In this study, we demonstrated that herbal formula SQDB and two constituent herbs Cinnamomum cassia bark (CIN) and Panax ginseng root (GS) significantly extended lifespan in C. elegans. The observation that herbal formula HLXL did not prolong lifespan validates the C. elegans model for evaluating efficacy of herbs in an organism, because the herbal formula SQDB has been used for age-related fatigue and improving wellness, whereas HLXL is more specific for anti-inflammation and used to treat arthritis.
is a relevant model system for evaluating the efficacy of life span extending herbs due to the high conservation of biochemical pathways from worms to humans, and because of the similarity of many aspects of aging shared by nematodes and humans. Sarcopenia, the loss of muscle mass, occurs in both humans and C. elegans as a result of advanced age and results in corresponding behavioral declines. Intervention with ginseng has been demonstrated to slow this decline in C. elegans 
. Though aging mechanisms have been well studied in model organisms such as yeast and nematodes, a feasible pharmaceutical intervention for humans has not yet been discovered. Because of the co-evolution of plants and animals, it is practical to evaluate natural compounds as potential therapies for aging and aging-related declines. Indeed, such compounds have been demonstrated to delay aging and age-related declines in model organisms 
Despite the common use of herbal supplements in the United States and elsewhere 
, the precise mechanisms associated with purported beneficial effects of these supplements are unknown. This lack of information is at least partially due to the fact that investigation of herbal formulas is complicated by the intricate nature of these mixtures, and an advanced method for studying individual herbs is necessary to facilitate understanding of their mechanisms. Well-characterized mutations of C. elegans
that promote longevity, including insulin signaling and stress response, have previously been utilized to evaluate the specific mechanisms of natural compounds 
. Similarly, multiple pathways were evaluated in this study for their involvement in lifespan-prolonging mechanistic targets of SQDB, GS, and CIN.
Oxidative stress has previously been demonstrated to be a major factor limiting lifespan in both C. elegans
and humans 
and as such, has gained widespread support as a preeminent theory of aging 
. SQDB, GS, and CIN all significantly reduced levels of reactive oxygen species. However, CIN did not extend lifespan in the mev-1
mutant (for summary see ) indicating that though this herb reduces oxidative stress in the wild type worm, it likely requires an endogenous oxidative response pathway to do so. SQDB extended lifespan in the ser-1
mutant, indicating that SQDB acts independently of serotonin signaling in prolonging lifespan. Additionally, GS extended lifespan in the daf-16
mutant, suggesting that GS does not require insulin signaling to promote longevity. However, we observed that CIN failed to promote longevity in ser-1
mutants (), indicating that serotonin and insulin signaling are both involved in the beneficial effects exerted by CIN. Cinnamon has previously been shown to activate insulin receptor kinase activity and autophosphorylation of the insulin receptor in rats 
. Combined with our data demonstrating that CIN requires a downstream member of the insulin signaling pathway in C. elegans
, it is rational to conclude that CIN acts on the insulin receptor to elicit a beneficial response in the nematode. Modulation of serotonin by antidepressants has been demonstrated to enhance lifespan in C. elegans 
and our results indicate that the serotonin pathway is required for CIN induced longevity. However, the manner in which biogenic amine neurotransmitters affect lifespan, and how CIN may modulate that mechanism requires further investigation. It is clear from our data that CIN has a pluripotent effect on lifespan in C. elegans
with multiple targets through which it exerts this effect.
Lifespan has been linked to stress response in C. elegans
, and in fact, long-lived worms are invariably stress resistant 
. It has been suggested that pharmaceutical interventions that enhance longevity will also have therapeutic benefits for age-related diseases such as cancer and neurodegenerative disorders 
, perhaps via activation of endogenous stress response pathways. Transgenic C. elegans
models have been used previously to investigate the pathological mechanisms of amyloid beta (Aβ), a contributing factor to the development and progression of Alzheimer's disease (AD) [15, 49, and 50]. In this study, we investigated the effects of SQDB, CIN, and GS on Aβ-induced paralysis in a transgenic C. elegans
. SQDB, CIN, and GS all delayed paralysis in this assay, indicating a potential beneficial effect of these herbs in an age-related disease. Additionally, we observed that these compounds significantly increased expression of heat shock protein, an endogenous stress response that has previously been demonstrated to diminish Aβ-induced pathological phenotypes in transgenic C. elegans
. These data support the theory that longevity promoting compounds may also provide a beneficial effect in age-related diseases, and we have identified several new herbal compounds that may be further investigated as potential AD therapeutics. These compounds present exciting and rational new leads because the stress response mediated by heat shock proteins is a highly conserved physiological process in both invertebrate and vertebrate models 
Cinnamon is one of the most widely used herbal medicines with diverse bioactive effects. Our study demonstrated its lifespan prolonging, anti-stress and anti-proteotoxicity effects in an organism. These observations are supported by recent in vitro
studies that demonstrated CIN to exert protection against oxidative stress 
and mitochondrial dysfunction 
, to have anti-tumor properties 
, and to inhibit Alzheimer disease associated tau aggregation 
. A systematic evaluation of more than 30 herb found that cinnamon is among the most potent antioxidant herbs 
and represents an important source of dietary antioxidants 
as well as a relevant source for candidate drugs in oxidative stress-related diseases 
. Furthermore, studies using type II diabetic animals reported anti-diabetic effects of cinnamon extract administered at 200 mg/kg for 6 weeks 
. Multiple human clinical trials have also provided evidence for anti-diabetic effects of cinnamon in patients and healthy subjects 
. Mechanistic studies of several biomarkers suggest that the effect of cinnamon is mediated via regulation of multiple genes involved in insulin sensitive and lipogenesis pathways 
. Our results provide further evidence for this view by providing genetic targets of cinnamon in an organism.
Though multiple genetic and pharmaceutical interventions have been discovered that increase lifespan in C. elegans
, the translation of these effects to humans remains unknown. Our data and that of several others illustrate the power of C. elegans
in screening for herb mechanisms 
. Further studies that use mammalian systems to build on the discoveries made in C. elegans
will lead to a better understanding of how these compounds apply to humans.
It should be noted that bioavailability studies can be done using C. elegans 
, though they were not performed in this study due to the complexity of the herbal formulas. Future studies may incorporate this type of assay to provide a better understanding of the pharmacokinetic properties of herbal compounds. It must also be noted that Zarse et al observed significant differences in lifespan between the liquid culture technique utilized in this study and solid media culture. It has been proposed that worms in liquid culture exist in a state of caloric restriction, though this conjecture has not been evaluated 
The present study validated a simple animal model of aging for mechanistic studies and evaluation of efficacy of herbal supplements. This model can be used for future investigation of longevity promoting herbs or compounds and concurrent mechanisms of action.