2.1 Nutritional profile
Collected biomass of S. alpini and S. emerici were appraised for proximate composition and mineral contents and results are showcased in Table 1. Dried biomass from both species presented moisture contents of 6.32 and 8% DW, for S. alpini and S. emerici, respectively. These values were lower than the results reported for Sarcocornia alpini, Sarcocornia perennis, and Sarcocornia pruinosa from the Iberian Peninsula where values were between 10.18 and 14.78% [33]. This parameter reflects the drying process’s ability (ambient air drying in our case) to remove residual water from dried plants, hence extending plant shelf life.
Table 1
Proximate and mineral compositions of Sarcocornia alpini and Salicornia emerici biomass
Parameters | Species |
Sarcocornia alpini | Salicornia emerici |
Moisture (%) | 6.32 ± 0.01a | 8 ± 0.06b |
Total ash (%) | 42.54 ± 0.05b | 53.84 ± 0.24a |
Total protein (%) | 10.42 ± 0.23a | 5.77 ± 0.08b |
Macro-elements (mg/g) | | |
Na | 157.17 ± 1.29b | 235.47 ± 1.20a |
K | 36.42 ± 2.98b | 49.61 ± 3.57a |
Ca | 17.09 ± 1.63b | 30.96 ± 2.27a |
Mg | 15.13 ± 0.74a | 13.83 ± 0.18a |
Micro-elements (µg/g) | | |
Cu | 5.95 ± 0.61b | 22.63 ± 0.18a |
Zn | 14.50 ± 0.29b | 85.9 ± 1.45a |
Fe | 205.9 ± 3.06b | 381.53 ± 1.64a |
Mn | 14.55 ± 0.66a | 15.60 ± 0.29a |
Data are presented as mean ± standard error of the mean (SEM), (n = 3). In the same row, values followed by different letters are significantly different (p < 0.05). |
The ash content is directly related to the amount of minerals present in the biomass [34], and reached 42.5 and 53.8% for S. alpini and S. emerici, accordingly, which were like the findings of Hayder et al. for the same species [32], as well as those for S. ramosissima collected from northern Portugal [35]. Castañeda-Loaiza et al. obtained lower levels for S. fruticosa collected from the wild in Spain and Portugal (28.9 and 37.3 g/100g, respectively) [36], and the same was observed for S. perennis alpini, Sarcocornia perennis perennis, and Salicornia ramosissima from Portugal [10]. Halophytes commonly have a higher ash content than other edible glycophytes which can be attributed to their ability to retain minerals, which is correlated with their adaptation mechanisms to the high levels of salinity in their natural habitat [37, 38].
S. alpini and S. emerici presented low protein levels of 10.4 and 5.7% accordingly, which were comparable to the values described by Hayder et al. (11.87 and 8.58% DW, for S. alpini and S. emerici, respectively) [32]. They were also akin to those reported for dried S. ramosissima [35]. The plant´s protein content is influenced by different factors namely species, fluctuations in temperature, salinity, and UV radiation [39], and can be boosted as a result of stress response [40].
Both species presented a high mineral content (Table 1) which could be related to their tolerance and capacity to survive in extreme conditions of high salinity and drought [10, 41, 42]. Overall, S. emerici presented higher mineral content which could be directly correlated to its higher ash content. The values disclosed by Hayder et al. for the same species showed lower Na, K, Ca, Mg, and Fe contents for S. emerici and higher amounts of Na, Ca, Mg, and Fe in S. alpini [32]. The macro-nutrients contents in S. alpini and S. emerici were higher than those reported for other edible Salicorniaceae, whereas Fe and Mn levels were lower [10]. S. alpini’s mineral content was lower than the values reported for wild S. fruticosa [36]. The same was observed for S. emerici except for Na, K, Ca, Cu, and Zn, where it presented similar or higher values. The mineral content in halophytes can be influenced not only by maturation stage and the studied part of the plant but also by environmental conditions related to water and soil characteristics, pollutants, and climate [38, 43]. Therefore, the elevated mineral levels in S. emerici might be attributed, at least in part, to the continuous immersion of the plant in seawater and its maturation stage, as the salt content in halophytes tends to increase with age [44].
Like previously reported for different Salicornia and Sarcocornia species, Na was the prevalent macro-element in both S. alpini and S. emerici (157.17 and 235.47 mg/g, respectively). This mineral is deemed essential for maintaining various physiological functions, including heart and nerve functions, muscle contraction, and the regulation of the body's water balance [45]. However, as high sodium intake has been positively associated with increased blood pressure and proved to be a major risk factor of cardiovascular diseases
[46], numerous studies proposed the substitution of table salt with halophytes to prevent such health repercussions. This recommendation is primarily based on their potassium and magnesium content, contributing to vascular protection. Knowing that replacing salt by the same amount of dried Sarcocornia perennis resulted in a decrease of more than 70% of Na content, S. alpini and S. emerici could be used as salt substitute while providing other beneficial elements like K, Mg, and Fe [28]. Besides, they could be considered flavor enhancers with various health benefits attributes.
Iron was the predominant micro-element in both species with values of 205.9 and 381.53 µg/g for S. alpini and S. emerici, respectively. Thus, their biomass could be further investigated as potential source of iron to be incorporated in functional food as part of the diet in case of anemia, a common blood disorder resulting from iron deficiency [47].
The presence of all identified essential minerals that are vital for the maintenance of various biochemical and physiological processes in the human body in relevant amounts, adds nutritional value to both halophytic species and enhances their value as functional compounds reservoir [48, 49].
2.2 Chemical profile
To adapt to harsh environmental conditions, particularly high levels of salinity and UV exposure, halophytes produce significant amounts of bioactive secondary metabolites such as phenolic acids, flavonoids, and tannins [18, 50, 51]. These compounds exhibit a wide range of health benefits, including antioxidant, anti-inflammatory, anti-cancer, and anti-diabetic activities [50, 52–55]. Having this in mind, the total phenolics, flavonoids, and condensed tannins contents of S. alpini and S. emerici extracts were quantified and results are presented in Table 2.
Table 2
Total phenolics (TPC), flavonoids (TFC) and condensed tannin contents (CTC) of aqueous ethanolic extracts of Sarcocornia alpini and Salicornia emerici
Extracts | TPC mg GAE/g DW | TFC mg QE/g DW | CTC mg CE/g DW |
Sarcocornia alpini | 15.4 ± 0.13a | 3.81 ± 0.07a | 0.94 ± 0.18a |
Salicornia emerici | 4.7 ± 0.13b | 0.91 ± 0.03b | 1.45 ± 0.13a |
Data are presented as mean ± standard error of the mean (SEM), (n ≥ 5). In the same column, values followed by different letters are significantly different (p < 0.05). |
Overall, S. alpini had higher TPC and TFC (15.4 mg GAE/g DW and 3.81 mg QE/g DW, respectively) while S. emerici presented higher CTC (1.45 mg CE/g DW). The TPC of S. alpini exceeded the levels previously reported in methanolic extracts of S. alpini gathered from different locations across the Iberian Peninsula, which ranged from 3.43 to 3.77 mg GAE/g DW [33]. For the same species collected from Portugal, Lopes et al. (2023) reported higher values (38.2 mg GAE/g DW) [19]. S. alpini exhibited higher TPC, similar TFC, and lower CTC than wild S. fruticosa [36]. The TPC of S. emerici (4.7 mg GAE/g DW) was comparable to the value described for Salicornia patula extracts collected in Spain [16], but lower than that reported for S. ramosissima extracts [35]. Hayder et al. relayed lower TFC and CTC contents for the same species from southern Tunisia [32]. Such variations can be due to several factors related to species, sites of collections, drying processes, and extraction methods [13, 35].
2.3 HPLC-ESI-MS analysis
The results of the phenolic compositions of S. alpini and S. emerici extracts identified with HPLC-ESI-MS method are summarized in Fig. 1, 2 and Table 3. Among the 31 tested standards, 11 compounds were identified in S. alpini: six phenolic acids (quinic, chlorogenic, 4-O-caffeoylquinic, syringic, p-coumaric and trans-ferulic acids) and five flavonoids (rutin, hyperoside (quercetin-3-O-galactoside), naringin, quercetin and cirsiliol). Whereas in S. emerici, only five phenolic compounds were detected (quinic, p-coumaric and trans-ferulic acids, naringin and cirsiliol). Even though several studies have described the phytochemical profiles of Salicornia and Sarcocornia species, data concerning the phenolic composition of S. alpini and S. emerici remains limited. As far as we know, this is the first time the phenolic profile of S. emerici has been disclosed.
Table 3
Phenolic compounds (mg/kg DW) identified in aqueous ethanolic extracts of Sarcocornia alpini and Salicornia emerici, by HPLC-ESI-MS
Compound | Formula | RT (min) | m/z | Sarcocornia alpini | Salicornia emerici |
Quinic acid | C7H12O6 | 2.129 | 191 | 148.46 | 229.15 |
Chlorogenic acid | C16H18O9 | 14.730 | 353 | 128.23 | - |
4-O- caffeoylquinic acid | C16H18O9 | 15.397 | 353 | 159.22 | - |
Syringic acid | C9H10O5 | 19.825 | 197 | 41.55 | - |
p-coumaric acid | C9H8O3 | 24.376 | 163 | 6.02 | 8.17 |
Transferulic acid | C10H10O4 | 26.974 | 193 | 6.93 | 1.72 |
Rutin | C27H30O16 | 28.439 | 609 | 87.70 | - |
Hyperoside | C21H20O12 | 28.900 | 463 | 53.22 | - |
Naringin | C27H32O14 | 30.448 | 579 | 212.43 | 46.28 |
Quercetin | C16H14O6 | 35.975 | 301 | 0.81 | - |
Cirsiliol | C17H14O7 | 39.290 | 329 | 98.25 | 51.30 |
Naringin, a flavanone glycoside known for its numerous biological activities such as anti-inflammatory, antioxidant, and anticancer [56], was the major compound in S. alpini (212.43 mg/kg) while quinic acid (cyclitol) was the dominant molecule in S. emerici (229.15 mg/kg). In fact, several studies described the multiple biological properties of quinic acid namely antioxidant [57], antidiabetic [58], anti-aging [59], and antitumor [60]. Chlorogenic and 4-O-caffeoylquinic acids were also prevalent in S. alpini alongside quinic acid. Previous studies reported the wide range of medicinal benefits of chlorogenic acid such as antioxidant, anti-cancer, anti-diabetic, and anti-inflammatory properties [61]. Plus, caffeoylquinic acids have been reported to exhibit antioxidant, antibacterial, antidiabetic, and anti-Alzheimer activities amongst others [62]. The levels of chlorogenic and p-coumaric acids, rutin and quercetin were lower than those reported for Salicornia ramosissima and S. perennis alpini [19]. The same was observed for chlorogenic acid and quercetin contents in S. alpini when compared to the results of wild S. fruticosa extracts from Spain and Portugal, while its rutin content was higher [36]. The observed variation in Salicornia and Sarcocornia species is noteworthy, as studies have suggested that the tolerance mechanism in halophytes is specific to each species [63]. This variability could elucidate the differential expression of phenolics as a protective mechanism against stressful environments. Variations in the collection sites and natural habitats of the two plants may also partially influence the production of bioactive phytochemicals as adaptive responses to challenging conditions. This could help to explain the observed differences, considering that S. emerici plants were submerged in seawater, while S. alpini biomass was collected at a distance from the tides [50, 64–67]. Furthermore, the harsh environmental conditions could, in part, account for the reduced diversity and quantities of phenolic compounds detected in S. emerici. In fact, studies indicate that beyond a specific threshold of high salinity, Salicornia and Sarcocornia species may either lose the ability to produce these compounds or shift to alternative protective mechanisms [25].
All the identified compounds are associated with diverse biological activities, including antioxidant, anti-inflammatory, anti-cancer, antimicrobial, and cardiovascular-protective properties [12, 13, 35]. Since halophytes present a rich source of polyphenols manifesting protective potential against various metabolic syndromes, including T2DM, hyperlipidemia, and cardiovascular disorders [68], our results underscore the significance of the edible S. alpini and S. emerici as sources of bioactive natural products.
2.4 In vitro toxicological evaluation
Because of their complex chemical profile, plants are prone to contain potentially harmful compounds. Therefore, it is advisable to assess potential toxicity before investigating their therapeutic properties to guarantee their safety for further nutritional and medicinal applications. Moreover, there is a lack of available data regarding the potential toxicity of S. alpini and to our knowledge, this is the first toxicity evaluation of S. emerici. Considering that these two species are being documented for the first time in southern Tunisia, it is crucial to investigate their toxicological characteristics. Different methods are used for this purpose including the assessment of acute toxic effects in vivo on Artemia salina and in vitro on mammalian cell lines. In this work, the toxicity of the tested extracts was evaluated towards three mammalian cell lines, namely murine RAW 264.7 macrophages, murine bone marrow stromal (S17), and human hepatocellular HepG2 carcinoma cells. No toxicity was observed after the application of the extracts (Fig. 3). In general, the application of the extracts resulted in cell viability near to 100% or higher in RAW 264.7 and HepG2 cells, and above 85% in S17 cells. The lack of cytotoxicity observed in in vitro models employing mammalian cell lines is regarded as an indicator of the low toxicity associated with a natural extract [69, 70]. Besides, various Salicornia and Sarcocornia species are not only edible but also traditionally consumed in fresh, boiled, or pickled forms. As such, it is logical to hypothesize that extracts from S. alpini and S. emerici could be deemed safe for consumption. This suggests a need for further exploration as potential novel and secure sources of functional ingredients with noteworthy biological attributes.
2.5 Antioxidant properties
In their native environment, halophytes encounter challenging abiotic conditions, including severe drought and salinity, leading to the generation of substantial quantities of reactive oxygen species (ROS) which are responsible for cellular damage [18]. To counteract ROS, halophytes have evolved effective antioxidant defense mechanisms, including the synthesis of enzymes and various categories of antioxidant compounds, particularly polyphenolics [71, 72]. To assess the antioxidant capacity of the S. alpini and S. emerici extracts, complementary methods were used based on their radical scavenging ability (RSA) towards ABTS and DPPH free radicals and iron reducing power (Table 4). In general, S. alpini, at 12.5 mg/mL, showed higher RSA against both DPPH (45.89%) and ABTS (73.44%) radicals and higher FRAP (EC50 = 1.32 mg/mL). S. emerici had no significant RSA (16.80 and 26.79% on DPPH and ABTS at 12.5 mg/mL, respectively) and showed a moderate capacity to reduce iron (EC50 = 7.26 mg/mL). To our best knowledge, there are no reports on the antioxidant properties of S. emerici. When compared to Sarcocornia perennis alpini, Sarcocornia perennis perennis and Salicornia ramosissima from Portugal, these three species exhibited higher DPPH radical scavenging ability and ferric reducing potential [10]. Wild Sarcocornia fruticosa collected from Spain and Portugal exhibited higher DPPH scavenging activity than both of our extracts with IC50 values of 6.10 and 8.19 mg/mL, respectively [36]. Typically, there is a correlation established between the phenolic and flavonoid content of extracts and their antioxidant activity. This correlation could explain the higher antioxidant activity of S. alpini, as it exhibited higher TPC and TFC than S. emerici. Furthermore, its individual phenolic profile displayed greater diversity, and the identified phenolic compounds are likely individually or in synergy responsible for the observed antioxidant effect [73, 77]. In fact, numerous identified phenolic acids such as quinic, chlorogenic and 4-O-caffeoylquinic acids, and flavonoids namely rutin and cirsiliol are renowned antioxidants [62, 75, 76]. Given that the extracts are a complex mixture of various phytochemicals, it is often difficult to predict exactly which compounds are responsible for the detected activity. These compounds can exhibit varying reactions to individual antioxidant mechanisms, contingent on their respective affinities, thus yielding distinct activity patterns [77, 78]. Hence, the use of several assays to evaluate the antioxidant activity of natural extracts is recommended [19, 79, 80]. Nevertheless, one should not exclude the potential effect of non-identified phytochemicals present in the extracts.
Table 4
Radical scavenging activity (RSA, %) on DPPH and ABTS free radicals and ferric reducing activity power (FRAP) (EC50, mg/mL) of aqueous ethanolic extracts of Sarcocornia alpini and Salicornia emerici
Extracts | RSA | FRAP |
DPPH | ABTS |
Sarcocornia alpini | 45.89 ± 5.18b | 73.44 ± 2.08b | 1.32 ± 0.03b |
Salicornia emerici | 16.80 ± 0.70c | 26.79 ± 1.68c | 7.26 ± 0.33c |
GA† | 80.96 ± 0.22a | 93.62 ± 0.10a | - |
BHA† | - | - | 0.16 ± 0.01a |
Data are presented as mean ± standard error of the mean (SEM), (n = 5). |
In the same column, values followed by different letters are significantly different at p < 0.05 (Tuckey HSD test). |
† Positive control at 1 mg/mL; GA: Gallic acid, BHA: Butylated hydroxyanisole. |
Table 5
Inhibitory activity (%) of aqueous ethanolic extracts of Sarcocornia alpini and Salicornia emerici on AChE, BuChE, α-amylase, α-glucosidase and tyrosinase
Extracts | AChE | BuChE | α-amylase | α-glucosidase | Tyrosinase |
Sarcocornia alpini | 14.19 ± 0.24c | 15.80 ± 0.54b | 10.05 ± 0.02b | 57.00 ± 1.04b | 51.3 ± 0.31b |
Salicornia emerici | 26.87 ± 4.24b | 8.00 ± 2.07c | 4.17 ± 0.29c | 62.82 ± 3.67b | 31.4 ± 0.06c |
Galanthamine† | 85.48 ± 0.95a | 74.99 ± 0.34a | - | - | - |
Acarbose† | - | - | 69.03 ± 0.19a | 78.12 ± 0.04a | - |
Arbutin† | - | - | - | - | 90.1 ± 0.0a |
Data are presented as mean ± standard error of the mean (SEM), (n = 3). |
In the same column, values followed by different letters are significantly different at p < 0.05 (Tuckey HSD test). |
† Positive control at 1 mg/mL. |
The findings from the in vitro antioxidant tests offered initial insights into the potential of the bioactive compounds within S. alpini and S. emerici extracts to function as scavengers of radicals and reducers of iron. Hence, it is advisable to conduct further investigations into the antioxidant attributes of both halophytes through supplementary in vitro assays, including metal chelating and cell-based methods, as well as in vivo studies. The outcomes of our study indicate that both species hold potential as rich and novel sources of natural antioxidants highlighting their consumption either in a traditional way or as functional ingredients sources in nutraceuticals and functional food.
2.6 Enzymatic inhibitory properties
This work assessed the inhibitory effects of S. alpini and S. emerici extracts against enzymes associated with various human disorders, namely AChE and BuChE (Alzheimer’s disease), α-amylase and α-glucosidase (type 2 diabetes mellitus, T2DM), and tyrosinase (hyperpigmentation disorders). Both extracts had low inhibitory activity toward AChE and BuChE, nil activity against α-amylase but relevant inhibition against α-glucosidase and tyrosinase (Table 4). To our best knowledge, there is no previous information of these enzymatic inhibitory properties for the two species.
Alpha-amylase and α-glucosidase are enzymes involved in carbohydrate digestion, therefore, their inhibition has the potential to curb the postprandial surge in blood glucose levels, consequently mitigating the hyperglycemia associated with T2DM which is a significant public health concern, given its substantial impact on patients' well-being, quality of life, and life expectancy [81, 82]. The extracts of S. alpini and S. emerici have shown potential as sources of α-glucosidase inhibitors, with inhibition rates of 57% and 62.82% at a concentration of 12.5 mg/mL, respectively. Both extracts were more active against α-glucosidase than α-amylase, in accordance with previous reports concerning plant extracts anti-diabetic effect [83]. S. ramossissima ethanolic extract at 10 mg/mL exhibited similar results (68.63%) but its raw water extracts showed lower α-glucosidase inhibition [30]. The observed enzymatic inhibition is likely attributed to the presence of various identified phenolic compounds and it's worth noting that the involvement of unidentified metabolites cannot be ruled out [84]. Further investigation is therefore required to fully explore the potential antidiabetic activity of both species, as products with a mild amylase inhibitory effect and strong glucosidase inhibitory action are sought after [85].
Tyrosinase is recognized as the key enzyme accountable for both mammalian melanogenesis and food oxidation. Hence, its inhibitors are sought after in medicine to treat skin hyperpigmentation disorders and cancers or as anti-browning compounds in agri-food industry [86, 87]. Given the interest of its diverse applications, the inhibitory activity of both S. alpini and S. emerici extracts against tyrosinase was evaluated and both extracts exhibited enzymatic inhibitory potential of 51.3 and 31.4%, respectively. Hulkko et al. reported higher tyrosinase inhibition activity (71.85%) for S. ramosissima EtOH extract at 10 mg/mL [30]. Considering that plant extracts with high TPC have a higher probability yielding tyrosinase inhibitors, this could explain in part the higher tyrosinase inhibition exhibited by S. alpini extract [85]. Furthermore, many identified phenolic compounds namely quinic and chlorogenic acids could individually or in synergism be behind melanogenesis suppression as previously disclosed [88].
This study reported the nutritional and phytochemical profiles, as well as the biological properties of the halophytes S. alpini and S. emerici species’ biomass collected from southern Tunisia. S. alpini presented higher phenolic contents and antioxidant activity, while S. emerici showed significant amounts of essential minerals, including Na, K, Mg, and Fe. Both species had no capacity to inhibit AChE and BuChE, exhibited similar capacity to inhibit α-glucosidase, while S. alpini was more active towards tyrosinase. Naringin was the major compound in S. alpini and quinic acid was the dominant molecule in S. emerici. The findings show that both species are valuable sources of nutrients, especially minerals, and underscore the potential of their use as a source of functional ingredients with beneficial biological attributes.
The sustainable harvesting and use of S. alpini and S. emerici biomass could offer economic advantages in Tunisia. This might be realized by either gathering this biomass from natural habitats or by developing saline agriculture systems with tailored growth conditions to improve their phenolic content and nutritional value. Additional research is necessary to elucidate the connection between the phytochemical composition of these plants and their health-promoting attributes, as well as to validate their consumption in a traditional way or as potential sources of functional ingredients with future applications in nutraceuticals and functional food.