# Seasonal Monitoring of Volatiles and Antioxidant Activity of Brown Alga _Cladosteplus spongiosus_
## 2 Results and Discussion
### Hydrodistillation Obtained Volatile Variations of C. spongiosus  
In the hydrodistillate, the percentages of the identified compounds ranged from 81.21% (Fr_July) to 89.11% (Dr_August) of the total compounds detected. These compounds were classified into four different groups: aliphatic compounds, sesquiterpenes, diterpenes, and others (Figure 5). Terpenes, especially sesquiterpenes and diterpenes, were the most abundant in all samples (36.09%, Fr_August--52.08%, Fr_May), except in the fresh sample in July (33.13%). Sesquiterpenes were more abundant only in May's fresh sample (30.47%), mainly because of the high proportion of germacrene D (9.63%) and epi-bicyclosesquiphellandrene (4.49%) and gleenol (11.58%) (Table 3). All three detected sesquiterpenes were the predominant sesquiterpenes in the hydrodistillate of brown alga _T. atomaria_ which belongs to the same subclass Dictyotophyceae [29]. When observing the seasonal changes in _H. scoparia_, which belongs to the same order of Sphacelariales, the highest proportion of germacrene D and gleenol in the hydrodistillate of the fresh sample was also found in May and later decreased each month until September [30]. Figure 4: Correlation (**a**) and score plots (**b**) of the dominant compounds from headspace volatiles (two different fibres obtained via headspace solid-phase microextraction (HS-SPME) with two fibres divinylbenzene/carboxene/polyddimethylsiloxane (f1) and polydimethylsiloxane/divinylbenzene (f2)) and analysed via gas chromatography–mass spectrometry (GC-MS): fresh (Fr) and dried (Dr) _Cladostephus spongiosus_ samples analysed together. Diterpenes were more abundant in the remaining samples (Figure 5). Diterpene alcohols phytol (4.42%, Fr_July--18.73%, Dr_August), pachydictylol A (3.18%, Fr_August--12.44%, Dr_May), (\(E\))-geranyl geraniol (1.40%, Fr_May--6.77%, Dr_July), and cembra-4,7,11,15-tetraen-3-ol (4.92%, Fr_May--16.04%, Dr_July) dominated. The lowest percentage of detected diterpenes was always found in the fresh samples and the highest in dry samples. Phytol content was the highest in August. A similar behaviour was observed in _H. scoparia_ where phytol was the predominant compound [30]. Phytol and its derivatives have a wide range of bioactive functions such as antioxidant, cytotoxic, anti-inflammatory, antimicrobial, autophagy- and apoptosis-inducing, immune- and metabolism-modulating, and anxiety-relieving activities [31]. Cembra-4,7,11,15-tetraen-3-ol was also found to be a very dominant terpene in _D. dichotomous_ and _H. scoparia_, as was pachydictyl A in _D. dichotomous_[30; 32]. Cembranoid-type diterpenes have good antitumor, antimicrobial, and neuroprotective activity [33], and pachydictyl A can be used as an antifouling paint, among other applications [34]. Monoterpenes were present only in the dry sample in May (\(<1\%\)) and in both the fresh and dry samples in June (\(<\)0.1%). In general, terpen are bioactive compounds and, as such, play an important role in human health, as well as food preservatives [35]. Figure 5: The volatile organic compounds (VOCs) from _Cladosteplus spongiosus_ sorted by structural groups isolated via hydrodistillation (HD) and analysed via gas chromatography–mass spectrometry (GC-MS): (**a**) fresh (Fr) samples; (**b**) dry (Dr) samples. \begin{table}
\begin{tabular}{c c c c c c c c c c c c c} \hline \hline \multirow{2}{*}{**No.