Season affects three insufficiently studied seaweed species (Bifurcaria bifurcata, Codium sp., Ericaria selaginoides): bioactivity alterations

ABSTRACT The biological activity and nutraceutical potential of two brown seaweed species (Bifurcaria bifurcata, Ericaria selaginoides) and one green seaweed (Codium sp.) were evaluated taking seasonal effects into account. The phenolic content of B. bifurcata was higher than those of the other two species regardless of season, 10.13–11.01 mg Gallic Acid Equivalent (GAE) g–1 dw seaweed. The ethanol extracts of B. bifurcata were more antioxidant as measured by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) technique than the equivalent extracts from E. selaginoides, 0.428–0.442 mg Ascorbic Acid Equivalent (AA Eq) g dw seaweed–1 vs 0.338–0.361 mg AA Eq g dw seaweed–1. Regarding Ferric Ion Reducing Antioxidant Power (FRAP), the highest activity was measured in Codium sp. extracts and no difference was detected between B. bifurcata and E. selaginoides. The 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) results differed from the FRAP values in that the ethanol extracts from Codium sp. were less antioxidant as measured by ABTS than those of the other two species. Summer E. selaginoides extracts were more antioxidant. The most anti-inflammatory extract was obtained from E. selaginoides harvested in Summer, exhibiting 94.2 ± 4.0% inhibition of cyclooxygenase-2 (COX-2). The extracts from Codium sp. had the lowest activity, 13.6–15.5%. For both B. bifurcata and E. selaginoides, Summer extracts were more anti-inflammatory than Winter extracts.


Introduction
There is an important and still largely untapped potential in seaweeds. These marine resources contain highly relevant bioactive compounds, such as specific carbohydrates and peptides or polyphenols (Pradhan et al., 2022). These substances may have antioxidant, antiinflammatory or antitumoral activities (Andrade et al., 2013;Rengasamy et al., 2020), which, in turn, may help in the prevention and cure of diseases or unhealthy conditions, such as high cholesterol, high blood pressure, and some kinds of cancer (Holdt & Kraan, 2011;Jimenez-Lopez et al., 2021;Sato et al., 2002). Accordingly, seaweeds can be employed in the pharmaceutical and nutraceutical industries, cosmetics, fertilizer, and food sectors (Pradhan et al., 2022). More specifically, phenolics from seaweeds can be used as natural ingredients in different industries (Jimenez-Lopez et al., 2021).
In this context, it should be noted that there are still many seaweed species that are poorly known, including species from temperate waters, such as those on European Atlantic shores. They include the selected seaweed species for the current study, Bifurcaria bifurcata R.Ross (Algaebase, 2018), Codium sp., and Ericaria selaginoides [(Novoa & Guiry, 2020), recently known as Carpodesmia tamariscifolia and previously as Cystoseira tamariscifolia (Hudson) Papenfuss (Algaebase, 2018)]. They are all from the Portuguese coast and relatively abundant -particularly on the coast near Lisbon, where they have a key ecosystem role. While B. bifurcata and E. selaginoides are brown seaweed species that belong to the order Fucales, Codium sp. is a green seaweed belonging to the order Bryopsidales (Algaebase, 2018). These three species are included in the category of edible seaweeds (Pereira, 2016), thus also justifying their study from the point of view of future nutraceutical applications of the whole algal biomass.
Moreover, fucoidans, a type of polysaccharide containing substantial percentages of L-fucose studied for their anticoagulant, anti-inflammatory, and antitumour activities, have been reported in different brown seaweeds (Li, Lu, Wei, & Zhao, 2008). For instance, these sulphated polysaccharides have been extracted from Sargassum henslowianum and Fucus vesiculosus (Ale, Maruyam, Tamauchi, Mikkelsen, & Meyer, 2011). They were able to limit the proliferation of melanoma cells in a dose-response way via induction of apoptosis with activation of caspase-3. Other biological activities of such polysaccharides have also been claimed, such as antioxidant, antithrombotic, immunoregulatory, and antiviral activities (Wang et al., 2019).
Concerning green seaweeds, in the genus Codium, a glycoprotein isolated from C. decorticatum displayed anticancer potential by inhibiting cell growth in breast, cervical, and lung cancer cells (Senthilkumar & Jayanthi, 2016). Furthermore, in an assessment of methanol extracts from 21 seaweed species, E. selaginoides was reported to have the highest acetylcholinesterase inhibitory capacity (85%, 10 mg ml -1 ), to be active against butyrylcholinesterase and tyrosinase and to protect SH-SY5Y cells against oxidative stress induced by H 2 O 2 (Custódio et al., 2016).
With respect to these biological activities and bioactive compounds, comparison across studies is often rendered more difficult by the use of different extracts, procedures, and measurement units. Literature indicates a wide variability of total phenolic contents in seaweed samples, including those of the same species. This may be related to geographical and seasonal variability as well as life stage and age of the specimens or the genetics of particular populations (Cotas et al., 2020;Domínguez, 2013). Polyphenol content discrepancies between studies may be related to UV radiation level variability (depending on location and season), since higher UV exposure may stimulate particular metabolic pathways that generate higher phenolic contents (Bischof et al., 2006). This is related to the protection from UV damage offered by these substances (Zubia, Robledo, & Freile-Pelegrin, 2007). Therefore, the aim of this study was to evaluate the biological activity of three insufficiently studied seaweed species, the brown seaweed species Bifurcaria bifurcata and Ericaria selaginoides and the green seaweed Codium sp., by the determination of antioxidant and antiinflammatory activities and also the analysis of total phenolic and fucose contents. Furthermore, this study was also designed to assess the effect of seasonality on these biological activities and bioactive contents.

Seaweed source, collection, and preparation
The studied brown seaweed species Bifurcaria bifurcata and Ericaria selaginoides and green seaweed species Codium sp. were identified and harvested at Cascais near Farol do Cabo Raso, Portugal (38°42ʹ38.7" N 9° 29ʹ09.7" W). The harvest was carried out in two tidal rock pools in the indicated area, removing the whole thallus of larger seaweed specimens. Care was taken to select the cleanest parts. Approximately 1 kg of biomass of each seaweed was collected and put into separate bags. Samples for the two seasons in the study, summer and winter, were collected on 22 August and 15 November 2020. It was observed that the abundance of seaweed biomass and length of the thallus were greater in summer than in winter, but, in spring, it was not possible to harvest due to the very early stage of growth of the seaweeds. After collection, samples were washed with seawater and immediately transferred to the Portuguese Institute for the Sea and Atmosphere (IPMA) in Lisbon. Thereafter, the fronds of the three seaweeds were rinsed with potable water to remove any remaining epiphytes and detritus. Finally, samples of each seaweed were frozen at -80°C, freeze-dried, ground, and stored individually at -80°C until further analysis.

Extract preparation
For the preparation of extracts, ethanol was chosen specifically as it was the most effective safe and environmentally acceptable solvent (others include 2-butanol, ethyl acetate, and isoamyl acetate). Accordingly, ethanol was added to freeze-dried seaweed biomass.
To determine antioxidant activity, approximately 0.5 g of freeze-dried brown seaweed (B. bifurcata/ E. selaginoides) biomass or 0.25 g of freeze-dried green seaweed (Codium sp.) biomass was used. The green seaweed biomass had a very low density, occupying much larger volumes than the other biomass, so was treated differently. The seaweed was weighed, homogenized with 10 or 5 ml of absolute ethanol, respectively, using a model Polytron PT 6100 homogenizer (Kinematica, Luzern, Switzerland) at a velocity of 30000 rpm for 1 min and agitated for 18 h on an orbital shaker. After centrifugation (5,000 × g at room temperature for 20 min), the supernatant was collected through a filter to a final volume of 10 ml (B. bifurcata/E. selaginoides) or 5 ml (Codium sp.), thereby corresponding to a concentration of 50 mg ml −1 . Extraction was carried out in quadruplicate from a single pool.
As to the anti-inflammatory activity, extracts were obtained from approximately 200 mg of freeze-dried seaweed homogenized with 2 ml of ethanol, using a model Polytron PT6100 homogenizer (Kinematica, Luzern, Switzerland) at a velocity of 30000 rpm for 1 min. The extracts were subjected to heat treatment (80°C for 1 h) and then centrifuged (3,000 × g at 4°C for 10 min) using a model Kubota 6800 centrifuge (KUBOTA, Tokyo, Japan). The supernatant was collected, and the solvent was evaporated under a nitrogen stream at room temperature using a model Reacti-Therm III n° 18,940 nitrogen evaporation unit (Pierce, East Lyme, CT, USA). The residue was directly dissolved in 100% dimethyl sulphoxide (DMSO) in order to prepare a stock solution with a concentration of 10 mg ml -1 . Extraction was carried out in duplicate from a single pool.

Total polyphenol content
Total polyphenol content was determined by the Singleton and Rossi method using the Folin-Ciocalteu reagent (Singleton & Rossi, 1965). Gallic acid (GA) was used as standard and phenolic content was expressed as gallic acid equivalents (mg GAE g dw -1 seaweed) through the calibration curve of gallic acid (Sigma, Steinheim, Germany). Analysis was done in triplicate.

Antioxidant activity as measured by the DPPH method
The antioxidant activity was measured through the determination of the radical scavenging activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Miliauskas, Venskutonis, & Van Beek, 2004). A volume of 1 ml of the extract was prepared in triplicate for each sample and 2 ml of DPPH (Sigma, Steinheim, Germany) 0.15 mM methanolic solution was added and thoroughly mixed. After 30 min of incubation at room temperature in the dark, absorbance was measured at 517 nm in a Helios Alpha model (Unicam, Leeds, UK) UV/visible light spectrophotometer. Ethanol was used as the blank.
Radical scavenging activity was calculated by the following formula: where: A 0 -Absorbance of the blank, A sample -Absorbance of the sample. Results were expressed in mg of ascorbic acid equivalents (AA Eq) per g dry weight of seaweed. Analysis was done in triplicate.

Antioxidant activity as measured by the FRAP method
The applied Ferric Ion Reducing Antioxidant Power (FRAP) method was a modified technique based on Benzie & Strain (1996) -absorbance was read at 595 nm instead of 593 nm. Results were expressed in μmol Fe 2+ equivalents per g dry weight of seaweed and compared with an ascorbic acid control. Analysis was done in triplicate.

Antioxidant activity as measured by the ABTS method
ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) radical scavenging activity was determined using the method described by Re et al. (1999). The ABTS radical scavenging activity of the samples was calculated as a percentage of inhibition: where:A 0 -Absorbance of the blank. A sample -Absorbance of the sample. Results were expressed in μmol of trolox equivalents (Trolox Eq) per g dry weight of seaweed. Analysis was done in triplicate.

Anti-inflammatory activity
The anti-inflammatory activity of the three seaweed species was tested at 1 mg ml −1 using a commercial cyclooxygenase (COX) inhibitory screening assay kit, Cayman test kit-560,131 (Cayman Chemical Company, Ann Arbor, MI, USA). A volume of 10 μl each of test extract or DMSO (blank) was used. Results were expressed as a percentage of inhibition of COX-2. Analysis was done in quadruplicate.

Fucose content
Free fucose was determined by the cysteine-sulphuric acid method for methyl pentoses. Triplicates (50 mg) of freeze-dried seaweed were placed into separate test tubes and mixed and homogenized at 30000 rpm with 1 ml of Milli-Q water using a model Polytron PT 6100 homogenizer (Kinematica, Luzern, Switzerland). Afterwards, samples were subjected to ultrasound treatment at 25°C for 15 min in a Sonorex Super 10 P model (Bandelin Electric, Berlin, Germany). Commercial L-fucose attained from Sigma (St. Louis, MO, USA) was used as the standard. About 4.5 ml of sulphuric acid (prepared by adding six volumes of concentrated sulphuric acid to one volume of water) was added into each tube (including tubes containing 1 ml of bioaccessible fraction of each seaweed) and mixed. Tubes were put into a boiling water bath for 3 minutes, then cooled and 0.1 ml cysteine hydrochloride solution (5% cysteine hydrochloride in Milli-Q water) was added to each tube and mixed. Absorbance was read at 396 nm and 427 nm, after zeroing the spectrophotometer with a water blank treated in the same way. Fucose-specific absorbance values were calculated according to the following expression: Absorbance = A396nm -A427nm (Dische & Shettles, 1948). Analysis was done in triplicate.

Statistical analysis
To test the normality and the homogeneity of variance of data, the Kolmogorov-Smirnov test and Levene's F-test, respectively, were used. Data were analysed by one-way ANOVA distribution using Tukey Honestly Significant Difference (HSD) to determine the difference in the constituents' contents/bioactivities between seaweed species or by a factorial ANOVA using the Tukey HSD to determine the difference in constituent contents/bioactivities between seaweed species and season. In the case of ABTS and FRAP, homogeneity of variance was not observed and non-parametric tests (Kruskal-Wallis test) were done. For all statistical tests, the significance level (α) was 0.05. All data analysis was performed using STATISTICA 6.

Total polyphenol content
The total polyphenol content of the ethanol extracts from the selected seaweed species (brown seaweed species: B. bifurcata and E. selaginoides; green seaweed species: Codium sp.) harvested in winter and summer is presented in Table 1.
B. bifurcata showed higher polyphenol levels than those from the other two species regardless of season. The phenolic content was 10.13-11.01 mg GAE g dw seaweed -1 . Codium sp. results were always the lowest (p < 0.05) and never exceeded 5 mg GAE g -1 dw. Regarding seasonality, while no effect was observed in the case of B. bifurcata, the other two seaweed species experienced an increase (p < 0.05) of the phenolic content in their biomass with the transition from Winter to summer. In particular, the phenolic content in Codium sp. increased (p < 0.05) from 3.88 ± 0.18 mg GAE g -1 dw in winter to 4.66 ± 0.29 mg GAE g -1 dw in summer and the phenolic content in E. selaginoides augmented (p < 0.05) from 6.44 ± 0.26 mg GAE g -1 dw in winter to 7.47 ± 0.34 mg GAE g -1 dw in summer.

Antioxidant activity
The antioxidant activity of the studied seaweed species (B. bifurcata, E. selaginoides, Codium sp.) harvested in winter or summer and as measured by the DPPH, FRAP, and ABTS techniques is shown in Tables 2-4, respectively.
With respect to DPPH, pigment interference in the extracts from Codium sp. prevented the application of this method to this seaweed species. The ethanol extracts from B. bifurcata were more antioxidant (p < 0.05) as measured by the DPPH technique than the equivalent extracts from E. selaginoides, 0.428- Values are presented as average ± standard deviation. Different lowercase letters within a row correspond to statistical differences (p < 0.05) between seaweed species. Different uppercase letters within a column correspond to statistical differences (p < 0.05) between seasons. 0.442 mg AA Eq g -1 dw vs 0.338-0.361 mg AA Eq g -1 dw. Season had effects on the antioxidant activity for both species, but in opposite directions. Indeed, whereas the DPPH value fell slightly by 3% from winter to summer in the case of B. bifurcata, it increased by 7% for the same seasonal determinations in E. selaginoides. A different assessment of the antioxidant power of the ethanol extracts was achieved with the FRAP method. In fact, the highest activity (p < 0.05) was measured in the extracts of Codium sp. and no difference (p > 0.05) was detected between B. bifurcata and E. selaginoides. Moreover, only Codium sp. showed a seasonal effect, with more antioxidant extracts (p < 0.05) in summer, 111.7 ± 1.2 μmol Fe 2+ Eq g -1 dw vs 89.9 ± 2.4 μmol Fe 2+ Eq g -1 dw. This represents an increase in excess of 20%.
The FRAP results contrast to the ABTS values. The antioxidant activity as measured by ABTS of the ethanol extracts from Codium sp. was much lower (p < 0.05) than that of the other two species. Moreover, differently from FRAP, the ABTS values of Codium sp. were lower (p < 0.05) in summer. In contrast, E. selaginoides was characterized by more antioxidant ethanol extracts (p < 0.05) in summer. Accordingly, ethanol extracts from Codium sp. harvested in summer had the lowest antioxidant values (p < 0.05), 8.9 ± 1.7 μmol Trolox Eq g -1 dw vs 42.7-45.0 μmol Trolox Eq g -1 dw.

Anti-inflammatory activity
The anti-inflammatory activity of the chosen seaweed species (B. bifurcata, E. selaginoides, Codium sp.) collected in the two studied seasons (winter and summer) is displayed in Table 5.

Fucose
The fucose levels in the studied seaweed species (B. bifurcata, E. selaginoides, Codium sp.) and seasons (Winter and Summer) are presented in Table 6.
This compound was not detected in Winter B. bifurcata. Almost all the other samples had similar low levels (p > 0.05), not exceeding 2 mg g dw seaweed -1 . The exception was summer Codium sp.

Total polyphenol content
The analysed phenolic contents broadly agree with the range reported in the literature for biomass rich in polyphenols, 1-50 mg GAE g -1 dw (Farasat, Khavari-Nejad, Nabavi, & Namjooyan, 2013;Mhadhebi, Mhadhebi, Robert, & Bouraoui, 2014). In any case, wider ranges of phenolic concentration in seaweed have been observed (Chkhikvishvili & Ramazanov, 2000). These authors found phenolic levels of less than Values are presented as average ± standard deviation. Different lowercase letters within a row correspond to statistical differences (p < 0.05) between seaweed species. Different uppercase letters within a column correspond to statistical differences (p < 0.05) between seasons. Values are presented as average ± standard deviation. Different lowercase letters within a row correspond to statistical differences (p < 0.05) between seaweed species. Different uppercase letters within a column correspond to statistical differences (p < 0.05) between seasons. Values are presented as average ± standard deviation. Different lowercase letters within a row correspond to statistical differences (p < 0.05) between seaweed species. Different uppercase letters within a column correspond to statistical differences (p < 0.05) between seasons.
Moreover, high extracting yields may also be achieved with "green" solvents, such as ethyl acetate (Vizetto-Duarte et al., 2016). In fact, a total phenolic content of 165.28 ± 1.92 mg GAE g -1 dw in C. tamariscifolia (currently E. selaginoides), an extremely high value, was determined by Vizetto-Duarte et al. (2016) using ethyl acetate as extracting agent.
UV exposure levels and their effects on seaweed may help explain seasonal variability. This may be relevant to our studied Codium sp. and E. selaginoides. It should be noted that studies on seasonality are scarce. In the case of Codium tomentosum, but cultivated and not harvested from the wild, Gonçalves (2018) detected differences between seasons, phenolic contents being higher in summer, approximately 3.50-5.00 mg GAE g -1 dw, than in Winter, 3.00-3.50 mg GAE g -1 dw. Celis-Plá et al. (2016) carried out a seasonal study of C. tamariscifolia (currently E. selaginoides), whose phenolic content results were not unequivocal. In fact, while in one year no seasonal effect on the total phenolic content (extracted with methanol) was observed, in another year the phenolic content increased steadily from summer to Spring, reaching values approximately twice the summer values, 64 mg Phloroglucinol Equivalent g dw -1 vs 28 mg Phloroglucinol Equivalent g -1 dw. This seems to contradict the possible influence of the UV irradiation and it is possibly related to the observation that peak phenol content may not occur summer, since nitrate levels may be limiting (Pavia & Åberg, 1996).

Antioxidant activity
As to DPPH, there was always substantial antioxidant activity in B. bifurcata and E. selaginoides, thereby broadly agreeing with relevant literature about seaweed and antioxidant activity (Fonseca et al., 2021;Regal et al., 2020), especially if compared to the ethanolic extracts. Vizetto-Duarte et al. (2016), also specifically for C. tamariscifolia (now E. selaginoides), obtained higher antioxidant activity as measured by DPPH, but in ethyl acetate extracts. Likewise, for B. bifurcata and methanol-water extracts, higher DPPH values have been measured (Le Lann et al., 2008). Seasonality of the antioxidant activity as measured by DPPH has been studied in the case of C. tamariscifolia (Celis-Plá et al., 2016). These authors did not find any difference between Summer and Winter, which compares to a very modest variation (< 10% increase) in the current study.
[Fucose] (mg g -1 dw) [Fucose] (mg g -1 dw) [ Values are presented as average ± standard deviation. N.D. -Not detected. Different lowercase letters within a row correspond to statistical differences (p < 0.05) between seaweed species. Different uppercase letters within a column correspond to statistical differences (p < 0.05) between seasons.
These values were obtained precisely with ethanol extracts. In the case of Codium sp., summer values surpassed to some extent the upper limit of this range, thereby highlighting a seasonal contrast to the Winter samples. The observed seasonal increase of ca. 20% paralleled the phenolic content increase in the same species (Table 1). In fact, the overall seasonal trend of increase from Winter to summer across the various species was mirrored in a relatively similar way by FRAP and total phenolic content. FRAP may correlate with the concentration of phenolic substances in the seaweed biomass due to the fact that phenolic compounds act as antioxidants through single electron transfer. Likewise, Alcalde, Granados, and Saurina (2019) have detected a high correlation between phenolic content and FRAP (R = 0.92), thus suggesting that the two parameters may be the result of similar redox phenomena.
All ABTS values do not depart much from results found for other seaweeds, which ranged between 5 and 60 μmol Trolox Eq g -1 dw (Campos et al., 2019) or between 16 and 37 μmol Trolox Eq g -1 dw (Fonseca et al., 2021). However, there are also studies with very different ABTS values for the same seaweed species. Agregán et al. (2018) observed very high values in water-ethanol (50:50, v/v) extracts from B. bifurcata, 538-549 μmol Trolox Eq g -1 dw. In addition, Raja et al. (2016) registered a higher level of ABTS inhibition by methanolic extracts from Codium fragile. Similarly, ethyl acetate extracts of C. tamariscifolia studied by Vizetto-Duarte et al. (2016) were shown to be more antioxidant as measured by ABTS than in this study. Of course, in all these three examples, different extracting solvents were used. Moreover, a comparable seasonal evaluation of ABTS was not found in the revised literature. There was an overall declining tendency from Winter to Summer that departs significantly from the total phenolic content variation. Hence, differently from FRAP, there was no good correlation between ABTS and total phenolic content.

Anti-inflammatory activity
Investigations of the anti-inflammatory activity of seaweed are rendered more difficult by the wide methodological variability, encompassing very different models (Campos et al., 2019;Jin et al., 2006;Kang et al., 2012a;Yang et al., 2010). A model used in different studies is based on the lipopolysaccharide-induced nitric oxide (LPINO) production. In this regard, a study on Sargassum siliquastrum (Kang et al., 2012a), a brown seaweed of the order Fucales, isolated a compound (sargaquinoic acid) that inhibited LPINO production in macrophages via modulation of specific pathways. Furthermore, Yang et al. (2010) verified a reduction in the lipopolysaccharide-induced expressions of inducible nitric oxide synthase and COX-2 at the protein level in a concentration-dependent manner by another brown seaweed, Petalonia binghamiae.
For those seaweed species chosen for the current study, no previous work concerning their antiinflammatory activity and using the same methodology has been found. However, the anti-inflammatory activity of B. bifurcata was studied through an in vitro model of inflammation consisting of macrophages stimulated with lipopolysaccharide (Santos et al., 2017). There was a reduction in NO production at a dichloromethane extract concentration of 50 μg ml -1 to levels similar to those usual in non-stimulated cells (Santos et al., 2017). This anti-inflammatory activity level was higher than that reported for other seaweed extracts, such as Saccharina japonica (Islam et al., 2013) or Undaria pinnatifida (Hwang, Y-S, & Lim, 2014). A similar methodology showed a substantial anti-inflammatory activity of a methanol extract of the green seaweed Codium fragile (Kang, Choi, Park, & Kim, 2012b). These authors reported that mRNA and protein expression of inducible NO synthase, COX-2 and Tumour Necrosis Factorα (TNF-α) were reduced by pre-treatment with the seaweed extract. In addition, Kang et al. (2012b) proposed that the methanol extract of Codium fragile downregulates the expression and secretion of inflammatory mediators by inhibition of Nuclear Factor kappa-Light-Chain-Enhancer of Activated B Cells (NF-κB) activity. As for Ericaria selaginoides, there are only studies on other species of Cystoseira (Mhadhebi et al., 2014). Mhadhebi et al. (2014) indicated an anti-inflammatory effect of aqueous extracts of Cystoseira compressa, Cystoseira crinita, and Cystoseira sedoides in the rat paw oedema test, that is similar to dexamethasone treatment, De La Fuente et al. (2021) reported a remarkable anti-inflammatory potential of ethanolic extracts from Cystoseira amentacea.
On the other hand, a comparison can be done for other seaweed (or, possibly, plant) species and extracts whose anti-inflammatory activity was assessed by an identical method based on inhibition of COX-2. Indeed, there are studies that also used 10%, w/v, seaweed extracts and that performed their tests at 1 mg ml −1 DMSO (Campos et al., 2019;Ripol et al., 2018). Ripol et al. (2018) applied the COX-2 inhibition method to aqueous extracts from green seaweeds and reported an inhibition between 31% and 45%. This is higher than what was measured with extracts from Codium sp., but near values measured for extracts from Winter B. bifurcata and E. selaginoides. Moreover, Campos et al. (2019) studied ethanol extracts from brown and red seaweeds, thereby detecting COX-2 inhibitory action in a single species, Halopteris scoparia, 79 ± 8%. A similar enzymatic inhibition methodology has been applied to plant extracts, such as 90%, v/v, ethanol extracts from Polygonum minus, at 100 μg ml -1 , yielding lower inhibition percentages, 25% (COX-2) (George et al., 2014).
There are many different groups of compounds displaying anti-inflammatory effects, such as phenolic compounds, carotenoids or quinones Máximo, Ferreira, Branco, Lima, & Lourenço (2018). However, there is no correlation between phenolic concentration (Table 1) in the ethanol extracts and the antiinflammatory activity. Hence, the anti-inflammatory activity of E. selaginoides and other seaweed species may be ascribed to other compounds, quinones being a possible candidate (García, Hernández, San Feliciano, & Castro, 2018;Máximo et al., 2018).

Fucose
It should be noted that there are already some studies concerning fucose in B. bifurcata. Fucoidans, whose main sugar in its polymer backbone is fucose, have been purified and characterized from Moroccan B. bifurcata (Bouissil et al., 2020). These authors found out that these fucoidans had 91% fucose and 6% galactose. No study on fucose in E. selaginoides was found, but fucans have been isolated from Cystoseira (a previous genus of Ericaria selaginoides) barbata harvested in Tunisia (Sellimi et al., 2014). Fucans were composed of 44.6% fucose. Nonetheless, quantification of seasonal variability of the fucose content in the three studied species has not been done previously. It should also be noted that within seaweeds fucoidans have only been found in brown seaweed (Zhao et al., 2018). Hence, detection of fucose in Codium sp. could result from an interference phenomenon, given the fact that the spectrophotometric detection of the analyte is not preceded by any fractionation or compound separation. Only future analytical work involving HPLC (Chen et al., 2018;Zou et al., 2021) could settle this issue.
In order to compare fucose levels obtained with those in the literature, other seaweed species may be considered. According to Ferreira et al. (2019), the amount of fucose recovered from F. vesiculosus varied between 0.911 mg g -1 dw and 50.75 mg g -1 dw, with the highest values being achieved when more drastic conditions are applied, such as 120°C for 4 h. These were harsher thermal treatment conditions than those applied in the current study, a boiling water bath for 3 minutes (see section Fucose content).
As a final remark, it must be stressed that further research is warranted regarding some of the studied biological activities. In particular, the relationship between antioxidant activity and specific phenolic compounds should be studied, entailing the determination of the phenolic profile in the extracts. In fact, the specific content of particular phenolic substances may determine the overall radical scavenging activity (Farvin & Jacobsen, 2013). The structure of each compound, namely, the amount and location of the hydroxyl groups, may be critical in determining the antioxidant activity (Brand-Williams, Cuvelier, & Berset, 1995). For instance, caffeic acid, with two hydroxyl groups, has a greater activity than coumaric acid, with only one hydroxyl group (Brand-Williams et al., 1995). On the other hand, the extremely high anti-inflammatory activity of the ethanol extracts from summer E. selaginoides is remarkable and also warrants further research, encompassing the importance of harvest and storage procedures (apparently optimal in the current study, since seaweed was carefully rinsed and quickly frozen at -80°C) as well as of particular compounds in the respective extract.

Disclosure statement
No potential conflict of interest was reported by the author(s).