## 3 Results
### Antibacterial Activities of Ethanolic and Aqueous Extracts  
The analysis of the results of the inhibition tests on the agar plate was conducted considering all the different extracts prepared (i.e., four ethanolic and four aqueous). As a first step, the results obtained with different extraction times (5 min, 10 min, 30 min, 1 h, and 24 h) were recorded. These demonstrated that with extraction times of 5 min, 10 min, and 24 h, the inhibition values are almost undetectable. Larger inhibition halos appear with an extraction time of 30 min. In all cases, noticeable halos are detected only in the ethanol extracts, indicating that the compounds that exhibit activity against the bacterial strains tested must be mainly insoluble in water and of a polar nature. Table 2 presents the inhibition halos for the 30 min extractions using ethanol as solvent. All null results were excluded.  
Figure 4: Quantification of total phenolic content (TPC, mg of GAE g\({}^{-1}\) extract) of the extracts obtained sequentially at different temperatures and using ethanol (**A**) or water (**B**) as a solvent. Values are presented as mean \(\pm\) SD (\(n\) = 4). If the values are not significantly different at 0.05 significance compared to the ones obtained from the highest value sample 4B/40, they are signaled with *; if the values are not significantly different at 0.05 significance compared to the ones obtained from the lowest value sample 2A/RT, they are signaled with #. Darker colored bars refer to the first and second extractions, with the lighter colored bars referring to the third and fourth extractions.  
The results of the growth inhibition tests on liquid media for both for ethanolic and aqueous extracts are shown in Figures 6 and 7, respectively. The percentage of growth inhibition of fish pathogenic bacteria in the presence of ethanolic extracts demonstrated higher mean values (66.2%) than the results in the presence of aqueous extracts (30.6%). Therefore, it is possible that the antimicrobial bioactive metabolites present are mostly extracted by ethanol. The highest percentages of inhibition were recorded in ethanol extracts against _A. hydrophila_ subsp. _hydrorophila_ (81.6%) and in _Y. ruckeri_ (78.41%), thus the least interesting values registered in the aqueous extracts against _A. hydrophila_ subsp. _hydrorophila_ (13.28%). In general, the growth inhibition in the presence of aqueous extracts occurs mainly in extracts obtained at higher temperatures (Figure 7, bars in shades of yellow/red), as the lowest temperatures (RT or 40 \({}^{\circ}\)C) are less effective in extracting compounds with antimicrobial activity against the strains in study.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Strain** & **2A** & **3B** & **4B** & **CHL** \\ \hline _Aeromonas hydrophila_ & - & 8 \(\pm\) 0.1 & - & 35 \(\pm\) 0.1 \\ _Aeromonas salmonica_ & 8 \(\pm\) 0.1 & 9 \(\pm\) 0.5 & - & 40 \(\pm\) 0.1 \\ _Edwardsial bisciida_ & – & 9 \(\pm\) 0.1 & - & 40 \(\pm\) 0.1 \\ _Photobacterium dansclae_ & 9 \(\pm\) 0.1 & 9 \(\pm\) 0.1 & 8 \(\pm\) 0.1 & 30 \(\pm\) 0.1 \\ _Vibrio angullarum_ & 8 \(\pm\) 0.1 & 8 \(\pm\) 0.1 & - & 35 \(\pm\) 0.1 \\ _Yersinia ruckeri_ & 8 \(\pm\) 0.1 & 9 \(\pm\) 0.1 & - & 35 \(\pm\) 0.1 \\ \hline \hline \end{tabular}
\end{table}
Table 2: Antimicrobial activity of _G. gracilis_ ethanolic fractions obtained after 30 min extraction at room temperature and chloramphenicol (CHL) against several fish pathogenic bacteria strains. The results are expressed as mean diameter of inhibition halos (mm) \(\pm\) standard deviation (\(n\) = 4).  
Figure 5: Linear correlation between the total polyphenic content (mg of GAE g\({}^{-1}\) extract) and DPPH antioxidant activity. Scatter plot diagram illustrating the correlation using ethanol (orange) or water (blue) as solvent.