The Anti-aging Effects Of Ludwigia Octovalvis On Drosophila Melanogaster And SAMP8 Mice Part 2

Chemical composition of LOE

To identify the mechanisms by which LOE  induces lifespan extension in flies, the chemical composition of LOE was analyzed by GC-MS. Seventeen major peaks were seen in the total ion chromatogram (Fig. 4) and were identified as the known compounds listed in Table 2.

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LOE attenuates acute oxidative damage

Since oxidative stress is the most popular theory for explaining how aging occurs at the molecular level, we decided to examine the DPPH radical scavenging activity of LOE as a critical indicator of its antioxidant capacity. In our assays, LOE had strong DPPH radical scavenging activity (IC50 3.15 μg/ml), in between the activity of ascorbic acid (IC50 1.31 μg/ml) and that of resveratrol (IC50 7.86 μg/ml), which were used as controls (Fig. 5a). However, DPPH radical scavenging activities of the two most abundant compounds identified in LOE, β-  sitosterol, and squalene were detected only at minimal levels (Fig. 5a). The antioxidant capacity of LOE could be attributable to its antioxidant-rich composition, since a  high level of polyphenols (146.3±3.1 mg of gallic acid equivalent per g of dry weight; Table 3) was present in our LOE preparation, and flavonoids were a major component among the polyphenols (90.2±2.6 mg of rutin equivalent per g of dry weight).

We therefore further tested the anti-oxidative properties of LOE by exposing 10-day-old female LOE-treated CS flies to paraquat (methyl viologen, a chemical known to generate reactive oxygen species). We found that flies receiving LOE supplementation in their food for 10 days showed increased resistance to paraquat-induced lethality (Fig. 5b). This result was further confirmed by analyzing the levels of oxidative damage in flies that had been treated with paraquat for 12 h. We used 4-hydroxynonenal (HNE), a protein adduct generated from products of lipid oxidation, as an indicator of oxidative damage. Although LOE supplementation showed no effect on the basal level of HNE in 10-day-old flies, it significantly reduced HNE accumulation in flies treated with 20 mM paraquat (Fig. 5c).

β-Sitosterol is a major component in LOE that contributes to the lifespan extension

We further examined the effects of β-sitosterol and squalene on the lifespan of flies. Intriguingly, β-  sitosterol induced lifespan extension in female CS  flies (Fig. 6a) in a dose-dependent manner. This pro-longevity effect, however, was not seen in female CS flies receiving the same doses of squalene (Fig. 6b).

The AMPK pathway has been shown to mediate lifespan extension by DR in both C. elegans (Apfeld et al. 2004; Greer et al. 2007) and in flies (Stenesen et al. 2013). We, therefore, examined whether LOE and β-sitosterol could regulate lifespan by modulating the AMPK pathway. Using Drosophila S2 cells, we found that LOE and β-sitosterol could significantly enhance AMPK phosphorylation (Fig. 6c). Similar observations were made when cells were maintained under DR  conditions (0.5× medium) (Fig. 6c). If AMPK had a  major role in β-sitosterol-induced lifespan extension in flies, loss of AMPK should blunt the longevity effect by β-sitosterol. We tested this hypothesis by knocking down AMPK expression, specifically in the fat bodies of adult flies (Fig. S1). Drosophila fat body is the fly equivalent of mammalian liver and adipose tissue and is responsible for metabolism. AMPK overexpression or knockdown in fat body cells has been associated with increased or decreased fly lifespan, respectively (Stenesen et al. 2013). We combined a UAS-AMPK.RNAi allele with an RU486-  inducible GeneSwitch GAL4 for the fat body (S106-GAL4). Adult flies carrying appropriate genetic elements were randomized to foods containing RU486 (the activating agent for the GeneSwitch-Gal4 drivers) and/or β-  sitosterol. We found that the β-sitosterol-induced lifespan extension was not seen when AMPK expression was knocked down specifically in the fat body of adult flies (Fig. 6d). Together, these data imply that LOE-derived β-  sitosterol may modulate lifespan, at least in part, through activating AMPK in the fat body of adult flies.

Discussion

Fruit flies have a relatively short lifespan, ranging from a few weeks to several months, depending on nutritional and other environmental conditions. For instance, altered calorie intake, mating conditions, and ambient temperature can all significantly change the fly's lifespan (Bauer et al. 2004). In well-defined conditions, we were able to demonstrate that food supplementation by LOE could extend lifespan in flies without any obvious negative tradeoff.

The development of DR mimetics has become an emerging field for anti-aging research. 2-Deoxyglucose, an inhibitor of glycolysis (Lane et al. 1998), and metformin, which stimulates AMPK (Anisimov et al. 2003),  are two molecules currently being explored as DR mimetics. However, long-term administration of 2-  deoxyglucose or metformin may produce unwanted side effects, including heart failure and lactic acidosis, making them unlikely candidates for use as DR mimetics (Ingram et al. 2004; Sinclair 2005). Flies receiving metformin also showed no beneficial effect on lifespan extension (Slack et al. 2012). Thus, plant-derived polyphenols such as resveratrol may provide an alternative option. Resveratrol has been shown to extend the lifespan of yeast, worms, and flies by mimicking DR  through a Sir2-dependent pathway (Wood et al. 2004). Although resveratrol did not extend the lifespan of mice fed ad libitum, it improved the health and survival of obese mice fed a high-calorie diet (Baur et al. 2006; Pearson et al. 2008). These results suggest that resveratrol may enhance resistance to high-calorie-induced stress, which is consistent with results seen in studies demonstrating that resveratrol conferred cellular protection from stresses including oxidative damage (Araki et al. 2004) and gamma radiation (Howitz et al. 2003).

Our data suggest that LOE may act in part as a DR  mimetic. We found that LOE significantly extended the lifespan of flies fed a high-calorie diet, but showed no additive effect under DR conditions (Fig. 1). LOE also attenuated age-related cognitive decline, keeping it to a  level similar to that seen in flies under DR conditions. We showed that these DR-like effects of LOE were not due to lower food consumption induced by LOE (Figs. 2 and 3). Neither were other typical DR-coupled physiological changes, such as reduced female fecundity or enhanced motor activity, observed in flies treated with LOE (Fig. 2). DR has been shown to increase mitochondrial activity (Nisoli et al. 2005; Lopez-Lluch et al. 2006), but to delay or reduce oxidative stress and damage in flies (Zheng et al. 2005), mice (Qiu et al. 2010), monkeys (Zainal et al. 2000), and women (Buchowski et al. 2012). This observed effect has been suggested to be a “mitohormesis” effect, a hypothesis that increased mitochondrial activity and ROS production can trigger an intrinsic defense program, which results in improved stress resistance and possibly lifespan extension (Schulz et al. 2007). We found that LOE had strong DPPH radical scavenging activity and that it reduced paraquat-induced oxidative damage and lethality in flies (Fig. 5), further supporting our hypothesis that LOE may act, at least in part, as a DR mimetic.

Our chemical analyses confirmed that LOE was a rich source of antioxidants, including polyphenolic compounds, phytosterols, and squalene (Fig. 4 and Table 2). It has been reported that β-sitosterol may up-regulate protein expression and antioxidant activity of manganese superoxide dismutase through the estrogen/phosphatidylinositol 3-kinase pathway in macrophages (Vivancos and Moreno 2005). Dietary squalene supplementation was also shown to attenuate age-related oxidative damage and to maintain the energy status of liver mitochondria in aged rats (Buddhan et al. 2007). Although β-sitosterol and squalene were the two most abundant compounds identified in LOE by GC-MS analyses, neither showed obvious DPPH radical scavenging activity in our assay system (Fig. 5). It is possible that the anti-oxidative activities of LOE could include free radical scavengers such as polyphenolic compounds, while β-  sitosterol and squalene could act through other pathways,  as discussed above. Other traditional and integrative medical practices such as R. rosea and C. longa have been shown to extend the lifespan of flies through pathways associated with anti-oxidation and regulation of known aging-related genes (Lee et al. 2010; Schriner et al. 2013, 2009). However, herbal extracts usually contain many compounds that require systematic biochemical analyses for identification. A mechanistic interpretation is therefore difficult without the identification of a pure compound. In the present study, we identified β-sitosterol as a major component that contributed to the lifespan-extending activity of LOE. Our data suggest that β-sitosterol-induced lifespan extension requires AMPK activation in the fat body of adult flies. This is largely in agreement with previous reports demonstrating that AMPK is the major nutrient sensor and mediates lifespan extension by DR in C. elegans (Apfeld et al. 2004; Greer et al. 2007) and flies (Stenesen et al. 2013).

It is desirable that longevity interventions not only extend lifespan but also delay the onset and morbidity of age-related diseases, that is, they increase both lifespan and health span. The precognitive effects of LOE that we describe in both flies and SAMP8 mice may also be of great interest to the aging field. It has been shown that SAMP8 mice exhibit age-related deficits in memory retention from 2 months of age, when tested on passive avoidance tasks (Miyamoto et al. 1986). In a footshock avoidance test, SAMP8 mice also show significant impairment in acquisition and memory retention from 6 to 9 months of age, compared to young SAMP8 mice (Flood and Morley 1993). Using novel object recognition and elevated-T maze tasks, we could observe clear age-related deficits in memory retention between 3- and 8-month-old SAMP8 mice, although there were no differences during the acquisition period of the elevated-T maze task (Fig. 3). These observations are further evidence that SAMP8 mice display an early onset of age-related cognitive deficits and that the impairment becomes worse with age. However, long-term LOE treatment (more than 5 months) in SAMP8 mice attenuated the age-related decline in memory retention without affecting physical activity, food intake, water consumption, or body weight (Fig. 3). We did not observe any side effects in mice receiving 5 months of LOE treatment (unpublished observations). These findings are largely in agreement with a recent report demonstrating that repeated administration of L. octovalvis for 28 days in mice (800 mg/kg≈2,400 μg/day vs. 400 μg/day used in our study) did not cause lethality or toxicological effects (Kadum Yakob et al. 2012).

In summary, we have identified LOE as a novel antiaging intervention, capable of extending the lifespan of flies and attenuating age-related cognitive decline in both flies and SAMP8 mice. GC-MS and other biochemical analyses suggest that LOE may exert its antiaging effects by attenuating oxidative damage and activating AMPK-related pathways.

Acknowledgments We would like to thank Chuan-Hao Lai and Hao-Wei Wang (Hsiehyu Biotech Company Ltd.) and Shang-Tse Lee (National Taiwan University) for their technical assistance. We would like to thank Fly Core in Taiwan for the fly stocks and reagents. Pei-Yu Wang is partially supported by the National Science Council (NSC 98-2320-B-004-003-MY2 and NSC 100- 2311-B-002-017-MY3), Taiwan.

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