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 2a and 2b 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/A0) 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. 2c). 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 (EC50) 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 (r2 = 0.78645 for CUPRAC, and r2 = 0.72591 for DPPH) (Fig. 1), but this relationship with flavonoids is not so evident (r2 = 0.45074 for CUPRAC, and r2 = 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.