Preparation And Antioxidant Activity Of Hydroxytyrosol From Olive Leaves

Olive（Olea europaea L）is a famous woody oil plant widely cultivated worldwide.The total plantation area of olive was 80,000 hm² in China and its fruits were mainly used to produce olive oil.With the continuous expansion of olive cultivation,the development and utilization of outstretched olive leaves are imperative.Olive leaves have attracted much attention because of their affluent phenolic components,among which the bioactivity of hydroxytyrosol is the highest..In this study,olive leaves were used to be hydrolyzed by acid for extracting hydroxytyrosol and macroporous resins were applied to enrich hydroxytyrosol from the hydrolysate.The antioxidant activities of hydroxytyrosol were evaluated through the free radical scavenging and reducing power in vitro,and Caenorhabditis elegans was applied to assess the antioxidant activities and explore the mechanism in vivo.This study is expected to provide theoretical basis and technical support for the high-value exploitation and utilization of olive leaves.The main invetigated results were as follows:1.The content of hydroxytyrosol in the esterification oleuropein was much higher than that in the free state in olive leaves,and the comparison of extraction methods showed that ultrasound assistance could improve the yield of hydroxytyrosol.Therefore,hydroxytyrosol was extracted from olive leaves by ultrasound-assisted hydrochloric acid hydrolysis.The optimal extraction condition was obtained:ultrasonic time was 120 min,hydrochloric acid concentration was 1.60 mol/L and the liquid-to-material ratio was 60.00 mL/g.Under the optimal extraction condition,the yield of hydroxytyrosol was 14.11±0.12 mg/g,which was not significantly different from the predicted value,indicating that the optimal process obtained by the optimization was stable and reliable.2.In contrast with the other seven types of macroporous resin of affinity for hydroxytyrosol,NKA-Πwas proved to be a suitable resin to enrich hydroxytyrosol with higher adsorption and desorption rate.250 mL of loading solution was loaded at the speed of 1.5 mL/min and the 40 mL eluent was 75%ethanol to elute hydroxytyrosol from resin at a flow rate of 1.0mL/min.The concentration of hydroxytyrosol changed from 2.27%to 9.25%after enrichment by macroporous resin and the recovery rate was 91.65%.3.The scavenging rate of hydroxytyrosol on DPPH·free radical was as high as 84.81%,and the reducing power also reached 89.95%of the same concentration Vc.Hydroxytyrosol had a good protective effect in H₂O₂-induced oxidative damage on CHO cells which could reduce the intracellular ROS and MDA content by 15.62%and 35.54%,and improve its cell vitality by 35.84%.Hydroxytyrosol showed strong antioxidant activity in vitro.4.Hydroxytyrosol could promote expression and nuclear translocation of DAF-16,increase expression of SOD-3::GFP and HSP-16.2::GFP,improve SOD and GSH-Px activity by42.86%and 35.86%,decrease ROS and MDA content by 21.79%and 36.00%,improve the survival rate by 15.79%of C.elegans under heat stress exerting strong antioxidant activity in vivo.Meanwhile,the result of qPCR showed that hydroxytyrosol could inhibit IIS signal,reduce the expression of upstream component daf-2 and age-1 to reduce the inhibition of downstream component daf-16,so that the expression of daf-16 and its target gene sod-3were up-regulated.It was suggested that hydroxytyrosol may regulate the ability to resist heat stress of C.elegans by affecting the IIS signaling pathway.