*3.1. HD and HT Enhance Health and Longevity of Wild type C. elegans*

Many studies reported the beneficial effects of polyphenols and flavonoids to improve health and extend lifespan in *C. elegans* [56,57]. Furthermore, healthy aging can be enhanced by several broad factors such as hormesis, autophagy and calorie restriction that increase stress resistance and longevity in *C. elegans* [58,59]. Healthspan is hard to define since it comprises the core features of health, that is physiological, cognitive, physical and reproductive function as well as a lack of disease [44]. To determine and compare the effects of treatments in terms of healthspan, it is necessary to operationalize features of health and thus, enable an objective way to measure healthspan. In this regard, the identification of predictive biomarkers and molecular pathways of health are mandatory to finally suggest applicable interventions, such as nutrition and exercise [44]. In the present study, we have demonstrated that the polyphenolic preparations HD and HT at a concentration of 250 μg/mL prolong lifespan and improve healthspan, determined via several physiological and functional parameters such as stress resistance, age pigment accumulation and swim behaviour in old wild type worms. The most powerful predictor of longevity and healthspan in old worms seems to be movement [60,61]. Similar to humans, the ability of *C*. *elegans* to move diminishes with aging [62]. Interestingly, HD also exerted optimal performances regarding the impact on locomotion in older wild type nematodes, which can be interpreted as an anti-ageing effect, while HT treatment did not show such evident improvements.

To treat *C. elegans* as naturally as possible, a live bacterial food was used throughout their life in this study. However, this protocol could also create some problems, which should be mentioned here. By adding these compounds to the living feeding bacteria, compound-bacteria interactions cannot be excluded. It is known that several plant polyphenols possess antibacterial activities [63]. These potential antimicrobial abilities could result in the inhibition of bacterial proliferation, which in turn would reduce the harmful intestinal accumulation of bacteria during ageing and prolong healthspan in *C. elegans* [64–66]. However, a recent study by Medina—Martínez et al. [67] showed, that at least 400 μg/mL HT were necessary to produce a perceptible growth deceleration of different *E. coli* strains. Furthermore, also olive leaf extract was shown to be only weakly active against *E. coli* bacteria [68]. On the other hand, it also cannot be excluded that compound-bacteria interactions lead to the degradation of the test compound by the bacteria. At least for hydroxytyrosol, this possibility can be neglected according to the study by Medina—Martínez et al. [67] who showed that degradation by *E. coli* was present to only a small extent. Nevertheless, in future studies the metabolic profile of *C. elegans* after the digestion of HT and HD in combination with alive *E. coli* should be checked for potential degradation products.
