Assessment of antioxidant capacity of Cystosphaera jacquinotii (Fucales, Ochrophyta) by two methods: DPPH and CUPRAC.

Assessment of antioxidant capacity of Cystosphaera jacquinotii (Fucales, Ochrophyta) by two methods: DPPH and CUPRAC. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Assessment of antioxidant capacity of Cystosphaera jacquinotii (Fucales, Ochrophyta) by two methods: DPPH and CUPRAC. Carolina Pena-Martín, Raquel Serrano, Guillermo Grindlay, Manuel B. Crespo This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4660453/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The present work was carried out to evaluate the antioxidant activity of Cystosphaera jacquinotii and to quantify its total phenolic content (TPC), compared with other fucal, Ericaria amentacea . Phenolic compounds occur in high quantities in brown algae and they have an essential paper in antioxidative action. Extracts were prepared with methanol dilution. The quantification methods used for the determination of the antioxidant capacity were CUPRAC and DPPH. From the obtained results it can be concluded that Cystosphaera jacquinotii presents a high TPC, which directly contributes to the strong antioxidant activity detected, even higher than Ericaria amentacea . Besides, this work evidences that the kinetics of the extracts in DPPH assays is a markedly variable factor that must be considered to assess the antioxidant capacity of seaweeds species. Furthermore, standardized extraction methods and antioxidant activity analyses are needed to properly compare data and get the optimal ones. In the same way, more studies on the role of different compounds in antioxidant activity are wanting, which will help to get the best performance. Antioxidant activity DPPH CUPRAC total phenolic content Cystosphaera jacquinotii Ericaria amentacea. Figures Figure 1 Figure 2 Figure 3 INDRODUCTION Seaweeds have been used in pharmacological and food systems due to their bioactive compounds, such as phenolic compounds, fucoxanthin and polysaccharides (Grina et al. 2020). Recently, seaweeds have received special attention as a source of natural antioxidants (Sabeena et al. 2013; Senthilkumar et al. 2013; Pinteus et al. 2017). In fact, although they are exposed to a combination of light and high oxygen concentrations which lead to the formation of free radicals and other strong oxidizing agents, they seldom suffer from any serious photodynamic damage in vivo because their cells have protective antioxidative mechanisms as well as antioxidative compounds (Park et al. 2004). According to that, and since antioxidant compounds play an important role against various diseases (e.g. atherosclerosis, chronic inflammation, cardiovascular disorders and cancer) and aging processes (Kohen and Nyska 2002; Senthilkumar et al. 2013), research on antioxidant capacity in seaweeds has been increasing in recent years. Among all seaweeds, Brown algae play a significant role, as they have high content on phenolic compounds such as flavonoids, phenolic acids, and phlorotannins (present only in Brown algae), which may directly participate in antioxidative action (Ferreres et al. 2012; Grina et al. 2020). Phenolic compounds are secondary metabolites involved in different protection mechanisms against grazers, epiphytes and UV radiation (Mannino et al. 2015). Audibert et al. (2010) concluded that the antioxidant activity of the extracts was related to the content of phlorotannins and to their molecular weight. Besides, the induced synthesis of phlorotannins is crucial for Brown macroalgae to tolerate environmental stress and, therefore, it can be regarded as a primary defence mechanism against episodic stress by elevated temperature and solar radiation (Flores-Molina et al. 2016). Specifically, species belonging to the order Fucales are particularly rich in antioxidants. In fact, many species of this group have already been evaluated in relation to their high antioxidant capacity (Ferreres et al. 2012; Jassbi et al. 2013; Ryabushko et al. 2014; Vizetto-Duarte et al. 2016; De los Reyes et al. 2020), as well as in studies focused on phenolic contents (e.g. Iken et al. 2009; Ferreres et al. 2012; Mannino et al. 2015). In this context, specimens of the Brown algae Cystosphaera jacquinotii (Mont.) Skottsb. were processed in the present study to evaluate their antioxidant activity and phenolic and flavonoids content. This is the only species of the order Fucales in Antarctica, and it is endemic to the Antarctic Peninsula and adjacent islands, with a circum-Antarctic distribution (Guiry & Guiry 2024). It is usually found in wave-exposed areas, and below 25 m at greater depths where ice scour is less common (Wiencke et al. 2007; Wiencke et al. 2014). This species seems to have a feed deterrence thanks to its phlorotannins (Wiencke et al. 2007; Iken et al. 2009). Wiencke et al. (2007) considered that in polar regions UVB radiation can be particularly harmful due to the thinning ozone layer, and so the phenolic compounds of C. jacquinotii should be increased as antioxidant defence as in low latitudes. Previous authors studied cytotoxic activity of this species (Martins et al. 2018; Frassini et al. 2019), but its antioxidant activity have not yet been studied. The available publications dealing with antioxidant activity in Fucales (e.g. Andrade et al. 2013; Dang et al. 2018; Grina et al. 2020) used different protocols for extraction and data analysis, which make results hardly comparable. Further, due to the scarcity of studies comparing marine natural products in distinct geographic areas (Costa-Leal et al. 2012), it might be interesting to compare the antioxidant capacity of Fucales along latitudinal gradients of distant territories, which might allow to determine eventual taxonomic or/and latitudinal variations to be taken into account in future studies and applications. Therefore, to facilitate data comparison with the Antarctic C. jacquinotii , the close relative Ericaria amentacea (C. Agardh) Molinari & Guiry, which occurs in the Mediterranean basin was analysed as well, since it and was rather accessible to the authors. E. amentacea is a Mediterranean endemism that is included in the Barcelona Convention (Annex II, COM/2009/0585 FIN). This is an outstanding species that forms marine forests in low intertidal and subtidal zones with high hydrodynamics; it is very sensitive to pollution, and changes are also expected in its distribution and abundance as a consequence of climate change (Bruno de Sousa et al. 2019). Macroalgae in the Mediterranean coasts are exposed to high doses of ultraviolet (UV) and photosynthetically active radiation, because of low latitude and water transparency. Therefore, E. amentacea is expected to develop a very effective antioxidant defence system related with higher UV radiation, as a more efficient photoprotective mechanisms to tolerate light stress than species from other biogeographical regions. In fact, UV radiation induces the promotion of antioxidant defence and because of these levels of phenolic compounds can be increased (Abdala-Diaz et al. 2006; Budhiyanti et al. 2012). In that context, the present study aims: (i) to evaluate the antioxidant capacity of C. jacquinotii extract in relation with other fucal species, E. amentacea , using CUPRAC and DPPH methods, and (ii) to assess the correlation of total phenolic content with his antioxidant activities. In addition, extraction and analysis methods will be assessed. MATERIALS AND METHODS Collection and preservation of seaweed material The Antarctic material was collected in Deception Island and Livingston Island, both in the Archipelago of South Shetland, in summer 2016, 2017, 2018 and 2019 (see Table 1 ). Samples were washed with sea water and stored in freezer at -20 ºC until further use. Collection of the Antarctic material was carried out in the framework of the project BLUEBIO (Bioactive marine natural products in an environmentally changing planet, University of Barcelona). The Mediterranean material was collected from the intertidal rocky shore of Cala Palmera (Alicante, Spain) in summer 2018 and 2019 (see Table 1 ). Samples were cleaned, washed with sea water and transported to the laboratory in a cooler box. In the laboratory, the material was washed with fresh water to completely remove any epiphytes, salts and sand, and stored in freezer at -20 ºC until further use. Gathering of both species was carried out during summer, according to latitude. This was done to match the collections with the highest water temperature in each site, since changes in temperature cause changes in Total Phenolic Content (TPC), as reported for Ericaria amentacea by Maninno et al. (2014). Seaweeds were identified by R. Martín (Antarctic material) and C. Pena-Martín (Mediterranean material), and voucher specimens were prepared and deposited in the herbarium ABH (University of Alicante). All samples were freeze-dried with a LYOQUEST – 55 PLUS lyophilizer (Telstar, Spain), ground to powder using a FRITSCH® pulverisette 6 blender (Oberstein, Germany), and stored in properly labelled polypropylene bottles until extraction. Table 1 Collections data of this study. Species Locality Temp(°C) Depth (m) Date Collectors Cystosphaera jacquinotii Wallers Bay, Deception Island (Antarctic) 62º58'49''S 60º33'20''W 3 27 19/01/2016 C. Ávila, C. Angulo, B. Figuerola Punta Fildes, Deception Island (Antarctic) 62º59'31S 60º3'33''W 26.5 9/02/2017 J. Giménez, C. Ávila, C. Angulo Pico Moores, Livingston Island (Antarctic) 62º43'01''S 60º25'23''W 2 23 1/02/2018 J. Vicente de Bobes, C. Angulo False Bay, Livingston Island (Antarctic) 62º41'15''S 60º18'53''W no data no data 17/01/2019 C. Ávila, C. Angulo, J. Giménez Ericaria amentacea Cala Palmera, Alicante (Spain) 38º35' N 0º24' W 25 0.5 15/07/2018 M.B. Crespo, C. Pena-Martín Cala Palmera, Alicante (Spain) 38º35' N 0º24' W 22 0.5 12/06/2019 J. Zarur-Matos, C. Pena-Martín Preparation of seaweed extract Powder seaweed was subjected to extraction preparing 0.5 g in a polypropylene bottle and adding 5 mL of methanol as extraction solvent (100 mg mL − 1 concentration). The mixture was then placed into an orbital shaker (Ivymen optic system, Comecta®, Spain) in darkness for 12h at room temperature. Then mixtures were filtered through Whatman® paper filters grade 1 and stored in properly labelled polypropylene bottles until analysis. Chemical analysis There is not a standard quantification method for the determination of the antioxidant capacity (Apak et al. 2007) so two different assays were used in this work for the assessment of the antioxidant capacity of the seaweed samples: DPPH (2,2-diphenyl-1-picrilhidrazilo) and CUPRAC (CUPric Reducing Antioxidant Capacity), which evaluate different antioxidant activity, in order to compare our results with others works. CUPRAC assay is an ET-based assay developed by Apak et al. (2004) which consist in a colorimetric method that measures the reducing power of antioxidants to convert Cu 2+ to Cu + ion. Briefly, the Cu 2+ -neocuproine complex (blue) can be reduced by antioxidants to Cu + -neocuproine (orange), which is a chromophore with a maximum absorption at 450 nm. By contrast, the DPPH assay is a HAT-based method, but DPPH radical can react with both electron and hydrogen donors. The electron transfer occurs first, and the mechanism is very quick, whereas the reaction with hydrogen is slower because it depends on the hydrogen bond accepting solvent used and the steric accessibility to the radical site (Apak et al. 2013; Pyrzynska & Pekal 2013). In DPPH assay, the radical is reduced by the antioxidants and its colour turns from purple to yellow of the corresponding hydrazine (DPPH 2 ). In this case, the reducing ability of the antioxidants can be evaluated by monitoring the decrease in the absorbance at 515–528 nm. CUPRAC assay 1 mL of CuCl 2 solution (10 mM), 1 mL of a Neocuproine solution (7.5 mM) in ethanol and 1 mL of NH 4 AC buffer solution were added to a test tube. Then 1 mL of the different seaweed extract dilutions (0.05–0.25 mg mL − 1 ) was added and the mixture was allowed to incubate for 1h in the dark at room temperature. Absorbance of the different solutions was measured at 450 nm against the reagent blank using a spectrophotometer UV-Visible Evolution™ 60S (ThermoFisher scientific, USA) The molar absorptivity of the CUPRAC method for each seaweed sample was obtained from the slope of the calibration curve prepared with the extract dilutions of each sample analysed (i.e. Cystoseira jacquinotii collected in 2016–2019 and Ericaria amentacea collected in 2018–2019). The same methodology was used with Trolox, which was selected as a standard reference to compare the antioxidant capacity. Results were expressed as Trolox Equivalents Antioxidant Capacity (TEAC: mg Trolox necessary to provide the same antioxidant capacity as 1 g dry weight of sample). DPPH assay The free radical scavenging (antioxidant activity) of the seaweed extracts were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical method according to a modified Brand-Williams method (Brand-Williams et al. 1995). A fresh solution of 0.1 mM DPPH was daily prepared in methanol and kept in the dark at room temperature when not used. Different dilutions were prepared (concentration range 0.5-3 mg mL − 1 ) for each seaweed extract (i.e. C. jacquinotii collected in 2016–2019 and E. amentacea collected in 2018 and 2019), in order to evaluate the reaction kinetics. Aliquots of 500 µL of each dilution of seaweed extracts were added to 2.5 mL of DPPH solution (0.1 mM DPPH) and the decrease in absorbance was measured at 517 nm during 30 seg at 0 min, and every 15 min until the reaction reached the steady state. The exact initial concentration of DPPH (A 0 ) in the reaction medium and the remaining DPPH concentration at the steady state (A F ) were calculated from a DPPH calibration curve (0.020–0.1 mM DPPH). In order to calculate the effective concentration (EC 50 ; amount of algal extract concentration necessary to decrease the DPPH concentration by 50%), the DPPH concentration in the steady state (A F ) was plotted against seaweed extract concentrations tested to obtain the equation: where b is the slope of the linear regression and C A is the concentration of the seaweed extract needed to reduce the initial DPPH concentration by 50% (A F = 50%). A lower EC 50 value indicates higher antioxidant capacity due to the extract concentration necessary to reduce the DPPH concentration by the 50% will be minor. For comparison purposes, Trolox was used as a standard to evaluate the antioxidant potential. So, 1 mM Trolox solution was prepared and diluted with methanol to give different solutions with concentrations ranging from 0.02–0.150 mM. These different solutions were used to determine EC 50 value of Trolox. Results were expressed in TEAC. The study of the DPPH reaction kinetics is usual in the determination of the antioxidant capacity of food and beverages to obtain more information about the composition of samples (Moon & Shibamoto 2009; Pyrzynska and Pekal 2013; Tomas et al. 2017). In the present work, we also evaluated the reaction kinetics for each C. jacquinotti (years 2016–2019) and E. amentacea (years 2018–2019) extract solution tested (0.5-3 mg mL − 1 ). Phytochemical estimation Quantitative phytochemical studies were carried out in order to evaluate the presence of several compounds with antioxidant activity: total phenolic content, and flavonoid. Estimation of total phenolic content The Total Phenolic Content (TPC) of the seaweed extracts was determined according a modified version of Singleton protocol (Singleton et al. 1999). Briefly, aliquots of 500 µL of diluted solutions of each seaweed extract (1 mg mL − 1 ) were mixed with 2.5 mL of 0.2N Folin-Ciocalteu reagent. After 5 min, 2 mL of sodium carbonate 7.5% w w − 1 solution was added, and the mixture was incubated for 2h in the dark at room temperature. Then, the absorbance was measured at 760 nm against the corresponding solvent as blank. Gallic acid was used as external standard (calibration curve in the range 10–60 mg Gallic acid L − 1 ) and results were expressed as mg of gallic acid equivalents (GAE) per g of dry seaweed material. Estimation of total flavonoid content The flavonoid content was determined by a modified Dowd method (Meda et al. 2005). The seaweed extract (5 mL, 1mg mL − 1 extract dilution) was mixed with 5 mL of 2% w w − 1 aluminium trichloride in methanol. After 10 minutes, absorbance was measured at 415 nm against the corresponding methanol solution as a blank. For the determination of flavonoids, Quercetin was used as a standard to prepare the calibration curve (1–7 mg Quercetin L − 1 ) and results were expressed as quercetin equivalents (µg Quercetin equivalents (QE) per mg of dry seaweed material). Statistical analysis Data analysis was performed with LibreOffice Calc 7.4.5.1. All measurements were performed in triplicate and results were expressed as mean ± standard deviation. Linear regression analysis were performed to estimate the relationship CUPRAC vs antioxidant compound and DPPH vs antioxidant compound, and significance of correlations were tested. Minimum significance level was established at P < 0.05 and the coefficient of determination (r 2 ) calculated. RESULTS Antioxidant activity During CUPRAC assay, Cystosphaera jacquinotii exhibited copper ion reduction ability that ranges from 30.7 ± 1.3 to 106 ± 5 mg Trolox g − 1 dw for the different years. The two highest values correspond to the Deception Island samples. As shown in Table 2 , values obtained for both samples of Ericaria amentacea are similar to those lower values obtained for C. jacquinotii samples, both from Livingston Island ( E. amentacea collected in 2018 and 2019, with 39 ± 3 and 23 ± 3 mg Trolox g − 1 dw, respectively). Data reported by Grina et al. (2020) for the same taxon are also similar (24.06 ± 0.02 mg TR g − 1 extract) (Table 2 ). Table 2 Antioxidant activity values (mean ± confidence interval; n = 15; 95% confidence level) obtained by means of DPPH and CUPRAC assays for Cystosphaera jacquinotii and Ericaria amentacea samples, and those reported in the literature. Specie DPPH CUPRAC (mg TR g − 1 dw) Ref. EC 50 (µg mL − 1 ) TEAC (mg TR 100g − 1 dw) Cystosphaera jacquinotii 2016 1960 ± 80 2140 ± 90 75 ± 12 Present work Cystosphaera jacquinotii 2017 930 ± 10 4500 ± 60 106 ± 5 Cystosphaera jacquinotii 2018 2500 ± 400 1700 ± 300 30.7 ± 1.3 Cystosphaera jacquinotii 2019 1770 ± 130 2400 ± 400 42.6 ± 0.5 Ericaria amentacea 2018 1930 ± 40 2210 ± 80 39 ± 3 Ericaria amentacea 2019 1250 ± 30 3300 ± 107 23 ± 3 Ericaria amentacea 78.7 ± 1.1 24.06 ± 0.02 Grina et al. 2020 Cystoseira humilis 121.1 ± 1.3 12.98 ± 0.03 Fucus spiralis 47.2 ± 3.8 55.24 ± 0.04 Ericaria tamariscifolia 580 Andrade et al. 2013 Treptacantha usneoides 1960 Treptacantha nodicaulis 4940 Sargassum sp. 1180 ± 40 Yangthong et al. 2009 Fucus ceranoides 460 Zubia et al. 2009 Ericaria tamariscifolia 490 Fucus serratus 5510 Ericaria crinita 20.0 ± 0.5 450.0 ± 0.3 Mhadhebi et al. 2014 Cystoseira sedoides 75.0 ± 0.8 120.0 ± 0.5 Cystoseira compressa 12.0 ± 0.7 750.0 ± 0.4 Fucus spiralis 141.2–224.9 Pinteus et al. 2017 Sargassum muticum 44.82–71.48 Ericaria tamariscifolia 87.3–136.8 Treptacantha usneoides 30.79–99.7 Sargassum vulgare 45.1–74.6 Sargassum vestitum 20950 ± 50 Dang et al. 2018 Sargassum linearifolium 1557 ± 80 Sargassum podocanthum 13700 ± 400 Regarding to the antioxidant capacity obtained by DPPH assay, values for both taxa are shown in Table 2 (TEAC, expressed as mg Trolox 100 g − 1 dw). For C. jacquinotii the sample collected in 2017 shown a higher value than the rest (4500 ± 60 mg Trolox 100g − 1 dw), which have values closer to each other (with values between 1700 and 2400 mg Trolox 100g − 1 dw), and closer to E. amentacea values too (2210 ± 80 and 3300 ± 107 mg Trolox 100g − 1 dw) (Table 2 ). The higher antioxidant capacity of C. jacquinotii collected in 2017 (from Punta Files) is not only evidenced by DPPH, but also by CUPRAC. Estimation of total phenolic content (TPC) Table 3 shows the total phenolic content for the studied samples. As it can be observed the total phenolic content for C. jacquinotti samples ranged from 20.5 ± 0.7 to 68 ± 11 mg GAE g − 1 dw. The higher value was registered for the C. jacquinotii sample collected in 2017 (68 ± 11 mg GAE g − 1 dw), which is consistent with the antioxidant capacity determined by CUPRAC and DPPH assays, while the rest of samples presented a phenolic content that ranges from ranges 20.5 ± 0.7 to 37.4 ± 1.8 mg GAE g − 1 dw. Regarding the E. amentacea samples, values obtained for both samples were lower than 20 mg GAE g − 1 dw. Table 3 Total phenolic content (TPC) (mg GAE g − 1 dry weight extract; mean ± confidence interval; n = 5; 95% con fidence level) for Cystosphaera jacquinotii and Ericaria amentacea samples, and those reported in the literature. Specie mg GAE g − 1 dw mg GAE 100 g − 1 dw Ref. Cystosphaera jacquinotii 2016 37.4 ± 1.8 3740 ± 180 Present work Cystosphaera jacquinotii 2017 68 ± 11 6800 ± 1100 Cystosphaera jacquinotii 2018 20.8 ± 0.6 2080 ± 60 Cystosphaera jacquinotii 2019 20.5 ± 0.7 2050 ± 70 Ericaria amentacea 2018 16.7 ± 0.5 1670 ± 50 Ericaria amentacea 2019 13.5 ± 0.3 1350 ± 30 Ericaria crinita 56.5 ± 0.4 Mhadhebi et al. 2014 Cystoseira sedoides 50.3 ± 0.1 Cystoseira compressa 61.0 ± 0.3 Fucus spiralis 397.23 ± 0.02 Pinteus et al. 2017 Sargassum muticum 71.69 ± 0.28 Ericaria tamariscifolia 57.52 ± 0.04 Treptacantha usneoides 86.81 ± 0.01 Sargassum vulgare 124.67 ± 0.01 Sargassum vestitum 141.91 ± 3.95 Dang et al. 2018 Sargassum linearifolium 47.06 ± 0.65 Sargassum podocanthum 48.13 ± 0.66 Fucus serratus 1200.8 ± 9.0 Sabeena Farvin et al. 2013 Fucus vesiculosus 1045.0 ± 45.8 Fucus distichus 862.3 ± 9.3 Fucus spiralis 752.5 ± 13.1 Estimation of total flavonoid content Total flavonoid content values obtained for the samples tested are shown in Table 4 . The C. jacquinotii samples collected in 2016 and 2017 presented the highest values with 0.63 ± 0.03 and 0.66 ± 0.02 µg QE mg − 1 dw, respectively. The sample of C. jacquinotii from 2018 presented the lowest flavonoid content for this specie, with 0.36 ± 0.02 µg QE mg − 1 dw. In the case of E. amentacea , the sample collected in 2018 showed a value similar to the Antarctic species (0.43 ± 0.03 µg QE mg − 1 dw) while the one from 2019 presented a lower flavonoid content (0.15 ± 0.02 µg QE mg − 1 dw). Table 4 Total flavonoid content (µg QE mg − 1 dry weight extract; mean ± confidence interval; n = 5; 95% confidence level) for Cystosphaera jacquinotii and Ericaria amentacea samples, and those reported in the literature (including other taxa of Fucales). Species µg QE mg − 1 dw extract Ref. Cystosphaera jacquinotii 2016 0.63 ± 0.03 Present work Cystosphaera jacquinotii 2017 0.66 ± 0.02 Cystosphaera jacquinotii 2018 0.36 ± 0.02 Cystosphaera jacquinotii 2019 0.56 ± 0.03 Ericaria amentacea 2018 0.43 ± 0.03 Ericaria amentacea 2019 0.15 ± 0.02 Ericaria amentacea 8.53 ± 0.02 Grina et al. 2020 Cystoseira humilis 6.72 ± 0.02 Fucus spiralis 26.9 ± 0.03 Correlation analysis between antioxidant activity and phenolic content Linear regression analysis between CUPRAC and DPPH vs antioxidant compound (TPC) is showed in Fig. 1 , where a correlation is evidenced. Ordinary least squares regressions provide high values for r 2 (0.78645 for CUPRAC and 0.72591 for DPPH), explaining the consequent increase in antioxidant activity (by any of both methods) the greater amount of polyphenols. DISCUSSION Eventual differences in antioxidant capacity values determined by CUPRAC and DPPH could be related with the nature and compositions of the antioxidant compounds in each seaweed sample (Brand-Williams et al. 1995). Antioxidants are bioactive compounds considered as effective chain breaking agents due to their capacity to neutralize chemically active metabolic products, such as free radicals (Kurek et al. 2022). One of the most important are the phenolic compounds, and among them flavonoids, due to their high antioxidant activity (Shahidi et al. 1992). On this regard, the total phenolic plus flavonoid content was evaluated and compared with the antioxidant capacity for the Brown algae samples of the Antarctic Cystosphaera jacquinotii and the Mediterranean Ericaria amentacea . Our analyses revealed the high antioxidant activity in samples of E. amentacea , in agreement with the reports of earlier studies (Grina et al. 2020) (Table 2 ), but even more in those of the Antarctic species C. jacquinotii , never reported before this work. In particular, values from samples of Punta Files (Deception) are markedly higher than the rest, mainly with CUPRAC, although no environmental differences between both Antarctic islands have been found that explain these results. In general, the antioxidant capacity determined by the CUPRAC assay for C. jacquinotii is higher than that obtained for E. amentacea . However, the values obtained in the DPPH assay showed that the antioxidant capacity for both seaweeds species is not very different, with the exception of the C. jacquinotii samples collected in Deception Island in 2017. The differences found between the results obtained with each method could be related to the mechanisms of action of the antioxidant compounds. While CUPRAC assay is an electron transfer (SET)-based assay that detects the capability of the antioxidant compounds to transfer one electron to reduce metal ions, DPPH is a mixed-mode assay consisting in the determination of the capability of the antioxidants to quench free radicals by means of the transfer of one electron (or the hydrogen atom transfer) from the antioxidant compounds to the DPPH radical (Apak et al. 2018). The H transfer is conditioned by the nature of the antioxidant compound (Brand-Williams et al. 1995) and the complexity of the sample (Carmona-Jiménez et al. 2014) which determines the velocity of the decrease in the absorbance and the time to reach the steady state. The study of the DPPH reaction kinetics is usual in the determination of the antioxidant capacity of food and beverages to obtain more information about the composition of samples (Pyrzynska & Pekal 2013; Tomas et al. 2017; Moon & Shibamoto 2009). In the present work, we also evaluated the reaction kinetics for each C. jacquinotti (years 2016–2019) and E. amentacea (years 2018–2019) extract solution tested (0.5-3 mg mL − 1 ). Figure 2 a and 2 b shows the evaluation of the decrease in the relative absorbance with time for the C. jacquinotti 2017 and E. amentacea 2019 samples. These samples were selected to represent the kinetic behaviour of a C. jacquinotti and E. amentacea extract solution. Similar results were obtained for the remaining extract solutions. Relative absorbance (A/A 0 ) is defined as the absorbance after a certain incubation time relative to the absorbance value obtained at the beginning of the reaction (i.e., time 0 min). For both, a fast decrease in the absorbance was observed in the first minutes (< 5 min) (corresponding to the electron transfer), followed by a slower decrease in the absorbance values (H transfer mechanism) until the reaction reached the steady state (120 min approximately). Similar kinetic behaviours were obtained for the rest of the samples. These results were also compared to those obtained for Trolox, used as a reference standard (Fig. 2 c). Trolox reacted very fast with the DPPH radical since this standard reached the steady state in a few minutes (6 approximately) which indicates a fast-kinetic reaction regarding the studied samples. The evidenced different velocity between our samples and Trolox indicates that the antioxidant compounds contained in our samples are more complex molecules and are more sterically hindered than Trolox. From reaction kinetic graphs, the percentage of DPPH remaining at the steady state was determined and the values were transferred onto another graph (not shown) showing the percentage of residual DPPH at the steady state as a function of the seaweed extract concentration tested in order to obtain the efficient concentration (EC 50 ) gathered in Table 2 which are related with the TEAC values. Although there is not a standard methodology to determine the antioxidant capacity because of the broad variety of antioxidant compound extractions procedures and the diversity of samples, the results obtained in the present work were comparable with some of those reported in the literature for both assays (Table 2 ). Regarding the CUPRAC assay, the antioxidant capacity values reported by Grina et al. (2020) were of the same order of magnitude that those obtained in our analysis for E. amentacea (Table 4 ). Interestingly, the values obtained for the C. jacquinotii samples were slightly higher than the values reported by those authors for the different Fucales samples evaluated. Concerning the DPPH assay results, the EC 50 values obtained for C. jacquinotii and E. amentacea in the present study were between 8 and 20-fold higher than those reported in the literature for E. amentacea and other Fucales which indicated that according to the DPPH assay, the antioxidant capacity of C. jacquinotii and E. amentacea was lower than that reported by Grina et al. (2020). Those differences observed are probably related mainly to the seaweed preparation procedure and the methodology used for the determination of the phenolic content. In the present work the extractant used to prepare the samples was methanol 100% (at room temperature, 12 hours), whereas Grina et al. (2020) prepared the extract with a solution composed by 70% ethanol (at 60ºC, but only 2 hours). Those methodological differences could affect directly to the compounds that were present in the extract obtained, due to the polarity of each extractant solution employed, the phenol units of the antioxidant compounds and the temperature conditions employed to carry out the extractions. Besides, protocols for measuring phenolic content were also different. Since extraction protocols used by Grina et al. (2020) got higher quantities of flavonoids for E. amentacea , it is expected that the application of these protocols to the Antarctic species C. jacquinotii also would lead to an optimization of the extraction of flavonoids. In this study, the selection of the extraction conditions was focused on three factors: (i) the extractant; (ii) the extraction temperature; and (iii) the extraction time. In the case of the extractant, preliminary experiments were carried out using water, ethanol, and methanol as extractants according to previous works reported in the literature (Mhadhebi et al. 2014; Dang et al. 2018; Grina et al. 2020). Water yielded an extract with a viscous texture that hampered its manipulation. Regarding the alcohols used, the extracts obtained with methanol presented higher antioxidant capacity values compared to those afforded by the ethanol ones. Thus, methanol was selected to prepare the extracts of the different samples. Concerning the temperature, it was maintained at room temperature (25ºC) during the extraction to avoid the degradation of the antioxidant compounds at higher temperatures. Finally, different extraction times were also tested, i.e. 2, 6, and 12h (overnight), obtaining the best results after 12h of extraction. Related with total phenolic content (TPC), the analysis revealed that C. jacquinotii exhibited higher values than E. amentacea (Table 3 ). Samples from Deception Island presented the higher phenolic content (68 ± 11 and 37.4 ± 1.8 µg GAE mg − 1 dw) that agreed with the antioxidant capacity obtained by those samples, especially in CUPRAC assays. It can be observed in Fig. 3 that the TPC values are directly proportional to the antioxidant capacity values obtained for each sample in CUPRAC assays, although DPPH assays did not show such a clear correlation, because of the arguments presented above. This fact revealed that the main responsible for the antioxidant properties of the seaweed evaluated are phenolic compounds, as previous authors already documented (cf. Karawita et al. 2005). This positive correlation is evidenced in the correlation analysis (Fig. 1 ), but they are not the sole compounds implicated. In fact, many researchers have identified several compounds as antioxidants in seaweeds, including protective enzymes, ascorbic acid, lipophilic antioxidants, phlorotannins and catechins (Mhadhebi et al. 2014). Total phenolic content obtained for samples tested (Antarctic and Mediterranean) are similar to those values reported in the literature for other genera of Fucales (i.e. Cystoseira s.l., Sargassum , Fucus ) from other locations (i.e. Mediterranean Sea, Atlantic Ocean, South Pacific Ocean, and Danish coasts) (see Table 3 ). Finaly, regarding the total flavonoid content, our data were compared to those reported by Grina et al. (2020), since they used a similar determination method for other Fucales (Table 4 ). In our case, values obtained for C. jacquinotii samples are higher than values for E. amentacea samples, but not significantly. In fact, correlation for the relationship of the antioxidant activity (by both methods) with the concentration of TPC is clear (r 2 = 0.78645 for CUPRAC, and r 2 = 0.72591 for DPPH) (Fig. 1 ), but this relationship with flavonoids is not so evident (r 2 = 0.45074 for CUPRAC, and r 2 = 0.16109 for DPPH). This fact evidences that other phenolic compounds participate in the antioxidant activity, and it is especially evident in DPPH, since this method also measures reduction by hydrogen, probably given by other compounds. CONCLUSIONS This study revealed that the Antarctic brown seaweed Cystosphaera jacquinotii presents a high TPC that directly contributes to the strong antioxidant activity detected. The comparison with Ericaria amentacea , the other Fucal analysed in this work with the same protocols, evidences the observed high antioxidant property. In the same way, our findings confirm other previous results related to brown algae and their bioactive compounds with antioxidant activity. Those extracts can be used as easily accessible source of natural antioxidants, and also as a possible food supplement or in pharmaceutical industry. The fact that samples from Deception Island exhibit a greater antioxidant capacity does not seem to be connected to distinct peculiarities in the ecological conditions of the site. However, new samplings and studies should be carried out in Antarctic Polar to check whether the higher antioxidant values in samples from Deception Island are unique or are also found in other places around. In any case, the factor or factors conditioning eventual differences might be identified. Furthermore, efforts should be focused on studying the kinetics of the extracts of the different species in DPPH assay, since it is a markedly variable factor that must be considered to assess the antioxidant capacity of each taxon, as shown in the present work. Additional studies are needed to better understand the role of the different antioxidant activity of various compounds in the studied brown seaweeds, and to find other proper parts and seasons for a given species to get higher antioxidant activity and improved extraction protocols. Therefore, further chemical investigations are needed to properly determine the antioxidant activity in different aqueous or polar extracts, through standardized extraction methods and antioxidant activity analyses. Declarations ACKNOWLEDGEMENTS Authors thank R. Martín for providing us with Antarctic samples, collected by himself and others collectors (C. Ávila, C. Angulo, J. Giménez, J Vicente de Bobes, B. Figuerola). Authors' contributions C. Pena-Martín: conceptualization, methodology, formal analysis, investigation, resources, writing, visualization and project administration. R. Serrano: methodology, formal analysis, investigation, resources, data curation and writing. G. Grindlay: methodology and resources. MB. Crespo: resources and writing. Availability of data and material The data required to support this research is made available within the manuscript. Samples from which fragments were analyzed for this study are conserved in the Herbarium of the University of Alicante (ABH-Algae). Competing interests The authors have no competing interests to declare that are relevant to the content of this article. Funding Declaration This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. References Abdala-Díaz R, Cabello-Pasini A, Márquez-Garrido A, López Figueroa F (2006) Variación intratalo de compuestos fenólicos, actividad antioxidante y actividad de la fenolsulfatasa en Cystoseira tamariscifolia (Phaeophyceae) del sur de España. Ciencias Marinas 40(1):01–09. Andrade PB, Barbosa M, Pedro Matos R, Lopes G, Vinholes J, Mouga T, Valentao P (2013) Valuable compounds in microalgae extracts. Food chemistry 138:1819–1828. Apak R, Capanoglu E, Shahidi F (Eds.) (2018) Measurement of antioxidant activity and capacity: Recent trends and applications. John Wiley and Sons Ltd. Oxford, UK. ISBN: 978-1-119-13535-7. Apak R, Gorinstein S, Böhm V, Schaich KM, Özyürek M, Glüçü K (2013) Methods of measurement and evaluation of natural antioxidant capacity/activity (IUPAC Technical report). Pure Appl Chem 85:957–998. Apak R, Güçlü K, Demirata B, Özyürek M, Çelik SE, Bektasoglu B, Berker KI, Özyurt D. (2007) Comparative evaluation of various total antioxidant capacity assays applied to phenolic compounds with the CUPRAC assay. Molecules 12:1496–1547. Apak R, Güçlü K, Özyürek M, Karademir SE (2004) Novel antioxidant capacity index for dietary polyphenols and vitamins C and E, using their Cupric Ion Reducing Capability in the presence of Neocuproine: CUPRAC method. J Agric Food Chem 52: 7970–7981. Audibert L, Fauchon M, Blanc N, Hauchardb D, Ar Gall E (2010) Phenolic Compounds in the Brown Seaweed Ascophyllum nodosum: Distribution and Radical-scavenging Activities Phytochem Anal 21:399–405. Brand-Williams W, Cuvelier M, Berset C. (1995) Use of free radical method to evaluate antioxidant activity. Lebensm Wiss Technol 28:25–30. Bruno de Sousa C, Cox CJ, Brito L, Pavão MM, Pereira H, Ferreira A, Ginja C, Campino L, Bermejo R, Parente M, Varela J (2019) Improved phylogeny of brown algae Cystoseira (Fucales) from the Atlantic-Mediterranean region based on mitochondrial sequences. Plos One 14(1): e0210143. Budhiyanti SA, Raharjo S, Marseno DW, Lelana IYB (2012) Antioxidant Activity of Brown Algae Sargassum species Extract from the Coastline of Java Island. Am J Agric Biol Sci 7(3):337–346. Carmona-Jiménez Y, García-Moren MV, Igartuburu JM, García Barroso C (2014) Simplification of the DPPH assay for estimating the antioxidant activity of wine ans wine by-products. Food Chem 165:198–204. Costa-Leal M, Madeira C, Brandao CA, Puga J, Calado R (2012) Bioprospecting of Marine Invertebrates for New Natural Products — A Chemical and Zoogeographical Perspective. Molecules 17:9842–9854. De los Reyes E, Sapin AB (2020) Bioprospecting of Turbinaria ornata (Fucales, Phaeophyceae) for cosmetic application: antioxidant, tyrosinase inhibition and antibacterial activities. J ISSAAS 26(2):30–41. Dang TT, Bowyer MC, Van Altena IA, Scarlett CJ (2018) Comparison of chemical profile and antioxidant properties of the brown algae. Inter J Food Sci Tec 53:174–181. Ferreres F, Lopes G, Gil-Izquierdo A, Andrade PB, Sousa C, Mouga T, Valentao P (2012) Phlorotannin Extracts from Fucales Characterized by HPLC-DAD-ESI-MS n : Approaches to Hyaluronidase Inhibitory Capacity and Antioxidant Properties. Mar Drugs 10(12): 2766-81. Flores-Molina MR, Rautenberger R, Muñoz P, Huovinen P, Gómez I (2016) Stress Tolerance of the Endemic Antarctic Brown Alga Desmarestia anceps to UV Radiation and Temperature is Mediated by High Concentrations of Phlorotannins. Photochem Photobiol 92(3):455–466. Frassini R, Da Silva YP, Moura S, Zambotti L, Paternostro A, Colepicolo P, Toyota M, Yokota NS, Pereira CM, Zingalli VR, Prgas JA, Roesch-Ely M (2019) Chemical Characterization and Cytotoxic Activity of Antarctic Macroalgae Extracts against Colorectal Cancer. Advances in Biological Chemistry 9: 167–177. Grina F, Ullah Z, Kaplaner E, Moujahid A, Eddoha R, Nasser B, Terzioglu P, Yilmaz MA, Erta A,Öztürk M, Essamadi A (2020) In vitro enzyme inhibitory properties, antioxidant activities, and phytochemical fingerprints of five Moroccan seaweeds. South African J Bot 128: 152–160. Guiry MD, Guiry GM. AlgaeBase. World-wide electronic publication, National University of Ireland, Galway. http://www.algaebase.org; accessed 1 February 2024. Iken K, Amsler CD, Amsler MO, McClintock JB, Baker BJ (2009) Field studies on deterrent properties of phlorotannins in Antarctic brown algae. Bot Mar 52:547–557. Jassbi AM, Mohabati M, Eslami S, Sohrabipour J, Miri R (2013) Biological activity and chemical constituents of red and brown algae from the persian gulf. Iran J Pharm Res 12(3):339–348. Karawita R, Siriwardhana N, Lee KW, Heo MS, Yeo IK, Lee YD, Jeon YJ (2005) Reactive oxygen species scavenging, metal chelation, reducing power and lipid peroxidation inhibition properties of different solvent fractions from Hizikia fusiformis. Eur Food Res. Technol. 220:363–371. Kohen R, Nyska A (2002) Invited Review: Oxidation of Biological Systems: Oxidative Stress Phenomena, Antioxidants, Redox Reactions and Method for their quantification. Toxicol Pathol 30(6):620–650. Kurek M, Benaida-Debbache N, Garofolic IE, Galic K, Avallone S, Voilley A, Waché Y (2022) Antioxidants and Bioactive Compounds in Food: Critical Review of Issues and Prospects. Antioxidants (Basel) 11(4): 742. Mannino AM, Vaglica V, Cammarata M, Oddo E (2015) Effects of temperature on total phenolic compounds in Cystoseira amentacea (C. Agardh) Bory (Fucales, Phaeophyceae) from southern Mediterranean Sea. Plant Biosyst 150(1):152–160. Martins RM, Nedel F, Guimarães VBS, da Silva AF, Colepicolo P, de Pereira CMP, Lund RG (2018) Macroalgae extracts from antarctica have antimicrobial and anticancer potential. Front. Microbiol. 9:412. Meda A, Lamien CE, Romito M, Millogo J, Naoulma OG (2005) Determination of the total phenolic, flavonoid and proline contents in Burkina Fasan honey, as well as their radical scavening activity. Food Chem 91:571–577. Mhadhebi L, Mhadhebi A, Robert J, Bouraoui A (2014) Antioxidant, anti-inflammatory and antiproliferative effects of aqueous extracts of three Mediterranean brown seaweeds of the genus Cystoseira. Ir J Pharm Res 13:207–220. Moon JK, Shibamoto T (2009) Antioxidant assays for plant and food components. J Agric Food Chem 57:1655–1666. Park PJ, Fereidoon S, You-Jin J (2004) Antioxidant activities of enzymatic extracts from an edible seaweed Sargassum horneri using ESR spectrometry. J Foods Lipids 11(1):15–17. Pinteus S, Silva J, Alves C, Horta A, FIno N, Rodrigues AI, Mendes SP (2017) Cytoprotective effect of seaweeds with high antioxidant activity from the Peniche coast (Portugal). Food Chem 218:591–599. DOI:10.1016/j.foodchem.2016.09.067 Pyrzynska K, Pekal A (2013) Application of free radical diphenilpicrylhydrazyl (DPPH) to estimate the antioxidant capacity of food samples. Anal Methods 5:4288–4295. Ryabushko VI, Prazukin AV, Popova EV, Nekhoroshev MV (2014) Fucoxanthin of the brown alga Cystoseira barbata (Stackh.) C. Agardh from the Black Sea. J Black Sea 20(2):108–113. Sabeena Farvin KH, Jacobsen C (2013) Phenolic compounds and antioxidant activities of selected species of seaweeds from the Danish coast. Food Chem 138:1670–1681. Senthilkumar K, Manivasagan P, Venkatesan J, Kim SK (2013) Brown seaweed fucoidan: Biological activity and apoptosis, growth signalling mechanism in cancer. Int J Biol Macromol 60:366–374. Singleton VL, Orthofer R, Lamuela-Reventós RM (1999) Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods Enzymol 299:152–178. Shahidi, F, Janitha, PK, Wanasundara, PD (1992) Phenolic antioxidants. Crit Rev Food Sci Nutr 32:67–103. Tomas M, Beekwilder J, Hall RD, Sagdic O, Boyacioglu D, Capanoglu E (2017) Industrial processing versus home processing of tomato sauce: Effects on phenolics, flavonoids and in vitro bioaccessibility of antioxidants. Food Chem 220:51–58. Vizetto-Duarte C, Custódio L, Barreira L, Moreira da Silva M, Rauter AP, Albericio F, Varela J (2016) Proximate biochemical composition and mineral content of edible species from the genus Cystoseira in Portugal. Bot Mar 59(4): 251–257. Wiencke C, Amsler CD, Clayton MN (2014) Macroalgae in Biogeographic atlas of the southern ocean. In: De Broyer C, Koubbi P, Griffiths HJ, Raymond B, Udekem d’Acoz C d’, et al. (eds.). Scientific Committee on Antarctic Research, Cambridge, pp 66–73. Wiencke C, Clayton MN, Gómez I, Iken K, Lüder U, Amsler CD, Karsten U, Hanelt D, Bischof K, Dunton K (2007) Life strategy, ecophysiology and ecology of seaweeds in polar water. Rev Environ Sci Biotechnol 6:95–126 66–73 Yangthong M, Hutadilok-Towatana N, Phromkunthong W (2009) Antioxidant activities of four edible seaweeds from the southern coast of Thailand. Plant Foods Hum Nutr 64:218–223. Zubia M, Fabre MS, Kerjean V, Le Lann K, Stiger-Pouvreau V, Fauchon M, Deslandes E (2009) Antioxidant and antitumoural activities of some Phaeophyta from Brittany coasts. Food Chem 116:693–701. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4660453","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":327876356,"identity":"996b8244-125a-4ea3-b202-915e36a1cc4e","order_by":0,"name":"Carolina Pena-Martín","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYLCCBwYwVgWxWhLgWs4QrQXGYGwjQjV/+9mHDxIK7sgzSDcfe/Bz3mF5Bvb2B3i1SJxJNzZIMHhm2CBzLN2wd9thwwaeMwZ4tRgwpLFJJBgcZmyQyDGT4N0GZuB3mAH/M7AW+waJ/G+Sf+cAGfLP8TvMQAJiSyLQcDZp3gYQgwG/wyRuPGMG+uVwcpvMMTNpoH+S23hy8Gvh709jfPDhz2HbfunmZ5Jvaqxt+9mP43cYHLBJwBjEqQc7kXilo2AUjIJRMMIAAI4zQYtCHqkjAAAAAElFTkSuQmCC","orcid":"","institution":"University of Alicante","correspondingAuthor":true,"prefix":"","firstName":"Carolina","middleName":"","lastName":"Pena-Martín","suffix":""},{"id":327876357,"identity":"9645180a-7ed7-48a6-8c24-b591c9c94c4d","order_by":1,"name":"Raquel Serrano","email":"","orcid":"","institution":"University of Alicante","correspondingAuthor":false,"prefix":"","firstName":"Raquel","middleName":"","lastName":"Serrano","suffix":""},{"id":327876358,"identity":"a2a808b8-e4d6-4439-9892-0c71dd9b2fed","order_by":2,"name":"Guillermo Grindlay","email":"","orcid":"","institution":"University of Alicante","correspondingAuthor":false,"prefix":"","firstName":"Guillermo","middleName":"","lastName":"Grindlay","suffix":""},{"id":327876359,"identity":"a3b0e711-c56c-49c5-9810-a3b34fe8184b","order_by":3,"name":"Manuel B. Crespo","email":"","orcid":"","institution":"University of Alicante","correspondingAuthor":false,"prefix":"","firstName":"Manuel","middleName":"B.","lastName":"Crespo","suffix":""}],"badges":[],"createdAt":"2024-06-29 19:10:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4660453/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4660453/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60999349,"identity":"b7a468be-4c3f-4b40-9698-46293b847097","added_by":"auto","created_at":"2024-07-24 12:49:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":40071,"visible":true,"origin":"","legend":"\u003cp\u003eLinear regression analysis: (a) CUPRAC \u003cem\u003evs\u003c/em\u003e total phenolic content (TPC); and (b) DPPH \u003cem\u003evs\u003c/em\u003e total phenolic content (TPC)\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4660453/v1/504e5af9b88a51c818fa7643.png"},{"id":60999351,"identity":"a3e3f5a4-fd13-4dd0-b3d4-d4276a9ab0be","added_by":"auto","created_at":"2024-07-24 12:49:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":86083,"visible":true,"origin":"","legend":"\u003cp\u003eKinetic curves of the scavenged DPPH\u003csup\u003e·\u003c/sup\u003e at the concentration of 0.5-3 mg mL\u003csup\u003e-1 \u003c/sup\u003efor: (a) \u003cem\u003eCystosphaera jacquinotii \u003c/em\u003e2018 sample; (b) \u003cem\u003eEricaria amentacea \u003c/em\u003e2018 sample; and (c) Trolox\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4660453/v1/cdd8bf076ca7294bd76ef935.png"},{"id":60999350,"identity":"06b57ce1-88aa-4745-8429-78d9ed0cf0ad","added_by":"auto","created_at":"2024-07-24 12:49:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":60886,"visible":true,"origin":"","legend":"\u003cp\u003eAntioxidant capacity obtained by DPPH and CUPRAC for \u003cem\u003eCystosphaera jacquinotii\u003c/em\u003e and \u003cem\u003eEricaria amentacea\u003c/em\u003e samples compared to the total phenolic content (TPC) (mean ± confidence interval; n=5; 95% confidence level)\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4660453/v1/54c9271305cb291668ffcfe1.png"},{"id":61000919,"identity":"d74e699e-78cb-4bc0-a4cb-70eb93d51601","added_by":"auto","created_at":"2024-07-24 13:07:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":910318,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4660453/v1/a612a0e8-5000-46d1-80ee-e2f1499bb9d0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Assessment of antioxidant capacity of Cystosphaera jacquinotii (Fucales, Ochrophyta) by two methods: DPPH and CUPRAC.","fulltext":[{"header":"INDRODUCTION","content":"\u003cp\u003eSeaweeds have been used in pharmacological and food systems due to their bioactive compounds, such as phenolic compounds, fucoxanthin and polysaccharides (Grina et al. 2020). Recently, seaweeds have received special attention as a source of natural antioxidants (Sabeena et al. 2013; Senthilkumar et al. 2013; Pinteus et al. 2017). In fact, although they are exposed to a combination of light and high oxygen concentrations which lead to the formation of free radicals and other strong oxidizing agents, they seldom suffer from any serious photodynamic damage \u003cem\u003ein vivo\u003c/em\u003e because their cells have protective antioxidative mechanisms as well as antioxidative compounds (Park et al. 2004). According to that, and since antioxidant compounds play an important role against various diseases (e.g. atherosclerosis, chronic inflammation, cardiovascular disorders and cancer) and aging processes (Kohen and Nyska 2002; Senthilkumar et al. 2013), research on antioxidant capacity in seaweeds has been increasing in recent years.\u003c/p\u003e \u003cp\u003eAmong all seaweeds, Brown algae play a significant role, as they have high content on phenolic compounds such as flavonoids, phenolic acids, and phlorotannins (present only in Brown algae), which may directly participate in antioxidative action (Ferreres et al. 2012; Grina et al. 2020). Phenolic compounds are secondary metabolites involved in different protection mechanisms against grazers, epiphytes and UV radiation (Mannino et al. 2015). Audibert et al. (2010) concluded that the antioxidant activity of the extracts was related to the content of phlorotannins and to their molecular weight. Besides, the induced synthesis of phlorotannins is crucial for Brown macroalgae to tolerate environmental stress and, therefore, it can be regarded as a primary defence mechanism against episodic stress by elevated temperature and solar radiation (Flores-Molina et al. 2016).\u003c/p\u003e \u003cp\u003eSpecifically, species belonging to the order Fucales are particularly rich in antioxidants. In fact, many species of this group have already been evaluated in relation to their high antioxidant capacity (Ferreres et al. 2012; Jassbi et al. 2013; Ryabushko et al. 2014; Vizetto-Duarte et al. 2016; De los Reyes et al. 2020), as well as in studies focused on phenolic contents (e.g. Iken et al. 2009; Ferreres et al. 2012; Mannino et al. 2015).\u003c/p\u003e \u003cp\u003eIn this context, specimens of the Brown algae \u003cem\u003eCystosphaera jacquinotii\u003c/em\u003e (Mont.) Skottsb. were processed in the present study to evaluate their antioxidant activity and phenolic and flavonoids content. This is the only species of the order Fucales in Antarctica, and it is endemic to the Antarctic Peninsula and adjacent islands, with a circum-Antarctic distribution (Guiry \u0026amp; Guiry 2024). It is usually found in wave-exposed areas, and below 25 m at greater depths where ice scour is less common (Wiencke et al. 2007; Wiencke et al. 2014). This species seems to have a feed deterrence thanks to its phlorotannins (Wiencke et al. 2007; Iken et al. 2009). Wiencke et al. (2007) considered that in polar regions UVB radiation can be particularly harmful due to the thinning ozone layer, and so the phenolic compounds of \u003cem\u003eC. jacquinotii\u003c/em\u003e should be increased as antioxidant defence as in low latitudes. Previous authors studied cytotoxic activity of this species (Martins et al. 2018; Frassini et al. 2019), but its antioxidant activity have not yet been studied.\u003c/p\u003e \u003cp\u003eThe available publications dealing with antioxidant activity in Fucales (e.g. Andrade et al. 2013; Dang et al. 2018; Grina et al. 2020) used different protocols for extraction and data analysis, which make results hardly comparable. Further, due to the scarcity of studies comparing marine natural products in distinct geographic areas (Costa-Leal et al. 2012), it might be interesting to compare the antioxidant capacity of Fucales along latitudinal gradients of distant territories, which might allow to determine eventual taxonomic or/and latitudinal variations to be taken into account in future studies and applications. Therefore, to facilitate data comparison with the Antarctic \u003cem\u003eC. jacquinotii\u003c/em\u003e, the close relative \u003cem\u003eEricaria amentacea\u003c/em\u003e (C. Agardh) Molinari \u0026amp; Guiry, which occurs in the Mediterranean basin was analysed as well, since it and was rather accessible to the authors. \u003cem\u003eE. amentacea\u003c/em\u003e is a Mediterranean endemism that is included in the Barcelona Convention (Annex II, COM/2009/0585 FIN). This is an outstanding species that forms marine forests in low intertidal and subtidal zones with high hydrodynamics; it is very sensitive to pollution, and changes are also expected in its distribution and abundance as a consequence of climate change (Bruno de Sousa et al. 2019). Macroalgae in the Mediterranean coasts are exposed to high doses of ultraviolet (UV) and photosynthetically active radiation, because of low latitude and water transparency. Therefore, \u003cem\u003eE. amentacea\u003c/em\u003e is expected to develop a very effective antioxidant defence system related with higher UV radiation, as a more efficient photoprotective mechanisms to tolerate light stress than species from other biogeographical regions. In fact, UV radiation induces the promotion of antioxidant defence and because of these levels of phenolic compounds can be increased (Abdala-Diaz et al. 2006; Budhiyanti et al. 2012).\u003c/p\u003e \u003cp\u003eIn that context, the present study aims: (i) to evaluate the antioxidant capacity of \u003cem\u003eC. jacquinotii\u003c/em\u003e extract in relation with other fucal species, \u003cem\u003eE. amentacea\u003c/em\u003e, using CUPRAC and DPPH methods, and (ii) to assess the correlation of total phenolic content with his antioxidant activities. In addition, extraction and analysis methods will be assessed.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCollection and preservation of seaweed material\u003c/h2\u003e \u003cp\u003eThe Antarctic material was collected in Deception Island and Livingston Island, both in the Archipelago of South Shetland, in summer 2016, 2017, 2018 and 2019 (see Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Samples were washed with sea water and stored in freezer at -20 \u0026ordm;C until further use. Collection of the Antarctic material was carried out in the framework of the project BLUEBIO (Bioactive marine natural products in an environmentally changing planet, University of Barcelona).\u003c/p\u003e \u003cp\u003eThe Mediterranean material was collected from the intertidal rocky shore of Cala Palmera (Alicante, Spain) in summer 2018 and 2019 (see Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Samples were cleaned, washed with sea water and transported to the laboratory in a cooler box. In the laboratory, the material was washed with fresh water to completely remove any epiphytes, salts and sand, and stored in freezer at -20 \u0026ordm;C until further use.\u003c/p\u003e \u003cp\u003eGathering of both species was carried out during summer, according to latitude. This was done to match the collections with the highest water temperature in each site, since changes in temperature cause changes in Total Phenolic Content (TPC), as reported for \u003cem\u003eEricaria amentacea\u003c/em\u003e by Maninno et al. (2014).\u003c/p\u003e \u003cp\u003eSeaweeds were identified by R. Mart\u0026iacute;n (Antarctic material) and C. Pena-Mart\u0026iacute;n (Mediterranean material), and voucher specimens were prepared and deposited in the herbarium ABH (University of Alicante). All samples were freeze-dried with a LYOQUEST \u0026ndash; 55 PLUS lyophilizer (Telstar, Spain), ground to powder using a FRITSCH\u0026reg; pulverisette 6 blender (Oberstein, Germany), and stored in properly labelled polypropylene bottles until extraction.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCollections data of this study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLocality\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTemp(\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDepth (m)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCollectors\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCystosphaera jacquinotii\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWallers Bay, Deception Island (Antarctic)\u003c/p\u003e \u003cp\u003e62\u0026ordm;58'49''S 60\u0026ordm;33'20''W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19/01/2016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC. \u0026Aacute;vila, C. Angulo, B. Figuerola\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePunta Fildes, Deception Island (Antarctic)\u003c/p\u003e \u003cp\u003e62\u0026ordm;59'31S 60\u0026ordm;3'33''W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9/02/2017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eJ. Gim\u0026eacute;nez, C. \u0026Aacute;vila, C. Angulo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePico Moores, Livingston Island (Antarctic)\u003c/p\u003e \u003cp\u003e62\u0026ordm;43'01''S 60\u0026ordm;25'23''W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1/02/2018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eJ. Vicente de Bobes, C. Angulo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFalse Bay, Livingston Island (Antarctic)\u003c/p\u003e \u003cp\u003e62\u0026ordm;41'15''S 60\u0026ordm;18'53''W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eno data\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eno data\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17/01/2019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC. \u0026Aacute;vila, C. Angulo, J. Gim\u0026eacute;nez\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEricaria amentacea\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCala Palmera, Alicante (Spain)\u003c/p\u003e \u003cp\u003e38\u0026ordm;35' N 0\u0026ordm;24' W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15/07/2018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eM.B. Crespo, C. Pena-Mart\u0026iacute;n\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCala Palmera, Alicante (Spain)\u003c/p\u003e \u003cp\u003e38\u0026ordm;35' N 0\u0026ordm;24' W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12/06/2019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eJ. Zarur-Matos, C. Pena-Mart\u0026iacute;n\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of seaweed extract\u003c/h2\u003e \u003cp\u003ePowder seaweed was subjected to extraction preparing 0.5 g in a polypropylene bottle and adding 5 mL of methanol as extraction solvent (100 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e concentration). The mixture was then placed into an orbital shaker (Ivymen optic system, Comecta\u0026reg;, Spain) in darkness for 12h at room temperature. Then mixtures were filtered through Whatman\u0026reg; paper filters grade 1 and stored in properly labelled polypropylene bottles until analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eChemical analysis\u003c/h2\u003e \u003cp\u003eThere is not a standard quantification method for the determination of the antioxidant capacity (Apak et al. 2007) so two different assays were used in this work for the assessment of the antioxidant capacity of the seaweed samples: DPPH (2,2-diphenyl-1-picrilhidrazilo) and CUPRAC (CUPric Reducing Antioxidant Capacity), which evaluate different antioxidant activity, in order to compare our results with others works. CUPRAC assay is an ET-based assay developed by Apak et al. (2004) which consist in a colorimetric method that measures the reducing power of antioxidants to convert Cu\u003csup\u003e2+\u003c/sup\u003e to Cu\u003csup\u003e+\u003c/sup\u003e ion. Briefly, the Cu\u003csup\u003e2+\u003c/sup\u003e-neocuproine complex (blue) can be reduced by antioxidants to Cu\u003csup\u003e+\u003c/sup\u003e-neocuproine (orange), which is a chromophore with a maximum absorption at 450 nm. By contrast, the DPPH assay is a HAT-based method, but DPPH radical can react with both electron and hydrogen donors. The electron transfer occurs first, and the mechanism is very quick, whereas the reaction with hydrogen is slower because it depends on the hydrogen bond accepting solvent used and the steric accessibility to the radical site (Apak et al. 2013; Pyrzynska \u0026amp; Pekal 2013). In DPPH assay, the radical is reduced by the antioxidants and its colour turns from purple to yellow of the corresponding hydrazine (DPPH\u003csub\u003e2\u003c/sub\u003e). In this case, the reducing ability of the antioxidants can be evaluated by monitoring the decrease in the absorbance at 515\u0026ndash;528 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCUPRAC assay\u003c/h2\u003e \u003cp\u003e1 mL of CuCl\u003csub\u003e2\u003c/sub\u003e solution (10 mM), 1 mL of a Neocuproine solution (7.5 mM) in ethanol and 1 mL of NH\u003csub\u003e4\u003c/sub\u003eAC buffer solution were added to a test tube. Then 1 mL of the different seaweed extract dilutions (0.05\u0026ndash;0.25 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was added and the mixture was allowed to incubate for 1h in the dark at room temperature. Absorbance of the different solutions was measured at 450 nm against the reagent blank using a spectrophotometer UV-Visible Evolution\u0026trade; 60S (ThermoFisher scientific, USA) The molar absorptivity of the CUPRAC method for each seaweed sample was obtained from the slope of the calibration curve prepared with the extract dilutions of each sample analysed (i.e. \u003cem\u003eCystoseira jacquinotii\u003c/em\u003e collected in 2016\u0026ndash;2019 and \u003cem\u003eEricaria amentacea\u003c/em\u003e collected in 2018\u0026ndash;2019). The same methodology was used with Trolox, which was selected as a standard reference to compare the antioxidant capacity. Results were expressed as Trolox Equivalents Antioxidant Capacity (TEAC: mg Trolox necessary to provide the same antioxidant capacity as 1 g dry weight of sample).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDPPH assay\u003c/h2\u003e \u003cp\u003eThe free radical scavenging (antioxidant activity) of the seaweed extracts were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical method according to a modified Brand-Williams method (Brand-Williams et al. 1995). A fresh solution of 0.1 mM DPPH was daily prepared in methanol and kept in the dark at room temperature when not used. Different dilutions were prepared (concentration range 0.5-3 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for each seaweed extract (i.e. \u003cem\u003eC. jacquinotii\u003c/em\u003e collected in 2016\u0026ndash;2019 and \u003cem\u003eE. amentacea\u003c/em\u003e collected in 2018 and 2019), in order to evaluate the reaction kinetics. Aliquots of 500 \u0026micro;L of each dilution of seaweed extracts were added to 2.5 mL of DPPH solution (0.1 mM DPPH) and the decrease in absorbance was measured at 517 nm during 30 seg at 0 min, and every 15 min until the reaction reached the steady state. The exact initial concentration of DPPH (A\u003csub\u003e0\u003c/sub\u003e) in the reaction medium and the remaining DPPH concentration at the steady state (A\u003csub\u003eF\u003c/sub\u003e) were calculated from a DPPH calibration curve (0.020\u0026ndash;0.1 mM DPPH). In order to calculate the effective concentration (EC\u003csub\u003e50\u003c/sub\u003e; amount of algal extract concentration necessary to decrease the DPPH concentration by 50%), the DPPH concentration in the steady state (A\u003csub\u003eF\u003c/sub\u003e) was plotted against seaweed extract concentrations tested to obtain the equation:\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eb\u003c/em\u003e is the slope of the linear regression and C\u003csub\u003eA\u003c/sub\u003e is the concentration of the seaweed extract needed to reduce the initial DPPH concentration by 50% (A\u003csub\u003eF\u003c/sub\u003e = 50%). A lower EC\u003csub\u003e50\u003c/sub\u003e value indicates higher antioxidant capacity due to the extract concentration necessary to reduce the DPPH concentration by the 50% will be minor. For comparison purposes, Trolox was used as a standard to evaluate the antioxidant potential. So, 1 mM Trolox solution was prepared and diluted with methanol to give different solutions with concentrations ranging from 0.02\u0026ndash;0.150 mM. These different solutions were used to determine EC\u003csub\u003e50\u003c/sub\u003e value of Trolox. Results were expressed in TEAC.\u003c/p\u003e \u003cp\u003eThe study of the DPPH reaction kinetics is usual in the determination of the antioxidant capacity of food and beverages to obtain more information about the composition of samples (Moon \u0026amp; Shibamoto 2009; Pyrzynska and Pekal 2013; Tomas et al. 2017). In the present work, we also evaluated the reaction kinetics for each \u003cem\u003eC. jacquinotti\u003c/em\u003e (years 2016\u0026ndash;2019) and \u003cem\u003eE. amentacea\u003c/em\u003e (years 2018\u0026ndash;2019) extract solution tested (0.5-3 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhytochemical estimation\u003c/h2\u003e \u003cp\u003eQuantitative phytochemical studies were carried out in order to evaluate the presence of several compounds with antioxidant activity: total phenolic content, and flavonoid.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of total phenolic content\u003c/h2\u003e \u003cp\u003eThe Total Phenolic Content (TPC) of the seaweed extracts was determined according a modified version of Singleton protocol (Singleton et al. 1999). Briefly, aliquots of 500 \u0026micro;L of diluted solutions of each seaweed extract (1 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were mixed with 2.5 mL of 0.2N Folin-Ciocalteu reagent. After 5 min, 2 mL of sodium carbonate 7.5% w w\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e solution was added, and the mixture was incubated for 2h in the dark at room temperature. Then, the absorbance was measured at 760 nm against the corresponding solvent as blank. Gallic acid was used as external standard (calibration curve in the range 10\u0026ndash;60 mg Gallic acid L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and results were expressed as mg of gallic acid equivalents (GAE) per g of dry seaweed material.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of total flavonoid content\u003c/h2\u003e \u003cp\u003eThe flavonoid content was determined by a modified Dowd method (Meda et al. 2005). The seaweed extract (5 mL, 1mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e extract dilution) was mixed with 5 mL of 2% w w\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e aluminium trichloride in methanol. After 10 minutes, absorbance was measured at 415 nm against the corresponding methanol solution as a blank. For the determination of flavonoids, Quercetin was used as a standard to prepare the calibration curve (1\u0026ndash;7 mg Quercetin L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and results were expressed as quercetin equivalents (\u0026micro;g Quercetin equivalents (QE) per mg of dry seaweed material).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData analysis was performed with LibreOffice Calc 7.4.5.1. All measurements were performed in triplicate and results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e \u003cp\u003eLinear regression analysis were performed to estimate the relationship CUPRAC \u003cem\u003evs\u003c/em\u003e antioxidant compound and DPPH \u003cem\u003evs\u003c/em\u003e antioxidant compound, and significance of correlations were tested. Minimum significance level was established at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and the coefficient of determination (r\u003csup\u003e2\u003c/sup\u003e) calculated.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAntioxidant activity\u003c/h2\u003e \u003cp\u003eDuring CUPRAC assay, \u003cem\u003eCystosphaera jacquinotii\u003c/em\u003e exhibited copper ion reduction ability that ranges from 30.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 to 106\u0026thinsp;\u0026plusmn;\u0026thinsp;5 mg Trolox g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dw for the different years. The two highest values correspond to the Deception Island samples. As shown in Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, values obtained for both samples of \u003cem\u003eEricaria amentacea\u003c/em\u003e are similar to those lower values obtained for \u003cem\u003eC. jacquinotii\u003c/em\u003e samples, both from Livingston Island (\u003cem\u003eE. amentacea\u003c/em\u003e collected in 2018 and 2019, with 39\u0026thinsp;\u0026plusmn;\u0026thinsp;3 and 23\u0026thinsp;\u0026plusmn;\u0026thinsp;3 mg Trolox g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dw, respectively). Data reported by Grina et al. (2020) for the same taxon are also similar (24.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 mg TR g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e extract) (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntioxidant activity values (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;confidence interval; n\u0026thinsp;=\u0026thinsp;15; 95% confidence level) obtained by means of DPPH and CUPRAC assays for \u003cem\u003eCystosphaera jacquinotii\u003c/em\u003e and \u003cem\u003eEricaria amentacea\u003c/em\u003e samples, and those reported in the literature.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSpecie\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eDPPH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCUPRAC\u003c/p\u003e \u003cp\u003e(mg TR g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dw)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRef.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(\u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTEAC\u003c/p\u003e \u003cp\u003e(mg TR 100g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dw)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCystosphaera jacquinotii 2016\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1960\u0026thinsp;\u0026plusmn;\u0026thinsp;80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2140\u0026thinsp;\u0026plusmn;\u0026thinsp;90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e75\u0026thinsp;\u0026plusmn;\u0026thinsp;12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003ePresent work\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCystosphaera jacquinotii 2017\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e930\u0026thinsp;\u0026plusmn;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4500\u0026thinsp;\u0026plusmn;\u0026thinsp;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e106\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCystosphaera jacquinotii 2018\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2500\u0026thinsp;\u0026plusmn;\u0026thinsp;400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1700\u0026thinsp;\u0026plusmn;\u0026thinsp;300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e30.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCystosphaera jacquinotii 2019\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1770\u0026thinsp;\u0026plusmn;\u0026thinsp;130\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2400\u0026thinsp;\u0026plusmn;\u0026thinsp;400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e42.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEricaria amentacea 2018\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1930\u0026thinsp;\u0026plusmn;\u0026thinsp;40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2210\u0026thinsp;\u0026plusmn;\u0026thinsp;80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e39\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEricaria amentacea 2019\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1250\u0026thinsp;\u0026plusmn;\u0026thinsp;30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3300\u0026thinsp;\u0026plusmn;\u0026thinsp;107\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e23\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEricaria amentacea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e78.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e24.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eGrina et al. 2020\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCystoseira humilis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e121.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e12.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFucus spiralis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e47.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e55.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEricaria tamariscifolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e580\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eAndrade et al. 2013\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTreptacantha usneoides\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTreptacantha nodicaulis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4940\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSargassum sp.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1180\u0026thinsp;\u0026plusmn;\u0026thinsp;40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYangthong et al. 2009\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFucus ceranoides\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e460\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eZubia et al. 2009\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEricaria tamariscifolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e490\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFucus serratus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5510\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEricaria crinita\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e450.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eMhadhebi et al. 2014\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCystoseira sedoides\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e120.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCystoseira compressa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e750.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFucus spiralis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e141.2\u0026ndash;224.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003ePinteus et al. 2017\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSargassum muticum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44.82\u0026ndash;71.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEricaria tamariscifolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e87.3\u0026ndash;136.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTreptacantha usneoides\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30.79\u0026ndash;99.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSargassum vulgare\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45.1\u0026ndash;74.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSargassum vestitum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e20950\u0026thinsp;\u0026plusmn;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eDang et al. 2018\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSargassum linearifolium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1557\u0026thinsp;\u0026plusmn;\u0026thinsp;80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSargassum podocanthum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e13700\u0026thinsp;\u0026plusmn;\u0026thinsp;400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eRegarding to the antioxidant capacity obtained by DPPH assay, values for both taxa are shown in Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (TEAC, expressed as mg Trolox 100 g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dw). For \u003cem\u003eC. jacquinotii\u003c/em\u003e the sample collected in 2017 shown a higher value than the rest (4500\u0026thinsp;\u0026plusmn;\u0026thinsp;60 mg Trolox 100g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dw), which have values closer to each other (with values between 1700 and 2400 mg Trolox 100g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dw), and closer to \u003cem\u003eE. amentacea\u003c/em\u003e values too (2210\u0026thinsp;\u0026plusmn;\u0026thinsp;80 and 3300\u0026thinsp;\u0026plusmn;\u0026thinsp;107 mg Trolox 100g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dw) (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The higher antioxidant capacity of \u003cem\u003eC. jacquinotii\u003c/em\u003e collected in 2017 (from Punta Files) is not only evidenced by DPPH, but also by CUPRAC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of total phenolic content (TPC)\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the total phenolic content for the studied samples. As it can be observed the total phenolic content for \u003cem\u003eC. jacquinotti\u003c/em\u003e samples ranged from 20.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 to 68\u0026thinsp;\u0026plusmn;\u0026thinsp;11 mg GAE g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dw. The higher value was registered for the \u003cem\u003eC. jacquinotii\u003c/em\u003e sample collected in 2017 (68\u0026thinsp;\u0026plusmn;\u0026thinsp;11 mg GAE g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dw), which is consistent with the antioxidant capacity determined by CUPRAC and DPPH assays, while the rest of samples presented a phenolic content that ranges from ranges 20.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 to 37.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 mg GAE g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dw. Regarding the \u003cem\u003eE. amentacea\u003c/em\u003e samples, values obtained for both samples were lower than 20 mg GAE g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dw.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTotal phenolic content (TPC) (mg GAE g \u0026minus;\u0026thinsp;1 dry weight extract; mean\u0026thinsp;\u0026plusmn;\u0026thinsp;confidence interval; n\u0026thinsp;=\u0026thinsp;5; 95% con fidence level) for \u003cem\u003eCystosphaera jacquinotii\u003c/em\u003e and \u003cem\u003eEricaria amentacea\u003c/em\u003e samples, and those reported in the literature.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecie\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003emg GAE g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dw\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003emg GAE 100 g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dw\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRef.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCystosphaera jacquinotii 2016\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e37.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3740\u0026thinsp;\u0026plusmn;\u0026thinsp;180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003ePresent work\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCystosphaera jacquinotii 2017\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e68\u0026thinsp;\u0026plusmn;\u0026thinsp;11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6800\u0026thinsp;\u0026plusmn;\u0026thinsp;1100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCystosphaera jacquinotii 2018\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e20.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2080\u0026thinsp;\u0026plusmn;\u0026thinsp;60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCystosphaera jacquinotii 2019\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e20.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2050\u0026thinsp;\u0026plusmn;\u0026thinsp;70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEricaria amentacea 2018\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e16.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1670\u0026thinsp;\u0026plusmn;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEricaria amentacea 2019\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e13.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1350\u0026thinsp;\u0026plusmn;\u0026thinsp;30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEricaria crinita\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e56.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eMhadhebi et al. 2014\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCystoseira sedoides\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e50.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCystoseira compressa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e61.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFucus spiralis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e397.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003ePinteus et al. 2017\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSargassum muticum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e71.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEricaria tamariscifolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e57.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTreptacantha usneoides\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e86.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSargassum vulgare\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e124.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSargassum vestitum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e141.91\u0026thinsp;\u0026plusmn;\u0026thinsp;3.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eDang et al. 2018\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSargassum linearifolium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e47.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSargassum podocanthum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e48.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFucus serratus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1200.8\u0026thinsp;\u0026plusmn;\u0026thinsp;9.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eSabeena Farvin et al. 2013\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFucus vesiculosus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1045.0\u0026thinsp;\u0026plusmn;\u0026thinsp;45.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFucus distichus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e862.3\u0026thinsp;\u0026plusmn;\u0026thinsp;9.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFucus spiralis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e752.5\u0026thinsp;\u0026plusmn;\u0026thinsp;13.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of total flavonoid content\u003c/h2\u003e \u003cp\u003eTotal flavonoid content values obtained for the samples tested are shown in Table \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The \u003cem\u003eC. jacquinotii\u003c/em\u003e samples collected in 2016 and 2017 presented the highest values with 0.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 and 0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;g QE mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dw, respectively. The sample of \u003cem\u003eC. jacquinotii\u003c/em\u003e from 2018 presented the lowest flavonoid content for this specie, with 0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;g QE mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dw. In the case of \u003cem\u003eE. amentacea\u003c/em\u003e, the sample collected in 2018 showed a value similar to the Antarctic species (0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 \u0026micro;g QE mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dw) while the one from 2019 presented a lower flavonoid content (0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;g QE mg \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dw).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTotal flavonoid content (\u0026micro;g QE mg \u0026minus;\u0026thinsp;1 dry weight extract; mean\u0026thinsp;\u0026plusmn;\u0026thinsp;confidence interval; n\u0026thinsp;=\u0026thinsp;5; 95% confidence level) for \u003cem\u003eCystosphaera jacquinotii\u003c/em\u003e and \u003cem\u003eEricaria amentacea\u003c/em\u003e samples, and those reported in the literature (including other taxa of Fucales).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026micro;g QE mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dw extract\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRef.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCystosphaera jacquinotii 2016\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003ePresent work\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCystosphaera jacquinotii 2017\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCystosphaera jacquinotii 2018\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCystosphaera jacquinotii 2019\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEricaria amentacea 2018\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEricaria amentacea 2019\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEricaria amentacea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e8.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eGrina et al. 2020\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCystoseira humilis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e6.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFucus spiralis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e26.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCorrelation analysis between antioxidant activity and phenolic content\u003c/h2\u003e \u003cp\u003eLinear regression analysis between CUPRAC and DPPH \u003cem\u003evs\u003c/em\u003e antioxidant compound (TPC) is showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, where a correlation is evidenced. Ordinary least squares regressions provide high values for r\u003csub\u003e2\u003c/sub\u003e (0.78645 for CUPRAC and 0.72591 for DPPH), explaining the consequent increase in antioxidant activity (by any of both methods) the greater amount of polyphenols.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eEventual differences in antioxidant capacity values determined by CUPRAC and DPPH could be related with the nature and compositions of the antioxidant compounds in each seaweed sample (Brand-Williams et al. 1995). Antioxidants are bioactive compounds considered as effective chain breaking agents due to their capacity to neutralize chemically active metabolic products, such as free radicals (Kurek et al. 2022). One of the most important are the phenolic compounds, and among them flavonoids, due to their high antioxidant activity (Shahidi et al. 1992). On this regard, the total phenolic plus flavonoid content was evaluated and compared with the antioxidant capacity for the Brown algae samples of the Antarctic \u003cem\u003eCystosphaera jacquinotii\u003c/em\u003e and the Mediterranean \u003cem\u003eEricaria amentacea\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eOur analyses revealed the high antioxidant activity in samples of \u003cem\u003eE. amentacea\u003c/em\u003e, in agreement with the reports of earlier studies (Grina et al. 2020) (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), but even more in those of the Antarctic species \u003cem\u003eC. jacquinotii\u003c/em\u003e, never reported before this work. In particular, values from samples of Punta Files (Deception) are markedly higher than the rest, mainly with CUPRAC, although no environmental differences between both Antarctic islands have been found that explain these results.\u003c/p\u003e \u003cp\u003eIn general, the antioxidant capacity determined by the CUPRAC assay for \u003cem\u003eC. jacquinotii\u003c/em\u003e is higher than that obtained for \u003cem\u003eE. amentacea\u003c/em\u003e. However, the values obtained in the DPPH assay showed that the antioxidant capacity for both seaweeds species is not very different, with the exception of the \u003cem\u003eC. jacquinotii\u003c/em\u003e samples collected in Deception Island in 2017. The differences found between the results obtained with each method could be related to the mechanisms of action of the antioxidant compounds. While CUPRAC assay is an electron transfer (SET)-based assay that detects the capability of the antioxidant compounds to transfer one electron to reduce metal ions, DPPH is a mixed-mode assay consisting in the determination of the capability of the antioxidants to quench free radicals by means of the transfer of one electron (or the hydrogen atom transfer) from the antioxidant compounds to the DPPH radical (Apak et al. 2018). The H transfer is conditioned by the nature of the antioxidant compound (Brand-Williams et al. 1995) and the complexity of the sample (Carmona-Jim\u0026eacute;nez et al. 2014) which determines the velocity of the decrease in the absorbance and the time to reach the steady state. The study of the DPPH reaction kinetics is usual in the determination of the antioxidant capacity of food and beverages to obtain more information about the composition of samples (Pyrzynska \u0026amp; Pekal 2013; Tomas et al. 2017; Moon \u0026amp; Shibamoto 2009). In the present work, we also evaluated the reaction kinetics for each \u003cem\u003eC. jacquinotti\u003c/em\u003e (years 2016\u0026ndash;2019) and \u003cem\u003eE. amentacea\u003c/em\u003e (years 2018\u0026ndash;2019) extract solution tested (0.5-3 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb shows the evaluation of the decrease in the relative absorbance with time for the \u003cem\u003eC. jacquinotti\u003c/em\u003e 2017 and \u003cem\u003eE. amentacea\u003c/em\u003e 2019 samples. These samples were selected to represent the kinetic behaviour of a \u003cem\u003eC. jacquinotti\u003c/em\u003e and \u003cem\u003eE. amentacea\u003c/em\u003e extract solution. Similar results were obtained for the remaining extract solutions. Relative absorbance (A/A\u003csub\u003e0\u003c/sub\u003e) is defined as the absorbance after a certain incubation time relative to the absorbance value obtained at the beginning of the reaction (i.e., time 0 min). For both, a fast decrease in the absorbance was observed in the first minutes (\u0026lt;\u0026thinsp;5 min) (corresponding to the electron transfer), followed by a slower decrease in the absorbance values (H transfer mechanism) until the reaction reached the steady state (120 min approximately). Similar kinetic behaviours were obtained for the rest of the samples. These results were also compared to those obtained for Trolox, used as a reference standard (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Trolox reacted very fast with the DPPH radical since this standard reached the steady state in a few minutes (6 approximately) which indicates a fast-kinetic reaction regarding the studied samples. The evidenced different velocity between our samples and Trolox indicates that the antioxidant compounds contained in our samples are more complex molecules and are more sterically hindered than Trolox. From reaction kinetic graphs, the percentage of DPPH remaining at the steady state was determined and the values were transferred onto another graph (not shown) showing the percentage of residual DPPH at the steady state as a function of the seaweed extract concentration tested in order to obtain the efficient concentration (EC\u003csub\u003e50\u003c/sub\u003e) gathered in Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e which are related with the TEAC values.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough there is not a standard methodology to determine the antioxidant capacity because of the broad variety of antioxidant compound extractions procedures and the diversity of samples, the results obtained in the present work were comparable with some of those reported in the literature for both assays (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Regarding the CUPRAC assay, the antioxidant capacity values reported by Grina et al. (2020) were of the same order of magnitude that those obtained in our analysis for \u003cem\u003eE. amentacea\u003c/em\u003e (Table \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Interestingly, the values obtained for the \u003cem\u003eC. jacquinotii\u003c/em\u003e samples were slightly higher than the values reported by those authors for the different Fucales samples evaluated. Concerning the DPPH assay results, the EC 50 values obtained for \u003cem\u003eC. jacquinotii\u003c/em\u003e and \u003cem\u003eE. amentacea\u003c/em\u003e in the present study were between 8 and 20-fold higher than those reported in the literature for \u003cem\u003eE. amentacea\u003c/em\u003e and other Fucales which indicated that according to the DPPH assay, the antioxidant capacity of \u003cem\u003eC. jacquinotii\u003c/em\u003e and \u003cem\u003eE. amentacea\u003c/em\u003e was lower than that reported by Grina et al. (2020). Those differences observed are probably related mainly to the seaweed preparation procedure and the methodology used for the determination of the phenolic content. In the present work the extractant used to prepare the samples was methanol 100% (at room temperature, 12 hours), whereas Grina et al. (2020) prepared the extract with a solution composed by 70% ethanol (at 60\u0026ordm;C, but only 2 hours). Those methodological differences could affect directly to the compounds that were present in the extract obtained, due to the polarity of each extractant solution employed, the phenol units of the antioxidant compounds and the temperature conditions employed to carry out the extractions. Besides, protocols for measuring phenolic content were also different. Since extraction protocols used by Grina et al. (2020) got higher quantities of flavonoids for \u003cem\u003eE. amentacea\u003c/em\u003e, it is expected that the application of these protocols to the Antarctic species \u003cem\u003eC. jacquinotii\u003c/em\u003e also would lead to an optimization of the extraction of flavonoids.\u003c/p\u003e \u003cp\u003eIn this study, the selection of the extraction conditions was focused on three factors: (i) the extractant; (ii) the extraction temperature; and (iii) the extraction time. In the case of the extractant, preliminary experiments were carried out using water, ethanol, and methanol as extractants according to previous works reported in the literature (Mhadhebi et al. 2014; Dang et al. 2018; Grina et al. 2020). Water yielded an extract with a viscous texture that hampered its manipulation. Regarding the alcohols used, the extracts obtained with methanol presented higher antioxidant capacity values compared to those afforded by the ethanol ones. Thus, methanol was selected to prepare the extracts of the different samples. Concerning the temperature, it was maintained at room temperature (25\u0026ordm;C) during the extraction to avoid the degradation of the antioxidant compounds at higher temperatures. Finally, different extraction times were also tested, i.e. 2, 6, and 12h (overnight), obtaining the best results after 12h of extraction.\u003c/p\u003e \u003cp\u003eRelated with total phenolic content (TPC), the analysis revealed that \u003cem\u003eC. jacquinotii\u003c/em\u003e exhibited higher values than \u003cem\u003eE. amentacea\u003c/em\u003e (Table \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Samples from Deception Island presented the higher phenolic content (68\u0026thinsp;\u0026plusmn;\u0026thinsp;11 and 37.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 \u0026micro;g GAE mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dw) that agreed with the antioxidant capacity obtained by those samples, especially in CUPRAC assays. It can be observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e that the TPC values are directly proportional to the antioxidant capacity values obtained for each sample in CUPRAC assays, although DPPH assays did not show such a clear correlation, because of the arguments presented above. This fact revealed that the main responsible for the antioxidant properties of the seaweed evaluated are phenolic compounds, as previous authors already documented (cf. Karawita et al. 2005). This positive correlation is evidenced in the correlation analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), but they are not the sole compounds implicated. In fact, many researchers have identified several compounds as antioxidants in seaweeds, including protective enzymes, ascorbic acid, lipophilic antioxidants, phlorotannins and catechins (Mhadhebi et al. 2014).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTotal phenolic content obtained for samples tested (Antarctic and Mediterranean) are similar to those values reported in the literature for other genera of Fucales (i.e. \u003cem\u003eCystoseira\u003c/em\u003e s.l., \u003cem\u003eSargassum\u003c/em\u003e, \u003cem\u003eFucus\u003c/em\u003e) from other locations (i.e. Mediterranean Sea, Atlantic Ocean, South Pacific Ocean, and Danish coasts) (see Table \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFinaly, regarding the total flavonoid content, our data were compared to those reported by Grina et al. (2020), since they used a similar determination method for other Fucales (Table \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In our case, values obtained for \u003cem\u003eC. jacquinotii\u003c/em\u003e samples are higher than values for \u003cem\u003eE. amentacea\u003c/em\u003e samples, but not significantly. In fact, correlation for the relationship of the antioxidant activity (by both methods) with the concentration of TPC is clear (r\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.78645 for CUPRAC, and r\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.72591 for DPPH) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), but this relationship with flavonoids is not so evident (r\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.45074 for CUPRAC, and r\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.16109 for DPPH). This fact evidences that other phenolic compounds participate in the antioxidant activity, and it is especially evident in DPPH, since this method also measures reduction by hydrogen, probably given by other compounds.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThis study revealed that the Antarctic brown seaweed \u003cem\u003eCystosphaera jacquinotii\u003c/em\u003e presents a high TPC that directly contributes to the strong antioxidant activity detected. The comparison with \u003cem\u003eEricaria amentacea\u003c/em\u003e, the other Fucal analysed in this work with the same protocols, evidences the observed high antioxidant property. In the same way, our findings confirm other previous results related to brown algae and their bioactive compounds with antioxidant activity. Those extracts can be used as easily accessible source of natural antioxidants, and also as a possible food supplement or in pharmaceutical industry.\u003c/p\u003e \u003cp\u003eThe fact that samples from Deception Island exhibit a greater antioxidant capacity does not seem to be connected to distinct peculiarities in the ecological conditions of the site. However, new samplings and studies should be carried out in Antarctic Polar to check whether the higher antioxidant values in samples from Deception Island are unique or are also found in other places around. In any case, the factor or factors conditioning eventual differences might be identified.\u003c/p\u003e \u003cp\u003eFurthermore, efforts should be focused on studying the kinetics of the extracts of the different species in DPPH assay, since it is a markedly variable factor that must be considered to assess the antioxidant capacity of each taxon, as shown in the present work.\u003c/p\u003e \u003cp\u003eAdditional studies are needed to better understand the role of the different antioxidant activity of various compounds in the studied brown seaweeds, and to find other proper parts and seasons for a given species to get higher antioxidant activity and improved extraction protocols. Therefore, further chemical investigations are needed to properly determine the antioxidant activity in different aqueous or polar extracts, through standardized extraction methods and antioxidant activity analyses.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors thank R. Mart\u0026iacute;n for providing us with Antarctic samples, collected by himself and others collectors (C. \u0026Aacute;vila, C. Angulo, J. Gim\u0026eacute;nez, J Vicente de Bobes, B. Figuerola).\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eC. Pena-Mart\u0026iacute;n: conceptualization, methodology, formal analysis, investigation, resources, writing, visualization and project administration.\u003c/p\u003e\n\u003cp\u003eR. Serrano: methodology, formal analysis, investigation, resources, data curation and writing.\u003c/p\u003e\n\u003cp\u003eG. Grindlay: methodology and resources.\u003c/p\u003e\n\u003cp\u003eMB. Crespo: resources and writing.\u003c/p\u003e\n\u003cp\u003eAvailability of data and material\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe data required to support this research is made available within the manuscript. Samples from which fragments were analyzed for this study are conserved in the Herbarium of the University of Alicante (ABH-Algae).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdala-D\u0026iacute;az R, Cabello-Pasini A, M\u0026aacute;rquez-Garrido A, L\u0026oacute;pez Figueroa F (2006) Variaci\u0026oacute;n intratalo de compuestos fen\u0026oacute;licos, actividad antioxidante y actividad de la fenolsulfatasa en \u003cem\u003eCystoseira tamariscifolia\u003c/em\u003e (Phaeophyceae) del sur de Espa\u0026ntilde;a. Ciencias Marinas 40(1):01\u0026ndash;09.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAndrade PB, Barbosa M, Pedro Matos R, Lopes G, Vinholes J, Mouga T, Valentao P (2013) Valuable compounds in microalgae extracts. Food chemistry 138:1819\u0026ndash;1828.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eApak R, Capanoglu E, Shahidi F (Eds.) (2018) Measurement of antioxidant activity and capacity: Recent trends and applications. John Wiley and Sons Ltd. Oxford, UK. ISBN: 978-1-119-13535-7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eApak R, Gorinstein S, B\u0026ouml;hm V, Schaich KM, \u0026Ouml;zy\u0026uuml;rek M, Gl\u0026uuml;\u0026ccedil;\u0026uuml; K (2013) Methods of measurement and evaluation of natural antioxidant capacity/activity (IUPAC Technical report). Pure Appl Chem 85:957\u0026ndash;998.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eApak R, G\u0026uuml;\u0026ccedil;l\u0026uuml; K, Demirata B, \u0026Ouml;zy\u0026uuml;rek M, \u0026Ccedil;elik SE, Bektasoglu B, Berker KI, \u0026Ouml;zyurt D. (2007) Comparative evaluation of various total antioxidant capacity assays applied to phenolic compounds with the CUPRAC assay. Molecules 12:1496\u0026ndash;1547.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eApak R, G\u0026uuml;\u0026ccedil;l\u0026uuml; K, \u0026Ouml;zy\u0026uuml;rek M, Karademir SE (2004) Novel antioxidant capacity index for dietary polyphenols and vitamins C and E, using their Cupric Ion Reducing Capability in the presence of Neocuproine: CUPRAC method. J Agric Food Chem 52: 7970\u0026ndash;7981.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAudibert L, Fauchon M, Blanc N, Hauchardb D, Ar Gall E (2010) Phenolic Compounds in the Brown Seaweed Ascophyllum nodosum: Distribution and Radical-scavenging Activities Phytochem Anal 21:399\u0026ndash;405.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrand-Williams W, Cuvelier M, Berset C. (1995) Use of free radical method to evaluate antioxidant activity. Lebensm Wiss Technol 28:25\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBruno de Sousa C, Cox CJ, Brito L, Pav\u0026atilde;o MM, Pereira H, Ferreira A, Ginja C, Campino L, Bermejo R, Parente M, Varela J (2019) Improved phylogeny of brown algae \u003cem\u003eCystoseira\u003c/em\u003e (Fucales) from the Atlantic-Mediterranean region based on mitochondrial sequences. Plos One 14(1): e0210143.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBudhiyanti SA, Raharjo S, Marseno DW, Lelana IYB (2012) Antioxidant Activity of Brown Algae Sargassum species Extract from the Coastline of Java Island. Am J Agric Biol Sci 7(3):337\u0026ndash;346.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarmona-Jim\u0026eacute;nez Y, Garc\u0026iacute;a-Moren MV, Igartuburu JM, Garc\u0026iacute;a Barroso C (2014) Simplification of the DPPH assay for estimating the antioxidant activity of wine ans wine by-products. Food Chem 165:198\u0026ndash;204.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCosta-Leal M, Madeira C, Brandao CA, Puga J, Calado R (2012) Bioprospecting of Marine Invertebrates for New Natural Products \u0026mdash; A Chemical and Zoogeographical Perspective. Molecules 17:9842\u0026ndash;9854.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe los Reyes E, Sapin AB (2020) Bioprospecting of Turbinaria ornata (Fucales, Phaeophyceae) for cosmetic application: antioxidant, tyrosinase inhibition and antibacterial activities. J ISSAAS 26(2):30\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDang TT, Bowyer MC, Van Altena IA, Scarlett CJ (2018) Comparison of chemical profile and antioxidant properties of the brown algae. Inter J Food Sci Tec 53:174\u0026ndash;181.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerreres F, Lopes G, Gil-Izquierdo A, Andrade PB, Sousa C, Mouga T, Valentao P (2012) Phlorotannin Extracts from Fucales Characterized by HPLC-DAD-ESI-MS\u003cem\u003en\u003c/em\u003e: Approaches to Hyaluronidase Inhibitory Capacity and Antioxidant Properties. Mar Drugs 10(12): 2766-81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlores-Molina MR, Rautenberger R, Mu\u0026ntilde;oz P, Huovinen P, G\u0026oacute;mez I (2016) Stress Tolerance of the Endemic Antarctic Brown Alga Desmarestia anceps to UV Radiation and Temperature is Mediated by High Concentrations of Phlorotannins. Photochem Photobiol 92(3):455\u0026ndash;466.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrassini R, Da Silva YP, Moura S, Zambotti L, Paternostro A, Colepicolo P, Toyota M, Yokota NS, Pereira CM, Zingalli VR, Prgas JA, Roesch-Ely M (2019) Chemical Characterization and Cytotoxic Activity of Antarctic Macroalgae Extracts against Colorectal Cancer. Advances in Biological Chemistry 9: 167\u0026ndash;177.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrina F, Ullah Z, Kaplaner E, Moujahid A, Eddoha R, Nasser B, Terzioglu P, Yilmaz MA, Erta A,\u0026Ouml;zt\u0026uuml;rk M, Essamadi A (2020) In vitro enzyme inhibitory properties, antioxidant activities, and phytochemical fingerprints of five Moroccan seaweeds. South African J Bot 128: 152\u0026ndash;160.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuiry MD, Guiry GM. AlgaeBase. World-wide electronic publication, National University of Ireland, Galway. http://www.algaebase.org; accessed 1 February 2024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIken K, Amsler CD, Amsler MO, McClintock JB, Baker BJ (2009) Field studies on deterrent properties of phlorotannins in Antarctic brown algae. Bot Mar 52:547\u0026ndash;557.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJassbi AM, Mohabati M, Eslami S, Sohrabipour J, Miri R (2013) Biological activity and chemical constituents of red and brown algae from the persian gulf. Iran J Pharm Res 12(3):339\u0026ndash;348.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarawita R, Siriwardhana N, Lee KW, Heo MS, Yeo IK, Lee YD, Jeon YJ (2005) Reactive oxygen species scavenging, metal chelation, reducing power and lipid peroxidation inhibition properties of different solvent fractions from Hizikia fusiformis. Eur Food Res. Technol. 220:363\u0026ndash;371.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKohen R, Nyska A (2002) Invited Review: Oxidation of Biological Systems: Oxidative Stress Phenomena, Antioxidants, Redox Reactions and Method for their quantification. Toxicol Pathol 30(6):620\u0026ndash;650.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKurek M, Benaida-Debbache N, Garofolic IE, Galic K, Avallone S, Voilley A, Wach\u0026eacute; Y (2022) Antioxidants and Bioactive Compounds in Food: Critical Review of Issues and Prospects. Antioxidants (Basel) 11(4): 742.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMannino AM, Vaglica V, Cammarata M, Oddo E (2015) Effects of temperature on total phenolic compounds in Cystoseira amentacea (C. Agardh) Bory (Fucales, Phaeophyceae) from southern Mediterranean Sea. Plant Biosyst 150(1):152\u0026ndash;160.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartins RM, Nedel F, Guimar\u0026atilde;es VBS, da Silva AF, Colepicolo P, de Pereira CMP, Lund RG (2018) Macroalgae extracts from antarctica have antimicrobial and anticancer potential. Front. Microbiol. 9:412.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeda A, Lamien CE, Romito M, Millogo J, Naoulma OG (2005) Determination of the total phenolic, flavonoid and proline contents in Burkina Fasan honey, as well as their radical scavening activity. Food Chem 91:571\u0026ndash;577.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMhadhebi L, Mhadhebi A, Robert J, Bouraoui A (2014) Antioxidant, anti-inflammatory and antiproliferative effects of aqueous extracts of three Mediterranean brown seaweeds of the genus Cystoseira. Ir J Pharm Res 13:207\u0026ndash;220.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoon JK, Shibamoto T (2009) Antioxidant assays for plant and food components. J Agric Food Chem 57:1655\u0026ndash;1666.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark PJ, Fereidoon S, You-Jin J (2004) Antioxidant activities of enzymatic extracts from an edible seaweed Sargassum horneri using ESR spectrometry. J Foods Lipids 11(1):15\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePinteus S, Silva J, Alves C, Horta A, FIno N, Rodrigues AI, Mendes SP (2017) Cytoprotective effect of seaweeds with high antioxidant activity from the Peniche coast (Portugal). Food Chem 218:591\u0026ndash;599. DOI:10.1016/j.foodchem.2016.09.067\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePyrzynska K, Pekal A (2013) Application of free radical diphenilpicrylhydrazyl (DPPH) to estimate the antioxidant capacity of food samples. Anal Methods 5:4288\u0026ndash;4295.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRyabushko VI, Prazukin AV, Popova EV, Nekhoroshev MV (2014) Fucoxanthin of the brown alga Cystoseira barbata (Stackh.) C. Agardh from the Black Sea. J Black Sea 20(2):108\u0026ndash;113.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSabeena Farvin KH, Jacobsen C (2013) Phenolic compounds and antioxidant activities of selected species of seaweeds from the Danish coast. Food Chem 138:1670\u0026ndash;1681.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSenthilkumar K, Manivasagan P, Venkatesan J, Kim SK (2013) Brown seaweed fucoidan: Biological activity and apoptosis, growth signalling mechanism in cancer. Int J Biol Macromol 60:366\u0026ndash;374.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingleton VL, Orthofer R, Lamuela-Revent\u0026oacute;s RM (1999) Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods Enzymol 299:152\u0026ndash;178.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShahidi, F, Janitha, PK, Wanasundara, PD (1992) Phenolic antioxidants. Crit Rev Food Sci Nutr 32:67\u0026ndash;103.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTomas M, Beekwilder J, Hall RD, Sagdic O, Boyacioglu D, Capanoglu E (2017) Industrial processing versus home processing of tomato sauce: Effects on phenolics, flavonoids and in vitro bioaccessibility of antioxidants. Food Chem 220:51\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVizetto-Duarte C, Cust\u0026oacute;dio L, Barreira L, Moreira da Silva M, Rauter AP, Albericio F, Varela J (2016) Proximate biochemical composition and mineral content of edible species from the genus Cystoseira in Portugal. Bot Mar 59(4): 251\u0026ndash;257.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWiencke C, Amsler CD, Clayton MN (2014) Macroalgae in Biogeographic atlas of the southern ocean. In: De Broyer C, Koubbi P, Griffiths HJ, Raymond B, Udekem d\u0026rsquo;Acoz C d\u0026rsquo;, et al. (eds.). Scientific Committee on Antarctic Research, Cambridge, pp 66\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWiencke C, Clayton MN, G\u0026oacute;mez I, Iken K, L\u0026uuml;der U, Amsler CD, Karsten U, Hanelt D, Bischof K, Dunton K (2007) Life strategy, ecophysiology and ecology of seaweeds in polar water. Rev Environ Sci Biotechnol 6:95\u0026ndash;126\u0026lt;?ColorInfoStart FFFFFF?\u0026gt; 66\u0026lt;?ColorInfoEnd FFFFFF?\u0026gt;\u0026ndash;73\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYangthong M, Hutadilok-Towatana N, Phromkunthong W (2009) Antioxidant activities of four edible seaweeds from the southern coast of Thailand. Plant Foods Hum Nutr 64:218\u0026ndash;223.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZubia M, Fabre MS, Kerjean V, Le Lann K, Stiger-Pouvreau V, Fauchon M, Deslandes E (2009) Antioxidant and antitumoural activities of some Phaeophyta from Brittany coasts. Food Chem 116:693\u0026ndash;701.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Antioxidant activity, DPPH, CUPRAC, total phenolic content, Cystosphaera jacquinotii, Ericaria amentacea.","lastPublishedDoi":"10.21203/rs.3.rs-4660453/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4660453/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe present work was carried out to evaluate the antioxidant activity of \u003cem\u003eCystosphaera jacquinotii \u003c/em\u003eand to quantify its total phenolic content (TPC), compared with other fucal, \u003cem\u003eEricaria amentacea\u003c/em\u003e. Phenolic compounds occur in high quantities in brown algae and they have an essential paper in antioxidative action.\u003c/p\u003e\n\u003cp\u003eExtracts were prepared with methanol dilution. The quantification methods used for the determination of the antioxidant capacity were CUPRAC and DPPH.\u003c/p\u003e\n\u003cp\u003eFrom the obtained results it can be concluded that \u003cem\u003eCystosphaera jacquinotii\u003c/em\u003e presents a high TPC, which directly contributes to the strong antioxidant activity detected, even higher than \u003cem\u003eEricaria amentacea\u003c/em\u003e. Besides, this work evidences that the kinetics of the extracts in DPPH assays is a markedly variable factor that must be considered to assess the antioxidant capacity of seaweeds species. Furthermore, standardized extraction methods and antioxidant activity analyses are needed to properly compare data and get the optimal ones. In the same way, more studies on the role of different compounds in antioxidant activity are wanting, which will help to get the best performance.\u003c/p\u003e","manuscriptTitle":"Assessment of antioxidant capacity of Cystosphaera jacquinotii (Fucales, Ochrophyta) by two methods: DPPH and CUPRAC.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-24 12:49:17","doi":"10.21203/rs.3.rs-4660453/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7de84005-0b9b-4bad-8206-36b356609421","owner":[],"postedDate":"July 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-24T13:06:50+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-24 12:49:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4660453","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4660453","identity":"rs-4660453","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}