# Seasonal Monitoring of Volatiles and Antioxidant Activity of Brown Alga _Cladosteplus spongiosus_
## 2 Results and Discussion
### Non-Target Screening of Non-Volatile Compounds in Ethanol Extract  
The ethanolic extract of the freeze-dried algal sample with the best antioxidant potential according to ORAC and FRAP assay (May, MAE) was analysed using UHPLC-ESI-HRMS. The major compounds were selected according to signal intensity (peak area in counts). From the extracted ion chromatograms (XICs) in positive ion mode, these compounds were identified based on the given elemental composition and MS/MS spectra with confidence level 2 (probable structure) and 3 (possible structure) [39]. Seventeen fatty acid derivatives, nine pigments and derivatives, and two steroid derivatives were found (Table 4). \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline
**No. ** & **Name** & **Monoisotopic** & **[M + H]\({}^{+}\)** & **Molecular** & **t\({}_{\mathbf{R}}\)** & **Mass Difference** & **Area** \\  & & **Mass (Da)** & **Formula** & **(min)** & **(ppm)** & **(Counts)** \\ \hline \multicolumn{8}{c}{**FATTY ACID DERIVATIVES**} \\
3 & 1-(9Z,12Z,15Z-Octadecatrienoy)l-3- & 514.31418 & 515.32146 & C\({}_{27}\)H\({}_{46}\)O\({}_{9}\) & 10.94 & 1.7 & \(1.73\times 10^{6}\) \\  & 1-(9Z,12Z,15Z-Octadecatrienoy)l-3- & 676.36701 & 677.37428 & C\({}_{33}\)H\({}_{56}\)O\({}_{14}\) & 10.94 & 3.0 & \(1.00\times 10^{6}\) \\  & & & & & & \\  & 1-(9Z,12Z-Octadecadienoy)l-3-O-(6\({}^{\prime}\)- & 678.38266 & 679.38993 & C\({}_{33}\)H\({}_{86}\)O\({}_{14}\) & 11.42 & 1.2 & \(1.39\times 10^{6}\) \\  & & & & & & & \\  & & & & & & & \\  & & & & & &  
Of the seventeen fatty acid derivatives, eight were fatty acid amides (FAs) (compounds 7-**10**, **13**, **19**, **24**), eight were glycerolipids (GLs) (compounds **3-**6**, **11**, **12**, **14**, **18**), and one was a fatty acid (FA) (compound **9**) (Table 4). The FAAs were dominant. Three C18 FAAs, derivatives of linoleic acid (linoleamide, no. **9**), oleic acid (oleamide, no. **13**), and stearic acid (stearamide, no. **17**), two C16 FAAs, derivatives of palmitic acid (palmitamide, no. **10**) and palmitoleic acid (palmitoleamide, no. **8**), and one C14, derivative of myristic acid (myristamide, no. **7**), C20 derivative of (11_Z_)-icos-11-enoic acid (gondamide, no. **19**), and C22 FAA derivative of erucic acid (erucamide, no. **24**) were detected. In our previous studies, we detected oleamide, erucamide, and palmitamide in the green alga _Dasycladus vermicularis_[40] and two others, palmitoleamide and linoleamide, in the brown algae _Ericaria crinita_ and _Ericaria amentacea_[28]. The FAAs detected and identified in _C. spongiosus_ belong to the group of primary fatty acid amides (PFAMs) with the structure R-CO-NH\({}_{2}\), where R represents a long-chain FA [41], and are bioactive signalling molecules. In general, PFFAs have a broad therapeutic spectrum including anticancer, antibiotic, anthelmintic and antidiabetic [42]. They play an important role in the nervous system of the mammals because they have the potential to bind to the receptors of many drugs, indicating effects on locomotion, angiogenesis, and sleep [43]. It has been reported that PFAMs have a great anticancer activity as they effect cell proliferation [44]. Oleamide is the most studied PFFA and has great potential against Alzheimer disease [45]. Certain plant essential oils are rich in PFFAs because they act as self-defence agents in plants [42], but data on the occurrence of PFAAs in marine algae are lacking. Among the GLs were four glycopolipids, more specifically monogalactosylmonoacylglycerols (MGMGs) (compounds **3-6**) and four monoacylglycerols (MG) (compounds **11**, **12**, **14**, **18**). All eukaryotes and prokaryotes can synthesise GLs which form membrane lipids [46]. Marine algae are known to be a great source of lipid molecules with wide-ranging bioactive properties [47]. Glycoglycerolipids are known for their antiviral, antitumor, antimicrobial, anti-inflammatory, and antioxidant properties [48].