Since there are several questions remaining concerning the anti-oxidative and lifespan-prolonging effects of myricetin in C. elegans, we investigated the kinetics of myricetin uptake in C. elegans, the modulation of heat induced intracellular ROS in general (fluorescent probe: DCF) and the effect of myricetin on lipofuscin accumulation, an autofluorescent pigment that is used as a biomarker of ageing. Furthermore, we analysed its effects on DAF-16 and SKN-1 and correlated the modulation of DAF-16 with anti-oxidative effects and ageing. To exclude the possibility that the effects of myricetin were mediated by caloric restriction, we finally analysed the effect of the flavonoid on food uptake by investigating pharyngeal pumping activity and body size.

Visualization of myricetin uptake in living C. elegans by use of the fluorescence enhancer 2-aminoethyl diphenylborinate (Naturstoffreagent A, NSRA) has already been reported and a concentration dependent increase in fluorescence was shown [20]. Here we were able to demonstrate a time dependent increase in myricetin fluorescence in C. elegans. Pharyngeal and intestinal fluorescence were clearly visible already after 30 min of myricetin treatment (Figure 2). An increase in fluorescence over time could be observed, with the brightest fluorescence 24 h after myricetin treatment (100 μM). These results indicate that myricetin is rapidly taken up by C. elegans and accumulates in the intestine of the nematode in a time dependent manner.

It is well-known that myricetin exerts anti-oxidative effects in various model systems [21]. To evaluate the anti-oxidative potency of myricetin, we first analysed the radical-scavenging capacity of this flavonoid using the cell free TEAC assay and, furthermore investigated anti-oxidative effects in a cellular system (Hct116 human colon carcinoma cells).

Next, we investigated if myricetin modulates the lifespan of C. elegans: Wild-type nematodes were treated with myricetin (100 μM) during their complete adult lifetime. As shown in Figure 7A and Table 1, myricetin extended the mean adult lifespan by 32.9%: lifespan of DMSO-treated nematodes was 18.7 ± 0.9 days, while myricetin-treated C. elegans had a mean lifespan of 24.8 ± 0.9 days. These data are in line with observations showing that myricetin prolongs lifespan in wild-type nematodes by 18% [20]. Several anti-oxidative polyphenols have already been identified to extend the lifespan of C. elegans, e.g., curcurmin [34], quercetin [11] and baicalein [32].

However, the myricetin-mediated prolongation of lifespan in wild-type C. elegans was completely abolished in daf-16 (mu86) mutant animals (Figure 7B and Table 1). This effect indicates that the transcription factor DAF-16 is responsible for myricetin mediated lifespan extension in wild-type nematodes, probably due to a modulation of antioxidant and stress responsive genes. Moreover, the data also indicate that lifespan extension by myricetin is largely independent of its radical scavenging properties. In contrast to these results, Grünz et al. [20] reported that myricetin mediates a lifespan extension independent of daf-16. This discrepancy could be explained by different experimental settings, which may alter the outcome of the experiment. In our experiments we used liquid culture medium and a temperature of 25 °C for the treatment, while Grünz et al. used agar plates and 20 °C for their experimental setup.

Since myricetin reduces oxidative stress and increases lifespan in C. elegans, we investigated if this flavonoid also mediates resistance to thermal stress. Thermal stress (37 °C) is known to be lethal in C. elegans. We analysed the effect of myricetin on the tolerance of C. elegans against thermal stress using the semi-automated SYTOX Green assay: 50% of the nematodes were counted dead after 3.75 h; after 5.25 h at 37 °C, no animal was alive (Figure 8A). In spite of the protective effects of myricetin against ROS accumulation, the flavonoid failed to elicit any protective effects against the lethal heat stress: Mean survival time of DMSO treated wild-type individuals was 3.94 ± 0.09 h compared to 4.01 ± 0.12 h survival time of myricetin-treated nematodes (Table 2). Using the daf-16 (mu86) mutant strain, similar results were obtained: myricetin again failed to protect against thermal stress (Figure 8B). Mean survival time of DMSO treated daf-16 (mu86) nematodes was 3.84 ± 0.12 h compared to 3.76 ± 0.09 h survival of animals treated with myricetin (Table 2). Taken together, in contrast to lifespan extending effects of myricetin (depending at least in part on DAF-16) this flavonoid failed to protect against heat stress in both wild-type and daf-16 (mu86) nematodes. These results show that an increase in lifespan and protection from ROS are not necessarily associated with a protection from heat stress in spite of the thermal-mediated generation of ROS.

It is well-known, that caloric restriction results in a prolongation of lifespan [35,36]. To exclude that the effects of myricetin on lifespan of C. elegans were mediated by a restriction in food uptake due to e.g., bitter taste of the compound, we determined food uptake of the nematodes by analysing the pharyngeal pumping activity. Since myricetin-treated nematodes showed no reduction in pharyngeal pumping activity compared to control nematodes (Figure 9A), we conclude that the myricetin induced effects were not due to caloric restriction. Further, this hypothesis is supported by an analysis of the body size of the nematodes, which is another indicator of caloric restriction. Myricetin had also no effect on the body size of C. elegans (Figure 9B). Therefore, we conclude that myricetin exerts its prolonging effect on lifespan independent of caloric restriction.

The strains used in this study were N2 var. Bristol, CF1038 [daf-16(mu86) I.], TJ356 [zIs356 IV (pdaf-16-daf-16::gfp; rol-6)] and LD001 [Is007 (skn-1::gfp; rol-6)]. Some strains were provided by the Caenorhabditis Genetics Center (CGC), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). Strains were maintained on nematode growth medium (NGM) agar plates at 20 °C containing a lawn of Escherichia coli var. OP50 (provided by the CGC) as the food source, as described elsewhere [37]. Treatment of C. elegans with the test compound was performed in 2 mL of liquid NGM containing 1% (w/v) bovine serum albumin (Sigma, Deisenhofen, Germany), 50 μg/mL streptomycin (Sigma) and 1 × 10⁹ OP50-1/mL (provided by the CGC) in 35 mm petri dishes (Greiner Bio-One, Frickenhausen, Germany). Myricetin (Extrasynthese, Genay, France) stock solution was prepared with the solvent DMSO (Merck) in a concentration of 100 mM. In all assays, myricetin was used in a final concentration of 100 μM and 0.1% (v/v) DMSO was used as the solvent control. Age synchronous animals were obtained by bleaching of gravid adults. Briefly, gravid adults were rinsed off NGM agar plates with liquid NGM, collected in 0.5 mL liquid NGM and mixed with 0.5 mL bleaching solution (50% 5 M NaOH/50% NaClO). Worms were then incubated at room temperature for three minutes, occasionally vortexed, pelleted by centrifugation (5000 rpm/4 °C/1 min) and the supernatant was discarded. The worm pellet was washed three times with liquid NGM and transferred to fresh NGM agar plates (containing OP50 lawn) and maintained for three days at 20 °C to obtain an age synchronous population of mainly L4 larvae.

In vivo visualization of myricetin in C. elegans was performed with slight modifications as described elsewhere [20]. Briefly, randomly picked 4 d old young adult animals were placed in liquid NGM ± 100 μM myricetin and 2% heat killed E. coli OP50 for the indicated time (0 min, 5 min, 30 min, 1 h, 2 h, 4 h, 6 h and 24 h) at 20 °C. Then, worms were transferred into liquid NGM containing 2% heat killed E. coli OP50 and 0.2% (w/v) 2-aminoethyl diphenylborinate (Naturstoffreagent A, NSRA) (Sigma, Deisenhofen, Germany) for 2 h. The enhanced fluorescence of myricetin in C. elegans was detected by fluorescence microscopy (excitation wavelength 460–495 nm; emission wavelength 510–550 nm; Olympus BX43). Experiments were repeated two times.

Over the lifetime of C. elegans, the autofluorescent pigment lipofuscin accumulates in gut granules and serves as an established marker of ageing [26]. Randomly picked L4 larvae were placed in liquid NGM ± 100 μM myricetin as described above and incubated for 72 h at 20 °C, followed by 24 h of incubation in compound free medium. During the incubation period, worms were transferred to fresh culture media daily. The lipofuscin fluorescence of seven days old worms was detected by fluorescence microscopy (excitation wavelength 360–370 nm; emission wavelength 420–460 nm; Olympus BX43, Olympus, Hamburg, Germany) and analysed densitometrically (ImageJ, National Institutes of Health, Bethesda, MD, USA). Therefore, the fluorescence of the whole body of each animal (except for head and tail regions) was determined and the background fluorescence was subtracted. The experiment was repeated four times.

Transgenic strain TJ356 [zIs356 IV (pdaf-16-daf-16::gfp; rol-6)] was used to detect the intracellular localization of GFP tagged DAF-16 protein. Therefore, embryos of this strain were placed in liquid NGM ± 100 μM myricetin directly after the synchronization procedure and incubated for 72 h at 20 °C. Subsequently, three days old larvae were placed on microscope slides, covered with cover slips and the cellular localization of DAF-16::GFP was detected by fluorescence microscopy (excitation wavelength 460–495 nm; emission wavelength 510–550 nm; Olympus BX43). The experiment was repeated four times. Strain LD001 [Is007 (skn-1::gfp; rol-6)] was used to observe intestinal nuclear localization of SKN-1::GFP. Three days old larvae and young adult animals were placed in liquid NGM ± 100 μM myricetin as described above for 24 h at 20 °C. Thereafter, animals were transferred on microscope slides, covered with cover slips and the cellular localization of SKN-1::GFP was detected by fluorescence microscopy (excitation wavelength 460–495 nm; emission wavelength 510–550 nm; Olympus BX43). Experiments were repeated four times.

Lifespan analyses were performed with N2 and CF1038 [daf-16(mu86) I]. About 30–50 L4 larvae per group and experiment were placed in liquid NGM ± 100 μM myricetin as described above and incubated at 25 °C. The starting day in liquid culture was considered as day 0 of the lifespan. Nematodes were transferred daily to new culture dishes during their fertile period to prevent overcrowding and to discriminate the test nematodes from their progeny. After the fertile period, worms were transferred to fresh medium every other day. Worms were scored as dead when they did not respond to gentle prodding with a flexible glass rod and when they showed no pharyngeal pumping movement. Lost worms and animals containing hatched larvae were censored. Experiments were repeated three times and Kaplan-Meier survival analysis was used to detect statistical differences (IBM SPSS 19).

The fluorescent probe H₂DCF-DA (2′,7′-dichlorodihydrofluorescein–diacetate; Sigma) was used to detect the intracellular ROS level in living individual nematodes. H₂DCF-DA is able to freely cross cell membranes, however, after entering the cell, non-fluorescent H₂DCF-DA becomes deacetylated to form the non-fluorescent derivative H₂DCF that is trapped within the cell. Then H₂DCF can quickly be oxidized by intracellular ROS to form fluorescent DCF that can be measured in a fluorescence spectrophotometer (excitation wavelength 485 nm; emission wavelength 535 nm). The fluorescence intensity correlates with the intracellular amount of ROS. The experiment was performed as described elsewhere [28]. Briefly, L4 larvae were incubated in liquid NGM ± 100 μM myricetin or 0.1% DMSO for 48 h at 20 °C. During the incubation period, worms were transferred to fresh culture media daily. After 48 h, all animals were transferred into PBST (PBS with 0.1% Tween 20) for one hour. Then single worms were transferred individually in 1 μL PBST into each well of a 384-well plate (384-well μClear® plate, Greiner Bio-One, Frickenhausen, Germany) containing 7 μL PBS. For measurement of the background fluorescence (without nematodes), 8 μL PBS were added into one column of the 384-well plate (16 wells). Subsequently, when all animals were transferred, 2 μL H₂DCF-DA (250 μM in PBS) were added into each well to obtain a final concentration of 50 μM H₂DCF-DA. A black backing tape (Perkin Elmer) was applied to the top of the plate to avoid evaporation. ROS accumulation was induced by thermal stress at 37 °C and recorded every 15 min for a period of 12 h in a fluorescence spectrophotometer (Wallac Victor² 1420 Multilabel-Counter, Perkin Elmer, Wellesley, MA, USA). The experiment was repeated four times.

Human colon carcinoma cell line Hct116 was maintained in DMEM high glucose (Gibco, Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat inactivated FCS and 100 U/mL penicillin/100 μg/mL streptomycin (Gibco) at 37 °C and 5% CO₂ in a humidified incubator (Binder, Tuttlingen, Germany).

The fluorescent probe H₂DCF-DA (2′,7′-dichlorodihydrofluorescein–diacetate; Sigma), was used to detect intracellular accumulation of ROS. Briefly, Hct116 cells were seeded into 6-well plates with a density of 5 × 10⁵ cells/well and allowed to attach for 24 h. Subsequently, Hct116 cells were treated with 50 μM myricetin or DMSO as solvent control in serum-free DMEM for 4 h. Then, cells were washed with medium, incubated with 10 μM H₂DCF-DA for 15 min, washed again and treated with 500 μM H₂O₂ to induce the generation of ROS. Afterwards, cells were washed with phosphate buffered saline (PBS) and the DCF fluorescence was determined by flow cytometry (Accuri C6, Accuri Cytometers, St. Ives, Cambs, UK).

Emission spectra of oxidized DCF (dichlorofluorescein; Sigma) in the presence of myricetin (10 μM) or the equal volumen DMSO (0.01%) as solvent control were determined in a monochromator-based microplate reader (Synergy Mx; BioTek, Bad Friedrichshall, Germany) to exclude quenching effects of the flavonoid in the DCF assay. A solution of oxidized DCF in PBS was prepared, then myricetin or DMSO were added to yield an end-concentration of 10 μM myricetin and 0.01% DMSO, respectively. Samples (100 μL) were transferred into black 96 well plates (Nalge Nunc International, Thermo Fischer Scientific Inc., Waltham, MA, USA) and the fluorescence emission spectra were recorded from 520 to 550 nm (1 nm intervals) with an excitation wavelength of 475 nm (n = 2).

The TEAC assay is a cell-free method for the measurement of radical scavenging properties of compounds [38]. The principle of this reaction is the reductive conversion of a stable, blue-green radical by an antioxidant. The solution becomes decolorized when an antioxidant is added and can be quantified photometrically. Substances without antioxidant activities show no decolorization of the radical solution. Thus, the decolorization of the radical solution indicates the anti-oxidative capacity of a compound which is compared to the potency of the reference substance Trolox (Calbiochem, Merck, Darmstadt, Germany), which is a synthetic vitamin E derivative. The radical solution was prepared the day before use and consists of a 1:1 mixture of 4.9 mM APS and 14 mM ABTS (Sigma, Deisenhofen, Germany) and is stored in the dark at room temperature. The absorption of this solution (1.4 at a wavelength of 734 nm) was adjusted by dilution with 80% (v/v) ethanol. The reference- and test-substances were measured in a concentration range from 0 to 25 μM by mixing 500 μL of the radical solution with 500 μL of the test solution. The radical scavenging activity was measured after two minutes at 734 nm with a spectrophotometer (Lambda 25 UV/VIS Spectrometer, Perkin Elmer, Wellesley, MA, USA). Three independent trials were performed.

Survival of individual nematodes at 37 °C was monitored with an assay described previously [39,40] with slight modifications. After treating L4 larvae for 48 h with 100 μM myricetin or 0.1% DMSO (daily transfer of the animals into fresh culture medium), worms were washed in PBST for 1 h and then individually transferred in 1 μL PBST into the wells of a 384-well plate (384-well μClear® plate, Greiner Bio-One, Frickenhausen, Germany) containing 9 μL PBS. Following the complete transfer of the nematodes, 10 μL of 2 μM SYTOX^® Green Nucleic Acid Stain (Molecular Probes Inc., Leiden, The Netherlands) in PBS were added to each well and the plate was sealed using black backing tape (Perkin Elmer, Wellesley, MA, USA) to avoid evaporation. SYTOX^® Green Nucleic Acid Stain is unable to pass the membranes of intact cells. However, thermal stress causes an impairment of the cellular membrane, thereby enabling the dye to enter the cells. There the dye binds to DNA and exerts a bright fluorescence that can be used as a marker for cellular damage and thus for the viability of individual nematodes [39]. The fluorescence intensity was determined with a fluorescence spectrophotometer (Wallac Victor² 1420 Multilabel-Counter, Perkin Elmer Wellesley, MA, USA) and was recorded every 15 min for 12 h (excitation wavelength 485 nm; emission wavelength 535 nm). The fluorescence curve of each nematode was calculated and the individual cut off value was determined by multiplying the average fluorescence of the first four measurements by the factor 3. The time point when the fluorescence exceeded the cut off value for each well was defined as the point of death of the respective nematode. The factor 3 in the calculation of the cut off value was previously shown to be adequate [39]. Survival curves and mean survival times were determined from these individual times of death (Kaplan-Meier survival analysis, IBM SPSS 19). Experiments were repeated at least three times.

For the uptake of food from the surrounding environment, C. elegans permanently shows pharyngeal pumping movements to filter bacteria and discard the remaining fluid. Therefore, the frequency of pharyngeal movements serves as a indicator of the feeding status of C. elegans; e.g., eat-2 mutants suffer from caloric restriction due to a reduced pharyngeal pumping activity [41]. For testing an influence of myricetin on the feeding behavior of the nematodes, wild-type L4 larvae were treated with 100 μM myricetin or 0.1% DMSO in liquid medium for 48 h at 20 °C. During the incubation period, worms were transferred to fresh culture media daily. Subsequent to the treatment, pharyngeal movement of worms was counted for 15 s and repeated three times with the corresponding worm, using a stereo microscope (Stemi 2000-C, Zeiss, Göttingen, Germany). Each experiment was performed with 5 nematodes per group and the experiment was repeated three times.

About 30 wild-type L4 larvae per experiment and group were randomly selected, transferred into liquid NGM ± 100 μM myricetin and incubated for 96 h at 20 °C. During the incubation period, worms were transferred to fresh culture media daily. Images of individual nematodes were prepared (Olympus BX43, Olympus, Hamburg, Germany) and the body size was determined by measuring the area of each worm (ImageJ, National Institutes of Health, Bethesda, MD, USA). The experiment was repeated three times.

Statistical analysis was performed with SPSS 19 (IBM) and Prism 5 (GraphPad) software and the results are presented as mean ± SD (in vitro experiments) and mean ± SEM (in vivo experiments). Statistical significance was determined by Student’s t-test, one-way ANOVA or two-way ANOVA with Bonferroni post-test. Lifespan analysis was performed using Kaplan-Meier survival analysis; animals that were lost, killed or showed internal hatching were censored. The minimum level of significance was set to p < 0.05.