Plant and fungal products that extend lifespan in Caenorhabditis elegans

The nematode Caenorhabditis elegans is a useful model to study aging due to its short lifespan, ease of manipulation, and available genetic tools. Several molecules and extracts derived from plants and fungi extend the lifespan of C. elegans by modulating aging-related pathways that are conserved in more complex organisms. Modulation of aging pathways leads to activation of autophagy, mitochondrial biogenesis and expression of antioxidant and detoxifying enzymes in a manner similar to caloric restriction. Low and moderate concentrations of plant and fungal molecules usually extend lifespan, while high concentrations are detrimental, consistent with a lifespan-modulating mechanism involving hormesis. We review here molecules and extracts derived from plants and fungi that extend the lifespan of C. elegans, and explore the possibility that these natural substances may produce health benefits in humans.

While many natural substances can extend the lifespan of nematodes, they act by regulating a small set of cellular pathways ( Table 1 and Figure 2). One of the main cellular pathways that control C. elegans lifespan is the insulin pathway induced by food intake [12,15]. This pathway consists of DAF-2 (the homolog of the human insulin receptor), several conserved protein kinases, and DAF-16 (the sole homolog of the FOXO family of transcription factors; Figure  2). In nematodes, insulin-like peptides bind to DAF-2 and induce intracellular signaling that leads to phosphorylation of DAF-16, thereby sequestering the transcription factor in the cytoplasm; in the absence of insulin-like peptides and DAF-2 signaling, as occurs when food is scarce, DAF-16 migrates into the nucleus where it induces expression of several genes including heat-shock proteins (HSPs) and antioxidant enzymes like superoxide dismutase (SOD) and catalase (CAT), as well as autophagy-related proteins (Figure 2) [12,15].
Phytochemicals were previously believed to produce beneficial effects on health and longevity mainly by acting as antioxidants that scavenge reactive oxygen species (ROS).  (GFP). FIB-1 is a marker of nucleolus size that negatively correlates with longevity across taxa [161], making the CGUIS-1 strain useful for screening natural products that may extend lifespan. In B, GFP auto-fluorescence is induced by ultraviolet light. The images are unpublished observations made by the authors.    However, several lines of evidence indicate that these molecules may act in other ways, notably by inducing stress resistance and anti-aging pathways [5,6]. Accordingly, the antioxidant properties of phytochemicals in vitro do not correlate with anti-aging effects in C. elegans [17]. Moreover, some phytochemicals can, instead, extend C. elegans lifespan by inducing ROS formation, which in turn leads to expression of SKN-1 and antioxidant enzymes that protect from oxidative stress by inactivating ROS [18]. For example, theophylline, a methylxanthine compound found in cocoa, chocolate, tea and guarana, slightly increases ROS levels in C. elegans, which prolongs lifespan and increases resistance to the ROS-producer juglone [19]. Plant molecules that induce ROS formation may activate c-Jun N-terminal kinase 1 (JNK-1) and DAF-16 (Figure 2). Other phytochemicals acti- The "Mechanism" column displays modulation of specific cellular components (e.g., DAF-16↑, SOD-1↑, ROS↓) or involvement of particular genes, proteins and enzymes (e.g., DAF-2, OSR-1, Sir-2.1). In the "Lifespan" column, the parentheses indicate that lifespan assays were performed in the presence of cellular stress such as high glucose, heat or paraquat; in some studies, extension of "median" lifespan was reported. Only the highest increase in mean, median or maximum lifespan is shown. Abbreviations: AAK-2, 5' adenosine-monophosphateactivated protein kinase catalytic subunit alpha 2; AGE-1, phosphatidylinositol 3-kinase age 1; AGEs, advanced glycation endproducts; AMPK, 5'-adenosine-monophosphate-activated protein kinase; CBP-1, calcineurin-binding protein-1; DAF, abnormal dauer formation protein; EGCG, epigallocatechin gallate; FOX, forkhead box; GLP-1, abnormal germ line proliferation; HSF-1, heat shock factor 1; HSP, heatshock protein; MDA, malondialdehyde; ND, not determined; NDGA, nordihydroguaiaretic acid; OSR-1, odd-skipped-related protein-1; ROS, reactive oxygen species; Sir, sirtuin; SKN-1, skinhead protein 1; SOD, superoxide dismutase; TOR, target of rapamycin; UPR mit , mitochondrial unfolded protein response.
vate SKN-1 and lead to reduction of ROS in a similar manner ( Table 1).
While several plant-derived compounds extend lifespan in nematodes, conflicting results have been obtained in some cases, possibly due to differences in study design or experimental conditions. For instance, the Caenorhabditis Intervention Testing Program, which aims to identify antiaging compounds that prolong lifespan in genetically diverse cohorts of C. elegans, reported that aspirin does not extend lifespan [20], contradicting the results of previous studies [21,22].

LIFESPAN EXTENSION OCCURS VIA HORMESIS
It has been proposed that many molecules derived from plants and fungi induce stress resistance and defense mechanisms via hormesis, i.e., which posits that cellular stress that is detrimental at high intensity can produce health benefits at low intensity [5,6,23]. By activating autophagy, mitochondrial biogenesis and expression of antioxidant and detoxifying enzymes, plant and fungal products reduce cellular damage and improve cellular functions, thus reducing aging and extending longevity [6]. This mechanism is consistent with the concept that, under conditions of stress such as CR, the organism allocates more energy for resistance and survival, instead of growth and reproduction [24].
Another observation suggesting that plant and fungal compounds extend lifespan via hormesis is the fact that stress resistance pathways are activated in the treated worms. Thus, many plant and fungal compounds that include 4,4'-dimethoxychalcone, glucosamine, nordihydroguaiaretic acid (NDGA), resveratrol and spermidine extend the lifespan of C. elegans by activating autophagy (Table 1 and Figure 2), which in itself is a typical cellular response to stress [6,28]. We also observed that polysaccharides isolated from the medicinal fungus G. lucidum extend the lifespan of C. elegans by inducing autophagy (unpublished data). In addition, several plant and fungal products increase the levels of HSPs and antioxidant and detoxifying enzymes ( Table 1), reflecting a cellular response that aims to maintain homeostasis in response to stress.
Plant and fungal compounds can also induce mitochondrial biogenesis via a process referred to as "mitohormesis" [29]. High levels of ROS usually induce cellular damage, but as mentioned above some phytochemicals can induce the formation of low levels of ROS which in turn induce stress resistance mechanisms. In this case, cells respond by forming new mitochondria which in turn may improve cellular function and longevity. Examples of natural compounds that act this way in nematodes include EGCG [27] and glucosamine [30] ( Table 1). Of note, excess intake of antioxidants such as vitamins C and E may reduce the health benefits of anti-aging interventions like exercise in humans by preventing mitohormesis [29].
In the studies consulted, plant and fungal extracts and molecules extend mean or median lifespan of nematodes by an average of 4 to 63% ( Table 1). These lifespan extensions are consistent with the hormetic effects observed in a large number of studies reporting the responses of microbes, plants and animals to various forms of biological stress, in which maximum effects of 20-90% above control were reported [31]. While hormetic responses may be relatively modest in magnitude, they are nevertheless highly significant in view of their overall impact on health and longevity.
Of note, only some plant or fungal substances increase maximum lifespan, producing increases ranging from 7 to 68% ( Table 1). While a description of the effects on maximum lifespan may have been omitted in some studies, this observation nonetheless suggests that the treatments may reduce the number of deaths in adult worms at some point in time but fail to extend the lifespan of old worms. Given that hormetic effects have been attributed to an overcompensation of homeostasis-regulating mechanisms and may thus rely on the capacity to maintain homeostasis [32], the absence of effects on maximum lifespan in some studies may indicate that very old individuals are unable to maintain homeostasis in response to biological stress, possibly due to a loss of resilience. Consistent with this possibility, feeding C. elegans with metformin late in life produces toxic effects and reduces lifespan by exacerbating agerelated mitochondrial dysfunction [33], unlike the lifespanenhancing effects of metformin seen in younger worms. Similarly, the lifespan-extension effects of EGCG decline with age [27]. This indicates that CR mimetics-and possibly other anti-aging interventions that work through hormesis-may be ineffective and even detrimental in very old individuals.

EFFECTS OF NATURAL PRODUCTS ON HEALTHSPAN VIA THE GUT MICROBIOTA
While studies in C. elegans have focused on extension of lifespan, many reports showed that natural substances that extend lifespan also produce beneficial effects on healthspan. For instance, plant-derived polyphenols such as chlorogenic acid, which is found in vegetables and coffee, improve insulin sensitivity and mobility in the treated worms [34]. Similarly, carnosic acid, a diterpene compound isolated from rosemary (Rosmarinus officinalis), improves mobility and aging-related pigmentation and neurodegeneration in nematodes [35]. These observations are consistent with the view that interventions that prolong lifespan may also improve physiological functions and reduce development of chronic disease.
Recent studies suggest that some of the beneficial effects on health and longevity in nematodes may take place via modulation of the gut microbiota. A key study showed that Escherichia coli mutants deficient in some biochemical components can extend nematode lifespan [36]. This study reported that production of the polysaccharide colanic acid by gut bacteria can extend lifespan and reduce age-related pathologies by inducing the unfolded protein response in the host. Similarly, metformin can extend lifespan and regulate host lipid metabolism via production of the metabolite agmatine by the gut microbiota [37]. Other studies showed that a strain of the probiotic Lactobacillus rhamnosus [38] or Weissella bacteria activated the DAF-16 pathway and extended C. elegans lifespan compared to feeding with E. coli [39]. However, these results may also be partially explained by the observation that E. coli becomes pathogenic for old worms and feeding with less pathogenic bacteria may therefore extend nematode lifespan [40]. Given that major differences exist between gut microbiota composition in C. elegans and humans-including the fact that the gut microbiota in nematodes studied in vitro usually consists of a single bacterial species provided as foodfurther studies are needed to assess the relevance of these observations in humans.

CHALLENGES AND OPPORTUNITIES
Our overview indicates that many plant and fruit extracts derived from blueberries to garlic, as well as plant molecules such as chlorophyll and caffeine, extend the lifespan of C. elegans (Table 1). Yet, many factors may partially limit the relevance of these findings for humans, including major differences in physiology and metabolism. Health and lon-gevity in humans depend on complex interactions between genetic background, lifestyle and diet, which can hardly be reproduced in experimental settings. It is likely that common lifestyle habits such as overeating, smoking, sedentarity, alcohol intake, stress and poor sleep, as well as environmental factors such as pollution, ultraviolet light and toxins, may reduce, suppress or even reverse the beneficial effects of phytochemicals and CR mimetics on health and longevity. Moreover, the appropriate concentrations and treatment schedule required to produce optimal health benefits remains largely unknown. The observations reported here also suggest that CR mimetics may become ineffective and even detrimental at very old age, therefore requiring the identification of optimal doses for older individuals and the development of new ways to monitor homeostasis and resilience.
Nonetheless, several epidemiological studies suggest that some of the plant-derived molecules described here may reduce human mortality and chronic diseases in humans. For instance, individuals who regularly consume coffee-arguably the highest source of polyphenols and caffeine in the human diet-live longer and show a reduced incidence of cancer, cardiovascular disease and Alzheimer's disease compared with non-consumers [41,42]. Similarly, people who regularly take metformin [43] or glucosamine [44,45], as well as those who have a higher dietary intake of spermidine [46], live longer than nonusers or controls. Finally, many CR mimetics derived from natural sources and studied in C. elegans, including quercetin, resveratrol and spermidine, have shown promising results in clinical trials [8]. It thus becomes a matter of when and how-as opposed to if-these plant and fungal molecules can be used in humans.

ACKNOWLEDGMENTS
We thank Dr. Szecheng J. Lo at Chang Gung University for helpful discussions and for providing reagents to study the effects of natural products in C. elegans. The authors' work is supported by Primordia Institute of New Sciences and Medicine, by grant MOST 109-2311-B-182-001-MY2 from Taiwan's Ministry of Science and Technology, and grant NMRPD1K0031 from Chang Gung University.