## 4 Discussion
The possibility that many of the observed effects may be caused by other compounds found in the investigated extract (Table S1) with unknown yet biological activity should not be overlooked. Our results revealed that _P. papyraceum_ extract exhibits antibacterial activity against both Gram-negative and Gram-positive bacteria, with a more pronounced effect on the Gram-negative strains. Probably, this activity is due to the compounds diversionol (**14**), torularhodin (**63**), tanikolide (**70**), oligomycin C (**78**), and azithromycin (**84**), reported previously as antibacterial agents [31; 32; 34; 35; 36; 37; 38]. Taking into account that many pathogenic bacteria develop resistance to the classical antibiotics, the cyanobacteria can be an alternative source of compounds with antibacterial activity. For many compounds, fragment spectra were not available in data-dependent MS/MS experiments. For others, the quality was poor due to inappropriate collision energy setting or spectra overlay due to the fragmentation of more than one ion. The quadrupole isolation window was set to 2 Da. In such complex samples, the isolation of just one molecular ion is rarely possible within a reasonable chromatographic separation time and resolution. For these, most of the spectra are overlayed. Within the non-polar fraction, more than 1,200 compounds were detected, but we were able to determine only 112 putative compounds. Half of these components are known to have some biological activity. For example, compounds **104**, **105**, and **111** inhibit reverse transcriptase, showing anti-HIV activity [39; 40; 41; 42]. Antitumour activity has been reported for the compounds **6**, **16**, **18**, **41**, **63**, **71**, **101**, and **111**[37; 42; 43; 44; 45; 46; 47; 48; 49; 50; 51]. Nahuoic acid A (**29**), oleamide (**37**), erucamide (**52**), oligomycin C (**78**), and plakevulin A (**101**) are known as inhibitors of different enzymes [52; 53; 54; 55]. Some of identified compounds (**17**, **18**, **34**, **37**, **41**, **59**, and **111**) are characterized by anti-inflammatory or antimalarial properties [56; 57; 58; 42; 43; 49]. In addition, other compounds in _P. papyraceum_ extract have been used for treatment of neuropathic pain (gabepentin, **1**) [59] or for preparation of liposomes of mixed lipid composition such as 1-(9Z-octadecenyl)-2-hexadecanoyl-3-beta-D-galactosyl-sn-glycerol (**104**) [60]. Among the putatively determined compounds given in Table S1, some were chosen to support the observed bioactivities based on biological properties previously reported by other researchers. As this has a supporting role, we provide putative identifications that we do not claim to be confirmed, but which do exhibit some degree of probability. FISh scoring was used to rank the probability. mzCloud similarity and molecular formula elucidation based on isotopic pattern (predicted composition) were actively used as well. Biological possibility and chromatographic behaviour were also taken into account for the selected candidates. In many cases, there is a huge number of possible structures with absolutely the same exact mass, molecular formula, and almost the same fragmentation pattern, which makes it difficult to precisely identify a compound without a reference substance. In most untargeted workflows, there are no standard substances available, and thus all identifications are putative and should be perceived as candidate structures with certain probability, but further elucidation is required. These putative structures are a good starting point for new work and/or have referential value. Exact identification and separation of the bioactive compounds is the major limitation for most researchers investigating natural products. Due to the complex chemical structures of natural compounds, it is not easy to use them effectively. On the one hand, their synthesis is difficult or impossible, and on the other hand, isolation and purification are very expensive and time-consuming processes.