# Impact of Frost on the Morphology and Chemical Composition of _cv_. [PERSON]
## 3 Results and Discussion
### Tocopherols, Total Phenols Content, and Antioxidant Activity  
Table 3 shows the mean concentrations of tocopherols in the oil extracted from the pulp and for both TPC and DPPH inhibition percentages (antioxidant activity) in the pulp methanolic extracts. The results showed that frost had a significant negative effect (\(p\)-value \(<\) 0.05) on the majority of the contents of the olives' lipophilic and hydrophilic antioxidant tocopherols and TPC, as well as on the radical scavenging activity (antioxidant activity). In fact, frost imposed a decrease in the mean concentrations of \(\alpha\)-, \(\beta\)-, and \(\gamma\)-tocopherol (an average reduction of 18, 12, and 6%, respectively). This slight reduction could be explained by the minor oxidative stress observed in the fatty acid profile [15,16]. Contrarily, the TPC concentration was severely affected by the frost events. An average reduction of 70% was observed (Table 3). These results are in accordance with those reported by [PERSON] et al. [14], who also observed a decrease in the concentration of phenolic compounds in olive oils extracted from olives collected after frost. The phenols'  
Figure 2: Differentiation of _cv._ Santulhana olives not subjected to frost (\(\blacksquare\)) and subjected to frost (\(\bullet\)): 2D PCA biplot based on the fatty acids profile (C\({}_{14:0}\), C\({}_{16:0}\), C\({}_{16:1}\), C\({}_{17:0}\), C\({}_{17:1}\), C\({}_{18:0}\), C\({}_{18:1}\), C\({}_{18:2}\), C\({}_{20:0}\), C\({}_{18:3}\), C\({}_{20:1}\), C\({}_{22:0}\) and C\({}_{24:0}\)). marked decrease can be partially attributed to the olive tissue injuries due to the water crystallization and the abrupt decrease of temperatures during the frost event, followed by a temperature rise after frost, which promotes the oxidative activity of enzymes, thereby leading to a degradation of phenolic compounds [15]. The potentially direct exposure of the inner pulp to oxygen during the frosting periods due to the micro-fissures created from crystal damage cannot be excluded, with a direct impact on phenolics' oxidative damage. Additionally, due to their water solubility, the phenolic compounds can also be affected by the water movements in the fruit pulp, increasing their probability of damage. The decrease of the antioxidant activity of the olives' extracts due to the frost phenomenon can be easily correlated with the decrease in the bioactive contents in the olives after frost, with an average significant reduction of 42% on the DPPH inhibition being observed. Finally, the data on the bioactive compounds and the related antioxidant activity were used to evaluate the possibility of being used as biomarkers for differentiating _cv._ Santulhana olives subjected or not subjected to frost. Figure 3 (2D-PCA biplot, with the two first PCs explaining 81.1% of the data variability) clearly shows that the tocopherols and TPC together with the DPPH inhibition percentages enabled an almost full differentiation of the olives affected or not affected by frost, pointing out visually that olives not subjected to frost had a richer composition of the evaluated bioactive compounds and a higher antioxidant activity. Figure 3: Differentiation of _cv._ Santulhana olives not subjected to frost (\(\blacksquare\)) and subjected to frost (\(\bullet\)): 2D PCA biplot based on the tocopherols content (\(\alpha\)-, \(\beta\)- and \(\gamma\)-), total phenol content (TPC), and DPPH radical scavenging inhibition.