# 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
#### 4.1.3 The Effect of Maturity on the Different Classes of Phenolic Compounds  
With reference to Figure 5, TPC was found to vary throughout the maturation of the olive. This has been attributed to enzymatic and chemical reactions occurring during the ripening process [49]. Olive oil waste consists of a wide range of phenolic compounds resulting from secondary plant metabolism, including phenolic alcohols (hydroxytrosol and tyrosol), phenolic acids (vanillic acid and caffe acid), flavonoids (luteolin and apigenin) and secoriidoids, which are the most abundant portion [50,51]. The aglycone forms of ligstroside and oleuppein are the most abundant in secoriidoids [52]. During the maturation process of olives, oleuppein and ligstroside accumulate [53]. As shown in Figure 13, any damages done to the fruit during the ripening process can result in the release of esters [54], which can hydrolyse ligstroside and oleuppein into a range of compounds [55]. Oleuppein aglycone and ligstroside aglycone are formed by hydrolysis. Furthermore, the aglycones can undergo a further ester hydrolysis in order to produce hydroxytrosol or tyrosol, elenolic acid and glucose, each of which can potentially reduce the Folin reagent [56]. Figure 12: Chemical structures of ortho-diphenolic compounds highlighting the ortho-diphenolic structures. In contrast to the results obtained, both [PERSON] et al. [58] and [PERSON] et al. [59] showed that there was an apparent decrease in the concentration of phenolic compounds during the maturation of olive fruits. In fact, [PERSON] al. [59] states that the highest concentration was seen in the first collected cultivar, with a concentration of 5.79 mg GAE/g, whilst the lowest concentration was found from waste collected a few months later and had a concentration of 0.55 mg GAE/g. The main key for phenolic biosynthesis is L-Phenylalanine Ammonia Lyase (PAL). When there is a decrease in the enzymatic activity of PAL, the TPC of olive drupes during the ripening stage can decrease [60]. As seen in Figure 5, flavonoid content is also affected by maturation. This can also be seen in the literature, where [PERSON] and [PERSON] [61] reported that the glucoside \begin{table}
\begin{tabular}{c c c c} \hline \hline  & \multicolumn{3}{c}{**Correlation Analysis**} \\ \hline \multicolumn{3}{c}{**Methanolic Extracts**} & **Acid Hydrolysis** & **Alkaline Hydrolysis** \\ \hline
**TPC** & & & \\ TPC–TFC & 0.67 & \(-\)0.09 & 0.51 \\ TPC-TdOPC & 0.67 & 0.23 & 0.57 \\ TPC-FRAP & 0.78 & 0.80 & 0.76 \\ TPC-CUPRAC & 0.69 & 0.57 & 0.93 \\ TPC-\(DPPH\) & \(-\)0.73 & \(-\)0.50 & \(-\)0.47 \\ TPC–\(ABTS\) & \(-\)0.70 & \(-\)0.47 & \(-\)0.57 \\ TPC–\(H_{2}\)O\({}_{2}\) & 0.42 & 0.80 & 0.85 \\
**TFC** & & & \\ TPC-TdOPC & 0.95 & 0.77 & 0.96 \\ TPC-FRAP & 0.45 & \(-\)0.35 & 0.052 \\ TPC-CUPRAC & 0.84 & 0.48 & 0.63 \\ TPC-\(DPPH\) & \(-\)0.46 & 0.34 & 0.20 \\ TPC-\(ABTS\) & \(-\)0.77 & \(-\)0.21 & \(-\)0.67 \\ TPC–\(H_{2}\)O\({}_{2}\) & 0.45 & \(-\)0.60 & 0.55 \\
**TdOPC** & & & \\ TdOPC-FRAP & 0.46 & 0.06 & 0.25 \\ TdOPC-CUPRAC & 0.83 & 0.58 & 0.69 \\ TdOPC–\(DPPH\) & \(-\)0.43 & 0.15 & 0.17 \\ TdOPC–\(ABTS\) & \(-\)0.73 & \(-\)0.26 & \(-\)0.70 \\ TdOPC–\(H_{2}\)O\({}_{2}\) & 0.46 & 0.18 & 0.58 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Correlation analysis between all assays. Figure 13: A figure shown by [PERSON] et al. [57], showing the structures of oleupopein, ligstroside and other related hydrolysis products. forms of flavonoids, cyanidin-3-glucoside, luteolin-7-glucoside, quercetin-3-rutinoside and cyanidin-3-rutinoside, are abundant in the pulp of mature olive fruits. The possible reason why TFC increases as the maturity progresses from unripe to ripe is because flavonoids tend to participate in the protection of the photosynthetic apparatus against photoinhibition under excessive light [62]. In fact, [PERSON] et al. [63] reported that as the light intensity decreased, the accumulation of flavones was inhibited.