Samples of macroalgae C. jacquinotii, I. cordata, H. grandifolius, and P. endiviifolia were collected during the expeditions integrating the Brazilian Antarctic Program, between January and February 2012, in Admiralty Bay, at different sites on King George Island, Antarctica. Collected macroalgae were dried at room temperature and placed in plastic bags to protect them from light Figure 1. The project was supported by the Botanical Institute of São Paulo (Government of the state of São Paulo, Brazil). Dr. Toyota Fujii Mutue, an expert on phycology, identified the authenticity of the collected algae biomass and the material was compared to a database of Botanic Institute. Part of this collection was recently reported in the literature by Fujii et al. (2014).

At each sampling site, macroalgae were randomly collected from the intertidal region by a destructive method inside 1 m² quadrants along three transects of 100 m, parallel to the coast, during low tide. Temperature, salinity, and pH were monitored using a double channel multiprobe (HQ40D Hexis/Hach—Loveland CO USA) on three surface water samples for each sampling point (Pellizzari et al., 2017). Table 1 provides information on the macroalgae collected and used in this study. The seaweeds were washed with local seawater to remove all extraneous matter, such as epiphytes, sand particles, pebbles and shells, and transported to the laboratory in plastic bags.

The lyophilized biomass (5 g) of each macroalga was extracted sequentially with 150 mL of different solvents (i.e., hexane/chloroform/ethyl acetate/ethanol) using a Soxhlet apparatus for 6 h, at a temperature not exceeding the boiling point of the solvent. Procedure was repeated 2–3 times to obtain a sizable quantity of extract. The solvent was removed from each extract under reduced pressure, at 20–25°C. They were finally dried under a N₂ stream. Extracts were weighed and stored at −20°C until use. Yields of the extracts ranged between 1 and 2%.

Table 2 shows the results of antimicrobial screening of the extracts and drug standards (characterized by IC₅₀, MIC, and MMC rates, μg mL⁻¹) against a panel of selected microbial strains. EA of C. jacquinotii proved to have the highest antibacterial activity among the different extracts, with IC₅₀ = 46.75 μg mL⁻¹ and MIC = 500 μg mL⁻¹ against E. faecalis. None of the other extracts showed in vitro antibacterial activity against the tested bacterial strains at concentrations up to 500 μg mL⁻¹.

Further, EA of H. grandifolius exhibited the most notable antifungal activity of all the extracts studied. In addition, it showed not only fungistatic but also fungicidal activity. The ethyl acetate extract of H. grandifolius showed IC₅₀ rates ranging between 4.94 and 49.50 μg mL⁻¹ and MIC and MMC rates ranging between 31.25 and 500 μg mL⁻¹ against fungal strains. The other extracts showed only low or no activity against the fungal strains of the panel at concentrations up to 500 μg mL⁻¹.

Ethyl Acetate (EA) of H. grandifolius was the most active against the fluconazole-resistant strains C. albicans ATCC62342 and C. albicans clinical isolate 1. Although the IC₅₀ rates were lower when compared to those of fluconazole, MIC and MMC were better, with rates 500 and 250 μg mL⁻¹ against C. albicans ATCC62342 and C. albicans clinical isolate 1, respectively. These rates are >500 μg mL⁻¹ for fluconazole. The extract exhibited activity equal to fluconazole against C. albicans clinical isolate 3 (MMC 62.5 μg mL⁻¹) and C. albicans clinical isolate 4 (MIC and MMC 31.25 μg mL⁻¹). Furthermore, EA had an equal MIC (125 μg mL⁻¹) and better MMC (125 μg mL⁻¹) rates than fluconazole (MMC 250 μg mL⁻¹) against C. glabrata.

When the different fungal strains were compared, one could perceive that C. albicans clinical isolate 4 was the most susceptible and C. albicans ATCC62342 was the most resistant strain. Results demonstrate that the sensitivity of Candida to seaweed extracts varies according to the different strains and species of the genus.

The genus Candida is the most important opportunistic fungal pathogen (Spampinato and Leonardi, 2013). Further, increase in antifungal resistance to commercial drugs and the incidence of fungal infections cause serious public health problems (Souza et al., 2016). Therefore, there is an urgent need to develop new compounds featuring low cytotoxicity but high potency to treat mycoses.

H. grandifolius appears to be a particularly interesting algal species, showing activity against all the fungal strains tested, including the fluconazole-resistant samples. According to Sufian et al. (2013), plant extracts with MIC rates below 1,000 μg mL⁻¹ are considered noteworthy, and isolated compounds with MICs ≤ 100 μg mL⁻¹ are considered active. Therefore, based on this criterion, EA was clearly the most active extract, demonstrating equal or higher rates than fluconazole, the reference drug. Although MIC/MMC rates >69 μg mL⁻¹ are considered toxic for fluconazole (Clinical and Laboratory Standards Institute, 2008), EA did not display any cytotoxic effects at concentrations up to 500 μg mL⁻¹, demonstrating the capacity of selectively killing yeast cells, with little damage to normal cells.

Results from cytotoxicity screening of extracts against NIH/3T3 cell line are shown in Figure 2. In general, the extracts presented very low or no cytotoxicity, with an inhibitory rate lower than 50%. The exception was CE of H. grandifolius. As shown in Figure 2, at H. grandifolius CE concentrations of 250 and 500 μg mL⁻¹, the cytotoxicity increased to 48.3/57.1% (24/48 h) and 64.5/82.5% (24/48 h), respectively. Some extracts, such as the CE and EE of I. cordata were able to stimulate the growth of normal cell lines after exposure for 24 h.

Since most of the algal extracts tested showed very low or no cytotoxic effects on the normal cell line, other potentially important biological activities of these algal extracts should be explored in the future. Some extracts, such as the CE and EE of I. cordata, were able to stimulate the growth of the normal cell line. These data may be considered useful for other investigations, such as those exploring healing processes.

The cytotoxicity of the algal extracts against cancer cell line A-431 is shown in Figure 3. C. jacquinotii extracts had low cytotoxicity against the A-431 cell line, with an inhibitory rate lower than 50%. No significant difference was detected between different types and concentrations of algal extracts and exposure times (24 and 48 h). EA of I. cordata displayed the highest cytotoxic activity of all extracts tested against A-431 cells. The extract increased the inhibitory ratio to 91.1 and 95.6% after 24 and 48 h of exposure, respectively, at a concentration of 500 μg mL⁻¹. There was no significant difference between A-431 cells exposed to I. cordata extracts for 24 and 48 h. Another study on antitumor activity of Antarctic macroalgae, including I. cordata, against cells from the malignant tumor Glioblastoma Multiforme was also published (Pereira et al., 2015). Another study isolated and characterized sulfated polysaccharides (SPs) from I. cordata and evaluated their anticancer activity against PC-3 (prostate cancer), HeLa (cervical cancer), and HT-29 (human colon adenocarcinoma). Their antitumor activities, albeit weak, proved to be interesting for medical attention and application (Kim et al., 2016). H. grandifolius CE and EE extracts showed the highest inhibitory activity against A-431 cells. CE concentration 500 μg mL⁻¹ increased the cytotoxicity to 53.3 and 61.6% after 24 and 48 h of exposure, respectively, while the same concentration of EE increased cytotoxicity to 41% after a 48 h-exposure. However, no significant difference was detected between the two extracts. A previous study on the selective cytotoxic effect of an H. grandifolius hydroacetonic extract on epithelial tumor cell lines (A375, A549, Hep-2, HeLa) revealed the capacity to suppress proliferation and promote apoptosis-mediated cell death with the induction of initial stages of apoptosis in these cell lines (Gambato et al., 2014). At a concentration of 500 μg mL⁻¹, the HE of P. endiviifolia increased the inhibitory rate to 56.6 and 74.9% after 24 and 48 h-exposure times, respectively. However, no significant differences were found among P. endiviifolia extracts.

Non-melanoma skin cancer is the most frequently diagnosed cutaneous malignancy, accounting for over 90% of all incidences of skin cancer (Jiang et al., 2012; Pal et al., 2013). Although skin cancer is often treated by surgery, chemotherapy or radiation therapies, the risk of recurrence and metastasis remains a concern (Jiang et al., 2012). Therefore, continuous efforts have been made to pursue and develop new approaches to treat skin cancer more effectively. In the present study, the authors detected that EA of I. cordata showed very strong cytotoxicity against an epidermoid carcinoma cell line (A-431) at a concentration of 500 μg mL⁻¹.

Several authors have suggested that the production of bioactive substances varies within a species. Variability may be due to seasonal variations, geographic distribution and growth stage or sexual maturation (Manilal et al., 2009; Patra et al., 2009; Galal et al., 2011). Our samples are brown and red, and they were collected from the Antarctic in the summer. Our results would have been different if seaweed samples had been collected during other seasons or at different growth stages and sexual maturation.

The bioactivity of the seaweed extracts may be influenced by extraction protocols and extraction capacity of the solvents used. Although a variety of solvents have been employed in screening seaweed for biological activity, it is still uncertain what type of solvent is the most effective and suitable for the extraction of compounds from seaweeds. Some studies on the effectiveness of extraction methods highlight that methanol extracts exhibit higher antimicrobial activity than ethyl acetate (Manilal et al., 2009; Galal et al., 2011). Other studies report that ethyl acetate and chloroform are better than ethanol and methanol (Patra et al., 2009). According to our antimicrobial and anti-cancer experimental results, ethyl acetate yielded better results than hexane, chloroform and ethanol. Result could be related to the presence of bioactive metabolites that are soluble in ethyl acetate but have no or little solubility in other solvents used.

Current study was limited by the relatively small yield obtained from macroalgae which, for instance, impaired testing the antimicrobial effects of the samples' algal parts. This could affect the interindividual/intraindividual changes of biological activities in macroalga, as the algae were only tested with 5 g of the material.

Demonstrating the bioactive profile of seaweed extracts from Antarctica, data suggest that seaweeds from this region may be promising for the isolation of substances with important biological potential. Fractionation or isolation of the individual components of extracts that showed antimicrobial and antitumor activities is necessary to understand the activity of each individual component and the contribution of each component to the overall activity of the extract. Further research should be undertaken to explore the bioactive compounds of the studied seaweeds to ensure their successful application as potential therapeutic tools.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.