# The Effect of Hydrolysis on the Antioxidant Activity of Olive Mill Waste
## 4 Discussion
### Antioxidant Activity
#### 4.2.3 Hydrogen Peroxide and Superoxide Scavenging Activity  
Hydrogen peroxide is a weak oxidizing agent which inactivates few enzymes directly. It can also cross cell membranes rapidly; in fact, once it is in the cell, it can react with Fe2+ ions in order to form hydroxyl radicals [95]. In this study, the highest mean average of hydrogen peroxide scavenging activity was found in methanolic extracts, followed by acid hydrolysis and lastly, alkaline hydrolysis, all expressed in mg AAE/g. From the existing literature, methanolic extracts have been found to be more effective against hydrogen peroxide activity. A study carried out by [PERSON], [PERSON] and [PERSON] [96] on antioxidant activity and total phenolic and flavonoid content of various solvents in red clover showed that methanol extracts were found to be more efficient than hexane or ethyl acetate fractions. [PERSON] et al. [97] also stated that all solvents used had the ability to reduce the decomposition of hydrogen peroxide, but methanol extracts had a significantly higher (\(p\)-value = \(<\)0.05) ability to reduce the decomposition of hydrogen peroxide compared with the other solvents. This resulted in methanol exhibiting the highest extraction yield and a better antioxidant activity.  
Figure 11 shows the correlation of hydrogen peroxide with TPC, TFC and TdOPC. From these correlations, it can be observed that TPC, TFC and TdOPC have lower correlations with CUPRAC in methanolic extracts. However, a higher correlation was observed in hydrolysed extracts, except for the correlation of TFC and TdOPC with hydrogen peroxide in acid hydrolysis. [PERSON] et al. [98] discussed that phenolic derivatives recorded a higher antioxidant activity than their parent compounds. This indicates that the aminophenol in the two and four positions showed the highest hydrogen peroxide scavenging activity [98]. [PERSON], [PERSON] and [PERSON] [99] also stated that the ortho-diphenyl content and antioxidant capacities assessed using the hydrogen peroxide scavenging activity revealed a good correlation, within R\({}_{2}\) value of 0.96. The antioxidant activity of ortho-diphenolic-containing compounds was associated with their ability to form intra-molecular hydrogen bonds between phenoxylic radicals and the hydroxyl group. In fact, [PERSON] et al. [100] states that the presence of an ortho-diphenol enhances the ability of the phenolic compounds to act as an antioxidant. Thus, this shows that the ortho-diphenols of olive waste extracts might bemajor contributors to the antioxidant activities assessed using this method.  
The _Superoxide Radical Savenging_ activity assay was refined in such a way that the probe molecule was replaced by one which was able to be reduced by O\({}_{2}\)\({}^{-}\) radicals, but not by polyphenic compounds. For this assay, acid hydrolysis had the highest IC\({}_{50}\) of superoxide radicals; in fact, it had an average mean value of 10.17 \(\pm\) 2.18, followed by methanolic extracts, with an average of 13.90 \(\pm\) 4.90, and lastly alkaline hydrolysis, with an average of 16.33 \(\pm\) 13.53. For _Superoxide Radical Savenging_ activity, only methanolic extracts correlated with TPC, and there were no correlations observed with TFC and TdOPC for all extracts used.