Essential Oil Alloaromadendrene from Mixed-Type Cinnamomum

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Essential Oil Alloaromadendrene from Mixed-Type Cinnamomum osmophloeum Leaves Prolongs the Lifespan in Caenorhabditis elegans Chan-Wei Yu,† Wen-Hsuan Li,† Fu-Lan Hsu,‡ Pei-Ling Yen,§ Shang-Tzen Chang,*,§ and Vivian Hsiu-Chuan Liao*,† †

Department of Bioenvironmental Systems Engineering, and §Department of Forestry and Resource Conservation, National Taiwan University, Number 1 Roosevelt Road, Section 4, Taipei 106, Taiwan ‡ Forest Chemistry Division, Taiwan Forestry Research Institute, 53 Nanhai Road, Taipei 100, Taiwan ABSTRACT: Cinnamomum osmophloeum Kaneh. is an indigenous tree species in Taiwan. The present study investigates phytochemical characteristics, antioxidant activities, and longevity of the essential oils from the leaves of the mixed-type C. osmophloeum tree. We demonstrate that the essential oils from leaves of mixed-type C. osmophloeum exerted in vivo antioxidant activities on Caenorhabditis elegans. In addition, minor (alloaromadendrene, 5.0%) but not major chemical components from the leaves of mixed-type C. osmophloeum have a key role against juglone-induced oxidative stress in C. elegans. Additionally, alloaromadendrene not only acts protective against oxidative stress but also prolongs the lifespan of C. elegans. Moreover, mechanistic studies show that DAF-16 is required for alloaromadendrene-mediated oxidative stress resistance and longevity in C. elegans. The results in the present study indicate that the leaves of mixed-type C. osmophloeum and essential oil alloaromadendrene have the potential for use as a source for antioxidants or treatments to delay aging. KEYWORDS: Cinnamomum osmophloeum, alloaromadendrene, antioxidant, lifespan, Caenorhabditis elegans, FOXO transcription factor DAF-16

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INTRODUCTION Essential oils are considered to be secondary metabolites and important for plant defense because they often possess antimicrobial properties.1 Today, many essential oils of plants have gained attention for various types of bioactivity, such as antibacterial,2 antiviral,3 antifungal,4 and antioxidant5 actions. For these reasons, essential oils from natural plants are considered to be important alternative source of drugs and natural food preservatives.6 Cinnamomum osmophloeum Kaneh. is an indigenous tree species in Taiwan.7 C. osmophloeum contains essential oils showing multiple biological activities, including antibacterial,8 antifungal,9 anti-inflammatory,10 and in vivo antioxidant11 activities. Other beneficial properties of the essential oils of C. osmophloeum, however, remain further explored. Among essential oils, aromadendrene is one of a group of naturally occurring sesquiterpenes structurally characterized by the fusion of a cyclopropane ring to a hydroazulene skeleton. Several studies have reported that aromadendrene has biological properties, including antifungal,12 antiviral,13 antibacterial,14 antifeedant and repellent,15 and cytotoxic16 properties. However, most of the potential beneficial effects, including the antioxidant and anti-aging properties of aromadendrene have not yet been evaluated. Aging research has rapidly gained increased scientific interest. The causes of aging remain largely unknown, although they are probably related to radical and mitochondrial activities.17 An increase in stress resistance has been linked with an increase in longevity in a variety of organisms, including the nematode Caenorhabditis elegans.18,19 C. elegans has become an informa© XXXX American Chemical Society

tive and popular model for stress and aging studies because of its small size, relatively short life cycle, and well-defined genetic and environmental factors that influence lifespan.20 The Forkhead box O (FOXO) transcription factors are evolutionarily conserved and have important roles in diverse cellular functions, including metabolism, cellular proliferation, defense against oxidative stress, and probably lifespan.21 Therefore, the association between oxidative stress and FOXO is of particular interest as a result of the possible role of FOXO in controlling lifespan, because aging has been suggested to result from cumulative free-radical-induced cellular and organismal damage.17,22 Herein, we use C. elegans as an in vivo model to determine whether extracts and bioactive compounds from C. osmophloeum leaves could (1) enhance oxidative stress resistance and also trigger mechanisms that lead to a longer lifespan in a whole organism and, if so, (2) what potential mechanism is involved.

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MATERIALS AND METHODS

Plant Material and Essential Oil Preparation. Leaves of C. osmophloeum were collected, and the species was identified as previously described.23 Fresh leaves from a single plant were divided into three batches (200 g/batch) and then hydrodistilled in a Clevenger-type apparatus for 6 h, followed by determination of the Received: February 8, 2014 Revised: June 8, 2014 Accepted: June 11, 2014

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dx.doi.org/10.1021/jf500417y | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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essential oil contents. The essential oils were stored in airtight containers prior to further analysis. Gas Chromatography−Mass Spectrometry (GC−MS) and Gas Chromatography−Flame Ionization Detection (GC−FID) Analyses of Essential Oils. Essential oils were analyzed by a Trace GC Ultra (Thermo, Austin, TX) gas chromatograph coupled with a Polaris Q (Thermo) ion-trap mass spectrometer, equipped with a 30 m × 0.25 mm × 0.25 μm DB-5ms column (Agilent J&W Scientific, Santa Clara, CA). The mass spectrometer was operated in the electron-impact mode, with ionization energy of 70 eV. The oven temperature started at 60 °C for 1 min and then was programmed as follows: from 60 to 220 °C at 4 °C/min, held for 2 min, then increased to 250 °C at 20 °C/min, and held for 3 min. The injector temperature was 250 °C, and the flow rate of helium (the carrier gas) was 1.0 mL/ min in a 1:10 split ratio. Samples were diluted (1.0 μL, 1:104, v/v, in ethyl acetate) and then manually injected in the split mode. Kovats indices were determined for all volatile constituents using a homologous series of C9−C19 n-alkanes on the DB-5ms column. Quantification was determined by percentage peak area calculations using GC−FID, and the major constituents were identified by coinjection with standards (wherever possible) and confirmed by using the Wiley (version 7.0) and National Institute of Standards and Technology (NIST) version 2.0 GC−MS libraries and the Kovats indices provided in the literature.24 The relative concentration of each compound was quantified by integrating the peak area of the chromatograms. C. elegans Strains and Handling. The nematodes used in this study were Bristol wild-type N2 and GR1307 daf-16 (mgDf50). C. elegans strains and Escherichia coli OP50 were obtained from the Caenorhabditis Genetics Center (CGC), University of Minnesota, Minneapolis, MN. C. elegans were maintained and assayed (unless otherwise stated) at 20 °C on nematode growth medium (NGM) agar plates carrying a lawn of E. coli OP50. Phytochemicals were dissolved in dimethyl sulfoxide (DMSO). All chemicals in NGM plates and liquid are presented as the final concentrations. C. elegans Oxidative Stress Assays. Oxidative stress assays were performed essentially as previously described.25 Briefly, synchronized L1 larvae were treated with chemical or 0.1% DMSO (Wako, Saitama, Japan) as the solvent control for 72 h, followed by 250 μM juglone (5hydroxy-1,4-naphthoquinone) (Sigma, St. Louis, MO) exposure for 3 h,11 and then scored for viability. The survival of nematodes was determined by touch-provoked movement as previously described.19 At least three independent biological replicates were performed. C. elegans Lifespan Assays. C. elegans lifespan analyses were performed in the same manner for all treatments at 20 °C. Synchronized L1 larvae (wild-type N2 and daf-16 mutant) were transferred to NGM plates in the absence or presence of 100 μM alloaromadendrene, and nematodes were allowed to develop to adulthood. Surviving and dead animals were counted daily (starting on the first day of adulthood) until all nematodes had died. Animals that did not move when gently prodded (with a platinum wire) were scored as dead. During the reproductive period, adult C. elegans was transferred to fresh NGM plates (in the absence or presence of 100 μM alloaromadendrene) every day during the progeny production period and then every other day thereafter. To examine antibacterial effects of alloaromadendrene, lifespan assays were performed with live and ultraviolet (UV)-killed E. coli OP50 according to Kaeberlein et al.26 At least three independent biological replicates were performed. Data Analysis. For the lifespan assay, animal survival was plotted using Kaplan−Meier survival curves and analyzed by log-rank test using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). Survival curves resulting in p values of < 0.05 relative to the untreated control were considered significantly different. Statistical analysis was performed using SPSS Statistics 17.0 Software (SPSS, Inc., Chicago, IL). The results are presented as the mean ± standard error of the mean (SEM). The statistical significance of differences between the C. elegans populations was determined using one-way analysis of variation (ANOVA) and the least significant difference (LSD) post-hoc test. A two-factor analysis of variation (twoway ANOVA) was performed to determine significance and

interactions between factors in the wild-type N2 lifespan assays. Differences were considered significant at p < 0.05 (see the figures).

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RESULTS AND DISCUSSION Chemical Constituents of the Essential Oils. Table 1 shows the main components and their relative contents of Table 1. Major Components from Mixed-Type C. osmophloeum Leaves compounda

content (%)a

Kovats indexb

L-bornyl acetate T-cadinol α-cadinol caryophyllene oxide alloaromadendrene coumarin cadalene (−)-α-copaene γ-muurolene 4-allylanisole trans-calamenene γ-cadinene 1,10-di-epi-cubenol α-muurolene spathulenol α-terpineol β-selinene α-calacorene (+)-cyclosativene L-borneol β-bourbonene (−)-nerolidol trans-cinnamyl acetate (+)-aromadendrene cis-verbenol D-(+)-camphor benzyl benzoate

16.6 14.2 8.8 5.5 5.0 4.3 4.1 2.8 2.7 1.9 1.9 1.9 1.2 1.1 1.0 0.7 0.6 0.6 0.5 0.4 0.4 0.4 0.3 0.3 0.2 0.2 0.2

1285 1641 1654 1581 1461 1433 1671 1377 1475 1198 1520 1512 1613 1497 1576 1195 1488 1541 1370 1175 1385 1561 1444 1439 1144 1151 1765

a

Compounds presented here are the main components (%) in the mixed-type essential oils from C. osmophloeum leaves according to a GC−MS analysis. bKovats index relative to n-alkanes (C9−C19) on a DB-5ms column.

essential oils distilled from leaves of C. osmophloeum. The relative amounts of each component were determined by GC− FID analysis. At least 27 chemicals were identified, comprising about 78% of the total amount. The remaining was either unidentified or of very low amount. The major compounds were L-bornyl acetate (16.6%) with 95% purity, T-cadinol (14.2%) with > 95% purity, α-cadinol (8.8%) with > 95% purity, caryophyllene oxide (5.5%) with 95% purity, and alloaromadendrene (5.0%) with 97% purity (Table 1). It has been suggested that the constituents and their relative contents of essential oils from the same plant species may vary because of ecological and plant growth factors.27 On the basis of the differences in the chemical constituents of leaf essential oils, C. osmophloeum was classified into nine types: cassia, cinnamaldehyde, coumarin, linalool, eugenol, camphor, terpineol-4-ol, linalool-terpineol, and mixed-type.28 On the basis of this classification, the indigenous C. osmophloeum tree, which was analyzed in this study, belongs to the mixed-type C. osmophloeum because of the lack of a dominant compound. B



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concentrations (10 and 20 μg/mL) of extract did not further enhance the antioxidant resistance of the nematodes. The extract contains various chemical compounds that might exert different biological activities. In addition, it is possible that higher concentrations of extracts might be toxic, thereby reducing the antioxidant effects. Alloaromadendrene Is the Key Compound That Contributes to the Oxidative Stress Resistance in C. elegans. We next examined which chemical constituents from the leaves of the mixed-type C. osmophloeum tree contributed to the observed oxidative stress resistance in Figure 1. Five major compounds (Figure 2A and Table 1), L-bornyl acetate, αcadinol, T-cadinol, caryophyllene oxide, and alloaromadendrene, were selected for further oxidative stress resistance assays. Initially, synchronized wild-type N2 L1 larvae were pretreated with 100 μM of each chemical and 0.1% DMSO (solvent control) for 72 h before juglone exposure. Except for alloaromadendrene, none of chemicals demonstrated significantly enhanced juglone-induced oxidative stress resistance in C. elegans (Figure 2B). Given that alloaromadendrene estimated at ∼5% in the extracts and as low as 5 μg/mL extracts from the leaves of mixed-type C. osmophloeum was able to protect C. elegans from juglone-induced oxidative stress, we tested levels of alloaromadendrene [molecular weight (MW) of 204.35] as low as 1.5 μM, which is approximately 0.3 μg of alloaromadendrene/mL of extracts. Figure 2C show that all examined concentrations of alloaromadendrene (1.5, 20, 100, and 200 μM) pretreatment produced a significant increase in the survival of nematodes exposed to juglone-induced oxidative stress (Figure 2C). This suggests that minor (alloaromadendrene, 5%) but not major chemical components in the extract might have played a key role in the observed oxidative stress resistance in Figure 1. This unexpected finding differed from trans-cinnamaldehyde and D-(+)-camphor, which represent the major compounds in the cinnamaldehyde- and camphor-type trees, respectively, and which exerted significant in vivo antioxidant activities against juglone-induced oxidative stress in C. elegans.11 Alloaromadendrene Prolongs the Lifespan of WildType C. elegans. Aromadendrene is a member of a group of naturally occurring sesquiterpenes, and it has been shown to exhibit toxicity in a number of applications.12−16 However, most of its potential beneficial effects remain largely unexplored. In addition, most of the biological activities of aromadendrene were examined in cell cultures or with in vitro systems and not with in vivo studies. Stress resistance has been linked to an increase in lifespan in C. elegans.18,19 Similarly, an increased lifespan has been shown to be closely associated with enhanced survival under conditions of oxidative stress or heat in C. elegans.19 Moreover, oxidative stress has been shown to be a major determinant of the aging process.32 We evaluated whether alloaromadendrene has an effect on the lifespan of C. elegans by comparing the lifespan of untreated (control) and alloaromadendrene-treated wild-type N2 nematodes. Synchronized wild-type L1 larvae were transferred to alloaromadendrene-containing plates and exposed to alloaromadendrene throughout normal aging. In Figure 2C, because 100 μM alloaromadendrene exerted the most significantly oxidative stress resistance, 100 μM alloaromadendrene was chosen as the working concentration for further experiments.

In light of the existence of chemical polymorphisms of leaf essential oils from various geographical indigenous cinnamon (C. osmophloeum),29 chemical compositions in mixed-type C. osmophloeum were varied. However, it is difficult to predict or conclude the chemical compositions in mixed-type C. osmophloeum simply based on geographical or environmental factors. Therefore, it is of great concern to use the plant extract instead of pure compounds because of the possible variability of the components in the same plant group. Extracts from the Leaves of Mixed-Type C. osmophloeum Enhance the Oxidative Stress Resistance in Wild-Type C. elegans. Our previous study showed that essential oils from leaves of cinnamaldehyde- and camphor-type C. osmophloeum trees exhibited antioxidant activities in C. elegans.11 To investigate whether essential oils from the leaves of the mixed-type C. osmophloeum tree have protective effects against oxidative stress in C. elegans, wild-type N2 nematodes were pretreated with extracts immediately followed by exposure to juglone-induced oxidative stresses. Wild-type N2 synchronized L1 larvae were pretreated with 0, 1, 5, 10, and 20 μg/mL extracts and 0.1% DMSO as the solvent control for 72 h before being exposed to juglone (250 μM) and then were incubated for 3 h. Juglone was used as the oxidative stress generator because it is a redox-active quinone capable of intracellular redox cycling with superoxide radical O2• − production in C. elegans30 and is commonly used to generate the intracellular oxidative stress in C. elegans.25,31 The results show that pretreatment with 5, 10, and 20 μg/ mL extracts from the leaves of the mixed-type C. osmophloeum tree significantly increased the survival of C. elegans exposed to juglone-induced oxidative stress (Figure 1). It was observed that 5 μg/mL extract exerted the maximal effect, whereas higher

Figure 1. Essential oils from the leaves of mixed-type C. osmophloeum enhance the oxidative stress resistance of wild-type C. elegans. Synchronized wild-type L1 larvae were pretreated with 0, 1, 5, 10, and 20 μg/mL essential oils or 0.1% DMSO as the solvent control for 72 h. Adult worms were then immediately subjected to oxidative stress assays. Essential-oil-treated and untreated control adult nematodes were exposed to 250 μM juglone for 3 h and then scored for viability. At least three independent biological replicates were performed, and approximately 60−80 worms were scored in each experiment. Error bars represent the standard error, and differences compared to the control (0 μg/mL, 0.1% DMSO) were considered significant at (∗) p < 0.05, (∗∗) p < 0.01, and (∗∗∗) p < 0.001 by one-way ANOVA and the LSD post-hoc test. C



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conditions at 20 °C have a mean lifespan of 12.0 ± 0.8 days, with a maximum lifespan of 18 days (age at death of the oldest animals) (Figure 3 and Table 2). On media containing 100 μM

Figure 3. Alloaromadendrene prolongs the lifespan of wild-type C. elegans. Synchronized wild-type L1 larvae were incubated on NGM plates with dead or live E. coli OP50 in the absence (0 μM, 0.1% DMSO) or presence of alloaromadendrene (100 μM), and nematodes were allowed to develop to adulthood. Surviving and dead nematodes were counted daily until all nematodes had died. Survival curves are of the untreated control (0 μM, 0.1% DMSO) compared to alloaromadendrene-treated nematodes. Day 1 refers to the first day of adulthood. At least three independent biological replicates were performed. Approximately 60 worms were scored in each experiment. Statistical significance of the difference between the curves (treated versus untreated control) was demonstrated by the log-rank test using the Kaplan−Meier survival analysis. Differences at the p < 0.05 level were considered significantly different.

alloaromadendrene, the mean lifespan of wild-type animals was 13.4 ± 0.6 days, with a maximum lifespan of 20 days (log rank; p = 0.047; untreated versus treated) (Figure 3 and Table 2), indicating that alloaromadendrene is capable of extending the lifespan of wild-type C. elegans. This suggests that the observed delayed aging by alloaromadendrene might be due to its antioxidant activity in C. elegans. One factor contributing to late-age mortality in C. elegans is the adverse effect of proliferating bacteria that produce harmful metabolites.33 To evaluate whether the lifespan extension by alloaromadendrene treatment was regulated by the inhibition of bacterial growth, nematodes were fed with UV-killed bacteria in the absence and presence of 100 μM alloaromadendrene. The results showed that UV-killed (non-proliferating) E. coli bacteria increased the mean lifespan from 12.0 ± 0.8 to 14.0 ± 0.3 days in the absence of alloaromadendrene, whereas exposure to 100 μM alloaromadendrene even further and significantly increased the lifespan to 16.1 ± 0.3 days (log rank; p = 0.0265; untreated versus treated) (Figure 3 and Table 2). We further performed a two-way ANOVA to determine significance and interaction between factors (with alloaromadendrene untreated versus treated and live versus dead bacteria as factors) in the wild-type N2 lifespan assays. Two-way ANOVA analysis showed that there is no statistically significant interaction between alloaromadendrene and the food source bacteria (p = 0.663), suggesting that the effect of alloaromadendrene on lifespan does not depend upon the bacterial load. Hence, the antibacterial property of alloaromadendrene is not an important factor for the lifespan extension. A summary of C. elegans lifespan data is present in Table 2. Several studies have reported that C. elegans responds to various natural compounds with increased stress resistance and/or extended lifespan.34,35 Although translation of lifespan extension from C. elegans to more complex organisms remains

Figure 2. Effects of major compounds on the oxidative stress resistance in C. elegans. (A) Structure of five major compounds from leaves of C. osmophloeum. (B) Oxidative stress assays were performed as described in extracts, except that L1 larvae were pretreated with 100 μM L-bornyl acetate, α-cadinol, T-cadinol, caryophyllene oxide, and alloaromadendrene. (C) Oxidative stress assays were performed as described in extracts, except that L1 larvae were pretreated with 1.5, 20, 100, and 200 μM alloaromadendrene. At least three independent biological replicates were performed. Approximately 60−80 worms were scored in each experiment. Error bars represent the standard error, and differences compared to the control (0 μM, 0.1% DMSO) were considered significant at (∗) p < 0.05, (∗∗) p < 0.01, and (∗∗∗) p < 0.001 by one-way ANOVA and the LSD post-hoc test.

The results showed that adult wild-type C. elegans fed with live E. coli OP50 and grown under our standard laboratory D



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Table 2. Lifespan of Alloaromadendrene Treatment on C. elegans N2

daf-16 a b

control (live bacteria) alloaromadendrene (live bacteria) control (dead bacteria) alloaromadendrene (dead bacteria) control (dead bacteria) alloaromadendrene (dead bacteria)

median lifespan (day)

mean lifespan (day)

12 14 14.5 16 10 9

± ± ± ± ± ±

12.0 13.4 14.0 16.1 10.0 9.8

0.8 0.6 0.3 0.3 0.7 0.6

log-rank testa

maximum lifespan (day) 18 20 22 25 17 17

b

p = 0.047 (untreated versus treated) p = 0.0265b (untreated versus treated) p = 0.9876 (untreated versus treated)

Statistical significance of the difference between the survival curves was demonstrated by the log-rank test using the Kaplan−Meier survival analysis. p < 0.05.

to be elucidated, studies have shown that an increase in stress resistance has been linked with an increase in longevity in a variety of organisms, including the nematode C. elegans.18,19 Moreover, several lines of experimental evidence suggest that drugs prolonging lifespan in C. elegans are very effective in the treatment of age-related diseases, such as cancer and neurodegenerative diseases.36,37 Therefore, C. elegans provides a useful tool for identifying genes and compounds that can prolong lifespan in higher animals, perhaps including humans. Alloaromadendrene Enhances Oxidative Stress Resistance and Longevity Depending upon DAF-16. One potential mechanism that might be involved in alloaromadendrene-mediated oxidative stress resistance and longevity was further explored. In C. elegans, many signaling pathways, including the insulin like/IGF-1, JNK, the sensory neurons signaling, and the germline signaling pathways, converge at the FOXO transcription factor DAF-16, which is the only FOXO in C. elegans.38,39 DAF-16 integrates signals from multiple pathways and regulates its downstream target genes to control diverse cellular functions, including metabolism, stress resistance, and longevity.38,39 Therefore, DAF-16 is regarded as a key regulator to enhance stress resistance and longevity in C. elegans and mammals.38,39 Herein, to analyze the involvement of DAF16 in alloaromadendrene-mediated oxidative stress resistance and longevity, we examined the loss of function of the daf-16 mutant for its ability to resist oxidative stress and prolong lifespan during alloaromadendrene exposure. The premise of these experiments was that alloaromadendrene treatment would not increase oxidative stress resistance or extend lifespan in daf-16 mutant nematodes. Oxidative stress and lifespan assays for the daf-16 mutant were performed essentially the same as those for wild-type nematodes. For the oxidative stress-resistance test, unlike wildtype N2 C. elegans, the survival of alloaromadendrene-treated (100 and 200 μM) daf-16 mutants did not show a significant difference compared to untreated daf-16 mutants (Figure 4A). This suggests that DAF-16 is required for alloaromadendrenemediated oxidative stress resistance in C. elegans. For lifespan assays, 100 μM alloaromadendrene-treated daf16 mutant nematodes did not exhibit a significant difference in lifespan in compared to that of untreated animals (log rank; p = 0.9876; Figure 4B and Table 2). The mean lifespans for both alloaromadendrene-treated and untreated daf-16 mutant animals are 10.0 ± 0.7 and 9.8 ± 0.6 days, respectively, with a maximum lifespan of 17 days (Figure 4B and Table 2). This indicates that DAF-16 is required for alloaromadendreneenhanced lifespan in C. elegans. Taken together, DAF-16 is required for alloaromadendrene-mediated oxidative stress resistance and longevity in C. elegans. In conclusion, the results obtained from this study reveal that minor (alloaromadendrene, 5%) but not major chemical

Figure 4. Alloaromadendrene enhances oxidative stress resistance and longevity depending upon DAF-16. Oxidative stress and lifespan assays for daf-16 mutants were performed essentially the same as wild-type nematodes. (A) For oxidative stress assays, daf-16 mutants were treated with 100 and 200 μM alloaromadendrene and at least three independent biological replicates were performed. Approximately 60− 80 worms were scored in each experiment. Error bars represent the standard error, and differences compared to the control (0 μM, 0.1% DMSO) were considered significant at (∗) p < 0.05, (∗∗) p < 0.01, and (∗∗∗) p < 0.001 by one-way ANOVA and the LSD post-hoc test. n.s. = non-significant. (B) For lifespan assays, daf-16 mutants were treated with 100 μM alloaromadendrene and at least three independent biological replicates were performed. Approximately 60 worms were scored in each experiment. Statistical significance of the difference between the curves (treated versus untreated control) was demonstrated by the log-rank test using the Kaplan−Meier survival analysis. Differences at the p < 0.05 level were considered statistically significant.

components from the leaves of mixed-type C. osmophloeum have key roles in oxidative stress resistance. We demonstrate that the essential oil alloaromadendrene not only acts protective against oxidative stress but also prolongs the lifespan of C. elegans. Additionally, DAF-16 is required for alloaromadendrene-mediated oxidative stress resistance and longevity in C. elegans. We show that alloaromadendrene can E



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significantly prolong lifespan and enhance resistance to oxidative stress in a whole organism. Although further detailed investigations are needed, our results provide the first tantalizing insights on the alloaromadendrene function in longevity and antioxidative stress in vivo. The results in the present study indicate that alloaromadendrene has the potential for use as a source for antioxidants or treatments to delay aging.

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AUTHOR INFORMATION

Corresponding Authors

*Telephone: +886-2-33664626; E-mail: [email protected]. *Telephone: +886-2-33665239. Fax: +886-2-33663462. E-mail: [email protected]. Funding

This work was financially supported in part by grants (Career Development Project (101R7845/102R7845) from the National Taiwan University to Vivian Hsiu-Chuan Liao. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS All nematode strains used in this work were provided by the Caenorhabditis Genetics Center (CGC), which is funded by the National Center for Research Resources (NCRR), National Institutes of Health (NIH).

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Essential Oil Alloaromadendrene from Mixed-Type Cinnamomum

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