## 2 Results and Discussion
Data are presented as mean value of 3 replicates and analysed through Pearson’s correlation. Variations were considered significant (*) with \(0<p\)-value \(\leq 0.3\) while strongly significant (**) with \(p>0.7\). For the second application, the extraction protocol was used to screen 13 Antarctic sponges and seven selected protists (three dinoflagellates, three diatoms and one green microalga) for identification of potential anti-infective candidates. After fractionation on SPE/HR-X, the small group of samples was tested against both the pathogenic fungus _Candida albicans_ and a panel of infective bacteria (_Staphylococcus aureus MSSA_, _Streptococcus pyogenes_, _Enterococcus faecium_, _Moraxella catharralis_, wild and hyperpermeable strains of _Escherichia coli_) at concentrations ranging from 1 to 256 \(\mathrm{\SIUnitSymbolMicro mL}\) for both extracts and SPE-fractions. One third of the samples showed antimicrobial activity in the range from 3 to 64 \(\mathrm{\SIUnitSymbolMicro mL}\) (Figure 4a). In agreement with the literature [24,25], antifungal activity was detected in the dinoflagellate _Amphidinium carterae_ (CCMP121) containing Amphidinol-18 (**4**), whereas another species of this genus, namely _Amphidinium massartii_ (ICB-BCL19), revealed inhibitory activity (Minimum Inhibitory Concentration, MIC) against both groups of microorganisms (study in progress). Except for _A. carterae_, the anti-infective activity of these samples was detectable only after fractionation by SPE, thus further proving the reliability of the novel protocol. The three samples of Antarctic sponges (ICB-BA83, ICB-BA98 and ICB-BA99) revealed mild comparable activity against _Streptococcus pyogenes_ (MIC 64, 37.5 and 18 \(\mathrm{\SIUnitSymbolMicro mL}\), respectively) associated to the fraction eluted by \(\mathrm{CH_{3}CN/H_{2}O}\). To verify the finding, the material from the most active species ICB-BA99 was processed according to standard bioassay-guided fractionation to give a mixture of lysoplatelet-activating factors (Lyso-PAF), (Figure 5) with MIC ranging between 12.5 and 9.4 \(\mathrm{\SIUnitSymbolMicro mL}\). Analogous fractionation of the other two species gave  
Figure 4: Score hit over 20 marine samples tested for antimicrobial activity. (**a**) Response distribution between sponges and protists: inactive sponge extracts (50%, blue), inactive protist extracts (15%, pale blue), antibacterial activity from sponges only after fractionation (15%, orange), antifungal (_Candida albicans_) protist extracts (5%, green), antibacterial activity from protists only after fractionation (9%, dark orange), antifungal activity from protists only after fractionation (6%, red); (**b**) increase of antibacterial activity during fractionation of sponge extracts. MIC (\(\mathrm{\SIUnitSymbolMicro mL}\)) of the Antarctic sponge extract ICB-BA99, its corresponding SPE/HR-X fractions (A–E) and the pool of LysoPAF analogs **6a–6h** against _Streptococcus pyogenes_ (L49). Van = Vanomycin; Gen = Gentamicinyn. similar activity (results not shown). The antimicrobial activity of Lyso-PAF from marine sponges has been already reported [26; 27; 28; 29; 30] and is comparable to the activity found for the analogs **6a-6h** (Figure 4b).