From Threat to Opportunity: Harnessing the Invasive Carpobrotus edulis (L.) N.E.Br for Nutritional and Phytotherapeutic Valorization Amid Seasonal and Spatial Variability

Carpobrotus edulis (L.) N.E.Br. (Hottentot-fig) is a problematic invasive species found in coastal areas worldwide. Mechanical removal is a common control method, leaving the removed biomass available as a possible source of natural phytochemicals with prospective commercial applications. While the Hottentot-fig’s vegetative organs have been studied previously, this work establishes for the first time a seasonal and spatial comparative analysis of its nutritional, chemical, and bioactivity profiles (in three locations over four seasons). Proximate and mineral contents were assessed, along with its phenolic composition and in vitro antioxidant and anti-inflammatory properties. Hottentot-fig’s biomass offered a good supply of nutrients, mainly carbohydrates, proteins, and minerals, with a tendency for higher concentrations of the most relevant minerals and proteins in autumn and winter, and in plants from sites A (Ria de Alvor lagoon) and B (Ancão beach). The extracts were rich in polyphenolics, with higher levels in spring and summer, especially for luteolin-7-O-glucoside and salicylic and coumaric acids. The extracts were also effective antioxidants, with stronger radical scavenging activities in spring and summer, along with anti-inflammatory properties. Our results suggest that the usually discarded plant material of this invasive halophyte could be valuable as a source of natural products with potential biotechnological applications in the food and nutraceutical industries.


Introduction
Coastal environments act as transitional zones between land and sea ecosystems.They harbor specialized fauna and flora while providing crucial ecological services, such as water filtration, flood control, and storm protection, but they are highly vulnerable to natural and human-induced changes [1][2][3].One such change is the presence of invasive species that results in detrimental effects on ecosystem functioning and services, leading to a significant loss of biodiversity and the suppression of native species [4].Additionally, the economic costs of biological invasions are substantial, with estimates based on models suggesting that in 2020, at least EUR 18.3 billion was required across Europe for reliable observed costs alone [5].Moreover, managing invasive species is challenging and expensive, with overall costs reaching billions of Euros annually [6].
The phytochemical composition of invasive plants must be diverse and with high bioactive potential to endow them with coping mechanisms to adapt to new habitats, particularly regarding competitive advantage (e.g., allelopathy) and resistance to predators (e.g., deterrents) [4].Furthermore, their rapid growth characteristics make them promising candidates for sustainable utilization as feedstock in diverse industries.Harnessing the biotechnological potential of biomass from invasive species is, therefore, considered a valuable strategy since it not only helps mitigate the negative impacts of these plants but also enables the production of beneficial bioactive products with potential commercial applications [4,7].
Carpobrotus edulis (L.) N.E.Br.(Aizoaceae), commonly known as ice plant, sour fig, or Hottentot-fig, is a South African native plant inhabiting coastal areas in many parts of the world, including most Mediterranean countries.It can be found on coastal rocks, sand dunes, and sea cliffs, spreading widely while displacing several native species, and is considered a problematic invasive species with negative impacts on the biodiversity of the invaded ecosystems [8,9].This perennial succulent halophyte has been introduced in all five continents for ornamental and soil stabilization purposes but has proved to be an aggressive competitor of local flora, one that is able to change soil physicochemical properties and geochemical processes [4,9,10].Traditionally, Hottentot-fig has been considered a medicinal and edible plant.It has been used to treat a variety of ailments, including inflammation, dysentery and diarrhea, tuberculosis, diabetes, sinusitis, blood pressure, coughs and colds, throat and mouth infections, digestive problems, and skin conditions including irritation, wounds, chilblains, eczema, and burns, has been employed as an antiseptic, and even to manage common infections in HIV patients [4,9].Additionally, it is consumed raw or used in jams, jellies, and other preserves; its leaves are used as a vegetable, either raw or cooked, and its fruits have a sweet, tangy flavor.There are also reported uses as a food additive (for flavoring) and preservative (antimicrobial) [9,10].
While Hottentot-fig has been the focus of various studies concerning its bioactivities and chemical composition, only Chokoe et al. [17] considered spring versus autumn while analyzing antibacterial activity.Seasonal and spatial analysis have not been considered, to the best of our knowledge.This information can be useful to characterize this invasive plant targeting its potential biotechnological applications, while establishing seasonal and location profiles for optimizing the exploration of discarded plant material.Hence, this work establishes a comparative analysis of nutritional, chemical, and bioactivity profiling of Hottentot-fig samples from three different locations in southern Portugal, throughout the four seasons of the year (Figures 1 and 2).The locations selected (Figure 2) correspond to three "hotspots" of high biomass density of Hottentot-fig on the Algarve coast: two lagoons of different characteristics (~70 km apart) and one beach (adjacent to the bigger lagoon).Proximate and mineral compositions were assessed, along with their phenolic composition (spectrophotometrically and by High-Performance Liquid Chromatography-Diode Array Detection (HPLC-DAD)) and in vitro antioxidant and anti-inflammatory properties.

Nutritional Profiling
The Hottentot-fig's nutritional composition varied significantly between seasons and locations (Table 1).The levels of moisture, ash, crude protein, and crude fat showed an overall trend of higher values in autumn and winter, and levels were also higher in plants from site A. Conversely, carbohydrates and fiber showed higher levels in spring and summer, with levels being higher in plants from site A for fiber and from site B for carbohydrates.Such a pattern seems less apparent for minerals, although a tendency for higher concentrations of Ca, Mg, Na, Fe, Mn, and Zn in autumn and winter is discernible.Plants from sites A and B have higher levels overall of Ca, Na, and Fe, but the plants from site C showed greater amounts of the other minerals.The association between spring and summer, and between autumn and winter, is depicted in the dendrogram of the seasonal and spatial nutritional profile of Hottentot-fig (Figure 3).The dotted line in Figure 3 represents the automatic truncation, leading to three clusters; within each cluster, spring and summer are more closely linked, as are autumn and winter in the second and third clusters.Additionally, each cluster groups the data according to location, implying that spatial variability may have more impact on the nutritional profile of this plant than

Nutritional Profiling
The Hottentot-fig's nutritional composition varied significantly between seasons and locations (Table 1).The levels of moisture, ash, crude protein, and crude fat showed an overall trend of higher values in autumn and winter, and levels were also higher in plants from site A. Conversely, carbohydrates and fiber showed higher levels in spring and summer, with levels being higher in plants from site A for fiber and from site B for carbohydrates.Such a pattern seems less apparent for minerals, although a tendency for higher concentrations of Ca, Mg, Na, Fe, Mn, and Zn in autumn and winter is discernible.Plants from sites A and B have higher levels overall of Ca, Na, and Fe, but the plants from site C showed greater amounts of the other minerals.The association between spring and summer, and between autumn and winter, is depicted in the dendrogram of the seasonal and spatial nutritional profile of Hottentot-fig (Figure 3).The dotted line in Figure 3 represents the automatic truncation, leading to three clusters; within each cluster, spring and summer are more closely linked, as are autumn and winter in the second and third clusters.Additionally, each cluster groups the data according to location, implying that spatial variability may have more impact on the nutritional profile of this plant than

Nutritional Profiling
The Hottentot-fig's nutritional composition varied significantly between seasons and locations (Table 1).The levels of moisture, ash, crude protein, and crude fat showed an overall trend of higher values in autumn and winter, and levels were also higher in plants from site A. Conversely, carbohydrates and fiber showed higher levels in spring and summer, with levels being higher in plants from site A for fiber and from site B for carbohydrates.Such a pattern seems less apparent for minerals, although a tendency for higher concentrations of Ca, Mg, Na, Fe, Mn, and Zn in autumn and winter is discernible.Plants from sites A and B have higher levels overall of Ca, Na, and Fe, but the plants from site C showed greater amounts of the other minerals.The association between spring and summer, and between autumn and winter, is depicted in the dendrogram of the seasonal and spatial nutritional profile of Hottentot-fig (Figure 3).The dotted line in Figure 3 represents the automatic truncation, leading to three clusters; within each cluster, spring and summer are more closely linked, as are autumn and winter in the second and third clusters.Additionally, each cluster groups the data according to location, implying that spatial variability may have more impact on the nutritional profile of this plant than season variability, as the data are more closely linked by location (across seasons) than by season (across locations).
0.6 ± 0.0 b 0.6 ± 0.0 b 0.9 ± 0.0 a 0.6 ± 0.0 b Cr 0.1 ± 0.0 b 0.0 ± 0.0 de 0.0 ± 0.0 de 0.1 ± 0.0 c 0.1 ± 0.0 b 0.0 ± 0.0 cd 0.1 ± 0.0 a 0.0 ± 0.0 cd 0.1 ± 0.0 b 0.0 ± 0.0 de 0.0 ± 0.0 cde 0.0 ± 0.0 e Ni 0.2 ± 0.0 ab 0.1 ± 0.0 efg 0.1 ± 0.0 ef 0.2 ± 0.0 de 0.1 ± 0.0 gh 0.1 ± 0.0 h 0.1 ± 0.0 fg 0.1 ± 0.0 h 0.2 ± 0.0 cd 0.2 ± 0.0 ab 0.2 ± 0.0 a 0.2 ± 0.0 bc Cd <LOQ Values represent mean ± standard deviation (SD) (n = 3).NDF: neutral detergent fiber; DW: dry weight; LOQ: limit of quantification.LOQs: 1 Cd = 0.02 mg/100 g DW.For each line, different letters represent significant differences (p < 0.05).The observed seasonal and spatial variability in the nutrient content of Hotten may have implications for its potential uses.The nutritional profile of halophyte may change with environmental fluctuations as a strategic adaptation to p The observed seasonal and spatial variability in the nutrient content of Hottentot-fig may have implications for its potential uses.The nutritional profile of halophyte plants may change with environmental fluctuations as a strategic adaptation to preserve intracellular ionic balance, avoiding excessive salt accumulation and protecting cellular structures from excessive oxidative stress [18].For example, the accumulation of carbohydrates contributes to osmotic adjustment [18,19]; thus, a higher carbohydrate concentration in spring and summer could be a strategy to cope with salt stress, which can increase in periods of higher temperatures and lower rainfall, as was the case in these seasons (Table 2).Moreover, higher carbohydrate levels in plants from site B, a beach, could imply higher stress in such a location than in the other sites (lagoons).On the other hand, higher values of moisture, ash, protein, fat, and most minerals in autumn and winter could be attributed to favorable meteorological conditions (e.g., higher humidity and rainfall, or lower salinity) that are usual in these seasons (Table 2), which allow their uptake and subsequent allocation for growth.In fact, some authors report that protein content decreases with increased salinity, which is typical in hotter seasons such as spring and summer (as shown in Table 2) [18,20].Other factors, such as the plant developmental/growth stage, may also influence nutrient content changes throughout the year [21,22].For instance, fiber content changes over the seasons according to growth stage and temperature, being lower in cool weather (associated with vegetative states) and increasing with warmer temperatures [23].This was the case for the fiber content of Hottentot-fig, with higher levels in the warmer spring and summer seasons (as depicted in Table 2).As for spatial variability, soil composition can vary widely between locations.Sites A and C are in lagoons with different characteristics and are of considerably different sizes (site A: ~15 km 2 small shallow estuary [24]; site C: ~100 km 2 multi-inlet barrier system [25]), while site B is a sandy beach.These differences are probably reflected in the soil composition of each location and, since soil properties are the major influence on plant nutrient contents [26], that fact alone may explain the differences in the nutrient contents of Hottentot-fig from the distinct sites.Reports on the Hottentot-fig's nutritional composition are scarce, and, to the best of our knowledge, no other studies have been published concerning seasonal and spatial analyses of its nutritional profile.However, an analogous study with the halophyte Cladium mariscus [27] showed some similarities in the seasonal variation of nutrient content, such as higher levels of ash in autumn and of fiber in summer, which are possibly related to the plants' reproductive stages.Hottentot-fig is a succulent halophyte [29], its succulence (specialized water-storing tissues) being an important characteristic for surviving in harsh conditions, such as drought and high salinity [30].Therefore, high levels of moisture in the Hottentot-fig's aerial parts (85.2-94.3g/100 g, Table 1) were expected and were comparable to those obtained in leaves of this species (92.3 g/100 g) [15], while they were higher than reported in its fruits (77.6 g/100 g) [31].In most edible vegetables, the moisture content is above 80%, and is sometimes more than 90%, as in spinach, cabbage, or lettuce [32].Higher moisture levels make vegetables more appealing to consumers as it is perceived as showing freshness [32,33].Ash content is associated with the minerals that plants retain and is largely correlated with salts concentration in the soil, meaning that halophytes are rich in this nutrient [33].This is corroborated by the present findings (18.6-27.5 g/100 g, Table 1), wherein values are similar to those found in different Hottentot-fig organs (21.4-32.0g/100 g) [13,15], but are higher than in its fruits (10.1 g/100 g) [31].Despite being rich in ash, levels in different halophyte species can vary from lower contents, such as 10.5 g/100 g in Atriplex nitens [34], to higher contents, such as 43.3 g/100 g in Sarcocornia fruticosa [35].Comparatively speaking, Hottentot-fig's ash contents can be considered high, even more so when compared to most vegetables, where the ash content usually ranges between 5 and 10 g/100 g [36], as found in tomatoes (7.1 g/100 g) or potatoes (10.4 g/100 g), or the ash-rich spinach (17.3-22.3g/100 g) [37].Crude protein, an estimation of the protein content in the samples, ranged between 1.6 and 6.2 g/100 g (Table 1), values similar to those in other reports for the Hottentot-fig's organs (4.6-5.4 g/100 g) [13,15]; its fruits, however, are described as a much better protein source at 23.5 g/100 g [31].Likewise, other halophytes have been considered good protein sources, with values varying between 5 g/100 g (Climacoptera lanata or Salicornia ramosissima) and 13 g/100 g (Arthrocnemum macrostachyum or Atriplex nitensi) [33,38], much like the values for regular edible vegetables, which can range from 0.65 g/100 g (cucumbers) to 5.42 g/100 g (peas) [32].Vegetables typically possess low levels of fat [32], but the crude fat content in the Hottentot-fig's aerial parts was particularly low, varying between 0.6-1.3g/100 g (Table 1), in comparison to those described in the Hottentot-fig's leaves, flowers, and fruits (1.7-4.4 g/100 g) [13,15,31].While halophyte species can contain higher crude fat levels, as, for example, 6.9-7.8 g/100 g for Mesembryanthemum nodiflorum [35], most common vegetables contain less than 1% fat, namely 0.12 g/100 g in asparagus or 0.39 g/100 g in spinach [32], which is lower than was currently found in the aerial parts.Conversely, carbohydrate contents were high (66.2-78.5 g/100 g, Table 1), as they are usually the second most abundant component after water [32], but carbohydrates were estimated by difference, which may have caused an overestimation of its contents.Nevertheless, the recorded values are analogous to those described for different Hottentot-fig's organs (61.4-70.2g/100 g) [13,31] and other halophytes, such as Crithmum maritimum (69.5 g/100 g) [39] or Juncus maritimus (73 g/100 g) [40], although they were higher than in most vegetables (e.g., leek at 14.2 g/100 g or yam at 27.9 g/100 g) [32].As for fiber, estimated in terms of neutral detergent fiber (NDF), comprising levels of cellulose, hemicellulose, and lignin [41], it is considered a good indicator of feed quality and is used as an assessment of insoluble dietary fiber [42].The values for the Hottentot-fig's aerial parts (17.1-26.0g/100 g, Table 1) are slightly lower than those reported for its leaves (30.4 g/100 g) [15], but overall, the results are akin to levels found in other halophytes, such as Salicornia and Sarcocornia species (20.8-22.5)[33], or other vegetables, such as carrot (18.2 g/100 g) or cauliflower (21.3 g/100 g) [43].
Overall, this invasive plant's aerial parts show adequate nutritional potential, akin to those from regularly consumed vegetables and edible halophytes, particularly as a source of carbohydrates, proteins, and minerals such as Na, Ca, Fe, Mn, and Zn.The best season and location for optimized nutrient exploration of the discarded plant material was not clear, but a tendency was noticeable for higher concentrations of the most relevant minerals and protein in autumn and winter and in plants harvested from sites A and B.

Chemical Profiling
The seasonal and spatial phenolic composition of Hottentot-fig extracts were assessed in terms of their phenolic groups, namely, total phenolics (TPC), flavonoids (TFC), and condensed tannins (CTC); the results are depicted in Figure 4.
Hottentot-fig extracts presented high levels of all phenolic groups, particularly of TPC, which was above 200 mg GAE/g extract DW (GAE-gallic acid equivalents) in all locations and seasons, considering the threshold of 20 mg/g that many authors apply to deem an extract rich in phenolic contents [51][52][53].Higher TPC values were noted in spring (site B; 311 mg GAE/g) and summer (site C; 312 mg GAE/g), while the lowest values were registered in winter (all sites; 213-254 mg GAE/g).These values fall within the wide range (6-378 mg GAE/g extract) reported by other authors for various plant organs and extract solvents [11][12][13]15,54].Tannins were also exceptionally high, varying from 125 to 250 mg CE/g extract DW (CE-catechin equivalents), especially when compared with other studies: 58-101 mg RE/g in its leaves (hexane, dichloromethane, ethyl acetate, and methanol) [15], or 0.4-20.3mg CE/g in Hottentot-fig's fruits (water, ethanol, and acetone extracts) [11].Following a similar trend to TPC, CTC values were higher in spring (site C; 235 mg CE/g) and autumn (sites B and C; 250 and 229 mg CE/g, respectively), and lower in winter (all sites; 125-144 mg CE/g).As for TFC, except for the higher content in summer (site A; 67 mg QE/g; QE-quercetin equivalents), no discernible trend was evident, and values were lower than described for leaves (298-587 mg CE/g) [12,15], but higher than those in fruits (0.01-1.6 mg QE/g) [11].Seasonal/location variability on polyphenolic composition would be expected, considering that phenolic content in plants can be influenced by factors such as weather, geography, and environmental stressors, in addition to the effect of organ/tissue and extraction methods [51,55].This can also help explain the differences in phenolic groups contents between the current results and those of other studies.Increased levels of total phenolics in spring and summer could result from the environmental challenges that are typical of these seasons (e.g., high salinity, UV exposure, drought, or elevated temperatures, as can be seen in Table 2) that induce the plant's production of these secondary metabolites as a coping strategy.For example, Parida et al. [20] describe significant increases in the levels of total polyphenols with increasing salinity.The same factors could account for location variations, as plants from sites B and C showed higher levels of polyphenols; these locations are closer in proximity, thus potentially experiencing more similar environmental challenges.Additionally, during spring, the aerial parts collected included the flowers (see Figure 1b), which could imply possible organ-related variations.Still noteworthy is the extracts' high tannin content as tannins hold important potential applications, namely, a role in sensory taste perception [56] and the prevention of cardiovascular disorders or other chronic diseases, such as diabetes mellitus [57].
The individual phenolic compounds present in Hottentot-fig's extracts throughout the seasons and sites were also identified and quantified by HPLC-DAD, as summarized in Table 3 (chromatograms in Figure 5).Out of the seven compounds identified, six were phenolic acids, namely gentisic, salicylic, chlorogenic, caffeic, coumaric, and ferulic acids, and one was a flavonoid, luteolin-7-O-glucoside (molecular structures in Figure 6).were lower for Fe (46-339 mg/100 g) [35].Vegetables such as beans, peas, chickpeas lentils, or spinach are excellent sources of Fe (1.0-11.1 mg/100 g), Mn (0.2-2.6 mg/100 g) and Zn (0.2-4.3 mg/100 g) [46], the levels of which are comparable to those determined in the Hottentot-fig's aerial parts.Moreover, potentially toxic elements such as Ni, Cd, and Pb were either below the reference value for tolerable upper intake levels (Ni, 1 mg/day) [49], below the legislated value (0.3 mg/kg wet weight for Pb) [50], or were not detected (Cd, below LOQs; Table 1).
Overall, this invasive plant's aerial parts show adequate nutritional potential, akin to those from regularly consumed vegetables and edible halophytes, particularly as a source of carbohydrates, proteins, and minerals such as Na, Ca, Fe, Mn, and Zn.The best season and location for optimized nutrient exploration of the discarded plant material was not clear, but a tendency was noticeable for higher concentrations of the most relevant minerals and protein in autumn and winter and in plants harvested from sites A and B.

Chemical Profiling
The seasonal and spatial phenolic composition of Hottentot-fig extracts were assessed in terms of their phenolic groups, namely, total phenolics (TPC), flavonoids (TFC), and condensed tannins (CTC); the results are depicted in Figure 4.      3.  3.  3.

Caffeic acid Chlorogenic acid Coumaric acid Ferulic acid
Gentisic acid Luteolin-7-O-glucoside Salicylic acid  3; adapted from Phenol-Explorer 2023 [58]).In agreement with the results of the TPC, the levels of individual phenolics were higher in spring and summer, although their sum was increased in autumn for sites B and C, mostly due to higher concentrations of the flavonoid being detected.In fact, luteolin-7-O-glucoside, together with salicylic acid, were the most abundant compounds in the samples from sites B and C, while in site A, the most abundant were salicylic and caffeic acids, together with the flavonoid.The prevalence of these compounds may be rooted in the role that phenolic compounds play in plants exposed to harsh environmental constraints, as is the case with halophytes.Luteolin derivatives seem to predominate in plants under full sun, as is usually the case with Hottentot-fig, and is even more predominant in tissues exposed to greater sunlight irradiance, likely serving photoprotective roles [59].Luteolin-7-O-glucoside is also reported to accumulate in response to soil salinity [59], which might contribute to its predominance in the current Hottentot-fig's samples; it was also a main compound found in Hottentot-fig from Tunisia  3; adapted from Phenol-Explorer 2023 [58]).
In agreement with the results of the TPC, the levels of individual phenolics were higher in spring and summer, although their sum was increased in autumn for sites B and C, mostly due to higher concentrations of the flavonoid being detected.In fact, luteolin-7-Oglucoside, together with salicylic acid, were the most abundant compounds in the samples from sites B and C, while in site A, the most abundant were salicylic and caffeic acids, together with the flavonoid.The prevalence of these compounds may be rooted in the role that phenolic compounds play in plants exposed to harsh environmental constraints, as is the case with halophytes.Luteolin derivatives seem to predominate in plants under full sun, as is usually the case with Hottentot-fig, and is even more predominant in tissues exposed to greater sunlight irradiance, likely serving photoprotective roles [59].Luteolin-7-O-glucoside is also reported to accumulate in response to soil salinity [59], which might contribute to its predominance in the current Hottentot-fig's samples; it was also a main compound found in Hottentot-fig from Tunisia (water and aqueous-ethanol extracts from leaves) [54].Salicylic acid is linked to stress tolerance in plants, namely, their adaptation to saline stress [60], having been recognized not only as a regulatory response to abiotic stress but also as a defensive reaction for plant immunity and a hormone in growth and development regulation [61].It was also found as a major phenolic in Hottentot-fig collected near location C (in the ethyl acetate extracts from leaves) [15].Caffeic acid is also involved in the plants' mechanisms of stress tolerance; for example, its higher concentrations increase salt tolerance in plants under salinity stress, enhances plant growth when under drought stress, and improves resistance to stress overall via antioxidant activity [62].Similarly to these two phenolic acids, gentisic acid has been reported as a signaling molecule in plant defense [63] and in response to salinity stress [64].Furthermore, several polyphenols are overall involved in cell wall thickening, which confers drought tolerance by mechanically reinforcing the plant tissues and boosting resistance to oxidative stress, which are both key features in the endurance of halophyte plants [59].For example, ferulic acid is a component of plant cell walls: it binds to lignin, polysaccharides, and proteins, acting as a universal connector between the cell wall polymers [65].Coumaric acid is also an essential cell wall component, being an important precursor to lignin and other polyphenols [66].Moreover, in stressful environments, strong antioxidants such as chlorogenic acid are produced to grant the plant protection against oxidative stress; this phenolic acid is also considered a facilitator in the abiotic stress-response mechanisms of plants [67,68].
Other phenolic compounds that have been reported in Hottentot-fig [9,[11][12][13][14][15]54] were not herein identified, while gentisic acid is, to the best of our knowledge, currently described in Hottentot-fig for the first time.Variations in the phenolic profile of extracts of a same species are likely due to different geographical, climatical, and/or genetic conditions, although the influence of different methodological procedures, such as sample preparation, extraction, and analysis should not be discarded.Nevertheless, it is worth mentioning that not all compounds in the extracts were identified, as can be seen in the chromatograms in Figure 5, due to the complexity of the extracts' mixtures.

Bioactivity Profiling
The seasonal and spatial variations in terms of the antioxidant activity of Hottentot-fig extracts are presented in Table 4. Considering the multifaceted aspects of antioxidants and their consequent reactivity, the extracts were assessed for radical scavenging and metal chelating activity using complementary in vitro methods, namely, radical-based (scavenging activity against radicals DPPH• [1,1-diphenyl-2picrylhydrazyl], ABTS•+ [2,2azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)], and NO (nitric oxide)) and metal-related (reducing iron capacity (FRAP), and chelating on iron (ICA) and copper (CCA)) assays.
Hottentot-fig extracts were overall effective radical scavengers as almost all samples showed EC 50 values that were significantly lower than those of the positive controls for the three tested radicals (DPPH•, EC 50 (BHA) = 0.6 mg/mL; ABTS•+, EC 50 (BHA) = 0.33 mg/mL; NO, EC 50 (ascorbic acid) = 1.71 mg/mL; Table 4).The higher activities toward DPPH• and ABTS•+ had EC 50 values ranging between 0.05 and 0.11 mg/mL in spring and summer for all locations (also in winter for sites A and B, for ABTS•+), while the stronger NO scavenging was between 0.18 and 0.26 mg/mL (IC 50 values in spring for all locations, plus winter in site C; Table 4).Other authors reported divergent radical scavenging activity (RSA) of this invasive halophyte.Against DPPH•, the leaves' extracts were either active only at > 1 mg/mL (in methanol and ethyl acetate extracts) [15], had 20-50% activity at 0.1 mg/mL (in water and aqueous ethanol extracts) [54], or showed IC 50 values of 0.11 mg/mL (in water extract) [12] and 0.02 mg/mL (in aqueous-ethanol extract) [13].Regarding ABTS•+, the RSA of extracts from Hottentot-fig's fruits varied with EC 50 values of 0.56 to 6.4 mg/mL (in water, ethanol, and acetone extracts) [11], while against NO, the leaves' extracts showed 20-50% activity at 0.1 mg/mL (in water and aqueous ethanol extracts) [54].Notwithstanding the diverse reports in the literature, the current RSA results were comparable to the best ones described by other authors.The extracts were also strong iron reducers since samples with the highest activity (EC 50 = 0.10-0.29 mg/mL in spring and autumn for all sites) were as potent as the positive control BHA (EC 50 = 0.16 mg/mL; Table 4).Comparatively, the water extracts from leaves showed stronger activity (EC 50 = 0.03 mg/mL) [12], while the methanol extracts were weaker (45% at 1 mg/mL) [15].As for the chelating properties of Hottentot-fig extracts, they were moderate for copper, compared with the positive control EDTA (EC 50 = 0.16 mg/mL), and were low for iron (EC 50 > 10 mg/mL; Table 4).The best samples in CCA showed EC 50 values from 1.84 to 1.99 mg/mL (spring in sites B and C; Table 4), which are comparable to those obtained with hexane and dichloromethane extracts from leaves (45-50% activity at 1 mg/mL) [15].Differences between the present results and the literature reports on the antioxidant activity of Hottentot-fig can most likely be pinpointed to the use of different extraction solvents/methods and/or a different sampling season, not discarding environmental variations, all these being factors that can influence the antioxidant profile of natural extracts [81].Values represent the mean ± SD of at least three experiments performed in triplicate (n = 9).For each assay (column), different letters represent significant differences (p < 0.05).
Overall, there was a clear tendency toward higher RSA in spring and summer and higher metal-related capacities in spring and autumn for all locations.Conversely, RSA in autumn and metal-related capacities in winter were weaker.As for spatial variation, there is no significant discrimination between locations, which is an opposite pattern to that observed for the nutritional profile, where spatial variability seemed to have more impact (Figure 3).Nevertheless, the closer association of spring and summer, and of autumn and winter, seemed to be constant, having been depicted throughout the nutritional, chemical, and bioactivity profiles.The higher antioxidant activity noted in spring and summer can probably be attributed to the higher phenolic content that was also perceived in this period, considering that the relationship between antioxidant activity and TPC is frequently reported [11,27,29,35,52,53,67,82,83].Moreover, individual phenolics also had higher levels overall in spring and summer.Phenolic compounds are powerful antioxidants that play a fundamental role against oxidative stress, not only physiologically in plants but also pharmacologically in terms of human health.They can protect cellular components against oxidative deterioration, limiting the onset of oxidative stress-associated diseases [84,85].
The extracts were further assessed for their anti-inflammatory activity by determining their capacity to decrease nitric oxide (NO) production when stimulating RAW 264.7 macrophages to produce NO (with bacterial lipopolysaccharide, LPS) in a chronic inflammatory state.The effect of seasonal and spatial variations on the anti-inflammatory activity of Hottentot-fig extracts is represented in Figure 7.
The extracts were further assessed for their anti-inflammatory activ determining their capacity to decrease nitric oxide (NO) production when stim RAW 264.7 macrophages to produce NO (with bacterial lipopolysaccharide, LP chronic inflammatory state.The effect of seasonal and spatial variations on th inflammatory activity of Hottentot-fig extracts is represented in Figure 7. Hottentot-fig extracts (tested at 100 μg/mL) were able to reduce NO producti although no clear tendency can be depicted, a higher percentage of NO decrea registered in summer in site B (69.5%) and in autumn in site A (67.5%), with activity to that of the positive control, L-Name (73.4%, tested at 200 μg/mL).Addit all extracts were able to reduce NO production by more than 50%, except the ext samples from sites A and C in spring and site B in autumn (44.7-47.9%NO decrea anti-inflammatory activity of Hottentot-fig had previously been reported by Oliveira et al. [14] in a hydro-ethanolic extract that inhibited NO by 50% at 237 μg/mL (EC50 value), which can be considered less active than has presentl demonstrated.Of the phenolic compounds identified in Hottentot-fig extracts (T all seven of them have recognized anti-inflammatory activity, as described in the lit [65,66,70,74,77,79,80], which potentially explains the results that were obtained.O chlorogenic acid (5-caffeoylquinic acid) was also identified by Garcia-Oliveira et in their Hottentot-fig samples.Nevertheless, as previously stated, not all compo the extracts were identified; therefore, other molecules in the samples could als contributed to the anti-inflammatory effects that were observed.Hottentot-fig extracts (tested at 100 µg/mL) were able to reduce NO production and, although no clear tendency can be depicted, a higher percentage of NO decrease was registered in summer in site B (69.5%) and in autumn in site A (67.5%), with similar activity to that of the positive control, L-Name (73.4%, tested at 200 µg/mL).Additionally, all extracts were able to reduce NO production by more than 50%, except the extracts of samples from sites A and C in spring and site B in autumn (44.7-47.9%NO decrease).The anti-inflammatory activity of Hottentot-fig had previously been reported by Garcia-Oliveira et al. [14] in a hydro-ethanolic extract that inhibited NO by 50% at 237.9 ± 5.8 µg/mL (EC 50 value), which can be considered less active than has presently been demonstrated.Of the phenolic compounds identified in Hottentot-fig extracts (Table 4), all seven of them have recognized anti-inflammatory activity, as described in the literature [65,66,70,74,77,79,80], which potentially explains the results that were obtained.Of these, chlorogenic acid (5-caffeoylquinic acid) was also identified by Garcia-Oliveira et al. [14] in their Hottentotfig samples.Nevertheless, as previously stated, not all compounds in the extracts were identified; therefore, other molecules in the samples could also have contributed to the anti-inflammatory effects that were observed.
Lastly, the seasonal and spatial variation of the bioactivities and polyphenolic contents, together with the nutritional profile, were correlated using Principal Component Analysis (PCA) (Figure 8), to uncover a pattern for the observed results.The PCA discriminated between seasons, placing spring and summer in the lower quadrants and autumn and winter in the upper quadrants (except site B in winter); this distribution in the vertical F2 axis accounts for 25.5% of the variation.Autumn and winter were also clearly separated, the first to the left and the latter to the right of the PCA, while spring and summer were evenly distributed; this horizontal distribution (F1) accounts for 37% of the variation.Overall, this placing of seasons by the PCA confirms the closer link between spring and summer, and autumn and winter, as portrayed in the nutritional, chemical, and bioactivity profiles.Of note, spring and summer seem to have a greater influence from the polyphenolic groups (TPC and TFC) and autumn (site A) from most minerals.
clearly separated, the first to the left and the latter to the right of the PCA, while spring and summer were evenly distributed; this horizontal distribution (F1) accounts for 37% of the variation.Overall, this placing of seasons by the PCA confirms the closer link between spring and summer, and autumn and winter, as portrayed in the nutritional, chemical, and bioactivity profiles.Of note, spring and summer seem to have a greater influence from the polyphenolic groups (TPC and TFC) and autumn (site A) from most minerals.

Sample Collection, Processing, and Extraction
Hottentot-fig's aerial parts (leaves, shoots, and flowers when present) (voucher code XBH26, XtremeBio laboratory herbarium, Faro, Portugal) were harvested throughout the year (2020; meteorological values are given in Table 2) in winter (January), spring (May), summer (July), and autumn (November), in 3 locations in southern Portugal: (A) the Ria de Alvor lagoon near Portimão (37 Freshly collected biomass was dried in a ventilated oven (105 • C, 16 h) to determine moisture content.Previously dried samples were analyzed for contents: of ash, by incineration (600 • C, 2 h) in a muffle furnace [86]; of crude protein, by assessing the total nitrogen (CHN Elemental Analyzer Vario EL III) and following the macro-Kjeldahl method (N × 6.25) [87]; of total lipids (crude fat), using a modified Bligh and Dyer method [88]; of total carbohydrates, calculated by difference.The results are conveyed as g/100 g DW.Neutral detergent fiber (NDF) content was assessed following the directive of the International Organization for Standardization (ISO), used to analyze animal feedstuffs (ISO 16472:2006), and the results are expressed as g/100 g DW.

Mineral Composition
Dried biomass was also assessed for mineral content: samples were digested with a Microwave Digestion System (Discover SP-D 80, CEM Corp., Matthews, NC, USA) with 67% nitric acid (4 min ramp temperature to 200 • C, holding for 3 min), diluted with ultrapure water (1:10), and analyzed with a Microwave Plasma-Atomic Emission Spectrometer (MP-AES; Agilent 4200 MP-AES, Agilent, Victoria, Australia), as described by Pereira et al. [89].The results are conveyed as g/100 g DW for macro and as mg/100 g DW for trace elements.The total phenolic (TPC), flavonoid (TFC), and condensed tannin (CTC) contents of the extracts were assessed via colorimetric assays, as fully described by Oliveira et al. [27].The Folin-Ciocalteu reagent was used for TPC determination, measuring the absorbance at 725 nm (Biochrom EZ Read 400, Santa Clara, CA, USA), and using gallic acid as the standard for the calibration curve (1 µg/mL-1 mg/mL, R 2 = 0.999).The aluminum chloride (AlCl 3 ) assay was used in TFC evaluation, measuring the absorbance at 415 nm, and using quercetin as standard (1 µg/mL-1 mg/mL, R 2 = 0.999).The DMACA method was used in CTC assessment, measuring absorbance at 640 nm, and using catechin as standard (1 µg/mL-1 mg/mL, R 2 = 0.998).The results are expressed as mg of standard equivalents (GAE, QE, and CE, correspondingly) per g of extract DW.

Bioactivity Profiling
Extracts were tested in serial dilution concentrations (from 10-0.01 mg/mL) to allow calculation of the half-maximal effective concentrations (EC 50 values).Absorbances were measured using a microplate reader (Biochrom EZ Read 400, Santa Clara, CA, USA).Results were calculated as an activity percentage (%) in relation to a negative control (the solvent in redissolved samples), except for the ferric-reducing antioxidant power (FRAP) assay that was in relation to the positive control, and were expressed as EC 50 values (mg/mL) whenever possible.

In Vitro Anti-Inflammatory Activity
The extracts' anti-inflammatory properties were tested using lipopolysaccharide (LPS) to stimulate the RAW 264.7 macrophages to produce nitric oxide (NO), as described by Rodrigues et al. [90].The murine leukemic macrophage cell line (RAW 264.7) was kindly provided by the Mountain Research Center (CIMO, Bragança Polytechnic Institute, Portugal).Cells were cultured in RPMI medium, supplemented with 10% heat-inactivated fetal bovine serum, 1% L-glutamine (2 mM), and 1% penicillin (50 U/mL)/streptomycin (50 µg/mL), and were kept at 37 • C in a 5% CO 2 humidified atmosphere.First, cells seeded at 10 × 10 3 cells/well were incubated for 24 h with extracts at 100 µg/mL (diluted in culture medium) to evaluate cellular viability using the MTT assay.Non-treated cells in a culture medium containing DMSO (0.4%, in the same proportions as in the extracts) were used as a negative control.Once established that extracts at 100 µg/mL allowed more than 80% of cell viability (non-cytotoxic), they were incubated for 24 h with cells seeded at 2.5 × 10 5 cells/well (in a serum-and phenol-free medium) and LPS (25 µg/mL).L-Name was used as a positive control.The Griess method was followed to determine the NO production, using sodium nitrite as a standard in the calibration curve.Non-treated, LPS-stimulated cells, in a culture medium containing DMSO (0.4%), were used as the negative control.

Statistical Analysis
All experiments were performed in triplicate.The results are expressed as mean ± standard deviation (SD).GraphPad Prism 8.4.3 for Mac (GraphPad Software, San Diego, CA, USA) was used to curve fit data and obtain EC 50 values.Statistical differences (p < 0.05), analyzed using the XLSTAT trial version for Mac (Addinsoft 2023, New York, NY, USA), were assessed with a one-way analysis of variance (ANOVA), followed

Figure 5 .
Figure 5. HPLC-DAD analysis (320 nm) of C. edulis (Hottentot-fig) extracts throughout the seasons and locations.Peak numbers refer to the compounds in Table3.

21 Figure 5 .
Figure 5. HPLC-DAD analysis (320 nm) of C. edulis (Hottentot-fig) extracts throughout the seasons and locations.Peak numbers refer to the compounds in Table3.

Figure 5 .
Figure 5. HPLC-DAD analysis (320 nm) of C. edulis (Hottentot-fig) extracts throughout the seasons and locations.Peak numbers refer to the compounds in Table3.

Table 2 .
[28]orological values of the mean temperatures (minimum and maximum) and total precipitation registered in the collection months (source: IPMA 2020)[28].
* X min.temp.: mean of the minimum temperature; X max.temp.: mean of the maximum temperature.

Ria Formosa Lagoon nº (min) Winter Spring Summer Autumn Winter Spring Summer Autumn Winter Spring Summer Autumn
RT: retention time; -: not detected.Peaks are numbered according to retention time.