# The Effect of Hydrolysis on the Antioxidant Activity of Olive Mill Waste
## 4 Discussion
### Antioxidant Activity
#### 4.2.2 Metal Ion Reducing Activity  
Both FRAP and CUPRAC assays are electron transfer (ET) based assays [88]. ET-based assays measure the capacity of an antioxidant using the reduction of chromogenic oxidant, which changes the colour when reduced. The degree of colour change is correlated with the concentration of antioxidants present in the samples [89]. The FRAP assay is carried out under acidic pH conditions in order to maintain iron solubility and drive electron transfer. This increases the redox potential, causing a shift in the dominant reaction mechanism [90] and forming an intense blue-coloured ferrous complex [FeII(TPTZ)]2+. From the results obtained, alkaline hydrolys extracts had the highest reducing activity, with a mean of 15.66 \(\pm\) 9.52. This was followed by acidic hydrolysis, with an activity of 12.92 \(\pm\) 6.56, and lastly methanolic extracts, with an activity of 7.26 \(\pm\) 5.12, all expressed in mg AAE/g. From these results, it can be clearly seen that hydrolysis affects the antioxidant capability of olive waste. This corroborates the study conducted by [PERSON] et al. [91], where it was stated that the FRAP values of bound phenolics from alkaline hydrolysis were higher than those from acid hydrolysis, with a \(p\)-value of less than 0.01.  
Figure 10 shows the correlation of FRAP with TPC, TFC and TdOPC. From the correlations obtained, it was observed that FRAP correlated similarly with TPC for all extractions used, while there was a lower correlation when FRAP was correlated with TFC. The outcomes may reveal that phenolic components could be the main ingredients responsible for the antioxidant capacities of olive waste; whilst flavonoid compounds might contribute to the antioxidant capacities of olive waste, they were not the main contributors. This is in line with a study conducted by [PERSON] et al. [92] where similar results were seen. The correlation between FRAP and TdOPC was also low, indicating that FRAP was more closely related to total phenolic content.  
The CUPRAC method consists of a chromogenic oxidizing reagent which is known as bis(neocuproine) copper (II) cation. Upon its reduction, the reactive Ar-OH groups of the polyphenic antioxidants are oxidized to the corresponding Ar=O (quinones), whilst Cu (II)-Nc is reduced to the orange-yellow coloured Cu(Nc)2+ chelate copper (I) [89]. From the results obtained in this study, it was found that CUPRAC in alkaline hydrolys extracts had the best activity, with a mean of 12.25 \(\pm\) 6.07. This was followed by methanolic extracts, with an activity of 10.14 \(\pm\) 3.20, and lastly acid hydrolysed extracts, with an activity of 8.91 \(\pm\) 3.67, all expressed in mg GAE/g. This is in line with a study conducted by [PERSON], [PERSON] and [PERSON] [93] on the impact of alkaline and acid digestion on antioxidant activity of rice hull extracts. In this study, it was stated that the extracts obtained after alkaline digestion of the hulls presented a higher reducing capacity of cupric ions.  
Figure 10 shows the correlation of CUPRAC with TPC, TFC and TdOPC. From the correlations obtained, it can be observed that the correlation of CUPRAC with both TFC and TdOPC was stronger in the methanolic extracts compared to acid and alkaline hydrolysed extracts. This suggests that with regards to copper reducing activity, flavonoid compounds containing an ortho-diphenolic group preferentially reduce Cu2+ ions compared to other phenolic structures present in the extract. This corroborates a study conducted by [PERSON] et al. [94]. In this study, it was stated that the correlation between CUPRAC and TPC was low; in fact, in order for flavonoid glycosides to fully exhibit their antioxidant potency, they required acid hydrolysis to their corresponding aglycons. Slow reacting antioxidants need elevated temperatures in order to complete their oxidation with the CUPRAC reagent. As flavonoid glycosides are hydrolyzed to their corresponding aglycons, their CUPRAC antioxidant capacities significantly increase [94].