in 20 samples from different populations of Sarcocornia and Arthrocnemum in the southwest and interior region of the Iberian Peninsula.
Other authors in localities in Portugal have reported values of 20 mg G.A.E./g plant dw for S. alpini, of S. perennis in populations in Castro Marim, and of 49 mg G.A.E./g plant dw for A. macrostachyum collected in Faro, notably both localities in Portugal [8].
This difference can be due to several factors related to the culture conditions of the fresh plant, including the salt stress conditions and environmental changes. Halophytes live in extremely harsh environments with high salinities and UV radiation, and these stressful conditions lead to the production of secondary metabolites such as the phenolic compounds in different concentrations [25]. The mere fact of detecting these total phenolic compounds emphasizes the antioxidant capacity of the halophytes in the study [26][27][28] and demonstrates that Sarcocornia and Arthrocnemum have a potential food use.

Phenolic Acids
Trans-cinnamic acid was significant in the species of the genus Sarcocornia, analyzed particularly in S. alpini and S. perennis, which were collected in the estuary of the Tinto River and in the mouth of the Guadiana River.
Trans-cinnamic acid was very scarce in Arthrocnemum macrostachyum, with a proportion of a little over 10%; the abundance of this acid differs between these genera.
Trans-cinnamic acid reduces adipogenesis and lipogenesis, emphasizing its potential for treating obesity [29]. Among monounsaturated fatty acids, the study material presented oleic acid with values between 20% and 10%, particularly in S. alpini and S. perennis.
Linoleic acid was the predominant polyunsaturated fatty acid in the genus Sarcocornia, with values between 24% and 18% in S. alpini, 17% in S. pruinosa, and between 21% and 17% in S. perennis. This acid decreased to a relative content of 10% in A. macrostachyum.
Finally, linolenic acid was present only in S. perennis between 22% and 17%, and in A. macrostachyum with a relative content of between 16% and 10%.
Arachidonic and behenic acids were found in all the samples of A. macrostachyum and S. alpini, with values ranging from 15% to 5%, whereas in S. pruinosa and S. perennis, the content varied between the different samples.
Lignoceric acid was identified in the samples of S. pruinosa and S. perennis from the estuary of the Tinto River (samples 6, 8, 11, 13, and 14), but was absent in S. alpini. This fatty acid was found in all the material of A. macrostachyum in proportions of between 11% and 9% relative percentage. This difference can be due to several factors related to the culture conditions of the fresh plant, including the salt stress conditions and environmental changes. Halophytes live in extremely harsh environments with high salinities and UV radiation, and these stressful conditions lead to the production of secondary metabolites such as the phenolic compounds in different concentrations [25]. The mere fact of detecting these total phenolic compounds emphasizes the antioxidant capacity of the halophytes in the study [26][27][28] and demonstrates that Sarcocornia and Arthrocnemum have a potential food use.

Phenolic Acids
Trans-cinnamic acid was significant in the species of the genus Sarcocornia, analyzed particularly in S. alpini and S. perennis, which were collected in the estuary of the Tinto River and in the mouth of the Guadiana River.
Trans-cinnamic acid was very scarce in Arthrocnemum macrostachyum, with a proportion of a little over 10%; the abundance of this acid differs between these genera.
Salicylic acid was the predominant phenolic acid in S. pruinosa with over 55%; however, its content ranged between 30% and 25% in S. perennis. Salicylic acid accounted for over 60% of the relative content in the material of A. macrostachyum from the Tinto River. This acid is involved in regulating plants' response to drought through the genetic expression of the genes PR1 and PR2. The induction of these genes increases the accumulation of salicylic acid as a protection mechanism at times of water stress [30].
Veratric acid was significant in the samples that were not affected by the influence of the tides and occupied drier environments in the salt marshes. This was the case of the populations of S. alpini and S. pruinosa in the southwest of the Iberian Peninsula, and the populations of A. macrostachyum in the interior and southwest. Veratric acid has antibacterial, anti-inflammatory, and anti-hypertensive activities [31].
A slightly greater diversity of phenolic acids has been shown more in A. macrostachyum than in species of the genus Sarcocornia. There is a clear difference in the content of coumaric acid in these genera within the Iberian Peninsula. Five out of fifteen samples of the genus Sarcocornia (samples 1, 5, 7, 9, and 10) were identified as having a relative content between 10% and 4%, while this acid was detected in the six samples analyzed of Arthrocnemum, with content ranging between 20% and 10%. This acid has been described in Salicornia patula Duval-Jouve on the Iberian Peninsula, where it was determined to be infrequent, as it was found in only two samples of the thirteen evaluated [10].
Caffeic acid is the lowest phenolic acid in the Sarcocornia material, as indicated by other authors such as Bertin et al. [32] and Costa et al. [33], with data of 0.402 mg/g in S. ambigua from Brazil. This acid thickens the plant cell walls and increases resistance to the ionic toxicity of sodium and heavy metal stress [34], suggesting that the presence of caffeic acid in these halophytes may allow them to adapt to highly saline environments.
Ferulic acid was only identified in A. macrostachyum collected in the localities of La Rábida and Belchite, with contents of between 59% and 56%. This phenolic acid has been described in S. ambigua [33] and Salicornia europaea L. [35]. Deng et al. (2015) observed a positive correlation between the ferulic acid content in the cuticle of Limonium bicolor (Bunge) Kuntze and the speed of sodium secretion, suggesting that ferulic acid is directly involved in the secretion of salt through saline glands [36]. These glands have not been described in species of Arthrocnemum and Sarcocornia [3,37]; however, the detection of ferulic acid in two of our populations of Arthrocnemum may point to its implication in certain mechanisms of tolerance to salinity.
Additionally, it has been proven that plants exposed to environments with heavy metals produce a high diversity of secondary metabolites, such as phenolic acids [38]. In our study, the diversity of phenolic acids found seems to correspond more closely with the plant species used (S. alpini, S. pruinosa, S. perennis, and A. macrostachyum) rather than with the influence of a medium with a high content of heavy metals, such as the Tinto River. However, the subject really deserves a detailed study in this regard, especially concerning wild plants that grow under the influence of the Tinto River in the province of Huelva.

Flavonoids and Hydroxycinnamic Acids
All the samples of Sarcocornia presented luteolin, which was previously identified in other Salicornioideae such as S. europaea [39].
Most flavonoids are present in plants in the form of esters, glucosides, and polymers. The chemical structure of these flavonoids determines their range of intestinal absorption and confers their beneficial uses for halophytes as edible plants. Glycosylation guarantees selective absorption and endows these compounds with prebiotic actions [40]. The species of Sarcocornia studied, namely S. pruinosa and S. alpini, also contained a glycosylated flavonoid with greater molecular weight (cyanidin-3-O-arabinoside, luteolin-7-glucoside, and dihydroquercetin). p-Coumaroyl glucose was found in Arthrocnemum. The presence of these compounds could be explained by the fact that halophytes increase their antioxidant requirements as a defense against extreme environments, forming macromolecular antioxidants [41]. The detection of these compounds also highlights the value of their use as edible plants.
In addition, apigenin-7-glucoside or rutin were identified in the 20 samples of the analyzed genera Sarcocornia and Arthrocnemum from material from the Iberian salt marshes; these two antioxidant compounds were identified in the genus Salicornia and, in the case of rutin, are associated to its tolerance of salinity [10].

Fatty Acids
Fatty acid composition affects the ability to tolerate salt stress [42,43]. Ten different fatty acids were found in the samples from the genus Sarcocornia and eight in Arthrocnemum. These included saturated, monounsaturated, and polyunsaturated fatty acids that provide halophytes an adaptive advantage, as they prevent the oxidative damage caused by the saline stress that is habitual in these environments [24].
Our results show that the saturated fatty acid present in the highest proportion in all species of Sarcocornia and A. macrostachyum was palmitic acid, which may account for over 90%. Values of 20% were found in other species of Sarcocornia, such as S. ambigua [44] and in Arthrocnemum from Tunisia, with a content of between 19% and 11% [45]. Stearic acid was another important acid that was present in all the halophyte samples studied, with values ranging from 19% to 5%. Other authors have reported similar results between 18% and 12% in Sarcocornia from Alcochete in Portugal [46]. Custodio et al. [47] identified 6% stearic acid in A. macrostachyum, which was also collected in Faro, Portugal. These bioactive compounds prevent the development of cardiovascular disorders, reduce insulin resistance, and strengthen the immune system [48].
No palmitoleic acid was found in the material from Sarcocornia and Arthrocnemum in our study. This monounsaturated fatty acid has been identified by other authors in S. perennis and S. alpini from Portugal, with values of between 21% and 17% [8]. These authors describe a content of between 6% and 4%, while Custodio et al. [47] reported values of between 11% and 4% in A. macrostachyum from Portugal.
There was a notable content of polyunsaturated fatty acids, specifically linoleic and linolenic acid. in S. perennis and A. macrostachyum, with a content of between 22% and 7%, which was much higher than the value described by Barreira et al. [8] in S. perennis, namely between 2% and 0.81%. This group of fatty acids have been considered the most important compounds against saline stress and their action has been proposed as an antioxidant [49]. Polyunsaturated fatty acids are bioactive compounds with antifungal activity, in addition to inhibiting carcinogenesis and the progression of atherosclerosis [50].
Long-chain fatty acids, such as arachidonic, behenic, and lignoceric acid, have content values of 17% in the species studied from the genus Sarcocornia; these values are higher than those published by Barreira et al. [8], who reported data between 11% and 4% for S. perennis and S. alpini, for populations from Portugal.
In A. macrostachyum, long-chain fatty acids had a relative content of over 15%, notably higher than the values published by Barreira et al. [8] and Custodio et al. [47] for the same species in Portugal.

Nutritional Importance and Future Implications
Halophytic plants of the Salicornioideae subfamily are known as "sea asparagus", "glasswort", "samphire", and "pickleweed" [51,52]. The plants most consumed as gourmet foods are those annual species of the genus Salicornia, especially those named under S. europaea, which may include other species given the taxonomic complexity of this group [53]. In fact, the difficulty of distinguishing between these types of plants has led many European markets and restaurants to use these halophytes as a mixture of several species, both annual and perennial [18,51].
Perennial halophytes, such as some species of the genera Sarcocornia and Arthrocnemum (evolutionarily close to Salicornia), have also shown to possess interesting nutritional properties for consumption [8,14,19]. Sarcocornia and Arthrocnemum produce succulent shoots which can be used for food as green leafy vegetables, as fresh ingredients for salads, and for spicing or substituting salt considering their great sodium amounts [48].
In our study, we analyzed the phenolic compounds and fatty acids of perennial plants of the genera Sarcocornia (S. alpini, S. perennis and S. pruinosa) and Arthrocnemum (A. macrostachyum), reaffirming that they are halophytes that also present properties with authentic nutritional potential for its consumption (potential foods with antioxidant properties, contribution of essential fatty acids for the human diet, etc.). The selective introduction of these underused species in markets and in traditional and healthy cuisine represents a future challenge to be implemented.

Materials
The material was collected in the southwest of the Iberian Peninsula in several localities in the Tinto River basin, such as La Rábida, San Juan del Puerto, and the river estuary. Samples were also collected in localities in the mouth of the Guadiana River (Ayamonte and Castro Marim) and in other points of southeast Portugal (Tavira and Santa Luzia). Salt marshes in Madrid and Zaragoza were selected from the interior of the Iberian Peninsula. Table 3 shows the data for each of the species studied and fresh plants were collected as follows: Upon reception, a portion of fresh plants was stored in airtight plastic bags (anaerogen TM 3.5 L, Thermo Scientific, Waltham, MA, USA) for one day until its analyses were performed. Then all material were dehydrated in a recirculated air stove (MEMMERT) to 100 • C for six hours for the subsequent analysis of the bioactive compounds: Sarcocornia alpini (1-5), Sarcocornia pruinosa (6-11), Sarcocornia perennis (12)(13)(14), and Arthrocnemum macrostachyum (15)(16)(17)(18)(19)(20).

Determination of Humidity
Humidity was determined by drying in an oven (984.25-AOAC, 2005): 5 g of dried samples were weighed in previously dried and tared capsules, and it was placed in a dryer. The samples were placed in a recirculated air stove (MEMMERT) to 100 • C for six hours until the elimination of the water present in the sample (constant weighing).

Preparation of Methanol Extract
Five hundred milligrams of dried plant sample were extracted with a solution of 40 mL of methanol at 25 • C. It was kept in magnetic stirring for 60 min. The extracts were filtered using a Whatman No. 4 filter. The solid residue was recovered and extracted with 40 mlf of methanol. The extracts were filtered again, combined, and evaporated (35 • C under vacuum of the methanolic extracts). Redissolve with methanol to obtain a 30 mg/mL of extract solution, from which different dilutions were made (from 0.03125 mg/mL to 16 mg/mL), was conducted. The extractions were performed in triplicates and were stored at 4 • C until the execution of the analyses. The total phenolic content was determined by the Folin-Ciocalteu method [54] using gallic acid as the recommended standard [55]. An 0.5 mL of aliquot of methanolic extract was taken from the extracts obtained previously and 2.5 mL of the Folin-Ciocalteu reagent was added and left to react for 3 min. Then, 2 mL of Na 2 CO 3 solution was added and mixed in a Heidolph shaker (Berlin, Germany). The solution was incubated at a temperature of 40 • C and stored in the dark for 1 h. The absorbance was measured at 765 nm with a spectrophotometer and the results were expressed as gallic acid equivalents (G.A.E.).

Gas Chromatography Coupled with Mass Spectrometry (GC-MS) for Phenolic and Fatty Acid Analysis
Chromatographic separation was performed as follows: Methanol extracts were brought to dryness in a Rotavapor Fischer rotary evaporator (USA) and later in the Telstar lyophilizer (Barcelona, Spain). The amount of the total sample obtained was weighed. The samples were then subjected to derivatization with a 0.2 N methanolic solution of m-trifluoromethylphenyl trimethylammonium hydroxide Meth Prep II (Fisher, Loughborough, UK). This one-step reagent simplifies the transesterification of triglycerides to methyl esters. In total, 5 µL was injected into GC/MS Agilent 6120 (Santa Clara, CA, USA). All standards were from Sigma Aldrich (Sant Louis, MI, USA) at ≥95.0% (HPLC).
The chromatography-mass spectrometry was carried out with the Interdepartmental Research Service at the Universidad Autónoma de Madrid (UAM).

High-Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry (HPLC-MS/ESI) for Flavonoid and Hydroxycinnamic Acid Analysis
Flavonoids were determined using a HPLC-MS/ESI Agilent 1100 (Santa Clara, CA, USA) in a C20 column ACE 3 C18 PFP, 150 mm × 4.6 mm, which was maintained at 35 • C. The solvent system used was a gradient of water (solvent A) and formic acid 0.1% (solvent A), and the acetonitrile and formic acid 0.1% (solvent B) as follows.

Conclusions
The bioactive compounds (phenolic compounds and fatty acids) present in S. alpini, S. pruinosa, S. perennis, and A. macrostachyum from different territories of Spain and Portugal were described. Samples of the genus Sarcocornia highlighted the presence of veratric acid material from dryer environments. A slightly greater diversity of phenolic acids was shown in A. macrostachyum than in species of the genus Sarcocornia. Ferulic acid was also detected in two of the samples from this genus but was not present in the genus Sarcocornia. The composition of the flavonoids detected in these species showed glycosylated structures that conferred prebiotic properties of these halophytes. The material from S. alpini, S. pruinosa, and A. macrostachyum contained macromolecular antioxidants, namely cyanidin-3-O-arabinoside, luteolin-7-glucoside, dihydroquercetin, and p-Coumaroyl glucoside, thus increasing their antioxidant requirements as a defense against extreme environments. The lipid profile revealed palmitic, linoleic, and oleic acids as the main fatty acids in the genus Sarcocornia, while the palmitic, linolenic, and stearic acid content was particularly notable in the genus Arthrocnemum. The presence of these compounds in different halophytes confirms their value for survival in conditions of extreme salinity and drought, and also adds to their value for consumption.