*3.1. HT and OLE Boost the Health, but not Necessarily the Lifespan, of Wild Type C. elegans*

Lifespan analyses in *C. elegans* treated with plant polyphenols and other natural compounds were often reported with positive outcome (reviewed in Collins, et al. [70] and Pallauf, et al. [71]). The mechanisms behind this beneficial action are discussed in different directions, ranging from antioxidant, pro-oxidant, hormetic, direct pathway targeting, or caloric restriction mimetic effects, to recall only a few [72–76]. In the present study, HT was shown to improve the lifespan of *C. elegans* as well as the accumulation of age-pigment and swim behaviour in old worms. Thus, health and lifespan were targeted in parallel as expected. Interestingly, OLE also exerted very good performances regarding the impact on locomotion, stress resistance and age-pigment accumulation, yet in the absence of a life extending effect. However, since longer life does not automatically indicate healthier life [77,78], the suggestion that vice versa improved health is not a guarantee for a longer life, is not unreasonable.

Healthspan is hard to define and a lot of parameters need to be considered, but, simplifying, it can be described as the period of life in which the individual is functionally independent and free from serious diseases [79,80]. Uncoupling of the correlation between lifespan and healthspan was discussed in detail for *daf-2 C. elegans* knockout mutants, in which the lifespan-extending inhibition of insulin signalling did [60,81] or did not [78] increase healthspan in parallel. However, this discussion is not restricted to worms, but also takes place for flies, mice and humans [77,82,83]. Thus, it remains unclear how and to what extent healthspan and lifespan correlate. Furthermore, the different outcomes in the labs as exemplified by *daf-2* underline that healthspan-related features should be tested in a standardized way; not only to maintain comparability between results from different labs, but also to fully characterize the potential relationship between lifespan and healthspan in an objective manner.

Nevertheless, here the question arises, why OLE only affected healthspan but not lifespan? The stability of OLE in aqueous and ethanol solutions was shown to be better than other polyphenols [84,85] and during UV-induced degradation, the life-extending hydroxytyrosol is one of the main end products of its metabolism. Thus, an elevated level of OLE degradation during long-lasting lifespan analysis is not a sufficient explanation, also because such an effect should be much stronger for other, less stable polyphenols like quercetin (reviewed in Wang, et al. [86]), which it is not [87,88]. Another explanation is provided by the mode of action of the green tea ingredient epigallocatechin 3-gallate (EGCG). Brown et al. [89] and Zhang et al. [90] reported that, in spite of several health benefits in EGCG-treated worms, including prolonged survival under stress, no survival advantages were monitored during standard culture conditions. The antioxidant capacity of EGCG was emphasized as the main biochemical mechanisms responsible for the improvement of diverse health attributes. Due to the known strong antioxidant characteristics of OLE [91], this could also be true for OLE; accordingly, the missing lifespan prolongation by EGCG and OLE is not unexpected considering that Pun et al. [92] observed that antioxidant actions do not lead to longevity in *C. elegans*. Based on this consideration, the action of HT needs to be reconsidered. It must be concluded that the lifespan extension by HT is probably independent of its antioxidant power.

Finally, it needs to be mentioned that all tests were performed in the presence of 5-fluoro-2-deoxyuridine (FUdR). Since FUdR was shown to have (mainly positive) influences on the stress resistance and lifespan in *C. elegans* [93–95], it cannot be excluded that this could lead to false-negative results. This could be an alternative explanation for the missing life-prolonging effects in the OLE-treated group.
