Whole apple extracts increase the life span, health and resistance to stress of Caenorhabditis elegans

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J Funct Foods. Author manuscript; available in PMC 2014 Jul 1

Published in final edited forms such as:

PMCID: PMC3714114

NIHMSID: NIHMS471550

abstract

Regular consumption of fruits and vegetables is associated with reduced risk of age-related functional decline and chronic diseases such as cancer and cardiovascular disease. These effects are mainly attributed to phytochemicals, plant compounds with a wide range of biological activities and health benefits. Apples, the major contributor to fruit phenolics in American diets, have high antioxidant, antiproliferative and chemopreventive activity in vitro and in vivo. However, little is known about their effect on aging. The purpose of this study was to determine the effect of whole apple-phytochemical extracts on life span, health span, and resistance to various stresses. in vivo With the help of C. elegans as a model. The mean and maximum life expectancy of animals treated with 2.5, 5 and 10 mg / ml whole apple extracts increased significantly in a dose-dependent manner by up to 39 and 25%, respectively. Healthspan also improved markedly as indicated by improved motility and reduced lipofuscin accumulation. Animals pretreated with whole apple extracts were more resistant to stresses such as heat, UV radiation, paraquat-induced oxidative stress and pathogenic infection, suggesting that cellular defense and immune system functions were also enhanced. Our findings show that i C. elegans, whole apple extracts slow aging, prolong life, improve health and increase resistance to stress.

Keyword: Apple, phytochemical, antioxidant, aging, health bucket, Caenorhabditis elegans

1 Introduction

Age is an important risk factor for many chronic diseases. Epidemiological studies have consistently shown that regular consumption of fruits and vegetables is associated with a markedly reduced risk of developing such diseases, including various types of cancer, cardiovascular disease and diabetes (Willett, 1994; Joshipura et al. 2001; Lock et al. 2005). E.g Doll and Peto (1981) estimated that at least one-third of all cancers could be prevented by dietary modification. We and others have suggested that the health benefits of fruits and vegetables stem from additive and synergistic interactions between complex mixtures of phytochemicals, the bioactive non-nutrient plant compounds that have been associated with reducing the risk of major chronic diseases (Liu, 2003).

As an organism ages, it experiences a gradual deterioration of body functions over time, increasing both the sensitivity to environmental challenge and the risk of disease and death (Kirkwood, 2005). The non-parasitic nematode Caenorhabditis elegans is a popular model in aging research because these animals decline behaviorally and physiologically with age in a manner similar to that of higher mammals, including humans. As they grow older, animals move more slowly and exhibit sarcopenia, the gradual deterioration of muscle tissue. They become infertile. They accumulate oxidized proteins and lipofuscin, the hallmark of aging common to many species (Class, 1977; Johnson, 2003). At the gene and protein levels C. elegans share up to 80% homology with human genes and a conserved protein network involved in aging (Braeckman & Vanfleteren, 2007; Bell et al. 2009). In addition to being a biologically relevant aging model, these nematodes are easy to grow and have a short life cycle, allowing for rapid replication of experimental treatments. In addition, standard assay conditions for investigating the pharmacology of drugs and interactions with genes are described in this model organism (Rand & Johnson, 1995). Specifically, human pharmacological interventions ranging from vitamins and clinical drugs to antioxidant supplements and phytochemicals are known to prolong life or delay physiological aging in C. elegans (Collins et al. 2006; Lucanic et al. 2012). Eg. Extends anticonvulsant drugs: ethosuximide, trimethadione, and 3,3-diethyl-2-pyrrolidinone longevity and delays age-related degenerative changes by modulating neuromuscular activity (Kornfeld & Evason, 2006). Similarly, extracts of Ginkgo biloba, components of green tea and blueberry prop phenols, extend health and longevity and enhance stress resistance through a variety of mechanisms (Wu et al. 2002; Zhang et al. 2009; Wilson et al. 2006; Gong et al. 2012). However, despite these and other studies, little is known about the possible anti-aging effects of frequently consumed fruits and vegetables.

Apples are a popular, widely available and economically significant fruit. In the United States, they are the major contributor of phenolics, a major class of biologically significant phytochemicals associated with a wide range of bioactivities and health benefits both in vitro and in vivo (Boyer & Liu, 2004). Whole apple extracts have powerful antioxidant effects and antiproliferative activity against colon, liver and breast cancer cells in vitro in a dose-dependent manner (Eberhardt et al. 2000; Sun et al. 2002). In MCF-7 human breast cancer cells, these extracts inhibit activation of the transcription factor NFKB, thereby promoting resistance to chemotherapeutic drugs against cancer and regulating the cell cycle by inducing G1 arrest and modulating the expression of key cell cycle proteins (Yoon & Liu, 2007; Sun & Liu, 2008). Previously, our group isolated new, structurally different compounds from the apple scales from Red Delicious apples, and showed that these compounds have potent antioxidant and antiproliferative activity against MCF-7 breast cancer cells and HepG2 liver cancer cells (He & Liu, 2007; 2008). Here, we explored the possibility that whole apple-phytochemical extracts from these apples modulate specific biochemical and immunological biomarkers of health that correlate with improved quality of life (health span) and increased longevity. in vivo. Specifically, our goal was to determine the effects of whole apple-phytochemical extracts on life span, health span, and stress resistance. in vivo With the help of Caenorhabditis elegans as a model for aging.

2 Materials and Methods

2.1 Chemicals and Reagents

Acetone was purchased from Fischer Scientific (Pittsburgh, PA, USA). Sodium carbonate was purchased from Mallinckrodt Baker, Inc. (Phillipsburg, NJ, USA) and gallic acid from ICN Biomedical Inc. (Aurora, OH, USA). 5-Fluoro-2-deoxyuridine (FUDR) and methyl violet dichloride hydrate (paraquat) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.2 Extraction of apples

Fresh apples of the variety Red Delicious were purchased from Cornell Orchards in September 2007 (Cornell University, Ithaca, NY, USA) and extracted using the method previously reported by our laboratory (Sun et al. 2002). Briefly, whole apples were sliced, mixed in ice-cooled 80% acetone, homogenized and rotary evaporated under vacuum at 45 ° C until approx. 90% of the filtrate and all acetone was evaporated (no solvent was left in the extract). The concentrate was suspended in water to a stock concentration of 100 mg / ml based on the dry weight of the extract, aliquoted and frozen at -80 ° C until use. Subsequent working concentrations were made from this stock. These extracts have been characterized based on bioactivity controlled fractionation and structure identification using HR-MS, 1D and 2D NMR and X-ray diffraction analysis using the methods we reported previously (He & Liu, 2007; 2008). Briefly, 29 compounds, including triterpenoids, flavonoids, organic acids and plant sterols, were isolated from Red Delicious apple peel. Of the flavonoids, the most important compounds were: quercetin-3ISLAND-p-D-glucopyranoside (82.6%), quercetin-3ISLAND-P-D-galactopyranoside (17.1%), quercetin 0.2%), (-) – catechin, (-) – epicatechin and quercetin-3ISLAND-a-L-arabinofuranoside (He & Liu, 2008). The major triterpenoids identified were: 2α-hydroxyursolic acid, 2α-hydroxy-3β- [(2[(2[(2[(2E3-phenyl-1-oxo-2-propenyl]oxy olean-12-one-28-oic acid and 3β-transp-coumaroyloxy-2a-hydroxyolean-12-ene-28-oic acid, ursolic acid, 2a-hydroxyursolic acid and 3β-transp-coumaroyloxy-2a-hydroxyolean-12-ene-28-oic acid and maslinic acid (He & Liu, 2007).

2.3 Strains, maintenance and cultivation of nematodes

Strains used were: Bristol N2 (wild type), daf-16 (mgDf47)) and age 1 (hx546). All strains were obtained from C. elegans Genetics Center (CGC) based at the University of Minnesota. Animals were maintained at 20 ° C on Petri dishes containing Nematode Growth Medium (NGM) inoculated with a live E. coli strain OP50 as the food source according to the general procedures outlined by Brenner (Brenner, 1974).

2.4 Assassin for longevity

Several pregnant adult nematodes were placed on NGM plates inoculated with E. coli strain OP50 and allowed to lay eggs at 20 ° C for approx. 6 hours to achieve a synchronous population. After 6 hours, the nematodes were removed and the plates were placed at 20 ° C until the offspring reached young adulthood (approximately 72 hours). On day 0 of the experiment, these young adult nematodes were transferred to 35 mm NGM petri dishes containing either no extracts or appropriate doses of dissolved whole apple extracts and 50 μM 5-fluoro-2-deoxyuridine (FUDR) to prevent progeny production. Plates were then dried in a sterile cap, seeded with 100 l 4-fold concentrated, saturated E. coli OP50 culture and dried again. Animals were transferred every other day to fresh extracts or control plates until adulthood 8. Animals were scored daily or every other day by gentle prodding with a platinum wire. The animals that could not move were judged as dead. Animals exhibiting sacking, exploding or crawling from the plates were censored. Statistical analyzes were performed using SPSS statistical software Kaplan-Meier Survival function; pvalues ​​were obtained using the log-rank test. The experiment was repeated several times and a representative experiment is shown. All experiments except P. aeruginosa pathogen droplet assay and heat shock treatment were performed at 20 ° C.

2.5 Healthspan analyzes

The animals were treated with whole apple extracts or grown on control plates as described under “Lifespan Assay” above.

2.5.1 Lipofuscin

Animals (N = 18 per group) were treated with whole apple extracts and at adult age 8, mounted on 2% agarose pads and immobilized in 20 µM sodium azide. Slides were visualized using the Leica DM5000B microscope (Bannockburn, IL) with the I3 cube filter (excitation 450/490, emission 510), and images were captured using a Hamamatsu ORCA-ER camera and OpenLab software. Image quantification of fluorescence intensity was performed densitometrically by tracking around each animal’s intestine and determining the average pixel intensity using ImageJ freeware (NIH) (Race Band, 1997).

2.5.2 Motility

The animals were treated as described above. On adulthood days 12, 14, 16 and 18, the animals were visualized using an Olympus SZ61 stereomicroscope (New York / New Jersey Scientific, Middlebush, NJ, USA). Motility classes were determined using the method reported by Golden et al., where ‘A’ animals move spontaneously and smoothly, leaving sinusoidal and symmetrical traces; ‘C’ animals only move the nose or tail when filled with a platinum wire; and ‘B’ animals represent any class of behavior in between (Herndon et al. 2002; Golden et al. 2008). N≥47 animals for days 10 and 12, N≥25 for day 16.

2.6 Stress Resistance Assays

For all stress resistance assays, animals were transferred to 35 mm NGM / OP50 plates with whole apple extracts or control plates at the young adult stage containing 50 µM FUDR, then incubated for 2 days followed by exposure to the stressor on the third adult day. All experiments were repeated at least 2-3 times and a representative study is shown.

2.6.1 Heat shock

Hot shock experiments were performed according to the method of Lithgow and colleagues (Lithgow et al. 1995). Animals were incubated at 35 ° C on the third adult life for 8 hours and then monitored every 30 minutes until ca. 50% of the controls were dead. Plates were removed from the incubator and scored to survive. Triplicate plates were used at each time point with N Group.

2.6.2 UV radiation

Animals were transferred to bacterial-free NGM plates on day three of adulthood and UV irradiated at 1200J / m2 with a UV Stratalinker 2400 (Stratagene, La Jolla, CA, USA) equipped with five 254nm UV bulbs (model 1800), each generating 15 watts. After UV irradiation, the animals were transferred to the standard NGM / OP50 plates without whole apple extracts and monitored daily to survive by gentle pushing with a platinum wire (Cypser & Johnson, 2002). N = 66, 82, 79, 72 animals for 0, 2.5, 5 and 10 mg / ml groups, respectively.

2.6.3 Pseudomonas aeruginosa infection

Animals were grown on NGM / OP50 plates at 25 ° C and transferred to plates with or without whole apple extracts for a period of two days. On the third adult day, animals were changed to modified NGM plates prepared according to the Tan method et al, containing P. aeruginosa strain PA14 at 25 ° C and scored for survival every 8-13 hours (Tan et al. 1999). Plates were seeded with 10 μL P. aeruginosaand allowed to dry overnight at 37 ° C and then at room temperature for an additional 24 hours. N = 82, 80, 73 and 76 animals for 0, 2.5, 5 and 10 mg / ml groups, respectively.

2.6.4 Oxidative stress

The oxidative stress paraquat assay on plates was performed using the methods described previously (Ishii et al. 1990; Gruber et al. 2007). On adulthood day three, animals were transferred to freshly prepared NGM / OP50 plates containing 10 mM paraquat and scored as above. N≥54 animals per animal. Group.

2.7 Bread size

The N2 animals were grown on NGM / OP50 plates until the late larval stage, L4, and transferred to the control plate or plates at different concentrations of whole apple extracts, one animal per day. Plate per Concentration, with N = 8 animals per day. Group. Animals were then transferred every 24 hours to the fresh control or whole apple extract plates until egg production had ceased. The total number of offspring growing from each animal was counted and the number of offspring for each concentration was calculated on average (Li et al. 2008).

2.8 Statistical analyzes

Survival data were analyzed using SPSS version 16 for Windows (SPSS Inc., Chicago, IL, USA) Kaplan-Meier Survival Function and log-rank test. All other analyzes were performed using Minitab statistical software (State College, PA, USA). Heat shock data were analyzed using two samples t-test (provided one’s variance); lipofuscin accumulation and strain size were analyzed using one-way ANOVA; motility classes were compared using logistic regression. Graphs were imaged with SigmaPlot version 10 for Windows software (Systat Software Inc., San Jose, CA, USA). One pvalue <0.05 was considered statistically significant.

3 results

3.1 Whole apple extracts Increase the life of the wild type C. elegans

Wild type adult C. elegans have a mean life of 2-3 weeks at 20 ° C. Under our standard laboratory conditions, wild-type control animals lived an average of 17.23 ± 0.30 days (maximum 24 days). After administration of 2.5, 5 and 10 mg / ml whole apple extracts, starting at the young adult stage, the mean lifespan increased to 20.46 ± 0.17 days (maximum 24 days), 21.38 ± 0.35 days (maximum 30 days) and 24.05 ± .53 days (maximum 30 days) in a statistically significant dose-dependent manner (p<0.05; ). These changes represent increases in lifetime of 18.7, 24.1 and 39.6%, respectively, compared to the control group ().

Effect of whole apple extracts on the lifespan of C. elegans. Day 1, young adult wild-type animals (N ≥120 in each group) were treated without (0 mg / ml) or with a low (2.5 mg / ml), moderate (5 mg / ml) and high (10 mg / ml). )) dose of standardized whole apple extracts containing 170 ± 4.6 mg of phenol per day. 100 g apples. Survival was monitored starting on adulthood day 1. Nematodes exposed to the entire apple extracts survived significantly longer than those who did not (p<0.05, log-rank test). The experiment was repeated several times and a representative experiment is shown: a) Effect of apple extracts on longevity; and b) Percentage increase in lifetime.

Table 1

Effects of whole apple extracts on the average lifespan of C. elegans.

Concentration of whole apple extracts (mg / ml) N Average longevity* (Days) % of control
0 120 17.23 ± 0.30a** 100.0
2.5 132 20.46 ± 0.17b 118.7
5 158 21.38 ± 35c 124.1
10 120 24.05 ± .53d 139.6

3.2 Whole apple extracts improve mobility and attenuate the accumulation of lipofuscin

Next, we examined whether the increase in longevity was accompanied by an overall improvement in health and vitality. We tested the motility of 12-, 14-, and 16-day-old animals treated with three concentrations of whole apple extracts. Motility was classified by movement spontaneity on petri dishes: Class A animals moved spontaneously; Class B animals required shots to stimulate movement of the entire body; and Class C animals only moved their heads or tails in response to gentle footing with a platinum wire (Herndon et al. 2002; Golden et al. 2008). The decrease in motility on days 12, 14 and 16 was significantly delayed in a dose-dependent manner in animals treated with low (2.5 mg / ml), moderate (5 mg / ml) and high (10 mg / ml) concentrations of whole apple extracts (p≤0.01; ). On day 12, most animals in all groups continued to move spontaneously, but there was already a small, significant difference in spontaneous (class A) movement among the control and treatment groups (p<0.05; ). By day 14, animals treated with low, moderate, and high doses had whole apple extracts significantly (p<0.05) more high motility (class A) individuals (63, 49 and 77%, respectively) than the control group (15%;). On day 16, the dose-dependent effects on motility were completely evident (), so that 92% of the control group would hardly move their heads after filling with a platinum wire (class C), while 49% of the high-dose treatment group still moved spontaneously (class A). We continued to follow the animals that remained alive until day 18; Interestingly, treatment groups still showed very significant dose-dependent differences in motility while most of the control animals were dead (p<0.01, data not shown).

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Effect of whole apple extracts on the movement of a) day 12, b) day 14 and c) day 16 animals. Motility was classified into three groups: Movement An animal moved spontaneously; Movement B animals required creature to stimulate movement; and Motion C animals only moved their heads in response to prodding. The decrease in motility on days 12, 14 and 16 was significantly delayed in a dose-dependent manner in animals treated with 2.5, 5 and 10 mg / ml apple extract. (N ≥ 47 animals per group on days 12 and 14; N ≥ 25 on day 16, p<0.05, logistic regression).

Lipofuscin is a by-product of lysosomal degradation that accumulates with age in most organisms, including C. elegans (Clokey & Jacobson, 1986). Wild-type N2 animals treated with 2.5 and 5 mg / ml whole apple extracts accumulated only about half as much lipofuscin as the N2 control animals (p<0.05; ). Animals that have a mutation in daf-16, a gene encoding the forkhead transcription factor, ages and accumulates lipofuscin at a faster rate than their wild type N2 counterparts, and was used as a positive control. As expected, this group accumulated more lipofuscin after 8 days than did N2 animals (p<0.05).

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Effect of whole apple extracts on lipofuscin accumulation i C. elegans (N = 18 animals per group). Wild type N2 animals treated with 2.5 and 5 mg / ml apple extracts accumulated only approx. half as much lipofuscin as the N2 control worms. daf-16 (mgDf47) Mutant animals without extract treatment, which grow at a faster rate, were used as a positive control. Columns without common letters are significantly different (p<0.05, one-way ANOVA).

3.3 Whole apple extracts increase resistance to heat stress, UV radiation and pathogenic infection

Increased life often correlates with increased stress resistance (Johnson et al. 1996; Lithgow & Walker, 2002). We treated young adults C. elegans with doses of 0, 2.5, 5 and 10 mg / ml whole apple extracts for two days and examined their response to various chemical and environmental stressors including heat shock, UV radiation and pathogen Pseudomonas aeruginosa.

The animals were placed in a 35 ° C heat shock chamber and monitored until approximately half (55%) of the wild-type N2 controls were dead (10.5 hours). At that time, 71.4, 87.1, and 89.73%, respectively, of 2.5, 5, and 10 mg / ml of apple-treated animals lived. Animals that have a mutation in age-1, the C. elegans PI3 kinase, more resistant to heat stress, was used as a negative control and survived at a rate of 96.13%; daf-16 animals more sensitive to heat stress were used as a positive control and had a survival rate of 28.96% (p<0.05; )

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Effect of pretreatment with whole apple extracts on resistance to stress i C. elegans. The animals were treated with different concentrations of whole apple extracts (0, 2.5, 5, or 10 mg / ml in each group) as young adults for 2 days at 20 ° C and exposed to a variety of stress factors on the third day of adulthood. Those who were pre-treated with whole apple extracts survived significantly longer a) 35 ° C heat shock (N ≥ 61, columns without common letters are significantly different) p<0.05, two-sample t-test). b) UV irradiation at 1200 J / m2 (N ≥ 66, p<0.05, log-rank test). c) infection with P. aeruginosa (N ≥ 73, p<0.05, log-rank test). d) exposure to 10 mM paraquat (N ≥ 54 animals, p<0.05, log-rank test). Each experiment is representative of three independent experiments. daf-16 (mgDf47) and age 1 (hx546) animals served as positive and negative controls, respectively.

In an analogous experiment, N2 animals pretreated with whole apple extracts survived significantly longer after UV irradiation than N2 control animals (p<0.05; ,). Survival time after irradiation for N2 animals pretreated with 2.5, 5 and 10 mg / ml whole apple extracts was increased by 22.6, 46.2, 61.5%, respectively. age-1 animals that are also more resistant to UV stress than N2 were used as a negative control and survived 74.4% longer than the N2 controls. daf-16 animals more sensitive to UV stress than N2 were used as a positive control and survived 12.8% less than the N2 controls.

Table 2

Effect of pretreatment with whole apple extracts on resistance to UV radiation i C. elegans.

Concentration of whole apple extracts (mg / ml) N Average longevity* (Days) % of control
0 66 3.92 ± 0.15a** 100.0
2.5 82 4.88 ± .18b 122.6
5 79 5.70 ± .18c 146.2
10 72 6.37 ± .18c, d 161.5
age-1 (neg control) 36 6.89 ± .32d 174.4
daf-16 (pos continued) 38 3.49 ± 0.19e 87.2

N2 animals treated with whole apple extracts showed increased resistance to the pathogen Pseudomonas aeruginosa. After they were transferred to pathogen-seeded plates on the third day of adulthood and monitored every 8-13. Hours, control animals survived an average of 82 ± 2.6 hours, while animals treated with 2.5, 5 and 10 mg / ml whole apple extracts had significantly increased mean lifespans of 100 ± 3.8, 111 ± 4.4 and 102, respectively. ± 4.4 hours (p<0.05; ,).

Table 3

Effect of pretreatment with whole apple extracts on resistance to infection by Pseudomonas aeruginosa in C. elegans.

Concentration of whole apple extracts (mg / ml) N Average longevity* (Hours) % of control
0 82 82.0 ± 2.62a** 100.0
2.5 80 100.0 ± 3.75b 122.0
5 73 110.9 ± 4.41c 135.2
10 76 102.1 ± 4.4c 124.5

3.4 Whole apple extracts increase resistance to oxidative stress induced by Paraquat

To test the antioxidant potential of whole apple extracts in vivo, we placed animals on NGM plates containing 10 mM of the superoxide-generating chemical paraquat. While control animals survived an average of 4.97 ± 0.19 days (maximum 7 days), those treated with 2.5, 5 and 10 mg / ml whole apple extracts survived an average of 7.71 ± 0.32 (max. 10 days), 9.41 ± 0.43 (max. 14 days) and 8.54 ± 0.39 days (max. 15 days, respectively). These represented statistically significant increases in the mean lifespan of 55.1, 89.3 and 71.8% above control (p<0.05; ,).

Table 4

Effect of pretreatment with whole apple extracts on resistance to chronic challenge with paraquat i C. elegans.

Concentration of whole apple extracts (mg / ml) N Average longevity* (Days) % of control
0 67 4.97 ± 0.19a** 100.0
2.5 58 7.71 ± .32b 155.13
5 54 9.41 ± .43c 189.3
10 61 8.54 ± 0.39c 171.83

3.5 Whole apple extracts do not affect the size of the bread

An increase in longevity is often correlated with a decrease in fertility. To test whether whole apple extracts negatively affect fecundity, we measured strain sizes of 8 N2 animals per day. Group: 0 (control), 2.5, 5 and 10 mg / ml. There were no significant differences between treatment groups and control (p= 0.321; ).

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Effect of whole apple extracts on stem size on C. elegans (N = 8 animals per group). There were no significant differences in the number of total offspring between whole apple extract-treated and untreated animals (p= .321, one-way ANOVA).

4 Discussion

Several studies have shown that compounds and extracts derived from plants can delay age-related decline and extend longevity and health span across different species. In the current study, we show for the first time that whole apple extracts prolong life and health stress in a dose-dependent manner in vivo. Average life of C. elegans increased by up to 39% and maximum lifespan by up to 25% when whole apple extracts were included in their diet. We also found similar improvements in several physiological and functional health team indicators. Previous work by our group has shown that whole apple extracts have antioxidant and antiproliferative activity in vitroand antitumor activity in vivo (Eberhardt et al. 2000; Sun et al. 2002; Liu et al. 2005). Our finding of anti-aging effects in vivo extends our knowledge of the biological effects of whole apple phytochemicals.

Specific apple fractions and phytochemicals have been shown to extend the average lifespan of C. elegans and Drosophila melanogaster, and to suppress amyloid-beta protein collection i C. elegans (Sunagawa et al 2011; Peng et al. 2011; Toda et al 2011). For example, Sunagawa and colleagues showed that procyanidins from Fuji apples prolonged the average life of wild type C. elegans by 12.1%, an effect that was dependent on SIR-2, a member of the sirtuin family of NAD + -dependent protein deacetylases. Likewise, apple polyphenols extended the average lifespan by 12.0% (Sunagawa et al. 2011). Peng and colleagues showed that polyphenols from the Bay of Red Fuji apples extend the average life of Drosophila by 10% and that this effect was at least partially mediated through SOD, CAT, MTH and Rpn11 activity (Peng et al. 2011). Other investigators demonstrated that quercetin and EGCG, two phytochemicals found in apples, prolonged average life span and health span C. elegans (up to 15% for quercetin and either 0, 5 or 10% for EGCG depending on dose and culture conditions) (Brown et al. 2006; Kampkotter et al. 2008; Zhang et al. 2009). Here, we asked whether phytochemicals from whole apple extracts have anti-aging and stress resistance activity and how this activity is compared to the activity of single apple phytochemicals and fractions previously published. We hypothesized that biological activity would be enhanced by synergistic interactions between several compounds across different phytochemical classes and chose the red delicious apple variety based on its high availability, popularity, and previously characterized antioxidant and biological activity. in vitro and in vivo. We find that the magnitude of the average lifetime extension (39%) is greater than single apple phytochemicals and apple fractions previously reported. Our findings suggest that interaction between different compounds is necessary to get the maximum effect. This hypothesis is consistent with our own preliminary data showing that quercetin-3-β-D-glucoside and 2-α-hydroxy-ursolic acid do not increase the mean C. elegans lifetime as much as whole apple extracts (data not shown).

In addition to living longer, the animals in our study showed an improved health span. Animals treated with whole apple extract accumulated half as much lipofuscin as untreated animals (). Treated animals also consistently showed more youthful and vigorous movement than control animals. Den største ændring blev observeret i post-reproduktiv, middelalder til sen alder 14 og 16 dyr. Vi fortsatte med at følge behandlede dyr gennem dag 18 (dyr med meget sen alder), selvom mere end halvdelen af ​​kontrol dyrene allerede var død. Mobiliteten hos mange dyr i æblebehandlede grupper på dag 18 lignede dem fra dag 2-8 kontroller (klasser ‘B’ og ‘A’) (data ikke vist).

Vi testede også dyrenes respons på forskellige stressfaktorer. Tidligere undersøgelser har vist en sammenhæng mellem længere levetid og modstand mod stress i C. elegans og andre dyr, inklusive pattedyr (Johnson et al. 1996; Lithgow & Walker, 2002; Gems & Pattridge, 2008). After pretreatment with 2.5, 5 and 10 mg/ml doses of whole apple extracts for the first two days of adulthood, the animals were challenged with a battery of stressors including heat shock, UV radiation, paraquat-induced oxidative stress, and the pathogen P. aeruginosa.

Pretreatment with whole apple extracts greatly improved survival following heat shock. This result is consistent with other studies in C. elegans that showed enhancement of both thermotolerance and lifespan by plant-derived extracts (Wilson et al. 2006; Benedetti et al. 2008). In our study, whole apple phytochemicals may have affected stress-signaling pathways through activating heat shock proteins (hsps). Alternatively, these compounds may have themselves acted as chemical chaperones that stabilize protein conformation and promote a general cellular stress response (Benedetti et al. 2008). This may explain why animals exposed to whole apple extracts had significantly improved survival. UV light can damage DNA (e.g. cyclobutane pyrimidine dimers and 6-4 photoproducts) and accelerate aging (Rittie & Fisher, 2002; Clancy, 2008;). In our experiments, pretreatment with whole apple extracts significantly improved survival following UV irradiation. Phytochemicals found in apples have been previously shown to repair damaged DNA. For example, EGCG and quercetin can modulate DNA repair genes and mechanisms, including: H2AX, histone acetylation, ATM/ATR, GADD, Chk1/2, Cdc25c, p53, and KAP1 (Rajendran et al. 2010). Whole apple phytochemicals could have acted through these or other mechanisms to increase resistance to UV damage and extend lifespan. Aging is also associated with an increase in oxidative damage to DNA, proteins and lipids (Bokov et al. 2004). Previously, our group showed that whole apple extracts have antioxidant activity in vitro (Eberhardt et al. 2000). Here, we used a paraquat-induced oxidative stress model to examine whether similar effects can be observed in vivo. After pretreating animals with whole apple extracts for two days, we found that their survival was significantly improved after exposure to 10mM paraquat. This result suggests a possible antioxidant mechanism underlying the anti-aging effects of whole apple phytochemicals. Animals were also more resistant to the pathogen P. aeruginosa. Infection with this bacterium occurs by ingestion and, under normal conditions, P. aeruginosa is lethal within 1–3 days. Animals we pretreated with whole apple extracts survived up to 35.2% longer after infection, indicating that apple phytochemicals might improve immune response.

5 Conclusions

In conclusion, we have shown for the first time that whole apple phytochemical extracts increase the mean and maximum lifespan and confer multiplex stress resistance in vivo in the C. elegans aging model. Our results indicate that whole apple extracts also improve healthspan, as measured by reduced lipofuscin accumulation and improved motility. Animals pretreated with whole apple extracts are more resistant to heat, UV irradiation, and paraquat-induced oxidative stress, suggesting that cellular defense and immune system functions are also improved. Studies are underway to determine the contributions of individual phytochemicals and elucidate the mechanisms by which they may synergize to produce the anti-aging and other health benefits we observed.

Highlights

  • Whole apple extracts increase the mean lifespan of C. elegans up to 39% in a dose-dependent manner.

  • Whole apple extracts improve healthspan as measured by motility and lipofuscin accumulation.

  • Whole apple extracts increased resistance to stress (e.g., heat shock, oxidative stress).

  • We suggest these benefits are due to synergistic interactions of multiple compounds across different phytochemical classes.

Acknowledgments

We thank Carolle Mok for technical assistance, Ben Hamilton, Atsushi Ebata, Nicole Liachko and Gizem Rizki for training and experimental troubleshooting, as well as Jason Neuswanger for critical editing of the manuscript.

Footnotes

Conflict of Interest Statement

Authors have no conflict of interest.

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