*4.6. Statistical Analysis*

All experiments were independently conducted at least two times. The Online Application for Survival Analysis (OASIS 2; https://sbi.postech.ac.kr/oasis2/) [114] was used for comparing survival differences between two conditions. Fluorescence intensities as well as swim behaviour parameters were calculated as mean ± SEM and statistical significance was calculated by two tailed t-test using GraphPad (https://www.graphpad.com/quickcalcs/). Chi-square test was used to compare the number of worms with intact and degenerated neurons in the UA44 strain.

## **5. Conclusions**

Both polyphenol treatments—pure hydroxytyrosol and the natural preparation 'Hidrox®'—were able to similarly improve the lifespan, stress resistance as well as age pigment accumulation in wild type nematodes. Furthermore, the beneficial locomotion effects of HD and HT are quite equally strong in the rotenone-stressed PD-model of *C. elegans*. However, the abilities of HD and HT also provide some differences: HD exposure led to much stronger beneficial locomotion effects in wild type worms compared to HT. Moreover, also the PD-model characterized by α-synuclein expression in muscles (strain OW13) did benefit significantly more from HD than HT. Only in the UA44 strain, which features α-synuclein expression in DA-neurons, the beneficial effects of HD and HT are rather weak with only one minor advantage of HD over HT. Thus, the hypothesis that HD features higher healthspan-promoting abilities than HT was only partly confirmed. Further studies are needed to uncover the molecular background mechanisms which led to this distribution of effects. Nevertheless, both polyphenolic treatments have the potential to partly prevent or even treat ageing-related neurodegenerative diseases and ageing itself. Future investigations including mammalian models and human clinical trials are needed to uncover the full potential of these olive ingredients.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/11/ 3893/s1.

**Author Contributions:** Conceptualization, V.C. and N.S.; methodology, N.S.; validation, N.S., V.C.; formal analysis, G.B., G.D.R.; investigation, G.B., G.D.R.; resources, V.C., M.S.; writing—original draft preparation, G.B., A.T.S., G.D.R. and N.S.; writing—review and editing, V.C., R.C., C.S.-L., M.S., E.J.C.; supervision, N.S., C.S.-L., and V.C.; funding acquisition, N.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This project has received funding from the European Union's Horizon 2020 research and innovation programme under Grant agreement No 633589 (Aging with Elegans). This publication reflects only the authors' views and the Commission is not responsible for any use that may be made of the information it contains.

**Acknowledgments:** We thank the Caenorhabditis Genetics Centre (CGC), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440) and the Caldwell laboratory, University of Alabama for the supply of the Caenorhabditis elegans strains. Furthermore, we thank Thea Böttcher and Shumon Chakrabarti for their support in the lab. Not least, a special thanks goes to Christian E.W. Steinberg who enabled this project and cooperation.

**Conflicts of Interest:** One of the authors, Roberto Crea, is the inventor of Hidrox® which is currently sold by Oliphenol LLC, California. The other authors declare no conflict of interest.
