*2.2. Hydroxytyrosol Prolonged the Lifespan of Wild Type* C. elegans

Several olive polyphenols and preparations have proven to be effective in extending the lifespan in *C. elegans* [57,58]. However, the olive oil polyphenols investigated in this study have not been tested so far in this sense.

Surprisingly, OLE treatment did not result in any significant lifespan enhancement (Figure 3A): The mean lifespan of wild type nematodes was only hardly noticeably increased by 2.7%, which

is probably the result of a minor, not significant, increase in the median lifespan from 22.55 days to 23.31 days (Table 1). However, the treatment with HT led to an increase of mean lifespan by 14.1% (Figure 3B). This life prolongation was not only visible in terms of mean and median lifespan, but was also reflected in terms of minimum and maximum lifespan (the time point, when 25% or 90%, respectively, of the individuals are dead) as well as in the time point when final death occurred (Table 1). No obvious side effects, such as extrusion of internal organs through the vulva or morphological alterations in movement in the polyphenols-treated groups compared to the controls were seen during lifelong observation.

**Figure 3.** Effect of OLE and HT on lifespan in *C. elegans*. The survival curves of controls and polyphenol-treated nematodes are shown. Survival is expressed as a percentage of the initial population per day. (**A**) The curve represents two independent experiments (*n* control = 184, *n* OLE = 111); (**B**) Representative survival curve of three independent experiments with control and HT-treated worms (*n* control = 250, *n* HT = 286). Statistical significance was calculated by log-rank test; differences compared to control were considered significant at *p* < 0.05 (\*) and *p* < 0.001 (\*\*). *n*: number of tested nematodes.


**Table 1.** Lifespan characteristics during OLE and HT treatment.

Differences compared to control were considered significant at *p* < 0.05 (\*) and *p* < 0.001 (\*\*). *p*-value determination was realized with log-rank test for the mean lifespan and Mann–Whitney U test for specific time points.

#### *2.3. Polyphenols Improved Age Pigment Accumulation and Locomotive Behaviour in Wild Type Nematodes*

The intestinal autofluorescence, one of the most prominent ageing and health biomarker in *C. elegans* [49], was surveyed to determine the overall health status. Observations with a fluorescence microscope fitted with a red filter set allowed to detect the accumulation of the "age pigment" in different age classes. As expected, the red fluorescent intensity increased with age (Figure 4), whereas the increase was only weakly pronounced at the 7th day of adulthood. Both polyphenolic compounds were able to reduce age-related gain in autofluorescence. The quantity of fluorescent pigments was slightly, yet significantly, diminished at the 12th day, but not at the 3rd or 7th day, of adulthood (Figure 4).

**Figure 4.** Age pigment quantification after OLE and HT treatment in *C. elegans*. Nematodes were observed by fluorescence microscopy in red spectrum at day 3, 7, and 12 of adulthood in two biological repeats. The bar charts (left) show the mean red fluorescence intensity of OLE (*n* A3 = 34; *n* A7 = 38; *n* A12 = 88) and DMSO (*n* A3 = 39; *n* A7 = 43; *n* A12 =84)-treated nematodes as well as HT (*n* A3 = 38; *n* A7 = 45; *n* A12 = 94)-treated nematodes and their respective water control (*n* A3 = 38; *n* A7 =44; *n* A12 = 95). Data are represented as mean ± SEM, and statistical differences compared to control were considered significant at *p* < 0.05 (\*). *n*: number of tested nematodes; A3, A7, A12: 3rd, 7th and 12th day of adulthood. In addition, example pictures (right) representing bright field and red fluorescence shots at the 12th day of adulthood in the control (DMSO and water) and polyphenol-treated (OLE and HT) groups are shown (all scale bars = 200 μm).

Since ageing is marked by physical decline, sarcopenia is considered a valuable parameter of health status in organisms of metazoans, including *C. elegans* [59,60]. Therefore, the ability of both polyphenols to boost the health of nematodes was additionally assessed with a swim assay. We measured the thrashing rate, the body wave number and the activity index to determine the physical performance at different ages. The thrashing rate is the number of body thrashes per minute as an indicator for the speed of movement whereas the activity index sums up the number of pixels that are covered by the body during the time needed for two strokes as an indicator for the vigorousness of bending [61]. Furthermore, the body wave number, a feature that increases with age, determines the waviness of the body at each time point. Indeed, the vigorousness and speed of movement of untreated worms declined with age, as indicated by the differences in both endpoints between the 3rd and 7th or 12th day of adulthood, respectively (Figure 5A,C). Moreover, the body wave number increased with age (Figure 5B), in agreement with the results from Restif et al. [61], thus verifying the correct performance of the test.

**Figure 5.** Swim behaviour characteristics in wild type *C. elegans* treated with OLE and HT. Locomotory performances were determined at day 3, 7, and 12 of adulthood in two independent repeats. The determination of locomotion differences comprises three parameters: (**A**) the thrashing rate, (**B**) the body wave number, and (**C**) the activity index. The number of analysed animals accounts for: DMSO control = 67 (A3), 70 (A7) and 117 (A12); OLE = 63 (A3), 70 (A7) and 106 (A12); water control = 76 (A3), 70 (A7) and 111 (A12); HT = 70 (A3), 70 (A7) and 104 (A12). Data are presented as mean ± SEM and differences compared to control were considered significant at *p* < 0.05 (\*) and *p* < 0.001 (\*\*). A3, A7, A12: day 3, 7, 12 of adulthood.

OLE treatment resulted in a remarkable increase of the number of thrashes per minute (Figure 5A) and of the activity index (Figure 5C) displayed by worms at all three tested stages, whereas the body wave number was decreased at the 7th and 12th day of adulthood (Figure 5B). The percentage increase was about 23% (A3), 89% (A7) and 64% (A12) and about 49% (A3), 69% (A7) and 61% (A12) for thrashing rate and activity index, respectively. The decrease of the body wave number reached its maximum at the 7th day of adulthood with a reduction of 39%. Surprisingly, HT was not able to enhance the thrashing rate of the nematodes at any adult-day (Figure 5A). However, at the 12th day of adulthood, an increase of 43% was detected by analysing the covered pixel per body and minute (Figure 5C). In addition, a decrease of 25% in the body wave number was found at A12 as well (Figure 5B). No polyphenol led to a reduction of motor performance in treated worms.
