## 3 Results
### PEF Impact as a Pre-Treatment for EO Extraction in Eucalyptus and Rosemary  
This experiment considers the analysis of different plant materials, under different PEF and HD time conditions; therefore, it was important to establish proper code names to easily know the conditions each sample referred to. The code names are defined by \"condition.plant material_HD duration_replica\", as described in Table 3. For example, for a sample of eucalyptus, with PEF application, 30 min of HD, and the first replica of this condition the code name would be \"P.E._30_1\"; when indicating the average results of different samples, under the same conditions, the \"replica\" code is not shown: for the average of the samples of eucalyptus, with PEF application and 30 min of distillation would be \"P.E._30\". All the different EOs were extracted by HD, during either 30 or 60 min, and the recovered volume, V, of EO was measured. The density of the extracted EOs, \(\rho\), was determined to calculate their mass, \(m\), that will allow for the determination of the extraction yield, \(\eta\), through (11). The average results are summarized in Table 4.  
\[\Delta\eta=\frac{\eta_{PEF}-\eta_{Control}}{\eta_{Control}}\cdot 100\;. \tag{11}\]  
Focusing first in the results from the eucalyptus EO shown both in Table 4 and Figure 3, there is evidence of a PEF effect for the HD during 30 min, having an average increase in the extraction yield of approximately 17%, when compared with the extracted without the pre-treatment. For the 60 min HD time, the average extraction yield using PEF is similar to the control assay. The results are based on two repetitions per condition, hence for the dispersion analysis, only the amplitude of the results was calculated, as seen in the vertical lines of Figures  
Looking at the results from rosemary presented in Table 4 and Figure 4, and similar to what is observed in eucalyptus, there is evidence of an average increase of 11% in the extraction yield upon PEF application and 30 min distillation. For the 60 min distillation time, there still is an average increase in yield when comparing with the traditional method, in the order of 7%. The reported average yield increase, when using PEF, lead to the following possible explanation for the EO yield variation observed, obtained by HD, with and without PEF pre-treatment:  
* In the case of eucalyptus, a more substantial effect with a shorter distillation time, i.e., 30 min, was observed, when PEF is used. Whereas, 60 min of HD is probably sufficient to extract almost all EO available in 50 g of eucalyptus leaves, as the yields obtained with and without PEF are very similar. * In the case of rosemary, a similar effect with both distillation times, i.e., 30 and 60 min, was obtained, where the yield was not enhanced substantially. Additionally, 60 min distillation might not be enough to extract all EO present in 50 g of rosemary leaves, as there is still some yield variation with and without using PEF. Unfortunately, during the present work, it was not possible to further investigate this possibility due to the reduced material available. The observed dispersions of the results, seen in the amplitude values, were unable to take a more definitive conclusion. Considering that PEF induces the formation of pores in the cell membrane, depending on the field strength and cell size, as described in [9], it seems that in the eucalyptus case, PEF induced a bigger cell disintegration than in the case of rosemary, in the cells where EOs are located. For validation of this hypothesis, further work should be developed addressing the mechanism behind the PEF effect on the cells, namely eucalyptus cells. From the results of EO extraction of the two plant materials discussed, it is possible to conclude that the use of PEF, as a pre-treatment before HD, with a field strength of 2 kV/cm and a specific energy of approximately 10 kJ/kg, is beneficial for increasing the extraction yield, when comparing to the traditional HD extraction.