Article pubs.acs.org/JAFC
Mechanism of Longevity Extension of Caenorhabditis elegans Induced by Pentagalloyl Glucose Isolated from Eucalyptus Leaves Yunjiao Chen,†,‡ Brian Onken,§ Hongzhang Chen,† Suyao Xiao,† Xiaojuan Liu,† Monica Driscoll,*,§ Yong Cao,*,† and Qingrong Huang*,‡ †
Department of Food Science, College of Food Science, South China Agricultural University, Guangzhou 510642, China Department of Food Science, Rutgers University, New Brunswick, New Jersey 08901, United States § Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854, United States ‡
S Supporting Information *
ABSTRACT: The multicellular model organism Caenorhabditis elegans (C. elegans) was used to identify the anti-aging effect of pentagalloyl glucose (PGG) isolated from Eucalyptus leaves at four different concentrations. For 160 μM PGG, the median lifespan of C. elegans was found to increase by 18%, and the thermal stress resistance was also increased. The anti-aging effect of PGG did not cause side effects on the physiological functions including the reproduction, pharyngeal pumping rate, age pigments accumulation, and locomotion ability. The life extension induced by PGG was found to rely on genes daf-16, age-1, eat-2, sir-2.1, and isp-1 but did not rely on genes mev-1 and clk-1. These findings suggested that the insulin/IGF-1 signaling pathway, dietary restriction, Sir-2.1 signaling, and mitochondrial electron transport chain became partly involved with the mechanism of lifespan extension mediated by PGG. Our results provided an insight into the mechanism of longevity extension mediated by PGG in C. elegans, which might be developed into a new generation of multitarget drug to prolong lifespan. KEYWORDS: Caenorhabditis elegans, pentagalloyl glucose, insulin/IGF-1 signaling pathway, median lifespan
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INTRODUCTION Nowadays the rapid increase of aging population is becoming a social and economical problem in many countries.1 The discovery of new botanical compounds with lifespan extension effect could prompt new strategies for treating age-related disease such as diabetes, cancer, and neurodegenerative disorders.2 Currently more and more research focuses on exploiting natural dietary compounds with the ability to prevent age-related diseases and promote healthy aging.2 The nematode Caenorhabditis elegans (C. elegans) is a wellestablished model system to study organismal aging and identify new pharmacological targets because of its relatively short life span, ease of cultivation, and well-known genetic pathways, which are conserved in diverse species including mammals.3 The insulin signaling pathway,4 dietary restriction,5 and rates of mitochondrial respiration6 are the three wellestablished mechanisms that modulate lifespan and work across species. It is demonstrated that 60−80% of human gene homologues have been identified in C. elegans.7 Caenorhabditis elegans has been increasingly applied to investigate the effect of pharmacologically active compounds on aging processes.7 A number of antioxidants including resveratrol, 8 EGCG,9 and coenzyme Q1010 have been found to extend the lifespan of C. elegans. PGG (pentagalloyl glucose) is a plant polyphenol widely enriched in many medicinal herbals.11 PGG has a wide spectrum of physiological and pharmacological functions, including anticancer, insulin-mimicking, antioxidative, and antiallergy activities.11 Our recent study also found that PGG isolated from Eucalyptus leaves exhibited a high antioxidant activity in both chemical-based (e.g., DPPH and ABTS assays) and cell-based (i.e., HepG2) antioxidant assays.12 Because of its © 2014 American Chemical Society
broad biological activities, PGG showed a great potential in the prevention and treatment of several major adult-onset diseases. However, little is known about its ability in promoting longevity in any organism. Therefore, the effect of PGG on the longevity of C. elegans needs to be explored. In this study, first, we investigated whether PGG intervened in the aging process at the organismal level for the first time and proved that the worms treated with PGG showed a significant survival curve and an 18% increase in median lifespan. Second, we evaluated whether PGG that induced longevity extension affected the normal physiological function and found that the lifespan extension mediated by PGG did not cause obvious physiological defect. Finally, we dissected the genetic requirements of lifespan extension induced by PGG and demonstrated that the insulin/IGF-1 signaling pathway, dietary restriction, Sir-2.1 signaling, and mitochondrial electron transport chain partly became involved with the mechanism. We believe these findings will further promote PGG to become a new generation of multitarget drug to extend longevity.
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MATERIALS AND METHODS
Materials. The following chemicals were used for the preparation of nematode growth medium (NGM): NaCl, peptone, streptomycin, agar, cholesterol, CaCl2, MgSO4, and Nystatin. NaOH and household bleach (The Clorox company, Oakland, CA, USA) were used for bleaching the worms. C. elegans mutant strains used: daf-16(mgDf50), age-1(hx546), CF1553 {muIs84 [pAD76 (sod-3::gfp)]}, eat-2Received: Revised: Accepted: Published: 3422
January 13, 2014 March 18, 2014 March 24, 2014 March 24, 2014 dx.doi.org/10.1021/jf500210p | J. Agric. Food Chem. 2014, 62, 3422−3431
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Figure 1. Effect of PGG treatment on the median lifespan of C. elegans. (A) Chemical structure of PGG. (B) Survival curves of wild-type (N2) worms raised at 20 °C on the plates containing either 0.2% DMSO (control) or final concentrations of PGG (40, 80, 120, or 160 μM). The survival curves treated with 120 or 160 μM PGG were significantly different by the Log-rank test (P < 0.05). Four independent trials were performed. (ad1116), sir-2.1(ok434), mev-1(kn1), isp-1(qm150), and clk-1(qm30). (The properties of the mutants are provided in Supporting Information.) All nematodes used in the study were supplied by the Caenorhabditis Genetics Center (CGC, University of Minnesota, Minneapolis, MN, USA). Preparation of Treatment Plates. PGG (the chemical structure was presented in Figure 1A) was isolated from Eucalyptus leaves in our lab and was identified by comparing its nuclear magnetic resonance (1H NMR and 13C NMR) data with the reported literature.12 PGG stock solution (80 mM in 100% DMSO) was diluted to the proper concentration. Then 5 μL of solution was added to 95 μL of Escherischia coli OP50, and the mixture was seeded onto the NGM plate (35 × 10 mm) and remained dry. To avoid autoxidation and polymerization of PGG, we analyzed the extraction of mixture with HPLC after it was seeded and found that there was a peak in the extraction that was identical with that of PGG. So PGG did not change before the treatment (the HPLC and MS of the PGG are provided in Supporting Information, Figures S1 and S2). The treatment plates contained different doses (final concentrations: 40, 80, 120, and 160 μM in 0.2% DMSO, respectively) of PGG or control solvent (0.2% DMSO). Lifespan Assay. Lifespan analysis was performed by the same assay for all strains. If not stated otherwise, eggs isolated with hypochlorite (bleached with 5% sodium hypochlorite and 2.5 M sodium hydroxide) were transferred to the treatment plates and maintained at 20 °C in a temperature-controlled incubator. Synchronization was performed at L4 larvae stage, and age synchronized young L4 larvae (20 worms per plate and total of 3 plates per treatment) were then transferred to fresh treatment plates. During the reproductive period, these worms were transferred daily to new treatment plates and thereafter about every other day. Live and dead C. elegans were counted daily (starting from the first day of hatching) until all individuals were dead. Nematodes that failed to respond to a gentle touch were scored as dead. Nematodes suffering from hatching of embryos within the adult hermaphrodite prior to eggs being laid (a so-called ‘internal hatch’) and those who escaped from the NGM agar were censored. Experiments were always performed in parallel with a control group. In the experiments assessing life span, reproduction, and pharyngeal pumping, treatments started from hatching unless otherwise stated. Reproduction Assay. This assay was performed according to the previously reported method.13 Wild-type C. elegans (10 individuals, 2 worms per plate) were shifted to fresh plates every day during the reproduction day, and the plates with eggs were left at 20 °C for 24 h to allow eggs to hatch. The number of progeny of each worm was counted at the L4 stage to verify that eggs were hatched. Assay for each experiment was performed triplicates. Pharynx Pumping Rate. Pharyngeal pumping was recorded on days 5, 10, and 15. Nematodes (10 individuals) were randomly selected, and the pumping assay was performed on NGM agar plate at
room temperature. The number of their pharyngeal contractions was counted for 30 s.13 All pumping rates were measured on a lawn of OP50 bacteria. The assay was repeated thrice with 10 worms per assay. Age Pigments Fluorescence Spectroscopy. Fluorescence intensities of age pigments were measured as described.13 Wild-type worms were raised from eggs on plates as in the lifespan assays. On days 5, 10, and 15 of life, 50 C. elegans were transferred to 50 μL of 10 mM NaN3 solution in a single well of a 96-well white FluoroNunc plate. The 96-well plate was scanned in a spectrofluorimeter (Fluorolog-3, JobinYvon Inc., Edison NJ, USA). The fluorescence peak intensities of age pigments were obtained by scanning through the range of excitation wavelengths from 280 to 410 nm and an emission wavelength of 430 nm. Locomotion Assay. C. elegans were raised from eggs similar to the lifespan assays. On days 5, 10, and 15, a total of 40 worms with or without PGG treatment were measured for body bend rate in liquid. Briefly, each time 4 worms were placed in 20 μL of M9 buffer on a glass slide. The locomotion of the worms was recorded by video for 30 s using a Qimaging Rotera-XR digital camera attached to a dissecting microscope and streamplx imaging software (ver. 3. 17. 2, NorPix Inc., Montreal, Quebec, Canada). Body bends in each frame of the 30 s film were analyzed by CeleST software. The assay was repeated three times with 40 worms per assay.13 Thermotolerance Assay. The thermotolerance assay was performed with hermaphrodites at 20 °C as for the aging assay. The adult worms with and without PGG treatment were shifted from 20 to 35 °C on day 4. Survivals were scored every hour after the temperature shift. Worms that died from desiccation on the sides of the plate were excluded.3 Accumulation of Reactive Oxygen Species (ROS). Accumulation of ROS was determined according to a modified protocol.14 H2DCF-DA (2,7-dichlorodihydrofluorescein diacetate) is a membrane-permeable compound and is converted to H2-DCF when entering the cells of the worm. This nonfluorescent probe would be oxidized by ROS to yield the fluorescent dye DCF. Briefly, the worms were maintained and treated as in the lifespan method. After treatment with PGG or control for 96 h, worms were placed on a NGM plate to remove the bacteria three times. Then 80 worms were transferred to 50 μL of M9 buffer in a 96-well plate. In the meantime, 50 μL of fresh 100 μM H2-DCF-DA solution in M9 buffer (100 mM stock solution in DMSO) was pipetted to the well (final concentration was 50 μM). On each plate, a control well containing H2-DCF-DA without worms was included. Emission fluorescence intensity at 528 nm with excitation at 485 was recorded every 15 min for 12 h at 25 °C using a Synergy HT multimode microplate reader (Bio Tek Instruments Inc., Winooski City, VT, USA). The results are presented as means ± SD of relative fluorescence units (RFU) of three independent experiments with 240 individual worms in total. Quantification of GFP-Labeled SOD-3 Expression. The GFP of the transgenic strain CF1553 {muIs84 [pAD76 (sod-3::gfp)]} can 3423
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Figure 2. Physiological functions experiments were performed with wild-type worms raised from eggs on plates containing either 0.2% DMSO (control) or 160 μM PGG at 20 °C. (A) Wild-type worms (10 individuals, 2 worms per plate) were shifted to fresh plates every day during the reproduction day, and the plates with eggs were left at 20 °C for 24 h to allow eggs to hatch. The number of progeny of each worm was counted after the eggs were developed to the L4 stage at 20 °C. (B) The number of their pharynx contractions was counted for 30 s. All pumping rates were measured on a lawn of OP50 bacteria. The assay was repeated thrice with 10 worms per assay. (C) Wild-type worms were transferred to 50 μL of 10 mM NaN3 solution in a single well of a 96-well white FluoroNunc plate on days 5, 10, and 15. The peak age pigments fluorescence intensity was scanned through a range of excitation wavelengths from 280 to 410 nm and an emission wavelength of 430 nm. (D) The locomotion of 4 worms placed in 20 μL of M9 buffer on a glass slide was recorded by video for 30 s on days 5, 10, and 15. The number of head thrash in each frame of the 30 s film for worms was analyzed by CeleST software. be induced by many environmental stresses, especially oxidative stress, and the expression of reporter GFP can reflect the level of sod-3.15
Worms were raised from eggs on plates as in the lifespan assays until day 5 of life. The level of GFP was measured using an in vivo 3424
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Figure 3. Effect of PGG treatment on thermal resistance and ROS accumulation. (A) Survival rates after being treated for 8 h after the worms were shifted from 20 to 35 °C on day 4. The different letters in the column denoted values that were significantly different (p < 0.05). (B) After treatment with PGG or control for 96 h, worms were placed on a NGM plate to remove the bacteria three times. Then 80 worms were transferred to 50 μL of M9 buffer in a 96-well plate and mixed with 50 μL of fresh 100 μM H2-DCF-DA (final concentration, 50 μM). Emission fluorescence intensity at 528 nm with excitation at 485 was recorded every 15 min for 12 h at 25 °C. (C) Survival curve of worms after the worms were shifted from 20 to 35 °C on day 4. spectrofluorometer (Fluorolog-3, Jobin Yvon Inc., Edison, NJ, USA). The results are the average fluorescence intensity levels of three independent trials. Statistical Analysis. All results were presented as mean ± SD (n = 3). Assays for each experiment were performed three or four times. Log-rank (Mantel-Cox) tests were performed to analyze the survival curve using Graph Pad Prism version 5.00 for Windows (GraphPad Software, Inc., San Diega, CA, USA). One-way analysis of Variance (ANOVA) was performed using SPSS software (SPSS Inc., Chicago, IL, USA).
worm started from embryo till death. We found that different concentrations of PGG had different performances. Of the four concentrations, worms raised on 160 μM PGG not only manifested a significant survival curve (Figure 1B) (P < 0.0001 by the Log-rank test) but also had a median lifespan of 21.5 ± 2.4 days with maximum lifespan of 40 days and increased by 18% (p < 0.05, ANOVA) in median lifespan. Worms treated with 40, 80, and 120 μM PGG did not show a significant increase in median lifespan, but their treatment showed a slightly increasing trend in median lifespan (4%, 3%, and 11% increase for 40, 80, and 120 μM PGG, respectively). Furthermore, worms cultured on 120 μM PGG displayed a significant different right-shifted survival curve (P = 0.0010 by the Log-rank test) when compared with the control, and the survival curve of 120 μM PGG was continuously above that of control (Figure 1B). Although worms treated with 120 μM PGG did not show significant increase in median lifespan, it was more effective than control in the survival curve. Taken together, these findings indicated that PGG supplementation could prolong median lifespan in C. elegans, and the optimal concentration was observed at 160 μM PGG (increased by 18% in median lifespan) during the tested concentrations. Therefore, 160 μM PGG was chosen for further study. Effect of PGG Treatment on Physiological Functions of C. elegans. The decline of physiological functions such as self-fertile reproduction, pharyngeal pumping, age pigments, and locomotion happened in the aging process. To determine whether PGG caused side effects on the physiological functions at the optimal concentration, we analyzed those physiological function changes under PGG treatment. First, it is critical to
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RESULTS AND DISCUSSION Effect of PGG Treatment on the Median Lifespan of C. elegans. To address the concern about the stability of the C. elegans system used, first, the anti-aging effect of resveratrol (100 μM) (National Institutes for Food and Drug Control, Beijing, China) was investigated because of its widely anti-aging effect across diverse species including C. elegans and well-known mechanism of action.8,16 We found that worms raised on 100 μM resveratrol not only manifested a significant survival curve (P < 0.0001 by the Log-rank test) but also showed a 13.2% increase in median lifespan (20.0 ± 1.0 days with resveratrol treatment vs 17.7 ± 0.6 days with control) (p < 0.05, ANOVA) (The survival curve for resveratrol is provided in Supporting Information, Figure S3), which was consistent with the results (approximately14%) obtained from the literature.8 These results indicated the determination of lifespan in C. elegans in our lab was stable and comparable. So we continued to analyze the anti-aging effect of PGG with high cellular antioxidant activity in C. elegans.12 To understand comprehensively the anti-aging effect, four different concentrations of PGG ranging from 40 to 160 μM were tested. The treatment of the wide-type 3425
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Figure 4. Effect of IIS pathway on the longevity extension induced by PGG. (A) Survival curves of daf-16(mgDf50) (Log-rank test, P = 0.2774). (B) Survival curves of age-1(hx546) (Log-rank test, P = 0.0003). (C) Worms of transgenic strain CF1553 [muIs84 [pAD76 (sod-3::gfp)]] were raised from eggs on plates as in the lifespan assays until day 5 of life. The level of expression of GFP in the transgenic strain was measured using an in vivo spectrofluorometer. The results are the average fluorescence intensity levels of three independent trials.
Locomotion ability declines on solid and liquid media with increasing age, which is correlated with the level of muscle deterioration.18 To determine whether PGG can enhance lifespan by extending locomotory ability, we tested the effect of the PGG treatment on the swimming vigor, defined as head thrash per unit time in a liquid environment, at early, middle, and late life stages (on days 5, 10, and 15, respectively). We found that head thrash of the worms declined with the increasing day and PGG treatment did not significantly change the swimming rates at the three stages (Figure 2D). These findings indicated PGG neither caused detrimental effects in locomotory ability nor increased swimming in a liquid environment. To summarize, PGG treatment prolonged the median lifespan without causing obvious defect in physiological functions including fertility, pharyngeal pumping rate, age pigments, and locomotion ability. So we continued to gain insight into the related mechanisms. Effect of PGG Treatment on Thermal Resistance and ROS Accumulation. Longevity extension is generally related with elevated survival under the conditions of stress.20 To exploit whether PGG enhanced heat stress resistance, worms treated with or without compound were shifted from 20 to 35 °C on day 4. The survival rates after 8 h at 35 °C were increased significantly by PGG treatment (increasd from 21% to 36%) (Figure 3A). Compared with the control, the group with PGG treatment showed a significantly different survival curve (P = 0.016 by the Log-rank test) (Figure 3C). The findings suggested that PGG supplementation could act
investigate progeny production because a few mechanisms that extend the longevity have an impact on it.17 We recorded the effect of PGG treatment on the progeny production daily to investigate whether PGG suppressed or delayed it. Worms cultured on medium containing 160 μM PGG showed neither slowed reproduction nor reduced total progeny production. These results demonstrated that median lifespan extension induced by PGG treatment was not obtained by suppressing or delaying progeny production (Figure 2A). One well-conserved feature of aging is the decline of pharyngeal pumping, which is rhythmic contraction in the neuromuscular organ.18 We investigated whether PGG had an effect on pharyngeal pumping rate on days 5, 10, and 15. We found that pharyngeal pumping decreased with age, but PGG treatment did not affect the pharyngeal pumping rate in the three stages (Figure 2B). So the possibility that PGG extended lifespan by reducing food intake that results into dietary restriction could be ruled out. Age pigments contain both intracellular and extracellular agerelated, fluorescent material and include advanced glycation end-products and lipofuscin, a heterogeneous mix of oxidized and cross-linked molecules (proteins, lipids, and carbohydrates) that accumulate in intestinal lysosomes.19 We measured the accumulation of age pigments in worms with PGG treatment in order to see the effect of PGG on the aging process. As shown in Figure 2C, age pigment fluorescence levels did not significantly lower or increase when the worms were cultured on 160 μM PGG at three stages. Therefore, PGG-induced lifespan extension was not associated with any significant change in the accumulation of age pigments . 3426
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protectively under the thermal stress and enhance resistance to heat shock in C. elegans. According to the free-radical theory of aging, an excess of reactive oxygen species (ROS) is responsible for the process of aging and the lifespan extension is associated with damage caused by ROS.21 Therefore, we measured the effect of PGG on the intracellular amount of ROS. The accumulation of ROS was measured using H2DCF-DA, which is converted to the fluorescent dye DCF intracellularly. In states of exposure to PGG for 96 h, the reduction of ROS accumulation induced by PGG was not significantly different (Figure 3B). Strikingly, PGG showed high antioxidant activity in both chemical-based and cell-based assays12 but did not exhibit the ROS scavenging activity in C. elegans. In fact, many factors and processes such as uptake, metabolism, and interaction need to be considered if the antioxidant activity needs to take effect in the whole organism. The reason for the failure in ROS scavenging ability could be that the antioxidant property of PGG was not sufficient to reduce the accumulation of ROS in C. elegans or the bioavailability of PGG was low in C. elegans. Effect of PGG on Median Lifespan of Mutants Involved with the IIS Pathway. The insulin/IGF signaling (IIS) pathway is well-known to be a central controller of longevity in C. elegans regulated via the phosphorylation situation of the transcription factor DAF-16.22 PGG could interact with DAF-2 (the proteins encoded by daf-2),11 the only homologue of insulin receptor in the C. elegans, which could reduce their activity and result in lifespan extension through triggering the IIS pathway. To clarify this possibility, we tested a daf-16(mgDf50) null mutant strain cultured on 160 μM PGG and found that PGG did not increase the median lifespan of the daf-16 mutant [16.7 days for the daf-16(mgDf50)] null mutant vs 17.3 days with PGG treatment (Figure 4A and Table 1). This finding suggested that PGG could extend median lifespan by acting through a mechanism that is dependent on DAF-16/ FOXO. Next, to further understand the genetic requirement for the IIS pathway in the action, we tested the effect of long-lived age-1(hx546) mutants treated with PGG. Interestingly, worms cultured on 160 μM PGG displayed a significantly different survival curve compared with daf-16(mgDf50) mutant (the control), but PGG did not extend the median lifespan of age1(hx546) (Figure 4B and Table 1). These findings suggested that PGG could be dependent on daf-16(mgDf50) and age1(hx546) to increase median lifespan. Considering that DAF-16 nuclear localization affects lifespan in the IIS pathway and sod-3 is one of the DAF-16 direct target genes and encodes a Fe/Mn superoxide dismutase, we tested the effect of PGG on the fluorescence intensity in worms of the transgenic strain CF1553, which is SOD-3::GFP reporter gene expression.15 However, the PGG-treated group did not demonstrate significant change in fluorescence intensity of the transgenic CF1553 on day 5 (Figure 4C). These results suggested that although age-1 and daf-16 were necessary for PGG-mediated median lifespan extension, the whole IIS pathway could not be a direct target of PGG. The possibility of this phenomenon was that PGG could act on another pathway that involved with daf-16 and age-1. It has been proven that multiple interaction partners are necessary for proper DAF-16 activity.23 Effect of PGG on Median Lifespan of Mutants Involved with Dietary Restriction and the Sir-2.1 Pathway. The other well-known mechanism considered could be dietary restriction.5 PGG also has the tannins’
Table 1. Genetic Requirement for Lifespan Extension Induced by PGG
genotype treatment daf-16
age-1
eat-2
sir-2.1
isp-1
mev-1
clk-1
0.2% DMSO 160 μM PGG 0.2% DMSO 160 μM PGG 0.2% DMSO 160 μM PGG 0.2% DMSO 160 μM PGG 0.2% DMSO 160 μM PGG 0.2% DMSO 160 μM PGG 0.2% DMSO 160 μM PGG
median lifespana (mean ± SD) (days)
maximum lifespana (mean ± SD) (days)
genetic requirement
16.7 ± 1.2
24.0 ± 0.0
yes
17.3 ± 1.2
23.3 ± 1.2
21.0 ± 1.8
44.8 ± 4.3
23.0 ± 3.2
37.8 ± 9.6
21.3 ± 1.5
36.7 ± 5.5
22.7 ± 2.5
36.0 ± 2.0
20.0 ± 0.0
29.3 ± 3.8
20.0 ± 0.0
31.7 ± 5.0
23.0 ± 0.0
38.0 ± 3.5
21.0 ± 2.0
39.3 ± 1.2
12.7 ± 1.0
18.5 ± 2.9
14.3 ± 1.2*
19.7 ± 0.8
17.7 ± 1.5
36.0 ± 4.6
23.0 ± 2.6*
37.3 ± 3.2
yes
yes
yes
yes
no
no
a
The median lifespan is the time at which fraction survival equals 50%. The maximum lifespan is the time at which fraction survival equals 0%. The data was analyzed by One way-ANOVA analysis (SPSS 13), and and asterisk (*) designates significantly different values (p < 0.05).
common characteristics of binding proteins by forming hydrogen and ionic bonds,11 which would inhibit digestive enzymes and cause effects that are similar to a dietary restriction. Therefore, to test whether PGG extended the lifespan by acting through dietary restriction, the effect of PGG on the lifespan of the eat-2(ad1116) mutant was investigated, which is a long-lived mutant and a genetic model for dietary restriction due to decreased pharyngeal pumping in C. elegans.5 PGG did not increase the median lifespan of the eat-2(ad1116) mutant (Figure 5A and Table 1). This indicated that PGG was depend on eat-2(ad1116), which could be consistent with the model that PGG acts by stimulating the DR pathway. However, one feature of dietary restriction in C. elegans is a reduction of total number of progeny or an extended period of progeny production.17 We found that PGG did not change the progeny production. To summarize, the results suggested that PGG did not act exactly through DR to increase median lifespan. Given the genetic indication that dietary restriction (DR) was involved with the mechanism and the botanical polyphenol antioxidant resveratrol, a DR mimetic, extends the lifespan by stimulating the activity of sir-2.1, which encodes a histone deacetylase-like protein that integrates metabolic situation with lifespan,24 we accordingly hypothesized that sir-2.1 might be also involved into the mechanism of action of PGG-mediated longevity because of the common properties of polyphenol and antioxidant. Given that possibility, we tested PGG effects on the sir-2.1(ok434) mutant. We found PGG did not extend the lifespan of a sir-2.1 null mutant (Figure 5B and Table 1). 3427
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Figure 5. Survival curves of mutants raised at 20 °C on plates containing 0.2% DMSO (control) or 160 μM PGG. (A) The P-value for eat-2(ad1116) was 0.1010 (Log-rank test). (B) The P-value for sir-2.1(ok434) was 0.0702 (Log-rank test). (C) The P-value for mev-1(kn1) was
