## 3 Metabolomics Applied to Marine Organisms in Cancer Studies
### Structure Elucidation of Bioactive Molecules from Algae
Assays on cells were carried out, testing not only the crude extract at increasing concentrations (25, 50, 100 ug/mL), but also three micro- and nano-particles loaded with the extracts, represented by PVA: Chitosan solution (PCH) and PVA: NaAlg solution (PNA) (produced through electrospraying techniques), and NPAL (obtained using the microemulsion method). The particles PCH and PNA were prepared by loading with concentrations of 70, 35 and 17.5 ug/mL, while NPAL particles obtained by the microemulsion method were loaded with 50, 25 and 12.5 ug/mL. Each particle was preliminarily tested on HUVEC cell lines (non-cancerous cells) in order to ensure absence of cytotoxicity against normal cells. Crude extracts were analysed with GC-MS. The results of the assay showed cytotoxic effects of both microalgal extracts and encapsulated microalgal extracts on two cancer cell lines while they did not have cytotoxic effects on healthy cells. This study showed that microalgal extracts have cytotoxic effects on cancer cells and did not lose their cytotoxic effects after encapsulation. In a similar study, [PERSON] et al. [124] analyzed the effects of crude extracts of _Tetraselmis suecica_ in conjunction with an innovative compound, through the application of silver nanoparticles adopted as a carrier, against MCF7, mammary carcinoma 4 T1 and normal Vero cell-lines. The cytotoxicity assays were carried out by separately testing the crude extract of the microalgae, the silver nanoparticles AgNPs. In conclusion, the co-application of the two. _T. suecica_ single application only showed the IC\({}_{50}\) of 46.77 ug/mL on MCF7 and 83.17 ug/mL on 4 T1 cells. The AgNPs single application displayed the highest cytotoxicity according to a dose-dependent pathway after 72 h treatments with an IC\({}_{50}\) of 5.3 ug/mL on MCF7, 17.78 ug/mL on 4 T1, and 25.11 ug/mL on Vero cells. Besides, the AgNPs-_T. suecica_ co-application reached the IC\({}_{50}\) of 6.60 ug/mL and 53.7 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, respectively, while the _T. suecia_-CHL single application only showed the IC\({}_{50}\) of 46.77 \(\mathrm{\SIUnitSymbolMicro g}\)/mL and 83.17 \(\mathrm{\SIUnitSymbolMicro g}\)/mL against MCF7 and 4 T1, respectively. Moreover, they analysed the crude extract of _T. suecia_ through GC-MS. In another study, [PERSON] et al. [125] demonstrated that the highest cytotoxic activity against MCF7 cells was exhibited by the synergic application of Tamoxifen (TMX, anti-estrogen drug) and _Nannochloropsis ocularata_'s water extract with IC\({}_{50}\) of 15.8 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, TMX-_T. suecia_'s ethanolic extract with IC\({}_{50}\) of 16.9 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, TMX-_Chlorella_ sp. 's chloroform extract with IC50 of 13.4 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, while TMX-_N. ocaluta_'s chloroform extract, TMX-_T. suecia_'s ethanolic extract and TMX-_Chroella_ sp. 's ethanolic extract showed cytotoxic effect against 4 T1 cells with IC\({}_{50}\) of 15.4, 13.8 and 16.9 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, respectively. Moreover, the synergistic application of TMX-algae's extracts maintains an antiproliferative effect on cancer cell lines and reduced the toxicity on normal Vero cells. In addition, after \({}^{1}\)H-NMR analyses, isoleucine was found only in the ethanolic extract of _Chlorella_ sp., glycerol only in the ethanolic extract of _T. suecia_ and in the chloroform extract of _Chlorella_ sp., while xanthine was found only in the chloroform extract of _Chlorella_ sp. These metabolites can help reduce the toxicity of non-cancerous Vero cells. [PERSON] et al. [126] tested various concentrations of hot methanolic extracts of _Spirulina platensis_ to identify the most active chemical compounds and also to check the cytotoxic effect on cancer cell lines L20 B (mice intestine carcinoma) and MCF7 (breast cancer) after 24 h and 48 h exposure. GC mass analysis showed that the active chemical compounds in the extracts contained alkaloids, terpen, phenols, resins, saponines, flavones, steroids, proteins and amino acids.