# Study of the Phenolic Compound Profile of _Arbutus unedo_ L.
## 3 Results and Discussion  
### Optimization of the Extraction Procedure of Phenolic Compounds  
In order to optimize the extraction conditions of the PC from the samples, different assays were performed, including the selection of the extraction solvent, the type and time of stirring, the number of extraction cycles and, finally, the sample/solvent ratio. These studies were carried out with ripe strawberry tree berries. A compromise between the values obtained for the TPC, the TAC, the RSA, the time, and the consumption of reagents in the extraction procedure was considered to select the most suitable extraction conditions. Firstly, the extraction solvent was evaluated under the following conditions: one extraction cycle, vortex stirring (1 min), and a 1:2 (\(w/v\)) sample/solvent ratio. The solvents tested were MeOH, EtOH, MeOH: H2O 1% HCl (6:1, \(v/v\)) and MeOH:water (70:30, \(v/v\)). These solvents were chosen according to the solubility of the analytes in organic and acidified media, with the aim of increasing the extraction of the PC. The results obtained (Figure 3a) showed values of TPC that were very similar for all the solvents tested, without significant differences among them. The RSA values achieved were also similar, except for the solvent MeOH:water (70:30, \(v/v\)), in which the RSA achieved was lower. For this reason, this solvent was directly discarded. The differences observed in the RSA values may be due to the extraction of non-phenolic constituents in the fruit sample extracts that can also show antioxidant activities, such as water-soluble vitamins. On the other hand, phenolic compounds can show different antioxidant activities depending on their structure, synergistic action, or concentration [17]. Consequently, the different solvents used in the extraction may be able to extract different types of phenolic compounds that influence the RSA. Among the other three solvents tested (MeOH, EtOH, and MeOH: H2O 1% HCl (6:1, \(v/v\))), MeOH was selected as the extraction solvent for further analysis, because although it provided similar extraction efficiency of TPC and RSA values than the other solvents, it seemed to provide slightly better extraction of TAC. Moreover, it was easier to use MeOH than the MeOH:water 1% HCl (6:1, \(v/v\)) solvent, which required more preparation to be used. The next assay was carried out to verify whether successive extraction cycles with MeOH could increase the extraction yield of the PC. It was observed that with two successive extraction cycles, the results for TPC, RSA, and TAC improved (Figure 3b). On the other hand, with three extraction cycles, only the values of TPC were significantly improved, whereas the RSA values decreased. Using three extraction cycles implied more consumption of reagents, as well as the possibility of the phenolics to be degraded, since performing three extraction cycles also increases the time of the assay, which may lead to higher exposure of the analytes to time and light. This can produce oxidation processes, so the analytes may consequently lose their antioxidant activity. Additionally, agitation with an ultrasonic bath (Elmasonic S 30, Elma, Singen, Germany) was tested for 5 min (Figure 3c). Although TPC values were similar, worse results were found for RSA when the ultrasound extraction was used, so vortex stirring for 1 min was selected as the optimal condition for agitation. The observed decrease in the RSA values in the ultrasound extraction may be due to the heat generated during the 5 min of extraction, which may have affected the RSA of the analytes. According to the results obtained, higher stirring times were not tested to avoid heating and oxidation, which may have led to the degradation of the PC. Finally, different sample/solvent ratios (1:2, 1:4, 1:8, 1:10, 1:12, and 1:20, \(w/v\)) were evaluated to determine which was the most suitable to obtain higher extraction efficiency.