## 4 Conclusions  
The use of the low-frequency, ultrasound-solvent extraction method yielded the highest concentration of oleupreein in wild olive leaf extract (WOLE-S) compared to extraction with microwaves and supercritical CO\({}_{2}\). This study proved the gastrointestinal instability of oleupreein from WOLE-S, especially after the gastric phase. In this sense, the use of microencapsulation techniques is necessary to protect the stability of oleupreein under digestive conditions and, thus, its biological potential. Pure oleupreein showed high antiproliferative activity against all tested cancer cell lines (T24, A549, A1235, and MD-MBA-123), while the antiproliferative activity of olive leaf extract was high against T24 cells and moderate against the other tested cell lines. WOLE-S caused a down-regulation of the expression of the inflammatory cytokine IL-6 in A549 cells. The results show the optimal effect of low-frequency ultrasound in combination with a suitable solvent extraction for a high yield of oleupreein in olive leaf extract. In addition, the gastrointestinal instability of oleupreein was demonstrated under simulated gastrointestinal conditions with human digestive enzymes, and a strong antiproliferative and anti-inflammatory effect of pure oleupreein and olive leaf extract was shown. However, this biological potential of oleupreeinas a dietary supplement or olive leaf extract or infusate should be protected by the use of microencapsulation techniques due to the gastrointestinal instability of oleuropein.  
The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app14020869/s1](https://www.mdpi.com/article/10.3390/app14020869/s1)]([https://www.mdpi.com/article/10.3390/app14020869/s1](https://www.mdpi.com/article/10.3390/app14020869/s1)), Figure S1: Calibration curve each of the 14 phenolic standards used in HPLC analysis, (**a**) hydroxytrosol, (**b**) 3,4-hydroxybenzoic acid, (**c**) tyrosol, (**d**) catechchin, (**e**) 4-hydroxybenzoic acid, (**f**) coffee acid, (**g**) \(p\)-cumaric acid, (**h**) \(t\)-ferulic acid, (**i**) \(o\)-cumaric acid, (**j**) oleuropein, (**k**) pinoresinol, (**l**) luteolin, (**m**) apigenin and (**n**) kaempfer. The amount of the standard is expressed in \(\mu\)g/mL.  
Conceptualization, B.S. and T.B.; methodology, B.S., T.B., J.G., I.L., V.C.C., A.B., P.B., I.S. and K.I.; formal analysis, B.S., T.B., J.G., I.L., V.C.C., P.B., I.S. and K.I.; investigation, B.S., T.B., J.G., I.L., V.C.C., A.B., P.B. and K.I.; resources, B.S., T.B., J.G., I.L., V.C.C., A.B., P.B., I.S. and K.I.; data curation, B.S. and T.B.; writing-original draft preparation, B.S.; visualization, B.S., T.B. and I.S.; writing--review and editing, B.S., T.B., J.G., I.L., V.C.C., A.B., P.B., I.S. and K.I.; supervision, B.S. All authors have read and agreed to the published version of the manuscript.  
This research received no external funding.  
The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of University of Hospital Centre Split (protocol code: 2181-147-01/06).B-16.2 and date of approval 19 May 2016).  
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study of gastrointestinal stability of oleuropein from WOLE-S. The approval for the collection of digestive juices was obtained from the Ethics Committee of the University of Hospital Centre Split.  
The data supporting our findings and analyses are contained in the article itself. Readers can access this data by referring to the article.  
The authors declare no conflicts of interest.