1. Introduction
Preserving edible plant species and improving their cultivation is a priority for the agri-food sector. This need is being driven by climate change, which is affecting the entire world. This challenge is being addressed in different ways, such as using varieties that can withstand water stress, increasing the cultivation of drought-resistant species, or those that can be irrigated with high saline water or even seawater. In addition, climate change is increasing land salinity. This problem affects an estimated 1 billion hectares of land, corresponding to 20% of the world’s cultivated soil [
1,
2], and 50% of the irrigated area [
3].
The problems that climate change is causing in crop production can be addressed by introducing or improving the cultivation of halophytes. Halophytes comprise more than 350 species in 20 orders and 256 families, representing 1–2% of the world’s flora [
4]. These plants are adapted to environments with salinity levels equal to or higher than 200 mM NaCl and do not require fresh water. This allows them to grow in areas where only saltwater is available. Some species are suitable for human consumption and could be good candidates for large-scale introduction as edible crops, especially in very arid regions where they could be the main (if not the only) feed resource available [
1,
5,
6,
7]. Halophytes are not only a good source of nutrients, but also of bioactive compounds. Some species have been used in traditional medicine to treat various diseases [
8,
9]. Some of the biological activities of halophytes that have been reported include antioxidant, anticholinesterase, anticancer, antimicrobial, anti-inflammatory, analgesic, astringent, diuretic, antipyretic, vasoactive, antibacterial, and cytotoxic [
7,
10,
11].
Sea fennel (
Crithmum maritimum) is one of the most abundant halophytes in Europe. It is commonly found along the coasts of Southern and Western Europe, and can also be found in other parts of the world [
12]. Sea fennel is capable of thriving in salt-free or low saline soils. The nutritional composition of sea fennel was recently described by Sánchez-Faure et al. [
13], who reported a high content of calcium and dietary fiber. Its tender leaves and stems are used in salads, pickles, appetizers, and condiments [
12,
14]. In addition, sea fennel is abundant in bioactive compounds. Recently, extracts from this plant have been used as a functional ingredient to enrich sunflower oil [
15]. Sea fennel has also been utilized to obtain essential oils and in traditional medicine [
12,
16,
17,
18]. The potential of this plant as a nutraceutical for preventing and treating certain human diseases has been documented due to the presence of antioxidant molecules [
19,
20], antimicrobials [
19,
21], antimutagenics [
19], vasodilators [
20], and cholinesterase inhibitors [
20], among others.
Seaside arrowgrass (
Triglochin maritima) is a common halophyte found in the marshlands of Northern Europe and in the coastal areas of Mediterranean countries. Their nutrient composition has also been recently documented [
13]. This plant contains molecules of interest such as fatty acids, amino acids, organic acids, mono- and disaccharides, polyols and polyphenols with health-promoting properties [
13,
22]. Likewise, there is limited knowledge about the beneficial effects that its consumption could have on human health. The utilization of this plant as a source of bioactive compounds has hardly been reported. A recent study by Boestfleisch and Papenbrock [
23] investigated the production of secondary metabolites with antioxidant capacity in seaside arrowgrass under different conditions of soil salinity. They found that the production of these metabolites was increased under conditions of high soil salinity. This suggests that seaside arrowgrass can be used as a source of antioxidants, which are of interest to protect cells from free radical damage.
Halophytes are exposed to high stress situations that result in the production of antioxidant compounds, including polyphenols [
24]. Polyphenols do not show significant toxicity or harmful effects on the human body and have promising therapeutic potential [
25]. Several polyphenols have the ability to inhibit angiotensin-converting enzyme (ACE), an enzyme linked to the development of hypertension [
26,
27,
28,
29,
30]. In addition, previous research has described the potential hypoglycemic capacity of polyphenols through their inhibitory effect on dipeptidyl peptidase IV (DPP-IV) [
28,
31,
32,
33]. Additionally, some polyphenols are effective inhibitors of prolyl endopeptidases (PEP) [
28,
34,
35,
36], a family of enzymes that can hydrolyze hormones such as vasopressin, thyrotropin-releasing hormone, or substance P. Some researchers have suggested that PEPs may present a viable pharmacological target for treating various neurological conditions linked to abnormally high serum PEP activity, including Alzheimer’s disease, schizophrenia, and depression, as well as other disorders like anorexia and bulimia [
37].
In certain areas where salinization of agricultural land and freshwater shortages are reducing crop yields, halophytes offer an alternative to traditional crops. Many studies have focused on evaluating the growth and yield of halophyte crops, as well as the impact of their cultivation on soil and water quality. Others have focused on characterizing their nutritional profile and the study of their potential for animal and human consumption. However, their potential as a potent and natural source of bioactive molecules is poorly documented. In this context, this study aims to explore the potential of two halophytes abundant in the European and Mediterranean coasts, seaside arrowgrass and sea fennel, as a source of molecules of nutraceutical interest against diseases of high prevalence in developed countries. More specifically, this study is focused on the analysis of the antihypertensive, nootropic, hypoglycemic and antioxidant potential of ethanolic extracts of these two plants, as well as on the isolation and identification of the polyphenols potentially responsible for these activities.
2. Materials and Methods
2.1. Chemicals
Folin–Ciocalteu reagent, gallic acid (GA), 2,2′-azino-bis-(3-ethylbenzothiazoline-6- sulphonic acid) (ABTS), 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ), ammonium iron (II) sulphate, human dipeptidyl peptidase-IV, angiotensin-converting enzyme from rabbit lung, and reagents used for HPLC analysis were purchased from Sigma-Aldrich, Co. (St. Louis, MO, USA). Seikagaku Corp. (Tokyo, Japan) provided prolyl endopeptidase from Flavobacterium. Chromogenic substrates were purchased from Bachem (Bubendorf, Switzerland). Ethylenediaminetetraacetic acid (EDTA) was from Leco Corp. (St. Joseph, MI, USA). Analytical-grade chemicals and reagents were purchased from Panreac Chemical Co. (Barcelona, Spain).
2.2. Plant Processing
The company Porto-Muiños S.L. (Cedeira, A Coruña, Spain) collected sea fennel and seaside arrowgrass on the coast of Galicia, in north-western Spain. The samples were transported at a low temperature (4 °C) to Madrid. The edible portions (leaves and stems) underwent a washing process involving distilled water and then were subjected to drying via forced air oven (FD 240, Binder, Tuttlingen, Germany) at 55 °C for 24 h. The dried plants were then stored at 4 °C until analysis.
2.3. Preparation of the Extract
The preparation of the extract was in accordance with the method of Calvo et al. [
28]. The dried samples (250 g) were homogenized in a 400 mL mixture of ethanol/water 1/1 (
v/
v) at pH 2, in a ratio of 1:2 (
w/
v) using an Ultra-Turrax homogenizer (Mod. T25D, IKA
®-Werke GmbH & Co. KG, Staufen, Germany) at 25,000×
g for 3 min at 25 °C. The homogenates were kept in an ice bath and then sonicated in 16 cycles at 90% amplitude, with 60 s of interval between each cycle of 1 min. After extraction, the samples were centrifuged at 12,000×
g for 10 min at 5 °C (Sorvall evolution, Thermo Fisher Scientific, Waltham, MA, USA). The supernatants were evaporated using a rotary evaporator (R-300, BÜCHI, Buchegg, Switzerland) until the ethanol was eliminated. Then, the extracts were freeze-dried and stored at 4 °C until analysis.
2.4. Determination of Total Phenol Content
The total phenolic content of the extracts was determined using the Folin–Ciocalteau assay, in accordance with the procedure outlined by Calvo et al. [
28]. The extracts were dissolved in distilled water to a concentration of 10 mg/mL (dry weight). The extracts were then mixed (1/1,
v/
v) with 500 µL of Folin reagent, 10 mL of 0.7 M Na
2CO
3, and 14 mL of distilled water. The mixtures were stirred for 1 h at room temperature, and the absorbance was then measured at 750 nm using a spectrophotometer Shimadzu UV-1601 (Kyoto, Japan). Gallic acid (0.05–0.3 mg/mL) was used as standard. The results were expressed as mg Eq GA/g (dry weight).
2.5. Scavenging Activity of ABTS Radical Cation
The radical scavenging activity of the fractions and the extract was performed according to Calvo et al. [
28]. First, an ABTS+ stock solution was prepared by mixing 7 mM aqueous ABTS+ and 2.45 mM K
2O
8S
2 in a 1:1 (
v/
v) ratio. After 16 h in the dark, the mixture was diluted in distilled water to reach an absorbance of 0.70 ± 0.02 at 734 nm. Then, 980 µL of ABTS+ working solution was mixed with 20 µL of sample diluted in distilled water (10 mg of extracts/mL or 1 mg of fraction/mL, dry weight). The mixture was incubated for 10 min at 30 °C in the dark. The absorbance was then measured at 734 nm using a spectrophotometer. Ascorbic acid (0.02–0.6 mg/mL) was used as standard. The results were expressed as mg Eq ascorbic acid/g (dry weight).
2.6. Ferric Reducing Antioxidant Power (FRAP)
The ferric-reducing antioxidant power of the fractions and the extract was analyzed following the protocol of Calvo et al. [
28]. To prepare the FRAP reagent, 25 mL of 0.2 M sodium acetate buffer (pH 3.6), 2.5 mL of 10 mM TPTZ (dissolved in 40 mM HCl), and 2.5 mL of 20 mM FeCl
3 were mixed. Afterwards, 900 µL of FRAP reagent was combined with 90 µL of distilled water and 30 µL of sample (10 mg of extract or 1 mg of fraction/mL, dry weight), and incubated at 37 °C for 30 min in the dark. The absorbance was measured at 595 nm using a spectrophotometer. Ammonium iron (II) sulphate, also known as Mohr’s salt, was used as a standard at concentrations ranging between 0.1 to 1 mM. The results were expressed as mEq Mohr’s salt per gram of dry weight.
2.7. Determination of ACE Inhibitory Activity
The ACE inhibitory activity was measured according to Sentandreu and Toldrá [
38] with some modifications. To prepare the enzyme, it was dissolved in a Tris-base buffer (150 mM, pH 8.3, containing 1.125 M NaCl). The final enzymatic activity was 15 mU/mL. The samples (final concentration of 0.5 mg of dried extract/mL or 0.1 mg of dried fraction/mL in the system) and the substrate (0.45 mM Abz-Gly-Phe(NO
2)-Pro) were also dissolved in the same buffer. A mixture consisting of 50 µL of either sample or buffer (control) and 50 µL of ACE was incubated at 37 °C for 15 min. The reaction was started by adding 200 µL of substrate and continued for 15 min. The fluorescence was measured at λexc 360 nm and λem 400 nm using a Sinergy Mx microplate reader (BioTeck, Colmar, France). The maximum linear increase in fluorescence per minute was determined for each well and inhibition activity was expressed as a percentage.
2.8. Determination of PEP Inhibitory Activity
PEP-inhibitory activity was measured in 96-well plates, as according to Sila et al. [
35]. In each well, 20 μL of enzyme (1 mU) was mixed with either 180 μL of 0.1 M sodium phosphate buffer pH 7 (assay buffer) or 150 μL of buffer, together with 30 μL of the sample diluted in buffer. The final concentration of sample in the system was 1 mg of dried extract/mL or 0.2 mg of dried fraction/mL. The plate was incubated at 37 °C for 15 min. Then, 100 μL of 0.01 mM Z-Gly-Pro-AMC was added. The microplate reader monitored fluorescence at λexc 340 nm and λem 450 nm every 1 min for 20 min. The inhibitory activity was expressed as a percentage of the inhibition, as outlined in
Section 2.7.
2.9. Determination of DPP-IV Inhibitory Activity
The DPP-IV-inhibition was determined as described by PEP-activity, but with 0.1 M Tris-HCl (pH 8) as buffer assay, following the protocol outlined by Sila et al. [
35]. The substrate used was 0.025 mM H-Gly-Pro-AMC·HBr. The inhibiting activity of the samples was calculated as indicated in
Section 2.7.
2.10. Chromatographic Fractionation of the Extracts
The extracts were fractionated following the method of Calvo et al. [
28]. The extracts were firstly reconstituted to a concentration of 80 mg/mL (dry weight) in a mixture of ethanol and water (1/1,
v/
v). A preparative HPLC system (Agilent LC PREP 1260 Infinity Series, Santa Clara, CA, USA) equipped with a diode array detector (DAD) and a Tracer Excel 120 ODS-A 25 × 0.78 cm preparative C18 column (Teknokroma, Barcelona, Spain) was used. Phase A was comprised of ultrapure water containing 5% acetonitrile and 0.1% formic acid, while phase B consisted of acetonitrile with 0.1% formic acid. The gradient used consisted of four steps: 0–15% B for the first 15 min, 15–50% B for the following 5 min, 50–65% B for the next 15 min, and finally 65–0% B for the last 10 min. The flow rate was maintained at 2 mL/min. Fractions were automatically collected using a fraction collector while detection was performed at 280 and 360 nm. The collected fractions underwent solvent evaporation in a Speed Vac concentrator (Thermo Fisher, Waltham, MA, USA). Then, the fractions were lyophilized and stored at 4 °C until use.
2.11. Tentative Identification of Polyphenols by HPLC-ESI-QTOF-MS
The fractions showing the highest bioactivity were selected and the polyphenols were extracted using Oasis
® HLB cartridges (Waters, Milford, MA, USA). The separation of the polyphenols was performed using high-performance liquid chromatography coupled with electrospray ionization and quadrupole time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS) as described by Sánchez-Faure et al. [
13]. The mass spectra were obtained by electrospray ionization in negative mode. The gas temperature was 325 °C and the drying gas flow was 12 L/min. A range of 100 to 1200 m/z was used to scan the acquisition for auto MS/MS. The mass spectra were analyzed with MassHunter Workstation version 4.0 software (Agilent Technologies, Santa Clara, CA, USA). The phenolic compounds were identified by comparing their experimental mass with the mass obtained in the negative mode analysis, allowing an error of 8 ppm. The fragmentation pattern and the relative abundance of fragment ions obtained for each compound were also compared with some databases such as MassBank, Food Database and Human Metabolome Database.
2.12. Statistical Analysis
A student’s t-test was used to compare the bioactivity of the extracts from sea fennel and seaside arrowgrass. The bioactivity of the fractions was compared using the one-way analysis of variance (ANOVA) method. The software program SPSS 27 (IBM Corporation, Armonk, NY, USA) was used to perform the statistical analysis. The level of significance was set at p ≤ 0.05. All measurements were taken three times to ensure accuracy.