# The Effect of Hydrolysis on the Antioxidant Activity of Olive Mill Waste
## 4 Discussion  
### Total Phenolic Content, Total Flavonoid Content and Total Ortho-Diphenolic Content  
#### 4.1.1 Effect of Hydrolysis on the Different Classes of Phenolic Compounds  
One of the most common solvents used for the extraction of phenolic content is methanol, as it leads to better phenolic content [33]. A study conducted by [PERSON] et al. [34] stated that methanol extracts were the most active compared with other extraction procedures used. Although methanolic extractions are common, the solvent may not allow a complete recovery of phenolic compounds that are linked by a glycosidic or ester bond to matrix components, such as cell wall polysaccharides and lipophilic structures. Thus, in order to break these bonds and achieve higher recoveries, a basic alkaline or acidic hydrolysis can be performed [23]. In fact, as seen in Figure 2, it can be noted that alkaline  
Figure 11: (**A**) Correlation between TPC and hydrogen peroxide scavenging activity. The \(\rho\)-value of methanolic extracts (\(\blacksquare\)) was 0.42, for acidic hydrolysis (+) it was 0.80 and for alkaline hydrolysis (\(\blacktriangle\)) it was 0.85. (**B**) Correlation between TFC and hydrogen peroxide scavenging activity. The \(\rho\)-value of methanolic extracts (\(\blacksquare\)) was 0.45, for acidic hydrolysis (+) it was \(-0.60\) and for alkaline hydrolysis (\(\blacktriangle\)) it was 0.55. (**C**) Correlation between TdOPC and hydrogen peroxide. The \(\rho\)-value of methanolic extracts was 0.46, for acidic hydrolysis (+) it was0.18 and for alkaline hydrolysis (\(\blacktriangle\)) it was 0.58.  
hydrolysed extracts produced the highest phenolic content compared with methanol and acid hydrolysis. Acid and alkaline hydrolysis are two methods which can be used to release bound phenolic compounds. In most of the studies conducted, alkaline hydrolysis is the most-used method for the liberation of bound phenolic compounds from the cell wall polysaccharidesides [35]. This is mainly attributed to the fact that alkaline hydrolysis is carried out at room temperature, without the need of excessive heat which might destroy thermolabile compounds [36]. [PERSON] et al. [37] studied the effects of different extraction methods, profiles and antioxidant abilities of free and bound phenolics of Sargassum polycystum from the South China Sea. In this study, it was found that alkaline hydrolysis releases a higher amount of bound phenolic compounds, which potentially results in higher TPC and antioxidant activity compared to that of acid hydrolysis. In contrast, a study conducted by [PERSON] et al. [38] reported that the use of acid hydrolysis results in a higher TPC. This could be due to the fact that acid hydrolysis is carried out at a higher temperature to release the bound phenolic compounds, which are trapped in the cores or conjugated to cell wall dietary proteins or fibres [36,39]. Furthermore, in this study it was shown that whilst TPC increased with the hydrolysis procedure, macerated extracts had the highest flavonoid content, as seen in Figure 3. This is in line with the results obtained by [PERSON], [PERSON] and [PERSON] [40], whereby it was shown that methanolic extracts had the highest level of flavonoid content compared to other polar solvents. Apart from being the best solvent for the extraction of flavonoids, [PERSON] and [PERSON] [41] showed that significant loss in the flavonoids' content occurs during boiling; in fact, it was stated that although flavanol aglycons, quercetin and kaempfer were detected, the quantities were lower than would be anticipated from the amount of kaempfer and quercetin glycosides in the non-hydrolysed samples. In fact, from the results obtained, it was evident that some loss of flavanols occurred during the process of hydrolysis. This study was further corroborated by [PERSON] et al. [42], whereby the sensitivity of flavonoids to high temperatures exceeding 75 \({}^{\circ}\)C resulted in the destruction of flavonoids.