Experimental Gerontology 69 (2015) 189–195
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Cranberry anthocyanin extract prolongs lifespan of fruit flies Lijun Wang, Yuk Man Li, Lin Lei, Yuwei Liu, Xiaobo Wang, Ka Ying Ma, Zhen-Yu Chen ⁎ Food and Nutritional Sciences Programme, School of Life Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China
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Article history: Received 13 April 2015 Received in revised form 25 June 2015 Accepted 26 June 2015 Available online 6 July 2015 Section Editor: Hally M. Brown Keywords: Cranberry Anthocyanins Antioxidants Insulin receptor Target of rapamycin
a b s t r a c t Cranberry is an excellent source of dietary antioxidants. The present study investigated the effect of cranberry anthocyanin (CrA) extract on the lifespan of fruit flies with focus on its interaction with aging-related genes including superoxide dismutase (SOD), catalase (CAT), methuselah (MTH), insulin receptor (InR), target of rapamycin (TOR), hemipterus (Hep), and phosphoenolpyruvate carboxykinase (PEPCK). Results showed that diet containing 20 mg/mL CrA could significantly prolong the mean lifespan of fruit flies by 10% compared with the control diet. This was accompanied by up-regulation of SOD1 and down-regulation of MTH, InR, TOR and PEPCK. The stress resistance test demonstrated that CrA could reduce the mortality rate induced by H2O2 but not by paraquat. It was therefore concluded that the lifespan-prolonging activity of CrA was most likely mediated by modulating the genes of SOD1, MTH, InR, TOR and PEPCK. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Free radical theory has been considered as one of the major aging theories even though it is still under debate (Peng et al., 2014; Speakman and Selman, 2011). It states that organisms become aged partially due to accumulation of oxidative damages caused by reactive oxygen species (ROS), namely hydroxyl radicals, superoxide anions and hydrogen peroxide. ROS are the byproducts of normal cellular metabolism of oxygen. Endogenous antioxidant enzymes in cells, including catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx), serve as a first line of defense against ROS, while exogenous antioxidants from diets such as plant flavonoids and vitamins function as a second line of defense in terminating the propagation of ROS reactions (Cutler, 1991; Willis et al., 2009). Recent research has demonstrated that metabolic signaling pathways can regulate aging and lifespan. Insulin receptors (InRs) play a key role in insulin/insulin peptide signaling (IIS) pathway. It has been reported that independent mutation of InR leads to reducing insulin signaling and extending lifespan in fruit flies (Tatar et al., 2001). Target of rapamycin (TOR) is a central controller of cell growth and influences Abbreviations: Cat, catalase; CrA, cranberry anthocyanins; DEPC, diethylpyocarbonate; dNTP, deoxy nucleotide triphosphate; Hep, hemipterous; GPx, glutathione peroxidase; InR, insulin receptors; JNK, C-jun N-terminal kinase; JNKK, C-jun N-terminal kinase kinase; MTH, methuselah; OR, oregon-R-C; PCR, polymerase chain reaction; PEPCK, phosphoenolpyruvate carboxykinase; ROS, reactive oxygen species; SOD, superoxide dismutase; SOD1, copper–zinc superoxide dismutase; SOD2, manganese superoxide dismutase; TOR, target of rapamycin. ⁎ Corresponding author. E-mail address: [email protected] (Z.-Y. Chen).
http://dx.doi.org/10.1016/j.exger.2015.06.021 0531-5565/© 2015 Elsevier Inc. All rights reserved.
aging (Wullschleger et al., 2006). Inhibition of TOR extends lifespan in both Drosophila melanogaster and Caenorhabditis elegans (Jia et al., 2004; Kapahi et al., 2004). Phosphoenolpyruvate carboxykinase (PEPCK) encodes a key enzyme that is involved in glucose and lipid metabolism (Chakravarty et al., 2005). Nectarine and plant extracts from Açai palm were reported to extend the lifespan of fruit flies partially due to down-regulation of PEPCK gene (Boyd et al., 2011; Sun et al., 2010). The Jun kinase (JNK) signaling pathway indirectly mediates the oxidative stress response in the cell and extends lifespan. Fruit flies with mutation in hemipterus (Hep), a Drosophila encoding gene of June kinase kinase (JNKK), were found more sensitive to oxidative stress and had a shortened lifespan (Wang et al., 2003). Age-related reduction in 26S proteasome activity is a key factor in the onset of neurodegenerative diseases. As one of the components in 26S proteasome, knocking down Rpn11 causes the accumulation of ubiquitinated proteins, and reduces 26S proteasome activity, and thus shortens lifespan (Tonoki et al., 2009). In contrast, over-expressing Rpn11 could reduce the age-related accumulation of ubiquitinated proteins, thus extending survival time of fruit flies (Tonoki et al., 2009). Methuselah (MTH) gene has been long recognized as one of the longevity-determined genes. It was reported that MTH mutant flies could live 35% longer than the wild strain and exhibited stronger resistance to exogenous oxidative stress (Lin et al., 1998). A recent study found that the wild-derived MTH Drosophila alleles such as, BF54/MTH△6, BF54/MTHR3, and S97/MTHR3 had a lifespan longer compared with S97/MTH△6 (Paaby and Schmidt, 2008). Interest in the relationship between diet and aging is increasing. Previous research have evidenced that both calorie restriction and some natural compounds in diet are able to extend the lifespan and delay the occurrence of age-related diseases in various aging models
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(Partridge et al., 2005; Peng et al., 2014). In this connection, fruit fly is one of the most commonly used models to investigate the effects of genetic modification and dietary intervention on aging (Lee et al., 2015). Cranberry has been regarded as a health fruit for its antioxidant, antimicrobial, anti-inflammatory, anti-cancer and anti-atherogenic properties (Duthie et al., 2006; Howell, 2007). Recent studies have found that cranberry extract could prolong the lifespan and increase heat shock tolerance of C. elegans or D. melanogaster (Guha et al., 2013, 2014; Wang et al., 2013). Given that anthocyanins are the most prevalent antioxidant polyphenols found in cranberry fruits, the present study was carried out to investigate the anti-aging activity of cranberry anthocyanin (CrA) extract with focus on its interaction with gene expressions of SOD1, SOD2, CAT, Rpn11, MTH, InR, TOR, Hep and PEPCK in D. melanogaster.
University, Bloomington, IN, USA). The control diet was prepared as we previously described (Peng et al., 2009). In brief, 1000 mL of diet contained 105 g cornmeal, 105 g glucose, 21 g yeast and 13 g agar. Ethyl 4-hydroxybenzoate (0.4%) was added in diet to prevent mold growth. Two experimental diets were similarly prepared except for adding 5 (CrA5) or 20 mg (CrA20) of cranberry anthocyanin extract per milliliter diet. For rearing the stocks, 15 mL of the control diet was poured and set into a vial, while for the experimental flies, 5 mL of the control or experimental diets was prepared per vial. All the flies in each vial were incubated at a controlled incubator maintaining at 25 ± 1 °C with 60–70% humidity. In this study, only male flies were used because hormone level could regulate Drosophila aging and there was less hormonal effect in the male than that in the female flies (Yamamoto et al., 2013).
2. Materials and methods 2.3. Effects of CrA extract on longevity 2.1. HPLC analysis of CrA extract CrA extract containing no vitamin C was obtained from Xi'an Realin Biotechnology Co., Ltd (Xi'an, China). The individual anthocyanins in CrA extract were separated on an Apollo C18 column (250 × 4.6 mm, 5 μm, Grace, Chicago, Illinois, USA) and quantified on a HPLC system with a UV detector at 520 nm. The flow rate was set at 1 mL/min, whereas the gradient mobile phase consisted of 5% acetic acid in water (Solvent A) and methanol (Solvent B). The gradient elution was programmed as following: 0–10 min, 40% B; 10–30 min, 40–45% B; 30–40 min, 45% B; and 40–50 min, 45–40% B. The peaks were identified according to the retention time and UV spectrum of authentic standards. HPLC analysis showed that the CrA extract used in the present study mainly contained cyanidin 3-galactoside (4.25%), cyanidin 3-arabinoside (4.75%), peonidin 3-galactoside (36.36%) and peonidin 3-arabinoside (4.72%) (Fig. 1). 2.2. Fruit fly strain and diets The wild type fly strain Oregon-R-C (OR) was obtained from Bloomington Drosophila Stock Center (Department of Biology, Indiana
Three-day old male flies were randomly divided into three groups with 200 flies each, and reared in 10 vials (20 flies per vial). The control group was maintained on the control diet, while the other two groups were raised on CrA5 and CrA20 diets, respectively. Every 2–3 days, the dead flies were counted and the remaining ones were transferred to a new vial containing the same diet. The feeding lasted 81 days. Another set of fruit flies were maintained as described above and were killed at days 0, 15, 25, 35, 45 and 55 to quantify the expression of SOD, CAT, Rpn11, MTH, InR, TOR, Hep and PEPCK.
2.4. Measurement of body weights Changes in body weights were used as an indicator of the food intake. To eliminate the possibility that the lifespan extension in survival assay might be due to effect of the dietary restriction, the body weights were recorded on days 0, 15, 25, 35, 45 and 55. Briefly, at selected days, 200 male flies in each group were anesthetized by carbon dioxide and then weighed on a balance. The mean body weights of the flies in each group were calculated.
Fig. 1. HPLC chromatogram of cranberry anthocyanin (CrA) extract. Peaks: 1, cyanidin 3-galactoside; 2, cyanidin 3-arabinoside; 3, peonidin 3-galactoside; and 4, peonidin 3-arabinoside.
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2.5. Gustatory assay Gustatory assay was an alternative method to measure food intake. In this assay, 120 newly eclosed male flies were collected (20 per vial) and reared on the standard diet for 7 days then they were starved for 24 h on Kimwipes paper soaked with distilled water. After that, flies were kept on the control diet (n = 60, 3 vials) or the CrA diets (n = 60, 3 vials) containing 0.2% sulforhodamine B sodium salt (Acid-red) for 2 h. Food intake was compared by scoring the degree of abdomen redness with a grading scale ranging from grade 0 (colorless abdomen) to grade 5 (fully red abdomen). The data were expressed as mean ± SD, n = 60. 2.6. Intensive paraquat challenge treatment
Real-time PCR amplification was carried out on a Fast Real-time PCR system 7500 (Applied Biosystems, Foster city, CA, USA). Nine target genes included SOD1 (NCBI Reference Sequence NM_057387.3), SOD2 (NCBI Reference Sequence NM_057577.2), CAT (NCBI Reference Sequence NM_080483.2), MTH (NCBI Reference Sequence NM_079147.2), Rpn11 (NCBI Reference Sequence NM_135061.2), TOR (NCBI Reference Sequence NM_080152.3), InR (NCBI Reference Sequence NM_079712.6), Hep (NCBI Reference Sequence NM_078587.3) and PEPCK (NCBI Reference Sequence NM_079060.3). The expression of the target genes was normalized with that of Rp49 (NCBI Reference Sequence NM_079843.2), a housekeeping gene used as an internal control. Gene expression was calculated on the basis of the comparative threshold cycle (CT) value. Levels of gene expression in all groups were expressed as a ratio of the control group value at day 0.
Dietary paraquat, chemically named 1, 1‵ -dimethy-4,4‵ -bipyridinium dichloride, (Sigma, St. Louis, Mo, USA), is able to produce superoxide anion radical. To investigate the effect of CrA extract on the paraquat-induced oxidative stress, the OR wild type flies (n = 400 in 20 vials) were reared on the control diet or CrA20 diet. At day 25, the two groups of fruit flies were first starved for 2 h and then transferred to new vials containing a filter paper saturated with 1 mL of 20 mM paraquat in a 6% glucose solution. The dead flies were counted every 4–6 h until all flies died.
2.10. Statistics
2.7. Hydrogen peroxide challenge test
3. Results
H2O2 is unstable and generates a hydroxyl radical (•OH). OR flies (n = 400 in 20 vials) were reared on the control diet or CrA20 diet for 25 days. Then the flies in the two groups were first subjected to starvation for 2 h and then placed in the vials containing a filter paper saturated with 1 mL of 30% H2O2 in a 6% glucose solution. The dead flies were counted every 4–6 h until all flies died.
3.1. Effect of CrA extract on longevity
2.8. Climbing ability test The climbing ability test was conducted to evaluate the effect of CrA extract on locomotor function in fruit flies. In brief, 10 male flies were placed in a plastic vial and given 20 s to climb up. At the end of each trial, the number of flies that could climb up to an 8 cm high or above vertical distance was recorded. The test was performed three times at days 0, 15, 25, 35, 45 and 55. 2.9. Real-time PCR Real-time PCR was used to quantify mRNA of SOD1, SOD2, CAT, MTH, Rpn11, TOR, InR, Hep and PEPCK. Briefly, total RNA was extracted using a commercial extraction agent TRIzol (Invitogen, Carlsbad, CA, USA). Flies (n = 15) were homogenized in 800 μL of TRIzol solution, and then centrifuged at 12,000g and 4 °C for 10 min. The supernatant was mixed with 160 μL of chloroform for 3 min, and then centrifuged at 12,000 g and 4 °C for 15 min. The upper layer was transferred to a new tube containing 400 μL isopropanol followed by incubation for 10 min at room temperature and centrifugation at 12,000 g and 4 °C for 10 min. The pellet was saved and washed in 1 mL of 75% ethanol solution followed by re-centrifugation. DEPC water (25 μL) was used to re-suspend the RNA pellet. The purity and concentration of RNA isolated were determined by measuring their absorbance at 260 nm and 280 nm, respectively. The cDNA was constructed by using a high capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). For each reaction, 2 μg RNA was mixed together with MgCl2, 10 × RT buffer, dNTP, random hexamers, RNase inhibitor and MultiScribe Transcriptase. The final volume was adjusted to 10 μL. The cDNA was synthesized in the thermocycler GeneAmp PCR system 9700 (Applied Biosystem) and stored at −20 °C.
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The Kaplan–Meier test with SPSS 20.0 (Statistical Package for the Social Sciences software, SPSS Inc., Chicago, USA) was used to assess the difference in the lifespan curves. Data were expressed as means ± SD. The significance of difference between means of stomach redness index was assessed using t-test and the others were assessed using two-way ANOVA. Difference were considered significant when P b 0.05.
CrA extract increased the mean lifespan of fruit flies. The 50% survival time of CrA20 group increased from 47 to 52 days compared with the control fruit flies (Fig. 2 and Table 1). Results showed that the mean lifespan in CrA5 and CrA20 groups were 51 and 53 days, respectively, which were significantly longer than that in the control fruit flies (P b 0.05; Fig. 2 and Table 1). Among the two experimental diets, CrA20 had the longevity activity greater than CrA5 diet (P b 0.01, Table 1). To simplify the presentation, only CrA20 diet was selected for the experiments hereafter. No difference in food intake was seen between the CrA20 and control group as reflected by measurements in the body weight and gustatory test (Fig. 3). Results on the climbing assay revealed that the climbing ability decreased with age. Compared with the control fruit flies, CrA20 group relatively increased the climbing activity (Fig. 4). mRNA SOD1 decreased significantly with age in both control and CrA20 groups (P b 0.01; Fig. 5) with mRNA SOD1 in CrA20 groups
Fig. 2. Lifespan curve of wild type fruit flies fed control diets (CTL) or diets containing 5 mg/mL (CrA5) and 20 mg/mL (CrA20) cranberry anthocyanin extract. Data were recorded until the last fly died. Mean lifespan and 50% survival time were calculated in 200 flies (Table 1). The Kaplan–Meier test was used to detect the significant differences among the three groups of flies, P b 0.05, CrA5 versus CTL; P b 0.01, CrA20 versus CTL.
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Table 1 Lifespan of OR wild type flies fed the control diet and the two experimental diets containing 5 (CrA5) and 20 mg (CrA20) cranberry anthocyanin extract per mL diet.
Control 5 mg/mL CrA 20 mg/ml CrA
Maximum lifespan of last fly (days)
50% survival (days)
Mean lifespan (mean ± SD, days)
78 78 81
47 50 52
48 ± 1 51 ± 1⁎ 53 ± 1⁎⁎
⁎ P b 0.05 vs. control. ⁎⁎ P b 0.01 vs. control.
being greater than that in the control fruit flies (P b 0.05). For SOD2, its mRNA also decreased with age in both control and CrA20 fruit flies (P b 0.01), however, no difference in mRNA SOD2 was seen between the control and CrA20 groups. mRNA CAT was up-regulated with age in both control and CrA20 fruit flies. However, no significant difference in mRNA CAT was observed between the control and CrA20 groups. In contrast, Rpn11 and Hep were down-regulated with age, with no differences in mRNA levels being seen between the control and CrA20 fruit flies. Regarding MTH, its mRNA level increased with ages, while addition of 20 mg CrA in diet suppressed the mRNA MTH compared with the control value. Concerning InR and TOR, their mRNA levels increased with age in both the control and CrA20 groups (P b 0.01). However, mRNA of InR and TOR in CrA20 group was relatively lowered compared with that in the control fruit flies (P b 0.05).
3.2. Effect of CrA on Paraquat and H2O2-challenged flies Results on H2O2 challenge test revealed that CrA20 group had a longer survival time than the control flies (P b 0.01, Fig. 6). The 50% survival time was increased from 15.0 h in the control to 16.5 h in CrA20 fruit flies. Intensive paraquat challenge test showed that CrA20 group had a longer 50% survival time compared with the control fruit flies, however, the mean survival time did not reach a significance level (P = 0.06; Fig. 6).
Fig. 3. Body weight (A, n = 200) and stomach redness index (B, n = 60) in fruit flies fed control diet or diet containing 20 mg/mL (CrA20) cranberry anthocyanin extract. Data were expressed as mean ± SD. The two-way ANOVA test was used to detect any significant difference in body weight between CTL and CrA20 groups. The t-test was used to detect any significant difference in stomach redness index between CTL and CrA20 groups. Df, degree of freedom; MS, mean squares; sig, significance.
Fig. 4. Climbing ability of fruit flies fed the control diet (CTL) and diet containing 20 mg/mL (CrA20) cranberry anthocyanin extract. Data were expressed as mean ± SD. CrA20 diet could significantly delay the age-dependent decline in climbing activity compared with the CTL diet, P b 0.01. df, degree of freedom; MS, mean squares; sig, significance.
4. Discussion The present study demonstrated clearly that CrA extract was effective against the aging in fruit flies. CrA supplementation at 20 mg/mL extended the mean lifespan by 10% compared with the control group (48 days versus 53 days, Table 1). The present results were in agreement with reports of Peng et al. (2012) and Zuo et al. (2012), who showed the blueberry and black rice anthocyanins could extend the mean lifespan of fruit flies by 10% and 14%, respectively. This prolonging effect of CrA was unlikely due to calorie restriction because the food intake was not altered by CrA supplementation, as reflected by the absence of significant changes in the body weights and gustatory stomach redness between the control and CrA fruit flies (Fig. 3). Lifespan-prolonging activity of CrA was most likely mediated by its interaction with gene expressions of SOD1, MTH, InR, TOR and PEPCK. Firstly, compared with the control, CrA in diet up regulated the expression of SOD1. It had been shown that transgenic fruit flies carrying three copies of SOD1 and CAT genes exhibited as much as one-third extension of lifespan, companied with a lower amount of protein oxidative damage and a delayed loss in physical performance (Orr and Sohal, 1994). In contrast, SOD1 null-mutants of flies conferred a hypersensitivity to paraquat and had a reduced longevity (Phillips et al., 1989; Sun et al., 2012). The up-regulation of SOD1 observed in the present study was consistent with the previous report of Deyhim et al. (2007), who demonstrated that cranberry supplementation was associated with a higher SOD activity in orchidectomized rats. Secondly, the lifespan prolonging activity of CrA was also likely associated with down-regulation of MTH, InR, TOR and PEPCK (Fig. 5). MTH encodes a G protein-couple receptor and is associated with longevity. MTH mutant flies had an extension of lifespan with increasing resistance to oxidative stress (Lin et al., 1998; Paaby and Schmidt, 2008). It had been also shown that apple polyphenols, blueberry anthocyanins, and curcumin could extend the lifespan via down-regulation of MTH in fruit flies (Lee et al., 2010; Peng et al., 2011, 2012). InR and TOR signaling pathway plays a key role in maintaining cellular and organismal homeostasis. The present study was the first to demonstrate that supplementation of CrA in diet was associated with down-regulation of InR and TOR compared with the control flies (Fig. 5). In fact, inhibition of InR and TOR had been shown to be associated with delayed age-related diseases, such as, type 2 diabetes, cancer, neurodegenerative and cardiovascular diseases and healthy lifespan (Goberdhan and Wilson, 2003; Katewa and Kapahi, 2011). In a human trial, it was found that supplementation of cranberry extracts for 12 weeks was effective in reducing atherosclerotic
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Fig. 5. Effect of 20 mg/mL (CrA20) cranberry anthocyanin extract on mRNA levels of copper–zinc containing superoxide dismutase (SOD1), manganese containing superoxide dismutase (SOD2), Rpn11, catalase (CAT), methuselah (MTH), insulin receptor (InR), target of rapamycin (TOR), hemipterus (Hep) and phosphoenolpyruvate carboxykinase (PEPCK) compared with those in the fruit flies fed the control diet (CTL). The wild type (OR) flies (n = 300/group, n = 20/vial) were incubated at for 0, 15, 25, 35, 45 and 55 days. Data are expressed as mean ± SD. df, degree of freedom; MS, mean squares; sig, significance.
cholesterol profiles in type 2 diabetic patients (Lee et al., 2008). PEPCK is a key enzyme which partially controls gluconeogenesis. Fenofibrate, a diabetic drug, was found to be effective in management of type 2 diabetes through suppressing PEPCK expression (Srivastava, 2009). In this regard, the present study found that PEPCK was significantly down-regulated in the fruit flies given the CrA diet compared with those given the control diet, suggesting that down-regulation of PEPCK could also mediate the lifespan prolonging activity of CrA extract.
It has been shown that cranberry polyphenols including anthocyanins are bioavailable and bioactive (McKay et al., 2015). It was also possible that the lifespan-prolonging activity of CrA was directly attributable to its antioxidant activity after absorption. In fact, the present study demonstrated that CrA supplementation significantly reduced the mortality rate induced by H2O2 (Fig. 6). The reduction in H2O2-induced mortality by CrA supplementation was largely associated with its direct antioxidant action to cause the decomposition of H2O2 rather than by regulating gene
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Fig. 6. Effect of hydrogen peroxide (H2O2) treatment and paraquat treatment on the survival time of the wild type (OR) flies fed the control diet (CTL) or diet containing 20 mg CrA/ml (CrA20) cranberry anthocyanin extract. The Kaplan–Meier test found CrA20 group survived better in the H2O2 treatment test compared with the control fruit flies (P b 0.01).
catalase as no significant difference in mRNA catalase was seen between the control and CrA20 groups (Fig. 5). Our result was in agreement with the study of Neto et al. (2005), who found that whole cranberry extract treatment could reduce H2O2-induced necrosis by 48%. It is generally believed that antioxidant can alleviate the neurodeterioration caused by free radicals and delay the aging process. Cranberry is an excellent source of antioxidants and abundant in anthocyanins. It had been shown that four anthocyanin isomers namely peonidin 3-galactoside, cyanidin 3-galactoside, cyanidin 3-arabinoside and peonidin 3-arabinoside accounted for more than 95% of the total anthocyanins in cranberry fruits (Prior et al., 2001). In this connection, cyanidin 3-galactoside has been shown to have an antioxidant activity being 3.9 times stronger than vitamin E (Yan et al., 2002), while peonidin 3-galactoside demonstrated to be equally effective as antioxidant as cyanidin 3-galactoside (Kähkönen and Heinonen, 2003). 5. Conclusions In conclusion, dietary cranberry anthocyanin extract in diet could extend the mean lifespan, alleviate H2O2-induced morality rate and partially reverse the age-related decline in locomotor activity in fruit flies. It was concluded that the anti-aging activity of cranberry anthocyanin extract was at least in part mediated by up-regulation of SOD1 while down-regulation of MTH, InR, TOR and PEPCK. References Boyd, O., Weng, P., Sun, X., Alberico, T., Laslo, M., Obenland, D.M., Kern, B., Zou, S., 2011. Nectarine promotes longevity in Drosophila melanogaster. Free Radic. Biol. Med. 50, 1669–1678. Chakravarty, K., Cassuto, H., Reshef, L., Hanson, R.W., 2005. Factors that control the tissuespecific transcription of the gene for phosphoenolpyruvate carboxykinase-C. Crit. Rev. Biochem. Mol. Biol. 40, 129–154. Cutler, R.G., 1991. Human longevity and aging: possible role of reactive oxygen species. Ann. N. Y. Acad. Sci. 621, 1–28.
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Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero
Cranberry anthocyanin extract prolongs lifespan of fruit flies Lijun Wang, Yuk Man Li, Lin Lei, Yuwei Liu, Xiaobo Wang, Ka Ying Ma, Zhen-Yu Chen ⁎ Food and Nutritional Sciences Programme, School of Life Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China
a r t i c l e
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Article history: Received 13 April 2015 Received in revised form 25 June 2015 Accepted 26 June 2015 Available online 6 July 2015 Section Editor: Hally M. Brown Keywords: Cranberry Anthocyanins Antioxidants Insulin receptor Target of rapamycin
a b s t r a c t Cranberry is an excellent source of dietary antioxidants. The present study investigated the effect of cranberry anthocyanin (CrA) extract on the lifespan of fruit flies with focus on its interaction with aging-related genes including superoxide dismutase (SOD), catalase (CAT), methuselah (MTH), insulin receptor (InR), target of rapamycin (TOR), hemipterus (Hep), and phosphoenolpyruvate carboxykinase (PEPCK). Results showed that diet containing 20 mg/mL CrA could significantly prolong the mean lifespan of fruit flies by 10% compared with the control diet. This was accompanied by up-regulation of SOD1 and down-regulation of MTH, InR, TOR and PEPCK. The stress resistance test demonstrated that CrA could reduce the mortality rate induced by H2O2 but not by paraquat. It was therefore concluded that the lifespan-prolonging activity of CrA was most likely mediated by modulating the genes of SOD1, MTH, InR, TOR and PEPCK. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Free radical theory has been considered as one of the major aging theories even though it is still under debate (Peng et al., 2014; Speakman and Selman, 2011). It states that organisms become aged partially due to accumulation of oxidative damages caused by reactive oxygen species (ROS), namely hydroxyl radicals, superoxide anions and hydrogen peroxide. ROS are the byproducts of normal cellular metabolism of oxygen. Endogenous antioxidant enzymes in cells, including catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx), serve as a first line of defense against ROS, while exogenous antioxidants from diets such as plant flavonoids and vitamins function as a second line of defense in terminating the propagation of ROS reactions (Cutler, 1991; Willis et al., 2009). Recent research has demonstrated that metabolic signaling pathways can regulate aging and lifespan. Insulin receptors (InRs) play a key role in insulin/insulin peptide signaling (IIS) pathway. It has been reported that independent mutation of InR leads to reducing insulin signaling and extending lifespan in fruit flies (Tatar et al., 2001). Target of rapamycin (TOR) is a central controller of cell growth and influences Abbreviations: Cat, catalase; CrA, cranberry anthocyanins; DEPC, diethylpyocarbonate; dNTP, deoxy nucleotide triphosphate; Hep, hemipterous; GPx, glutathione peroxidase; InR, insulin receptors; JNK, C-jun N-terminal kinase; JNKK, C-jun N-terminal kinase kinase; MTH, methuselah; OR, oregon-R-C; PCR, polymerase chain reaction; PEPCK, phosphoenolpyruvate carboxykinase; ROS, reactive oxygen species; SOD, superoxide dismutase; SOD1, copper–zinc superoxide dismutase; SOD2, manganese superoxide dismutase; TOR, target of rapamycin. ⁎ Corresponding author. E-mail address: [email protected] (Z.-Y. Chen).
http://dx.doi.org/10.1016/j.exger.2015.06.021 0531-5565/© 2015 Elsevier Inc. All rights reserved.
aging (Wullschleger et al., 2006). Inhibition of TOR extends lifespan in both Drosophila melanogaster and Caenorhabditis elegans (Jia et al., 2004; Kapahi et al., 2004). Phosphoenolpyruvate carboxykinase (PEPCK) encodes a key enzyme that is involved in glucose and lipid metabolism (Chakravarty et al., 2005). Nectarine and plant extracts from Açai palm were reported to extend the lifespan of fruit flies partially due to down-regulation of PEPCK gene (Boyd et al., 2011; Sun et al., 2010). The Jun kinase (JNK) signaling pathway indirectly mediates the oxidative stress response in the cell and extends lifespan. Fruit flies with mutation in hemipterus (Hep), a Drosophila encoding gene of June kinase kinase (JNKK), were found more sensitive to oxidative stress and had a shortened lifespan (Wang et al., 2003). Age-related reduction in 26S proteasome activity is a key factor in the onset of neurodegenerative diseases. As one of the components in 26S proteasome, knocking down Rpn11 causes the accumulation of ubiquitinated proteins, and reduces 26S proteasome activity, and thus shortens lifespan (Tonoki et al., 2009). In contrast, over-expressing Rpn11 could reduce the age-related accumulation of ubiquitinated proteins, thus extending survival time of fruit flies (Tonoki et al., 2009). Methuselah (MTH) gene has been long recognized as one of the longevity-determined genes. It was reported that MTH mutant flies could live 35% longer than the wild strain and exhibited stronger resistance to exogenous oxidative stress (Lin et al., 1998). A recent study found that the wild-derived MTH Drosophila alleles such as, BF54/MTH△6, BF54/MTHR3, and S97/MTHR3 had a lifespan longer compared with S97/MTH△6 (Paaby and Schmidt, 2008). Interest in the relationship between diet and aging is increasing. Previous research have evidenced that both calorie restriction and some natural compounds in diet are able to extend the lifespan and delay the occurrence of age-related diseases in various aging models
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(Partridge et al., 2005; Peng et al., 2014). In this connection, fruit fly is one of the most commonly used models to investigate the effects of genetic modification and dietary intervention on aging (Lee et al., 2015). Cranberry has been regarded as a health fruit for its antioxidant, antimicrobial, anti-inflammatory, anti-cancer and anti-atherogenic properties (Duthie et al., 2006; Howell, 2007). Recent studies have found that cranberry extract could prolong the lifespan and increase heat shock tolerance of C. elegans or D. melanogaster (Guha et al., 2013, 2014; Wang et al., 2013). Given that anthocyanins are the most prevalent antioxidant polyphenols found in cranberry fruits, the present study was carried out to investigate the anti-aging activity of cranberry anthocyanin (CrA) extract with focus on its interaction with gene expressions of SOD1, SOD2, CAT, Rpn11, MTH, InR, TOR, Hep and PEPCK in D. melanogaster.
University, Bloomington, IN, USA). The control diet was prepared as we previously described (Peng et al., 2009). In brief, 1000 mL of diet contained 105 g cornmeal, 105 g glucose, 21 g yeast and 13 g agar. Ethyl 4-hydroxybenzoate (0.4%) was added in diet to prevent mold growth. Two experimental diets were similarly prepared except for adding 5 (CrA5) or 20 mg (CrA20) of cranberry anthocyanin extract per milliliter diet. For rearing the stocks, 15 mL of the control diet was poured and set into a vial, while for the experimental flies, 5 mL of the control or experimental diets was prepared per vial. All the flies in each vial were incubated at a controlled incubator maintaining at 25 ± 1 °C with 60–70% humidity. In this study, only male flies were used because hormone level could regulate Drosophila aging and there was less hormonal effect in the male than that in the female flies (Yamamoto et al., 2013).
2. Materials and methods 2.3. Effects of CrA extract on longevity 2.1. HPLC analysis of CrA extract CrA extract containing no vitamin C was obtained from Xi'an Realin Biotechnology Co., Ltd (Xi'an, China). The individual anthocyanins in CrA extract were separated on an Apollo C18 column (250 × 4.6 mm, 5 μm, Grace, Chicago, Illinois, USA) and quantified on a HPLC system with a UV detector at 520 nm. The flow rate was set at 1 mL/min, whereas the gradient mobile phase consisted of 5% acetic acid in water (Solvent A) and methanol (Solvent B). The gradient elution was programmed as following: 0–10 min, 40% B; 10–30 min, 40–45% B; 30–40 min, 45% B; and 40–50 min, 45–40% B. The peaks were identified according to the retention time and UV spectrum of authentic standards. HPLC analysis showed that the CrA extract used in the present study mainly contained cyanidin 3-galactoside (4.25%), cyanidin 3-arabinoside (4.75%), peonidin 3-galactoside (36.36%) and peonidin 3-arabinoside (4.72%) (Fig. 1). 2.2. Fruit fly strain and diets The wild type fly strain Oregon-R-C (OR) was obtained from Bloomington Drosophila Stock Center (Department of Biology, Indiana
Three-day old male flies were randomly divided into three groups with 200 flies each, and reared in 10 vials (20 flies per vial). The control group was maintained on the control diet, while the other two groups were raised on CrA5 and CrA20 diets, respectively. Every 2–3 days, the dead flies were counted and the remaining ones were transferred to a new vial containing the same diet. The feeding lasted 81 days. Another set of fruit flies were maintained as described above and were killed at days 0, 15, 25, 35, 45 and 55 to quantify the expression of SOD, CAT, Rpn11, MTH, InR, TOR, Hep and PEPCK.
2.4. Measurement of body weights Changes in body weights were used as an indicator of the food intake. To eliminate the possibility that the lifespan extension in survival assay might be due to effect of the dietary restriction, the body weights were recorded on days 0, 15, 25, 35, 45 and 55. Briefly, at selected days, 200 male flies in each group were anesthetized by carbon dioxide and then weighed on a balance. The mean body weights of the flies in each group were calculated.
Fig. 1. HPLC chromatogram of cranberry anthocyanin (CrA) extract. Peaks: 1, cyanidin 3-galactoside; 2, cyanidin 3-arabinoside; 3, peonidin 3-galactoside; and 4, peonidin 3-arabinoside.
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2.5. Gustatory assay Gustatory assay was an alternative method to measure food intake. In this assay, 120 newly eclosed male flies were collected (20 per vial) and reared on the standard diet for 7 days then they were starved for 24 h on Kimwipes paper soaked with distilled water. After that, flies were kept on the control diet (n = 60, 3 vials) or the CrA diets (n = 60, 3 vials) containing 0.2% sulforhodamine B sodium salt (Acid-red) for 2 h. Food intake was compared by scoring the degree of abdomen redness with a grading scale ranging from grade 0 (colorless abdomen) to grade 5 (fully red abdomen). The data were expressed as mean ± SD, n = 60. 2.6. Intensive paraquat challenge treatment
Real-time PCR amplification was carried out on a Fast Real-time PCR system 7500 (Applied Biosystems, Foster city, CA, USA). Nine target genes included SOD1 (NCBI Reference Sequence NM_057387.3), SOD2 (NCBI Reference Sequence NM_057577.2), CAT (NCBI Reference Sequence NM_080483.2), MTH (NCBI Reference Sequence NM_079147.2), Rpn11 (NCBI Reference Sequence NM_135061.2), TOR (NCBI Reference Sequence NM_080152.3), InR (NCBI Reference Sequence NM_079712.6), Hep (NCBI Reference Sequence NM_078587.3) and PEPCK (NCBI Reference Sequence NM_079060.3). The expression of the target genes was normalized with that of Rp49 (NCBI Reference Sequence NM_079843.2), a housekeeping gene used as an internal control. Gene expression was calculated on the basis of the comparative threshold cycle (CT) value. Levels of gene expression in all groups were expressed as a ratio of the control group value at day 0.
Dietary paraquat, chemically named 1, 1‵ -dimethy-4,4‵ -bipyridinium dichloride, (Sigma, St. Louis, Mo, USA), is able to produce superoxide anion radical. To investigate the effect of CrA extract on the paraquat-induced oxidative stress, the OR wild type flies (n = 400 in 20 vials) were reared on the control diet or CrA20 diet. At day 25, the two groups of fruit flies were first starved for 2 h and then transferred to new vials containing a filter paper saturated with 1 mL of 20 mM paraquat in a 6% glucose solution. The dead flies were counted every 4–6 h until all flies died.
2.10. Statistics
2.7. Hydrogen peroxide challenge test
3. Results
H2O2 is unstable and generates a hydroxyl radical (•OH). OR flies (n = 400 in 20 vials) were reared on the control diet or CrA20 diet for 25 days. Then the flies in the two groups were first subjected to starvation for 2 h and then placed in the vials containing a filter paper saturated with 1 mL of 30% H2O2 in a 6% glucose solution. The dead flies were counted every 4–6 h until all flies died.
3.1. Effect of CrA extract on longevity
2.8. Climbing ability test The climbing ability test was conducted to evaluate the effect of CrA extract on locomotor function in fruit flies. In brief, 10 male flies were placed in a plastic vial and given 20 s to climb up. At the end of each trial, the number of flies that could climb up to an 8 cm high or above vertical distance was recorded. The test was performed three times at days 0, 15, 25, 35, 45 and 55. 2.9. Real-time PCR Real-time PCR was used to quantify mRNA of SOD1, SOD2, CAT, MTH, Rpn11, TOR, InR, Hep and PEPCK. Briefly, total RNA was extracted using a commercial extraction agent TRIzol (Invitogen, Carlsbad, CA, USA). Flies (n = 15) were homogenized in 800 μL of TRIzol solution, and then centrifuged at 12,000g and 4 °C for 10 min. The supernatant was mixed with 160 μL of chloroform for 3 min, and then centrifuged at 12,000 g and 4 °C for 15 min. The upper layer was transferred to a new tube containing 400 μL isopropanol followed by incubation for 10 min at room temperature and centrifugation at 12,000 g and 4 °C for 10 min. The pellet was saved and washed in 1 mL of 75% ethanol solution followed by re-centrifugation. DEPC water (25 μL) was used to re-suspend the RNA pellet. The purity and concentration of RNA isolated were determined by measuring their absorbance at 260 nm and 280 nm, respectively. The cDNA was constructed by using a high capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). For each reaction, 2 μg RNA was mixed together with MgCl2, 10 × RT buffer, dNTP, random hexamers, RNase inhibitor and MultiScribe Transcriptase. The final volume was adjusted to 10 μL. The cDNA was synthesized in the thermocycler GeneAmp PCR system 9700 (Applied Biosystem) and stored at −20 °C.
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The Kaplan–Meier test with SPSS 20.0 (Statistical Package for the Social Sciences software, SPSS Inc., Chicago, USA) was used to assess the difference in the lifespan curves. Data were expressed as means ± SD. The significance of difference between means of stomach redness index was assessed using t-test and the others were assessed using two-way ANOVA. Difference were considered significant when P b 0.05.
CrA extract increased the mean lifespan of fruit flies. The 50% survival time of CrA20 group increased from 47 to 52 days compared with the control fruit flies (Fig. 2 and Table 1). Results showed that the mean lifespan in CrA5 and CrA20 groups were 51 and 53 days, respectively, which were significantly longer than that in the control fruit flies (P b 0.05; Fig. 2 and Table 1). Among the two experimental diets, CrA20 had the longevity activity greater than CrA5 diet (P b 0.01, Table 1). To simplify the presentation, only CrA20 diet was selected for the experiments hereafter. No difference in food intake was seen between the CrA20 and control group as reflected by measurements in the body weight and gustatory test (Fig. 3). Results on the climbing assay revealed that the climbing ability decreased with age. Compared with the control fruit flies, CrA20 group relatively increased the climbing activity (Fig. 4). mRNA SOD1 decreased significantly with age in both control and CrA20 groups (P b 0.01; Fig. 5) with mRNA SOD1 in CrA20 groups
Fig. 2. Lifespan curve of wild type fruit flies fed control diets (CTL) or diets containing 5 mg/mL (CrA5) and 20 mg/mL (CrA20) cranberry anthocyanin extract. Data were recorded until the last fly died. Mean lifespan and 50% survival time were calculated in 200 flies (Table 1). The Kaplan–Meier test was used to detect the significant differences among the three groups of flies, P b 0.05, CrA5 versus CTL; P b 0.01, CrA20 versus CTL.
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Table 1 Lifespan of OR wild type flies fed the control diet and the two experimental diets containing 5 (CrA5) and 20 mg (CrA20) cranberry anthocyanin extract per mL diet.
Control 5 mg/mL CrA 20 mg/ml CrA
Maximum lifespan of last fly (days)
50% survival (days)
Mean lifespan (mean ± SD, days)
78 78 81
47 50 52
48 ± 1 51 ± 1⁎ 53 ± 1⁎⁎
⁎ P b 0.05 vs. control. ⁎⁎ P b 0.01 vs. control.
being greater than that in the control fruit flies (P b 0.05). For SOD2, its mRNA also decreased with age in both control and CrA20 fruit flies (P b 0.01), however, no difference in mRNA SOD2 was seen between the control and CrA20 groups. mRNA CAT was up-regulated with age in both control and CrA20 fruit flies. However, no significant difference in mRNA CAT was observed between the control and CrA20 groups. In contrast, Rpn11 and Hep were down-regulated with age, with no differences in mRNA levels being seen between the control and CrA20 fruit flies. Regarding MTH, its mRNA level increased with ages, while addition of 20 mg CrA in diet suppressed the mRNA MTH compared with the control value. Concerning InR and TOR, their mRNA levels increased with age in both the control and CrA20 groups (P b 0.01). However, mRNA of InR and TOR in CrA20 group was relatively lowered compared with that in the control fruit flies (P b 0.05).
3.2. Effect of CrA on Paraquat and H2O2-challenged flies Results on H2O2 challenge test revealed that CrA20 group had a longer survival time than the control flies (P b 0.01, Fig. 6). The 50% survival time was increased from 15.0 h in the control to 16.5 h in CrA20 fruit flies. Intensive paraquat challenge test showed that CrA20 group had a longer 50% survival time compared with the control fruit flies, however, the mean survival time did not reach a significance level (P = 0.06; Fig. 6).
Fig. 3. Body weight (A, n = 200) and stomach redness index (B, n = 60) in fruit flies fed control diet or diet containing 20 mg/mL (CrA20) cranberry anthocyanin extract. Data were expressed as mean ± SD. The two-way ANOVA test was used to detect any significant difference in body weight between CTL and CrA20 groups. The t-test was used to detect any significant difference in stomach redness index between CTL and CrA20 groups. Df, degree of freedom; MS, mean squares; sig, significance.
Fig. 4. Climbing ability of fruit flies fed the control diet (CTL) and diet containing 20 mg/mL (CrA20) cranberry anthocyanin extract. Data were expressed as mean ± SD. CrA20 diet could significantly delay the age-dependent decline in climbing activity compared with the CTL diet, P b 0.01. df, degree of freedom; MS, mean squares; sig, significance.
4. Discussion The present study demonstrated clearly that CrA extract was effective against the aging in fruit flies. CrA supplementation at 20 mg/mL extended the mean lifespan by 10% compared with the control group (48 days versus 53 days, Table 1). The present results were in agreement with reports of Peng et al. (2012) and Zuo et al. (2012), who showed the blueberry and black rice anthocyanins could extend the mean lifespan of fruit flies by 10% and 14%, respectively. This prolonging effect of CrA was unlikely due to calorie restriction because the food intake was not altered by CrA supplementation, as reflected by the absence of significant changes in the body weights and gustatory stomach redness between the control and CrA fruit flies (Fig. 3). Lifespan-prolonging activity of CrA was most likely mediated by its interaction with gene expressions of SOD1, MTH, InR, TOR and PEPCK. Firstly, compared with the control, CrA in diet up regulated the expression of SOD1. It had been shown that transgenic fruit flies carrying three copies of SOD1 and CAT genes exhibited as much as one-third extension of lifespan, companied with a lower amount of protein oxidative damage and a delayed loss in physical performance (Orr and Sohal, 1994). In contrast, SOD1 null-mutants of flies conferred a hypersensitivity to paraquat and had a reduced longevity (Phillips et al., 1989; Sun et al., 2012). The up-regulation of SOD1 observed in the present study was consistent with the previous report of Deyhim et al. (2007), who demonstrated that cranberry supplementation was associated with a higher SOD activity in orchidectomized rats. Secondly, the lifespan prolonging activity of CrA was also likely associated with down-regulation of MTH, InR, TOR and PEPCK (Fig. 5). MTH encodes a G protein-couple receptor and is associated with longevity. MTH mutant flies had an extension of lifespan with increasing resistance to oxidative stress (Lin et al., 1998; Paaby and Schmidt, 2008). It had been also shown that apple polyphenols, blueberry anthocyanins, and curcumin could extend the lifespan via down-regulation of MTH in fruit flies (Lee et al., 2010; Peng et al., 2011, 2012). InR and TOR signaling pathway plays a key role in maintaining cellular and organismal homeostasis. The present study was the first to demonstrate that supplementation of CrA in diet was associated with down-regulation of InR and TOR compared with the control flies (Fig. 5). In fact, inhibition of InR and TOR had been shown to be associated with delayed age-related diseases, such as, type 2 diabetes, cancer, neurodegenerative and cardiovascular diseases and healthy lifespan (Goberdhan and Wilson, 2003; Katewa and Kapahi, 2011). In a human trial, it was found that supplementation of cranberry extracts for 12 weeks was effective in reducing atherosclerotic
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Fig. 5. Effect of 20 mg/mL (CrA20) cranberry anthocyanin extract on mRNA levels of copper–zinc containing superoxide dismutase (SOD1), manganese containing superoxide dismutase (SOD2), Rpn11, catalase (CAT), methuselah (MTH), insulin receptor (InR), target of rapamycin (TOR), hemipterus (Hep) and phosphoenolpyruvate carboxykinase (PEPCK) compared with those in the fruit flies fed the control diet (CTL). The wild type (OR) flies (n = 300/group, n = 20/vial) were incubated at for 0, 15, 25, 35, 45 and 55 days. Data are expressed as mean ± SD. df, degree of freedom; MS, mean squares; sig, significance.
cholesterol profiles in type 2 diabetic patients (Lee et al., 2008). PEPCK is a key enzyme which partially controls gluconeogenesis. Fenofibrate, a diabetic drug, was found to be effective in management of type 2 diabetes through suppressing PEPCK expression (Srivastava, 2009). In this regard, the present study found that PEPCK was significantly down-regulated in the fruit flies given the CrA diet compared with those given the control diet, suggesting that down-regulation of PEPCK could also mediate the lifespan prolonging activity of CrA extract.
It has been shown that cranberry polyphenols including anthocyanins are bioavailable and bioactive (McKay et al., 2015). It was also possible that the lifespan-prolonging activity of CrA was directly attributable to its antioxidant activity after absorption. In fact, the present study demonstrated that CrA supplementation significantly reduced the mortality rate induced by H2O2 (Fig. 6). The reduction in H2O2-induced mortality by CrA supplementation was largely associated with its direct antioxidant action to cause the decomposition of H2O2 rather than by regulating gene
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Fig. 6. Effect of hydrogen peroxide (H2O2) treatment and paraquat treatment on the survival time of the wild type (OR) flies fed the control diet (CTL) or diet containing 20 mg CrA/ml (CrA20) cranberry anthocyanin extract. The Kaplan–Meier test found CrA20 group survived better in the H2O2 treatment test compared with the control fruit flies (P b 0.01).
catalase as no significant difference in mRNA catalase was seen between the control and CrA20 groups (Fig. 5). Our result was in agreement with the study of Neto et al. (2005), who found that whole cranberry extract treatment could reduce H2O2-induced necrosis by 48%. It is generally believed that antioxidant can alleviate the neurodeterioration caused by free radicals and delay the aging process. Cranberry is an excellent source of antioxidants and abundant in anthocyanins. It had been shown that four anthocyanin isomers namely peonidin 3-galactoside, cyanidin 3-galactoside, cyanidin 3-arabinoside and peonidin 3-arabinoside accounted for more than 95% of the total anthocyanins in cranberry fruits (Prior et al., 2001). In this connection, cyanidin 3-galactoside has been shown to have an antioxidant activity being 3.9 times stronger than vitamin E (Yan et al., 2002), while peonidin 3-galactoside demonstrated to be equally effective as antioxidant as cyanidin 3-galactoside (Kähkönen and Heinonen, 2003). 5. Conclusions In conclusion, dietary cranberry anthocyanin extract in diet could extend the mean lifespan, alleviate H2O2-induced morality rate and partially reverse the age-related decline in locomotor activity in fruit flies. It was concluded that the anti-aging activity of cranberry anthocyanin extract was at least in part mediated by up-regulation of SOD1 while down-regulation of MTH, InR, TOR and PEPCK. References Boyd, O., Weng, P., Sun, X., Alberico, T., Laslo, M., Obenland, D.M., Kern, B., Zou, S., 2011. Nectarine promotes longevity in Drosophila melanogaster. Free Radic. Biol. Med. 50, 1669–1678. Chakravarty, K., Cassuto, H., Reshef, L., Hanson, R.W., 2005. Factors that control the tissuespecific transcription of the gene for phosphoenolpyruvate carboxykinase-C. Crit. Rev. Biochem. Mol. Biol. 40, 129–154. Cutler, R.G., 1991. Human longevity and aging: possible role of reactive oxygen species. Ann. N. Y. Acad. Sci. 621, 1–28.
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