# Cellular Antioxidant Effects and Bioavailability of Food Supplements Rich in Hydroxyrosol
## 3 Results
### Bioassays
#### 3.2.4 Antiglycation Activity  
Advanced glycation end products (AGEs) are a heterogeneous group of substances that are formed in the human body during non-enzymatic glycosylation between the carbonyl group of a reducing sugar and a free amino group of a protein [30; 31]. AGEs play an important, albeit complicated, role in cellular aging processes, in which they are produced in large quantities, causing oxidative stress, inflammatory reactions, and chronic diseases such as diabetes and cardiovascular disease [32; 33]. Hyperglycemia, the accumulation of triosephosphates and ketone bodies, lipid peroxidation, and oxidative stress can increase AGEs formation [34]. The irreversible glycation of proteins in turn results in structural alterations and the accumulation of defective proteins in cells, which impactnormal physiological functions [35].  
Since increased intracellular and extracellular stress is a source of AGEs accumulation in vivo, and the antioxidant activities of natural phenolics may inhibit the AGEs production [36; 37], we investigated cellular AGEs to further support the potential of P-1 and P-2 in preventing the cellular stress.  
We have found that both products reduce the formation of new AGEs (Table 5).  
Relative values greater than 100 indicate cellular accumulation of AGEs (e.g., treatment with the AGE-inducer alone), while lower values indicate a potential for the sample to reduce the AGEs formation. Pre-incubation of the cells with each product, followed by the induction of AGEs formation, effectively cleanses the cellular AGEs, with respect to both the AGEs-inducer treatment alone and with the cells under normal culture conditions. The strong positive influence is similar for both pre-treatments with each olive-derived concentrates, and both show a nearly 50% reduction in AGEs formation in vitro.  
Our results are consistent with data from a recent study conducted with an olive leaf extract concentrated in hydroxytyrosol (54.5 mg/g), in which a reduction in AGEs produc  
\begin{table}
\begin{tabular}{c c c} \hline
**Treatment** & **8-epi PGF2\(\alpha\) (pg/mL)** & **Relative Isoprostanes (\%)** \\ \hline UTC & \(21.80\pm 1.95\) & 100.0 \\ AAPH & \(28.45\pm 0.76\) * & 130.5 * \\ P-1 & \(15.42\pm 1.18\) * & 70.7 * \\ P-2 & \(20.49\pm 0.60\) & 93.9 \\ Q & \(10.24\pm 2.23\) * & 47.0 * \\ \hline \end{tabular}
\end{table}
Table 4: Free isoprostanes released in culture after treatments. The relative values (third column) were normalized to the UTC, which represents the basal amount of isoprostanes under normal culture conditions, and multiplied by 100. UTC: untreated control cells; AAPH: lipid peroxidation initiator; Q: quercetin 100 uM. * \(p<0.05\) vs. UTC.  
\begin{table}
\begin{tabular}{c c c} \hline
**Treatment** & **\(\mu\)g AGEs/mg Protein** & **Relative AGEs (\%)** \\ \hline UTC & \(1.02\pm 0.006\) & 100 \\ + & \(2.64\pm 0.042\) & 258 \\ P-1 & \(0.52\pm 0.008\) & 50.9 \\ P-2 & \(0.47\pm 0.007\) & 46.0 \\ \hline \end{tabular}
\end{table}
Table 5: AGEs content in cell lysates after treatment with the extract products and an AGEs-inducer mix. UTC: untreated control cells; +: AGEs inducer solution. Relative AGEs values are normalized to the UTC, which represents the basal amount of AGEs in normal culture conditions, multiplied by 100.  
tion was demonstrated in HepG2 cells subjected to carbonyls-induced stress [38].These authors reported that, likely due to a synergistic effect of hydroxytyrosol and other minor compounds with similar polarities, the olive leaf extract exerts a wide antiglycative activity.