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Purple sweet potato anthocyanin attenuates fat-induced mortality in Drosophila melanogaster
Experimental Gerontology 82 (2016) 95–103
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Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero
Purple sweet potato anthocyanin attenuates fat-induced mortality in Drosophila melanogaster Lijun Wang, Yuk Man Li, Lin Lei, Yuwei Liu, Xiaobo Wang, Ka Ying Ma, Chengnan Zhang, Hanyue Zhu, Yimin Zhao, 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 4 September 2015 Received in revised form 16 June 2016 Accepted 17 June 2016 Available online 18 June 2016 Keywords: Purple sweet potato Anthocyanin High-fat diet Oxidative stress
a b s t r a c t A high fat diet induces the accumulation of lipid hydroperoxides (LPO), accelerates the ageing process and causes a greater mortality in Drosophila melanogaster. Purple sweet potato is rich in antioxidant anthocyanin. The purpose of the present study was to examine if supplementation of purple sweet potato anthocyanin (PSPA) could reduce the mortality of fruit flies fed a high-fat diet. Results showed that the mean lifespan of fruit flies was shortened from 56 to 35 days in a dose-dependent manner when lard in the diet increased from 0% to 20%. PSPA supplementation partially attenuated the lard-induced mortality. The maximum lifespan and 50% survival time were 49 and 27 days, respectively, for the 10% lard control flies, in contrast, these parameters increased to 57 and 30 days in the PSPA-supplemented fruit flies. Similarly, addition of lard into diet increased the total body LPO, while addition of PSPA partially attenuated its increase. Real-time PCR analysis indicated that PSPAsupplemented diet significantly up-regulated the mRNA of superoxide dismutase (SOD), catalase (CAT) and Rpn11, compared with the control lard diet. The western blot analysis also demonstrated that PSPA supplementation was associated with up-regulation protein mass of SOD1, SOD2, and CAT. In addition, PSPA supplementation could restore the climbing ability of fruit flies fed a 10% lard diet. We could conclude that the lifespanprolonging activity of PSPA was potentially mediated by modulating the genes of SOD, CAT and Rpn11. © 2016 Elsevier Inc. All rights reserved.
1. Introduction A high-fat diet has been associated with a shorter lifespan from Drosophila to mammals (Baur et al., 2006; Driver and Cosopodiotis, 1979; Silberberg and Silberberg, 1954; Woodcock et al., 2015). In humans, long consumption of high fat diets could induce obesity which facilitates the development of cardiovascular diseases, type 2 diabetes, hypertension, some types of cancers, and inflammationmediated diseases (Hariri and Thibault, 2010; Xia et al., 2010; Zhou and Pan, 2015). Increasing evidence has demonstrated that consumption of vegetables and fruits could promote health, prevent and delay age-related diseases. In this regard, some phytochemicals derived from vegetables and fruits have been shown to improve the survival
Abbreviations: Cat, catalase; Hep, hemipterous; InR, insulin receptors; JNK, C-jun Nterminal kinase; JNKK, C-jun N-terminal kinase kinase; MTH, methuselah; OR, oregon-RC; 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 at: School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong, China.
http://dx.doi.org/10.1016/j.exger.2016.06.006 0531-5565/© 2016 Elsevier Inc. All rights reserved.
rate of mice or fruit flies fed a high-fat diet (Baur et al., 2006; Boyd et al., 2011; Li et al., 2008; Sun et al., 2010). Purple sweet potato is becoming a popular vegetable worldwide and its flesh is rich in anthocyanin. Both in vitro and in vivo studies have shown that purple sweet potato anthocyanin (PSPA) could protect humans against colorectal cancer by inducing cell-cycle arrest, antiproliferative, and apoptosis (Lim et al., 2013). Zhang et al. (2013) claimed that supplementation of PSPA in diet could attenuate the insulin resistance via inhibiting the formation of reactive oxygen species (ROS) and blocking the ROS-mediated endoplasmic reticulum stress. In addition, PSPA is effective in ameliorating D-galactose-induced brain ageing and domoic acid-induced cognitive deficits (Lu et al., 2012; Lu et al., 2010). Fruit fly (Drosophila melanogaster) is an excellent model to investigate the longevity-promoting property of nutraceuticals or functional foods because it has a short life span, can easily grow, its full sequence of the genome is already known, and, more importantly, it conserves about 74% of human disease-causing genes (Bier and Bodmer, 2004). To test the hypothesis that dietary antioxidants possess the anti-ageing activity, this study demonstrated that PSPA supplementation could prolong the lifespan of fruit flies when they were given a high fat diet.
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2. Materials and methods 2.1. HPLC analysis of PSPA PSPA was obtained from Xi'an SR Bio-Engineering Co., Ltd (Xi'an, China). The individual components in PSPA were separated on an Apollo C18 column (250 × 4.6 mm, 5 μm, Chicago, Illinois, USA) and quantified on a HPLC system with a UV detector at 520 nm. The gradient mobile phase consisted of 5% acetic acid in water (Solvent A) and methanol (Solvent B). The gradient elution was programmed as following: 40% B, 0–10 min; 40–45% B, 10–30 min; 45% B, 30–40 min; 45–40% B, 40– 50 min. The flow rate was 1 mL/min. The peaks were identified according to the retention time of standards. PSPA used in the present study mainly contained cyanide-3-O-glucoside (Fig. 1). 2.2. Fly strain and diets The wild type D. melanogaster strain (Oregon-R-C) was obtained from Bloomington Drosophila Stock Center (Department of Biology, Indiana University, Bloomington, IN, USA). The standard diet was prepared according to the previously reported formula (Li et al., 2008; Wang et al., 2015). 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. To prepare a high fat diet, lard and emulsifier Tween-20 were added to the standard diet and thoroughly mixed. The final concentration of Tween-20 was 1% (v/v) of the 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 were prepared per vial. All the flies were incubated at a controlled incubator maintaining 25 ± 1 °C and 65% humidity with a 12-h light/dark cycle. In this study, only male flies were used because there was less hormonal effect in the male than in the female flies. 2.3. Effect of PSPA on lifespan of fruit flies fed a non-fat diet 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 standard diet, while the other two groups were raised on diet containing 5 (PSPA5) or 10 mg/mL PSPA (PSPA10). Every 2–3 days, the dead flies were counted and the remaining ones were transferred to a new vial containing the same diet. 2.4. Paraquat challenge treatment test Dietary paraquat, chemically named 1,1′-dimethy-4,4′-bi-pyridinium dichloride (Sigma, St. Louis, Mo, USA), is able to produce superoxide
anion radical. To investigate the effect of PSPA on the paraquat-induced oxidative stress, 400 (20 flies per vial) newly eclosed flies were reared on the control diet or PSPA10 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. 2.5. Hydrogen peroxide challenge test H2O2 is unstable and generates a hydroxyl radical (•OH). The newly eclosed flies (n = 400 in 20 vials) were reared on the control diet or PSPA10 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. 2.6. Dose effect of lard on lifespan Three-day old male flies were randomly divided into six groups with 200 flies each group rearing in 10 vials (20 flies per vial). The first group was maintained on the non-lard control diet (NCTL), while the remaining five groups were fed one of the five diets containing 2%, 5%, 10%, 15% and 20% lard. The final concentration of Tween-20 was 1% (v/v) in all of the six group diets. The dead flies were counted every 2–3 days, and the remaining alive flies were then transferred to a new vial containing the same diet. 2.7. Effect of PSPA on lifespan of flies fed a high fat diet New eclosed flies (three-day old flies) were randomly divided into four groups. The first group was maintained on NCTL diet. The second group was kept on a 10% lard control diet (LCTL). The other two groups were maintained on diets containing 10% lard with addition of 5 mg PSPA/mL (LPSPA5) or 10 mg PSPA/mL (LPSPA10). For each group, 200 flies were used with 20 flies per vial. The dead flies were counted every 2–3 days, and the remaining alive flies were then transferred to a new vial containing the same diet until all the flies died. Another set of the experiment described above was similarly conducted and the fruit flies were killed at days 0, 10 and 35 to quantify the expression of genes involved in ageing including copper-zinc superoxide dismutase (SOD1), manganese superoxide dismutase (SOD2), catalase (CAT), Rpn1 (encodes 26S proteasome regulatory subunit Rpn11), Methuselah (MTH), insulin receptor (InR), target of rapamycin (TOR), hemipterous (Hep), iron regulator protein 1B (Irp-1B), and phosphoenolpyruvate carboxykinase (PEPCK).
Fig. 1. HPLC chromatogram of purple sweet potato anthocyanin extract (PSPA). Peaks: 1, cyaniding-3-O-glucoside; 2, unknown.
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2.8. Measurement of body weights The changes in body weights could be used as an indicator of the food intake. The body weight of fruits flies was recorded on days 0, 10 and 35. In brief, at selected days, 100 flies (n = 3 × 100) 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. 2.9. Gustatory assay To exclude the possibility that lifespan extension observed in survival assay was caused by caloric restriction, gustatory assay was carried out to measure the food intake of flies (Bahadorani and Hilliker, 2008; Wang et al., 2015). In brief, 180 newly eclosed male flies were collected (20 flies per vial) and reared on the standard diet for 6 days and then starved for 24 h on Kimwipes paper soaked with distilled water. After that, flies were kept on the NCTL diet (n = 60, 3 vials), LCTL diet (n = 60, 3 vials), or the LPSPA10 diet (n = 60, 3 vials) containing 0.2% sulforhodamine B sodium salt (Acid-red) for 2 h. Food intake of the flies in the three groups was compared by scoring the degree of abdomen redness with a scale ranging from grade 0 (colorless abdomen) to 5 (fully red abdomen). The data were expressed as mean ± SD, n = 60. 2.10. Climbing ability test The climbing ability test was conducted to evaluate the effect of PSPA on locomotor function in fruit flies fed a 10% lard diet with or without addition of 10 mg/mL PSPA. 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, 10, 25, and 35 in the fruit flies fed one of three diets NCTL, LCTL and LPSPA10. 2.11. Measurement of lipid peroxide (LPO) LPO level was measured using a thiobarbituric acid reactive substance (TBARS) assay kit (Cayman Chemical, Ann Arbor, MI, USA), which measures the amount of malondialdehyde (MDA)-thiobarbituric acid (TBA) adduct. The MDA-TBA adduct formed was measured colorimetrically at 535 nm. The flies were maintained on NCTL, LCTL and LPSPA10 diets for a period of 0, 10 and 35 days. In each measurement, every 100 fruit flies (n = 3 × 100) were weighted and homogenized in a 0.8 mL of 1 × RIPA buffer. The mixture was centrifuged at 1600g for 10 min at 4 °C. The supernatant was collected for analysis according to the protocol described in assay kit. 2.12. Real-time PCR Gene expression of SOD1, SOD2, CAT, MTH, Rpn11, TOR, InR, Hep, Irp-1B, and PEPCK were measured as we previously described (Wang et al., 2015). Total RNA was extracted using the commercial extraction agent TRIzol (Invitrogen, Carlsbad, CA, USA). Flies (n = 15) were homogenized in 800 μL of TRIzol solution, centrifuged at 12,000g 4 °C for 10 min and then the supernatant was mixed with 160 μL of chloroform for 3 min. The mixture was then centrifuged at 12,000g 4 °C for 15 min. The upper layer was transferred to a new tube containing 400 μL isopropanol. After 10 min incubation at room temperature, the samples were subjected to centrifugation at 12,000g at 4 °C for 10 min. The pellet was saved and washed in 1 mL of 75% ethanol 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.
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The cDNA was constructed by using a high capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). In each reaction, RNA (2 μg) was mixed together with MgCl2, 10 × RT buffer, random hexamers, dNTP, 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. Real-time PCR amplification was carried out on a Fast Real-time PCR system 7500 (Applied Biosystems, Foster city, CA, USA). Ten 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), Irp-1B (NCBI Reference Sequence NM_079579.3) 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 shown as a ratio of the day 0 control group value.
2.13. Western blot analysis The western blot analysis was conducted as we previously described with some modification (Peng et al., 2009). In brief, 50 flies were homogenized in a 1.5 mL tube containing 500 μL homogenizing buffer (20 mM Tris-HCl, 2 mM MgCl2, and 0.2 M sucrose, pH = 7.5) and protease inhibitor cocktail (Roche, Mannheim, Germany). The extracts were then centrifuged at 13,000g for 5 min at 4 ⁰C and the supernatant was collected. Total protein concentration was measured using a protein concentration assay kit according to the manufacturer's instructions (Bio-Rad, Hercules, CA, USA). After adding 6× loading dye, samples were denatured at 95 °C for 5 min and the stored at −80 °C until use. The total protein (20 μg) was size-fractionated by 15% SDS-PAGE at 130 V for 160 min. After that the gel was cut to two parts. The upper portion (containing β-actin and CAT) and lower portion (containing SOD1 and SOD2) were then transferred to Hybond-P PVDF membranes (Millipore Corporation, Billerica, MA, USA) at 15 V for 60 min and 40 min, respectively. The membranes were blocked in 5% non-fat milk in 1 × tris-buffered saline containing 0.1% Tween-20 (1 x TBST) at room temperature and then in the same solution containing diluted anti-catalase/anti-actin/anti-SOD1/anti-SOD2 antibodies, respectively, at 4 °C overnight. The membranes were then washed in 1 × TBST for 4 × 15 min and incubated for 1 h at 4 °C in diluted horseradish peroxidase-linked goat anti-rabit IgG or anti-mouse IgG (Santa Cruz Biotechnology, Inc., California, USA). The washes mentioned above were repeated before the membranes were developed with ECL enhance chemiluminescence agent (Santa Cruz Biotechnology, Inc., California, USA) and subjected to autoradiography on ChemiDocTM Touch Imaging System (Bio\\Rad Laboratories, Hercules, USA). Densitometry was analyzed using ChemiDocTM Touch Imaging System Software. Protein concentrations of CAT, SOD1 and SOD2 were normalized with β-actin.
2.14. Statistics The Kaplan-Meier test was used to compare the difference between the lifespan curves using SPSS 20.0 (Statistical Package for the Social Sciences software, SPSS Inc., Chicago, USA). Data were expressed as means ± standard deviation (SD). The significance of difference between means was assessed using two-way ANOVA. Difference were considered to be significant when P ﹤ 0.05.
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3. Results
Table 1 Lifespan of fruit flies fed a non-lard control diet (NCTL) or two non-lard diets containing 5
3.1. Effect of PSPA on the lifespan and stress resistance ability of fruit flies fed a non-lard control diet
mg/mL purple sweet potato anthocyanin extract (PSPA5) and 10 mg/mL purple sweet potato anthocyanin extract (PSPA10).
To assess the effect of PSPA on the ageing and stress resistance of fruit flies, PSPA was added into the standard fly diet at 5 mg/mL and 10 mg/mL. Results showed that none of these concentrations of PSPA significantly affected the lifespan of fruit flies compared with the nonsupplemented standard diet (Fig. 2, Table 1). Similarly, supplementation of 10 mg/mL PSPA in diet did not change the survival rate of fruit flies treated with paraquat and H2O2 (Fig. 3). This indicated that PSPA supplementation up to 10 mg/mL is not sufficient to promote longevity and improve the stress resistance ability under the culture condition using the non-fat standard fly diet. 3.2. Effect of lards on the lifespan of fruit flies The present study demonstrated clearly that high-fat diets shortened the lifespan of fruit flies. Results showed that both maximum survival time and 50% survival time of the flies were inversely related to the amounts of lard added in the diet (Fig. 4, Table 2). The non-lard control group (NCTL, 0% fat) had a maximum lifespan of 56 days, whereas the 20% lard group had only 35 days; the lifespan was shortened by 37.5%. Similarly, 50% survival time also decreased with the increasing amounts of lard. The NCTL group had a 50% survival time of 34 days, while the 20% lard group had only 17 days. In addition, when the lard was increased from 0 to 20%, the mean lifespan of the flies was decreased from 35.6 to 17.7 days. Data are expressed as the maximum lifespan of the last fruit fly, 50% survival time and mean lifespan (n = 200 flies per group, n = 20 flies per vial). Means in the same column with different superscripts differ significantly at P b 0.01.
NCTL PSPA5 PSPA10
Maximum lifespan of last fly (day)
50% survival (day)
Mean lifespan (day)
67 70 70
44 45 47
45.0 ± 0.8 46.7 ± 0.7 46.5 ± 0.7
supplemented with 10 mg/mL PSPA (Fig. 5, Table 3). The 50% survival time for the LCTL group was 27 days. In contrast, it increased to 29 and 30 days in LPSPA5 and LPSPA10, respectively. The LCTL group had a mean lifespan of 27.8 days, whereas that of the LPSPA5 and LPSPA10 groups increased to 29.1 and 30.8 days, respectively. There was no significance difference in food intake was seen among the NCTL, LCTL and LPSPA10 groups as reflected by the measurements of gustatory assay (Fig. 6). No significant differences in the body weights among the NCTL, LCTL and LPSPA10 groups were also observed (Fig. 6). Results on the climbing assay revealed that the climbing ability decreased with the age in both the non-lard standard and 10% lard diet groups (Fig. 7). Compared with the NCTL group, LCTL fruit flies decreased the climbing activity. However supplementation of PSPA into a 10% lard diet completely reversed the high fat-induced declined climbing ability. The LPO level in the fruit flies was significantly increased during ageing. Compared with the NCTL group, incorporation of lard into the diet could induce more production of LPO, while PSPA supplementation reduced production of LPO by 3.1% and 9.3%, respectively, in fruit flies maintained on a 10% fat diet for 10 and 35 days (Fig. 8).
3.3. Effect of PSPA on the lifespan of fruit flies fed a high fat diet Supplementation of PSPA could partially reverse the fat-induced mortality. The maximum lifespan was 49 days for a 10% lard control group (LCTL), whereas it was increased to 57 days in LPSPA10 diet
Fig. 2. Lifespan curves of fruit flies fed a non-lard control (NCTL) diet or two non-lard diets containing 5 mg/mL purple sweet potato anthocyanin extract (PSPA5) or 10 mg/mL purple sweet potato anthocyanin extract (PSPA10). 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 showed that none of these concentrations of PSPA significantly affected the lifespan of fruit flies compared to NCTL diet.
Fig. 3. Effect of paraquat and hydrogen peroxide (H2O2) treatment on the survival time of the wild type (OR) fruit flies fed a non-lard control diet (NCTL) or an experimental diet containing 10 mg/mL purple sweet potato anthocyanin extract (PSPA10). The KaplanMeier test found PSPA10 did not affect the survival rate of fruit flies challenged with paraquat or H2O2.
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4. Discussion
Fig. 4. Lifespan curves of fruit flies fed a non-lard control diet (NCTL) or diets containing 2% to 20% lard (L). Data were recorded until the last fruit fly died. Mean lifespan and 50% survival time were calculated in 200 flies (See Table 2).
Data are expressed as the maximum lifespan of the last fruit fly, 50% survival time and mean lifespan (n = 200 flies per group, n = 20 flies per vial). a,b,c Means at the same column with different superscripts differ significantly at P b 0.01.
3.4. Changes in gene expression and protein level induced by PSPA supplementation To investigate the molecular mechanisms by which PSPA improved the survival time in fruit flies fed a high fat diet, the mRNA levels of genes involved in ROS eliminating, nutrient sensing and insulin-like signaling pathways were measured. As shown in Fig. 9, mRNA SOD1 and SOD2 decreased significantly with age in all of the three groups. Incorporation of lard into the diet lowered the mRNA SOD1 and SOD2. However, supplementation of PSPA increased the mRNA of SOD1 and SOD2. Compared with the NCTL group, LCTL could significantly down-regulated the gene expression of CAT and RPn11, while LPSPA10 diet partially restored the expression level of CAT and Rpn11. Addition of lard downregulated Hep, while supplementation of PSPA further decreased its mRNA level (Fig. 9). For MTH, InR, TOR, PEPCK and Irp-1B, no differences in their mRNA levels were seen between fruit flies fed the LCTL and LPSPA10 diets (Fig. 9). The Western blot data showed that protein mass of SOD1 and SOD2 increased whereas the protein mass of CAT decreased with age in NCTL group flies. Addition of lard to the diet significantly down-regulated the protein mass of SOD1, SOD2 and CAT. However, PSPA group had protein mass of SOD1, SOD2, and CAT greater than that in the high fat control (Fig. 10).
The purpose of the present study was to investigate the effect of PSPA on lifespan of fruit flies. We had the following major observations. First, addition of dietary fat into diets shortened the lifespan of fruit flies in a dose-dependent manner (Fig. 4). Second, PSPA could prolong the lifespan of fruit flies fed a high fat diet but not that of fruit flies given a non-fat diet (Figs. 2 and 5). This prolonging effect of PSPA was unlikely due to calories restriction because the food intake was not altered by PSPA supplementation, as reflected by the absence of significant changes in the body weights and gustatory stomach redness between the control and PSPA fruit flies (Fig. 6). Third, addition of fat into diet negatively affected the locomotor behavior of fruit flies, while supplementation of PSPA into diet could completely restore the declined climbing ability (Fig. 7). Fourth, PSPA supplementation into diet of fruit flies fed a high fat diet could favorably modulate the genes of SOD1, SOD2, CAT and Rpn11 with little or no effect on the mRNA of MTH, InR, TOR, PEPCK and Irp-1B (Fig. 9). Fifth, supplementation of PSPA was associated with up-regulation protein mass of SOD1, SOD2, and CAT (Fig. 10), accompanied by a decreased in LPO level in the body of fruit flies (Fig. 8). These data strongly suggested that anthocyanin from purple sweet potato possessed the anti-ageing activity at least in fruit fly model given a high fat diet. Incorporation of dietary fat could accelerate the ageing process in fruit flies. The present results were in agreement with that of Driver and Cosopodiotis (1979), who reported that incorporation of palmitic acid into diet could shorten the lifespan of fruit flies by 20%. Li et al. (2008) demonstrated that addition of a mixture of fatty acids derived from lard into diet significantly shortened the lifespan and caused a greater mortality in D. melanogaster, while addition of green tea and black tea extract could partially restore the lifespan of fruit flies. It is known that addition of dietary fat into diet could induce formation of lipid hydroperoxide (Li et al., 2008), thus leading to a shorter lifespan in fruit flies. Like tea antioxidants catechins and theaflavins, PSPA is also an excellent antioxidant (Yousuf et al., 2015). In fact, the present results clearly demonstrated that PSPA could inhibit the lipid oxidation, partially attenuate the formation of LPO and thus decrease the mortality of fruit flies given a high fat diet (Fig. 8). The lifespan-prolonging activity of PSPA in fruit flies given a high fat diet is most likely mediated by its interaction with the gene expression of endogenous antioxidant enzymes. The present results clearly demonstrated that the high fat diet shortened the lifespan while addition of
Table 2 Lifespan of fruit flies fed a non-lard control diet (NCTL) or diets containing 2, 5, 10, 15, and 20% lard.
NCTL 2% lard 5% lard 10% lard 15% lard 20% lard
Maximum lifespan of last fly (day)
50% survival (day)
Mean lifespan (mean ± SD, day)
56 47 44 40 35 35
34 30 23 20 18 17
35.6 ± 0.5a 30.4 ± 0.5b 24.7 ± 0.4c 21.6 ± 0.4d 19.3 ± 0.3e 17.7 ± 0.3f
Fig. 5. Lifespan curves of fruit flies fed a non-lard control diet (NCTL) or a 10% lard control diet (LCTL), with addition of 5 mg/mL purple sweet potato anthocyanin extract (LPSPA5) or 10 mg/mL purple sweet potato anthocyanin extract (LPSPA10). Data were recorded until the last fruit fly died. Mean lifespan and 50% survival time were calculated in 200 flies (Table 3).
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Table 3 Lifespan of fruit flies fed a non-lard control diet (NCTL), a 10% lard control diet (LCTL) and two 10% lard experimental diets containing 5 mg/mL PSPA (LPSPA5) and 10 mg/mL PSPA (LPSPA10).
NCTL LCTL LPSP5 LPSP10
Maximum lifespan of last fly (day)
50% survival (day)
Mean lifespan (day)
58 49 49 57
33 27 29 30
34.2 ± 0.6a 27.8 ± 0.5c 29.1 ± 0.6 bc 30.8 ± 0.3b
PSPA could up-regulate the mRNA and protein mass of SOD1, SOD2 and CAT in fruit flies. This is in agreement with the report of Freeman et al. (2014), who demontrated that a high fat diet could shorten the lifespan and accelerate the ageing. ROS, the most abundant free radicals in cells, are the byproducts of normal cellular metabolism of oxygen. ROS can cause lipid oxidation, DNA damage, and protein oxidation, thus leading to the ageing (Harman, 1956). The endogenous oxidative defense system in flies consists predominantly of SOD1, SOD2 and CAT. In this regard, fruit flies with over-expression of SOD and CAT genes had as much as one-third extension of lifespan (Orr and Sohal, 1994; Sun et al., 2004). The exogenous antioxidants may also play an important role in deactivating the ROS. In this connection, many natural antioxidants, such as apple polyphenols, tea extracts, anthocyanins enrich extracts and sesamin, have been found to up-regulate SOD1, SOD2 and
Fig. 6. Average body weights and stomach redness index of fruit flies fed a non-lard control diet (NCTL), or a 10% lard diet control diet (LCTL), or a 10% lard diet containing 10 mg/mL purple sweet potato anthocyanin extract (LPSPA10). Data were expressed as mean ± SD.
Fig. 7. Climbing ability of fruit flies fed a non-lard control diet (NCTL), or a 10% lard diet control diet (LCTL), or a 10% lard diet containing 10 mg/mL purple sweet potato anthocyanin extract (LPSPA10). Data were expressed as mean ± SD. The 10% lard control diet decreased the climbing ability, whereas, LPSPA10 diet could completely restore the climbing ability of fruit flies (P b 0.01). df, degree of freedom; MS, mean squares; sig, significance.
CAT gene expression levels and extend fruit fly lifespan (Peng et al., 2014). In contrast, deletion or reduction of SOD2 gene severely shortened the lifespan of fruit flies (Duttaroy et al., 2003; Paul et al., 2007; Phillips et al., 1989). The present study suggested that the lifespanprolonging activity of PSPA was mediated at least partially by up-regulation of SOD and CAT genes in fruit flies. Many genes and signaling pathways have been reported to regulate the lifespan in model organisms. Rpn11 is one of components in the 26S proteasome and is mainly responsible for “waste” protein degradation by 26S proteasome (Verma et al., 2002; Lee et al., 2009). It was reported that knock down of Rpn11 reduced 26S proteasome activity, caused the accumulation of ubiquitinated proteins and shortened lifespan, while over-expression Rpn11 reduced the ubiquitinated protein accumulation and extended the lifespan (Tonoki et al., 2009). Hep encodes JNKK (cJun. N-terminal kinase kinase), which locates in the upstream of the JNK signaling. It was reported that fruit flies gained resistance to paraquat and had a longer lifespan when JNK signaling was promoted by overexpression of Hep (Wang et al., 2003). PEPCK encodes a key enzyme that controls gluconeogenesis and lipid metabolism. Irp-1B is
Fig. 8. Lipid hydroperoxide level in fruit flies fed a non-lard control diet (NCTL), or a 10% lard diet control diet (LCTL), or a 10% lard diet containing 10 mg/mL purple sweet potato anthocyanin extract (LPSPA10). Data were expressed as mean ± SD.
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Fig. 9. 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) in the fruit flies fed a non-lard control diet (NCTL), a 10% lard control diet (LCTL) and a 10% lard diet with supplementation of 10 mg/mL purple sweet potato anthocyanin extract (LPSPA10) at 0, 15, and 35 days (n = 300/group, n = 20/vial). Data are expressed as mean ± SD. df, degree of freedom; MS, mean squares; sig, significance.
involved in iron metabolism. Down-regulation of both PEPCK and Irp1B genes could extend the lifespan (Boyd et al., 2011). MTH, a G-protein coupled receptor, has been long recognized as one of the longevity-determined genes. Mutant flies with a reducing expression level of MTH exhibited stronger resistance to oxidative stress and longer lifespan (Lin et al., 1998; Paaby and Schmidt, 2008). InR and TOR signaling pathways are evolutionarily conserved from invertebrates to mammals. Previous studies demonstrated that inhibition on InR and TOR signaling pathways could significantly extend the lifespan in many species, including yeast, worms, fruit flies and mammals (Verdaguer et al., 2012). The present study demonstrated that a high fat diet down-regulated, while addition of PSPA only up-regulated the mRNA of Rpn11
without having effect on mRNA of PEPCK, Irp-1B, InR, MTH and TOR (Fig. 9). 5. Conclusion The present study demonstrated clearly that addition of lard reduced the locomotor power, increased the LPO production and shortened the lifespan of fruit flies. PSPA supplementation partially attenuated the high fat-induced mortality, increased the climbing ability and reduced the LPO production of fruit flies. It was concluded that the anti-ageing activity of PSPA was at least in part mediated by up-regulation of gene and protein mass of SOD, CAT and Rpn11.
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Fig. 10. The relative immunoreactive mass of SOD1, SOD2, and CAT of fruit flies fed a non-lard control diet (NCTL), or a 10% lard diet control diet (LCTL), or a 10% lard diet containing 10 mg/ mL purple sweet potato anthocyanin extract (LPSPA10). The results were normalized with the values of day 0 being 1.0.
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Orr, W.C., Sohal, R.S., 1994. Extension of life-span by overexpression of superoxide dismutase and catalase in Drosophila melanogaster. Science 263, 1128–1130. Paaby, A.B., Schmidt, P.S., 2008. Functional significance of allelic variation at methuselah, an aging gene in Drosophila. PLoS One 3, e1987. Paul, A., Belton, A., Nag, S., Martin, I., Grotewiel, M.S., Duttaroy, A., 2007. Reduced mitochondrial SOD displays mortality characteristics reminiscent of natural aging. Mech. Ageing Dev. 128, 706–716. Peng, C., Chan, H.Y.E., Li, Y.M., Huang, Y., Chen, Z.Y., 2009. Black tea theaflavins extend the lifespan of fruit flies. Exp. Gerontol. 44 (12), 773–783. Peng, C., Wang, X., Chen, J., Jiao, R., Wang, L., Li, Y.M., Zuo, Y., Liu, Y., Lei, L., Ma, K.Y., 2014. Biology of ageing and role of dietary antioxidants. Biomed. Res. Int. 831834. Phillips, J.P., Campbell, S.D., Michaud, D., Charbonneau, M., Hilliker, A.J., 1989. Null mutation of copper/zinc superoxide dismutase in Drosophila confers hypersensitivity to paraquat and reduced longevity. Proc. Natl. Acad Sci. U.S.A. 86, 2761–2765. Silberberg, M., Silberberg, R., 1954. Factors modifying the lifespan of mice. Am. J. Phys. 177, 23–26. Sun, J.T., Molitor, J., Tower, J., 2004. Effects of simultaneous over-expression of Cu/ZnSOD and MnSOD on Drosophila melanogaster life span. Mech. Ageing Dev. 125, 341–349. Sun, X., Seeberger, J., Alberico, T., Wang, C., Wheeler, C.T., Schauss, A.G., Zou, S., 2010. Açai palm fruit (Euterpe oleracea Mart.) pulp improves survival of flies on a high fat diet. Exp. Gerontol. 45, 243–251. Tonoki, A., Kuranaga, E., Tomioka, T., Hamazaki, J., Murata, S., Tanaka, K., Miura, M., 2009. Genetic evidence linking age-dependent attenuation of the 26S proteasome with the aging process. Mol. Cell. Biol. 29, 1095–1106. Verdaguer, E., Junyent, F., Folch, J., Beas-Zarate, C., Auladell, C., Pallàs, M., Camins, A., 2012. Aging biology: a new frontier for drug discovery. Expert Opin. Drug Discovery 7, 217–229. Verma, R., Aravind, L., Oania, R., McDonald, W.H., Yates, J.R., Koonin, E.V., Deshaies, R.J., 2002. Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome. Science 298, 611–615. Wang, M.C., Bohmann, D., Jasper, H., 2003. JNK signaling confers tolerance to oxidative stress and extends lifespan in Drosophila. Dev. Cell 5, 811–816. Wang, L., Li, Y.M., Lei, L., Liu, Y., Wang, X., Ma, K.Y., Chen, Z.-Y., 2015. Cranberry anthocyanin extract prolongs lifespan of fruit flies. Exp. Gerontol. 69, 189–195. Woodcock, K.J., Kierdorf, K., Pouchelon, C.A., Vivancos, V., Dionne, M.S., Geissmann, F., 2015. Macrophage-derived upd3 cytokine causes impaired glucose homeostasis and reduced lifespan in Drosophila fed a lipid-rich diet. Immunity 42, 133–144. Xia, E.-Q., Deng, G.-F., Guo, Y.-J., Li, H.-B., 2010. Biological activities of polyphenols from grapes. Int. J. Mol. Sci. 11, 622–646.
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Contents lists available at ScienceDirect
Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero
Purple sweet potato anthocyanin attenuates fat-induced mortality in Drosophila melanogaster Lijun Wang, Yuk Man Li, Lin Lei, Yuwei Liu, Xiaobo Wang, Ka Ying Ma, Chengnan Zhang, Hanyue Zhu, Yimin Zhao, 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
i n f o
Article history: Received 4 September 2015 Received in revised form 16 June 2016 Accepted 17 June 2016 Available online 18 June 2016 Keywords: Purple sweet potato Anthocyanin High-fat diet Oxidative stress
a b s t r a c t A high fat diet induces the accumulation of lipid hydroperoxides (LPO), accelerates the ageing process and causes a greater mortality in Drosophila melanogaster. Purple sweet potato is rich in antioxidant anthocyanin. The purpose of the present study was to examine if supplementation of purple sweet potato anthocyanin (PSPA) could reduce the mortality of fruit flies fed a high-fat diet. Results showed that the mean lifespan of fruit flies was shortened from 56 to 35 days in a dose-dependent manner when lard in the diet increased from 0% to 20%. PSPA supplementation partially attenuated the lard-induced mortality. The maximum lifespan and 50% survival time were 49 and 27 days, respectively, for the 10% lard control flies, in contrast, these parameters increased to 57 and 30 days in the PSPA-supplemented fruit flies. Similarly, addition of lard into diet increased the total body LPO, while addition of PSPA partially attenuated its increase. Real-time PCR analysis indicated that PSPAsupplemented diet significantly up-regulated the mRNA of superoxide dismutase (SOD), catalase (CAT) and Rpn11, compared with the control lard diet. The western blot analysis also demonstrated that PSPA supplementation was associated with up-regulation protein mass of SOD1, SOD2, and CAT. In addition, PSPA supplementation could restore the climbing ability of fruit flies fed a 10% lard diet. We could conclude that the lifespanprolonging activity of PSPA was potentially mediated by modulating the genes of SOD, CAT and Rpn11. © 2016 Elsevier Inc. All rights reserved.
1. Introduction A high-fat diet has been associated with a shorter lifespan from Drosophila to mammals (Baur et al., 2006; Driver and Cosopodiotis, 1979; Silberberg and Silberberg, 1954; Woodcock et al., 2015). In humans, long consumption of high fat diets could induce obesity which facilitates the development of cardiovascular diseases, type 2 diabetes, hypertension, some types of cancers, and inflammationmediated diseases (Hariri and Thibault, 2010; Xia et al., 2010; Zhou and Pan, 2015). Increasing evidence has demonstrated that consumption of vegetables and fruits could promote health, prevent and delay age-related diseases. In this regard, some phytochemicals derived from vegetables and fruits have been shown to improve the survival
Abbreviations: Cat, catalase; Hep, hemipterous; InR, insulin receptors; JNK, C-jun Nterminal kinase; JNKK, C-jun N-terminal kinase kinase; MTH, methuselah; OR, oregon-RC; 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 at: School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong, China.
http://dx.doi.org/10.1016/j.exger.2016.06.006 0531-5565/© 2016 Elsevier Inc. All rights reserved.
rate of mice or fruit flies fed a high-fat diet (Baur et al., 2006; Boyd et al., 2011; Li et al., 2008; Sun et al., 2010). Purple sweet potato is becoming a popular vegetable worldwide and its flesh is rich in anthocyanin. Both in vitro and in vivo studies have shown that purple sweet potato anthocyanin (PSPA) could protect humans against colorectal cancer by inducing cell-cycle arrest, antiproliferative, and apoptosis (Lim et al., 2013). Zhang et al. (2013) claimed that supplementation of PSPA in diet could attenuate the insulin resistance via inhibiting the formation of reactive oxygen species (ROS) and blocking the ROS-mediated endoplasmic reticulum stress. In addition, PSPA is effective in ameliorating D-galactose-induced brain ageing and domoic acid-induced cognitive deficits (Lu et al., 2012; Lu et al., 2010). Fruit fly (Drosophila melanogaster) is an excellent model to investigate the longevity-promoting property of nutraceuticals or functional foods because it has a short life span, can easily grow, its full sequence of the genome is already known, and, more importantly, it conserves about 74% of human disease-causing genes (Bier and Bodmer, 2004). To test the hypothesis that dietary antioxidants possess the anti-ageing activity, this study demonstrated that PSPA supplementation could prolong the lifespan of fruit flies when they were given a high fat diet.
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2. Materials and methods 2.1. HPLC analysis of PSPA PSPA was obtained from Xi'an SR Bio-Engineering Co., Ltd (Xi'an, China). The individual components in PSPA were separated on an Apollo C18 column (250 × 4.6 mm, 5 μm, Chicago, Illinois, USA) and quantified on a HPLC system with a UV detector at 520 nm. The gradient mobile phase consisted of 5% acetic acid in water (Solvent A) and methanol (Solvent B). The gradient elution was programmed as following: 40% B, 0–10 min; 40–45% B, 10–30 min; 45% B, 30–40 min; 45–40% B, 40– 50 min. The flow rate was 1 mL/min. The peaks were identified according to the retention time of standards. PSPA used in the present study mainly contained cyanide-3-O-glucoside (Fig. 1). 2.2. Fly strain and diets The wild type D. melanogaster strain (Oregon-R-C) was obtained from Bloomington Drosophila Stock Center (Department of Biology, Indiana University, Bloomington, IN, USA). The standard diet was prepared according to the previously reported formula (Li et al., 2008; Wang et al., 2015). 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. To prepare a high fat diet, lard and emulsifier Tween-20 were added to the standard diet and thoroughly mixed. The final concentration of Tween-20 was 1% (v/v) of the 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 were prepared per vial. All the flies were incubated at a controlled incubator maintaining 25 ± 1 °C and 65% humidity with a 12-h light/dark cycle. In this study, only male flies were used because there was less hormonal effect in the male than in the female flies. 2.3. Effect of PSPA on lifespan of fruit flies fed a non-fat diet 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 standard diet, while the other two groups were raised on diet containing 5 (PSPA5) or 10 mg/mL PSPA (PSPA10). Every 2–3 days, the dead flies were counted and the remaining ones were transferred to a new vial containing the same diet. 2.4. Paraquat challenge treatment test Dietary paraquat, chemically named 1,1′-dimethy-4,4′-bi-pyridinium dichloride (Sigma, St. Louis, Mo, USA), is able to produce superoxide
anion radical. To investigate the effect of PSPA on the paraquat-induced oxidative stress, 400 (20 flies per vial) newly eclosed flies were reared on the control diet or PSPA10 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. 2.5. Hydrogen peroxide challenge test H2O2 is unstable and generates a hydroxyl radical (•OH). The newly eclosed flies (n = 400 in 20 vials) were reared on the control diet or PSPA10 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. 2.6. Dose effect of lard on lifespan Three-day old male flies were randomly divided into six groups with 200 flies each group rearing in 10 vials (20 flies per vial). The first group was maintained on the non-lard control diet (NCTL), while the remaining five groups were fed one of the five diets containing 2%, 5%, 10%, 15% and 20% lard. The final concentration of Tween-20 was 1% (v/v) in all of the six group diets. The dead flies were counted every 2–3 days, and the remaining alive flies were then transferred to a new vial containing the same diet. 2.7. Effect of PSPA on lifespan of flies fed a high fat diet New eclosed flies (three-day old flies) were randomly divided into four groups. The first group was maintained on NCTL diet. The second group was kept on a 10% lard control diet (LCTL). The other two groups were maintained on diets containing 10% lard with addition of 5 mg PSPA/mL (LPSPA5) or 10 mg PSPA/mL (LPSPA10). For each group, 200 flies were used with 20 flies per vial. The dead flies were counted every 2–3 days, and the remaining alive flies were then transferred to a new vial containing the same diet until all the flies died. Another set of the experiment described above was similarly conducted and the fruit flies were killed at days 0, 10 and 35 to quantify the expression of genes involved in ageing including copper-zinc superoxide dismutase (SOD1), manganese superoxide dismutase (SOD2), catalase (CAT), Rpn1 (encodes 26S proteasome regulatory subunit Rpn11), Methuselah (MTH), insulin receptor (InR), target of rapamycin (TOR), hemipterous (Hep), iron regulator protein 1B (Irp-1B), and phosphoenolpyruvate carboxykinase (PEPCK).
Fig. 1. HPLC chromatogram of purple sweet potato anthocyanin extract (PSPA). Peaks: 1, cyaniding-3-O-glucoside; 2, unknown.
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2.8. Measurement of body weights The changes in body weights could be used as an indicator of the food intake. The body weight of fruits flies was recorded on days 0, 10 and 35. In brief, at selected days, 100 flies (n = 3 × 100) 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. 2.9. Gustatory assay To exclude the possibility that lifespan extension observed in survival assay was caused by caloric restriction, gustatory assay was carried out to measure the food intake of flies (Bahadorani and Hilliker, 2008; Wang et al., 2015). In brief, 180 newly eclosed male flies were collected (20 flies per vial) and reared on the standard diet for 6 days and then starved for 24 h on Kimwipes paper soaked with distilled water. After that, flies were kept on the NCTL diet (n = 60, 3 vials), LCTL diet (n = 60, 3 vials), or the LPSPA10 diet (n = 60, 3 vials) containing 0.2% sulforhodamine B sodium salt (Acid-red) for 2 h. Food intake of the flies in the three groups was compared by scoring the degree of abdomen redness with a scale ranging from grade 0 (colorless abdomen) to 5 (fully red abdomen). The data were expressed as mean ± SD, n = 60. 2.10. Climbing ability test The climbing ability test was conducted to evaluate the effect of PSPA on locomotor function in fruit flies fed a 10% lard diet with or without addition of 10 mg/mL PSPA. 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, 10, 25, and 35 in the fruit flies fed one of three diets NCTL, LCTL and LPSPA10. 2.11. Measurement of lipid peroxide (LPO) LPO level was measured using a thiobarbituric acid reactive substance (TBARS) assay kit (Cayman Chemical, Ann Arbor, MI, USA), which measures the amount of malondialdehyde (MDA)-thiobarbituric acid (TBA) adduct. The MDA-TBA adduct formed was measured colorimetrically at 535 nm. The flies were maintained on NCTL, LCTL and LPSPA10 diets for a period of 0, 10 and 35 days. In each measurement, every 100 fruit flies (n = 3 × 100) were weighted and homogenized in a 0.8 mL of 1 × RIPA buffer. The mixture was centrifuged at 1600g for 10 min at 4 °C. The supernatant was collected for analysis according to the protocol described in assay kit. 2.12. Real-time PCR Gene expression of SOD1, SOD2, CAT, MTH, Rpn11, TOR, InR, Hep, Irp-1B, and PEPCK were measured as we previously described (Wang et al., 2015). Total RNA was extracted using the commercial extraction agent TRIzol (Invitrogen, Carlsbad, CA, USA). Flies (n = 15) were homogenized in 800 μL of TRIzol solution, centrifuged at 12,000g 4 °C for 10 min and then the supernatant was mixed with 160 μL of chloroform for 3 min. The mixture was then centrifuged at 12,000g 4 °C for 15 min. The upper layer was transferred to a new tube containing 400 μL isopropanol. After 10 min incubation at room temperature, the samples were subjected to centrifugation at 12,000g at 4 °C for 10 min. The pellet was saved and washed in 1 mL of 75% ethanol 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.
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The cDNA was constructed by using a high capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). In each reaction, RNA (2 μg) was mixed together with MgCl2, 10 × RT buffer, random hexamers, dNTP, 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. Real-time PCR amplification was carried out on a Fast Real-time PCR system 7500 (Applied Biosystems, Foster city, CA, USA). Ten 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), Irp-1B (NCBI Reference Sequence NM_079579.3) 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 shown as a ratio of the day 0 control group value.
2.13. Western blot analysis The western blot analysis was conducted as we previously described with some modification (Peng et al., 2009). In brief, 50 flies were homogenized in a 1.5 mL tube containing 500 μL homogenizing buffer (20 mM Tris-HCl, 2 mM MgCl2, and 0.2 M sucrose, pH = 7.5) and protease inhibitor cocktail (Roche, Mannheim, Germany). The extracts were then centrifuged at 13,000g for 5 min at 4 ⁰C and the supernatant was collected. Total protein concentration was measured using a protein concentration assay kit according to the manufacturer's instructions (Bio-Rad, Hercules, CA, USA). After adding 6× loading dye, samples were denatured at 95 °C for 5 min and the stored at −80 °C until use. The total protein (20 μg) was size-fractionated by 15% SDS-PAGE at 130 V for 160 min. After that the gel was cut to two parts. The upper portion (containing β-actin and CAT) and lower portion (containing SOD1 and SOD2) were then transferred to Hybond-P PVDF membranes (Millipore Corporation, Billerica, MA, USA) at 15 V for 60 min and 40 min, respectively. The membranes were blocked in 5% non-fat milk in 1 × tris-buffered saline containing 0.1% Tween-20 (1 x TBST) at room temperature and then in the same solution containing diluted anti-catalase/anti-actin/anti-SOD1/anti-SOD2 antibodies, respectively, at 4 °C overnight. The membranes were then washed in 1 × TBST for 4 × 15 min and incubated for 1 h at 4 °C in diluted horseradish peroxidase-linked goat anti-rabit IgG or anti-mouse IgG (Santa Cruz Biotechnology, Inc., California, USA). The washes mentioned above were repeated before the membranes were developed with ECL enhance chemiluminescence agent (Santa Cruz Biotechnology, Inc., California, USA) and subjected to autoradiography on ChemiDocTM Touch Imaging System (Bio\\Rad Laboratories, Hercules, USA). Densitometry was analyzed using ChemiDocTM Touch Imaging System Software. Protein concentrations of CAT, SOD1 and SOD2 were normalized with β-actin.
2.14. Statistics The Kaplan-Meier test was used to compare the difference between the lifespan curves using SPSS 20.0 (Statistical Package for the Social Sciences software, SPSS Inc., Chicago, USA). Data were expressed as means ± standard deviation (SD). The significance of difference between means was assessed using two-way ANOVA. Difference were considered to be significant when P ﹤ 0.05.
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3. Results
Table 1 Lifespan of fruit flies fed a non-lard control diet (NCTL) or two non-lard diets containing 5
3.1. Effect of PSPA on the lifespan and stress resistance ability of fruit flies fed a non-lard control diet
mg/mL purple sweet potato anthocyanin extract (PSPA5) and 10 mg/mL purple sweet potato anthocyanin extract (PSPA10).
To assess the effect of PSPA on the ageing and stress resistance of fruit flies, PSPA was added into the standard fly diet at 5 mg/mL and 10 mg/mL. Results showed that none of these concentrations of PSPA significantly affected the lifespan of fruit flies compared with the nonsupplemented standard diet (Fig. 2, Table 1). Similarly, supplementation of 10 mg/mL PSPA in diet did not change the survival rate of fruit flies treated with paraquat and H2O2 (Fig. 3). This indicated that PSPA supplementation up to 10 mg/mL is not sufficient to promote longevity and improve the stress resistance ability under the culture condition using the non-fat standard fly diet. 3.2. Effect of lards on the lifespan of fruit flies The present study demonstrated clearly that high-fat diets shortened the lifespan of fruit flies. Results showed that both maximum survival time and 50% survival time of the flies were inversely related to the amounts of lard added in the diet (Fig. 4, Table 2). The non-lard control group (NCTL, 0% fat) had a maximum lifespan of 56 days, whereas the 20% lard group had only 35 days; the lifespan was shortened by 37.5%. Similarly, 50% survival time also decreased with the increasing amounts of lard. The NCTL group had a 50% survival time of 34 days, while the 20% lard group had only 17 days. In addition, when the lard was increased from 0 to 20%, the mean lifespan of the flies was decreased from 35.6 to 17.7 days. Data are expressed as the maximum lifespan of the last fruit fly, 50% survival time and mean lifespan (n = 200 flies per group, n = 20 flies per vial). Means in the same column with different superscripts differ significantly at P b 0.01.
NCTL PSPA5 PSPA10
Maximum lifespan of last fly (day)
50% survival (day)
Mean lifespan (day)
67 70 70
44 45 47
45.0 ± 0.8 46.7 ± 0.7 46.5 ± 0.7
supplemented with 10 mg/mL PSPA (Fig. 5, Table 3). The 50% survival time for the LCTL group was 27 days. In contrast, it increased to 29 and 30 days in LPSPA5 and LPSPA10, respectively. The LCTL group had a mean lifespan of 27.8 days, whereas that of the LPSPA5 and LPSPA10 groups increased to 29.1 and 30.8 days, respectively. There was no significance difference in food intake was seen among the NCTL, LCTL and LPSPA10 groups as reflected by the measurements of gustatory assay (Fig. 6). No significant differences in the body weights among the NCTL, LCTL and LPSPA10 groups were also observed (Fig. 6). Results on the climbing assay revealed that the climbing ability decreased with the age in both the non-lard standard and 10% lard diet groups (Fig. 7). Compared with the NCTL group, LCTL fruit flies decreased the climbing activity. However supplementation of PSPA into a 10% lard diet completely reversed the high fat-induced declined climbing ability. The LPO level in the fruit flies was significantly increased during ageing. Compared with the NCTL group, incorporation of lard into the diet could induce more production of LPO, while PSPA supplementation reduced production of LPO by 3.1% and 9.3%, respectively, in fruit flies maintained on a 10% fat diet for 10 and 35 days (Fig. 8).
3.3. Effect of PSPA on the lifespan of fruit flies fed a high fat diet Supplementation of PSPA could partially reverse the fat-induced mortality. The maximum lifespan was 49 days for a 10% lard control group (LCTL), whereas it was increased to 57 days in LPSPA10 diet
Fig. 2. Lifespan curves of fruit flies fed a non-lard control (NCTL) diet or two non-lard diets containing 5 mg/mL purple sweet potato anthocyanin extract (PSPA5) or 10 mg/mL purple sweet potato anthocyanin extract (PSPA10). 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 showed that none of these concentrations of PSPA significantly affected the lifespan of fruit flies compared to NCTL diet.
Fig. 3. Effect of paraquat and hydrogen peroxide (H2O2) treatment on the survival time of the wild type (OR) fruit flies fed a non-lard control diet (NCTL) or an experimental diet containing 10 mg/mL purple sweet potato anthocyanin extract (PSPA10). The KaplanMeier test found PSPA10 did not affect the survival rate of fruit flies challenged with paraquat or H2O2.
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4. Discussion
Fig. 4. Lifespan curves of fruit flies fed a non-lard control diet (NCTL) or diets containing 2% to 20% lard (L). Data were recorded until the last fruit fly died. Mean lifespan and 50% survival time were calculated in 200 flies (See Table 2).
Data are expressed as the maximum lifespan of the last fruit fly, 50% survival time and mean lifespan (n = 200 flies per group, n = 20 flies per vial). a,b,c Means at the same column with different superscripts differ significantly at P b 0.01.
3.4. Changes in gene expression and protein level induced by PSPA supplementation To investigate the molecular mechanisms by which PSPA improved the survival time in fruit flies fed a high fat diet, the mRNA levels of genes involved in ROS eliminating, nutrient sensing and insulin-like signaling pathways were measured. As shown in Fig. 9, mRNA SOD1 and SOD2 decreased significantly with age in all of the three groups. Incorporation of lard into the diet lowered the mRNA SOD1 and SOD2. However, supplementation of PSPA increased the mRNA of SOD1 and SOD2. Compared with the NCTL group, LCTL could significantly down-regulated the gene expression of CAT and RPn11, while LPSPA10 diet partially restored the expression level of CAT and Rpn11. Addition of lard downregulated Hep, while supplementation of PSPA further decreased its mRNA level (Fig. 9). For MTH, InR, TOR, PEPCK and Irp-1B, no differences in their mRNA levels were seen between fruit flies fed the LCTL and LPSPA10 diets (Fig. 9). The Western blot data showed that protein mass of SOD1 and SOD2 increased whereas the protein mass of CAT decreased with age in NCTL group flies. Addition of lard to the diet significantly down-regulated the protein mass of SOD1, SOD2 and CAT. However, PSPA group had protein mass of SOD1, SOD2, and CAT greater than that in the high fat control (Fig. 10).
The purpose of the present study was to investigate the effect of PSPA on lifespan of fruit flies. We had the following major observations. First, addition of dietary fat into diets shortened the lifespan of fruit flies in a dose-dependent manner (Fig. 4). Second, PSPA could prolong the lifespan of fruit flies fed a high fat diet but not that of fruit flies given a non-fat diet (Figs. 2 and 5). This prolonging effect of PSPA was unlikely due to calories restriction because the food intake was not altered by PSPA supplementation, as reflected by the absence of significant changes in the body weights and gustatory stomach redness between the control and PSPA fruit flies (Fig. 6). Third, addition of fat into diet negatively affected the locomotor behavior of fruit flies, while supplementation of PSPA into diet could completely restore the declined climbing ability (Fig. 7). Fourth, PSPA supplementation into diet of fruit flies fed a high fat diet could favorably modulate the genes of SOD1, SOD2, CAT and Rpn11 with little or no effect on the mRNA of MTH, InR, TOR, PEPCK and Irp-1B (Fig. 9). Fifth, supplementation of PSPA was associated with up-regulation protein mass of SOD1, SOD2, and CAT (Fig. 10), accompanied by a decreased in LPO level in the body of fruit flies (Fig. 8). These data strongly suggested that anthocyanin from purple sweet potato possessed the anti-ageing activity at least in fruit fly model given a high fat diet. Incorporation of dietary fat could accelerate the ageing process in fruit flies. The present results were in agreement with that of Driver and Cosopodiotis (1979), who reported that incorporation of palmitic acid into diet could shorten the lifespan of fruit flies by 20%. Li et al. (2008) demonstrated that addition of a mixture of fatty acids derived from lard into diet significantly shortened the lifespan and caused a greater mortality in D. melanogaster, while addition of green tea and black tea extract could partially restore the lifespan of fruit flies. It is known that addition of dietary fat into diet could induce formation of lipid hydroperoxide (Li et al., 2008), thus leading to a shorter lifespan in fruit flies. Like tea antioxidants catechins and theaflavins, PSPA is also an excellent antioxidant (Yousuf et al., 2015). In fact, the present results clearly demonstrated that PSPA could inhibit the lipid oxidation, partially attenuate the formation of LPO and thus decrease the mortality of fruit flies given a high fat diet (Fig. 8). The lifespan-prolonging activity of PSPA in fruit flies given a high fat diet is most likely mediated by its interaction with the gene expression of endogenous antioxidant enzymes. The present results clearly demonstrated that the high fat diet shortened the lifespan while addition of
Table 2 Lifespan of fruit flies fed a non-lard control diet (NCTL) or diets containing 2, 5, 10, 15, and 20% lard.
NCTL 2% lard 5% lard 10% lard 15% lard 20% lard
Maximum lifespan of last fly (day)
50% survival (day)
Mean lifespan (mean ± SD, day)
56 47 44 40 35 35
34 30 23 20 18 17
35.6 ± 0.5a 30.4 ± 0.5b 24.7 ± 0.4c 21.6 ± 0.4d 19.3 ± 0.3e 17.7 ± 0.3f
Fig. 5. Lifespan curves of fruit flies fed a non-lard control diet (NCTL) or a 10% lard control diet (LCTL), with addition of 5 mg/mL purple sweet potato anthocyanin extract (LPSPA5) or 10 mg/mL purple sweet potato anthocyanin extract (LPSPA10). Data were recorded until the last fruit fly died. Mean lifespan and 50% survival time were calculated in 200 flies (Table 3).
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Table 3 Lifespan of fruit flies fed a non-lard control diet (NCTL), a 10% lard control diet (LCTL) and two 10% lard experimental diets containing 5 mg/mL PSPA (LPSPA5) and 10 mg/mL PSPA (LPSPA10).
NCTL LCTL LPSP5 LPSP10
Maximum lifespan of last fly (day)
50% survival (day)
Mean lifespan (day)
58 49 49 57
33 27 29 30
34.2 ± 0.6a 27.8 ± 0.5c 29.1 ± 0.6 bc 30.8 ± 0.3b
PSPA could up-regulate the mRNA and protein mass of SOD1, SOD2 and CAT in fruit flies. This is in agreement with the report of Freeman et al. (2014), who demontrated that a high fat diet could shorten the lifespan and accelerate the ageing. ROS, the most abundant free radicals in cells, are the byproducts of normal cellular metabolism of oxygen. ROS can cause lipid oxidation, DNA damage, and protein oxidation, thus leading to the ageing (Harman, 1956). The endogenous oxidative defense system in flies consists predominantly of SOD1, SOD2 and CAT. In this regard, fruit flies with over-expression of SOD and CAT genes had as much as one-third extension of lifespan (Orr and Sohal, 1994; Sun et al., 2004). The exogenous antioxidants may also play an important role in deactivating the ROS. In this connection, many natural antioxidants, such as apple polyphenols, tea extracts, anthocyanins enrich extracts and sesamin, have been found to up-regulate SOD1, SOD2 and
Fig. 6. Average body weights and stomach redness index of fruit flies fed a non-lard control diet (NCTL), or a 10% lard diet control diet (LCTL), or a 10% lard diet containing 10 mg/mL purple sweet potato anthocyanin extract (LPSPA10). Data were expressed as mean ± SD.
Fig. 7. Climbing ability of fruit flies fed a non-lard control diet (NCTL), or a 10% lard diet control diet (LCTL), or a 10% lard diet containing 10 mg/mL purple sweet potato anthocyanin extract (LPSPA10). Data were expressed as mean ± SD. The 10% lard control diet decreased the climbing ability, whereas, LPSPA10 diet could completely restore the climbing ability of fruit flies (P b 0.01). df, degree of freedom; MS, mean squares; sig, significance.
CAT gene expression levels and extend fruit fly lifespan (Peng et al., 2014). In contrast, deletion or reduction of SOD2 gene severely shortened the lifespan of fruit flies (Duttaroy et al., 2003; Paul et al., 2007; Phillips et al., 1989). The present study suggested that the lifespanprolonging activity of PSPA was mediated at least partially by up-regulation of SOD and CAT genes in fruit flies. Many genes and signaling pathways have been reported to regulate the lifespan in model organisms. Rpn11 is one of components in the 26S proteasome and is mainly responsible for “waste” protein degradation by 26S proteasome (Verma et al., 2002; Lee et al., 2009). It was reported that knock down of Rpn11 reduced 26S proteasome activity, caused the accumulation of ubiquitinated proteins and shortened lifespan, while over-expression Rpn11 reduced the ubiquitinated protein accumulation and extended the lifespan (Tonoki et al., 2009). Hep encodes JNKK (cJun. N-terminal kinase kinase), which locates in the upstream of the JNK signaling. It was reported that fruit flies gained resistance to paraquat and had a longer lifespan when JNK signaling was promoted by overexpression of Hep (Wang et al., 2003). PEPCK encodes a key enzyme that controls gluconeogenesis and lipid metabolism. Irp-1B is
Fig. 8. Lipid hydroperoxide level in fruit flies fed a non-lard control diet (NCTL), or a 10% lard diet control diet (LCTL), or a 10% lard diet containing 10 mg/mL purple sweet potato anthocyanin extract (LPSPA10). Data were expressed as mean ± SD.
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Fig. 9. 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) in the fruit flies fed a non-lard control diet (NCTL), a 10% lard control diet (LCTL) and a 10% lard diet with supplementation of 10 mg/mL purple sweet potato anthocyanin extract (LPSPA10) at 0, 15, and 35 days (n = 300/group, n = 20/vial). Data are expressed as mean ± SD. df, degree of freedom; MS, mean squares; sig, significance.
involved in iron metabolism. Down-regulation of both PEPCK and Irp1B genes could extend the lifespan (Boyd et al., 2011). MTH, a G-protein coupled receptor, has been long recognized as one of the longevity-determined genes. Mutant flies with a reducing expression level of MTH exhibited stronger resistance to oxidative stress and longer lifespan (Lin et al., 1998; Paaby and Schmidt, 2008). InR and TOR signaling pathways are evolutionarily conserved from invertebrates to mammals. Previous studies demonstrated that inhibition on InR and TOR signaling pathways could significantly extend the lifespan in many species, including yeast, worms, fruit flies and mammals (Verdaguer et al., 2012). The present study demonstrated that a high fat diet down-regulated, while addition of PSPA only up-regulated the mRNA of Rpn11
without having effect on mRNA of PEPCK, Irp-1B, InR, MTH and TOR (Fig. 9). 5. Conclusion The present study demonstrated clearly that addition of lard reduced the locomotor power, increased the LPO production and shortened the lifespan of fruit flies. PSPA supplementation partially attenuated the high fat-induced mortality, increased the climbing ability and reduced the LPO production of fruit flies. It was concluded that the anti-ageing activity of PSPA was at least in part mediated by up-regulation of gene and protein mass of SOD, CAT and Rpn11.
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Fig. 10. The relative immunoreactive mass of SOD1, SOD2, and CAT of fruit flies fed a non-lard control diet (NCTL), or a 10% lard diet control diet (LCTL), or a 10% lard diet containing 10 mg/ mL purple sweet potato anthocyanin extract (LPSPA10). The results were normalized with the values of day 0 being 1.0.
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