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Experimental Gerontology 44 (2009) 773–783
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Experimental Gerontology
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Black tea theaflavins extend the lifespan of fruit flies
Cheng Peng a, Ho Yin Edwin Chan a, Yuk Man Li b, Yu Huang c, Zhen Yu Chen a,*
a Department of Biochemistry, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, Chinab Li Ka Shing Institute of Professional and Continuing Education, Open University of Hong Kong, Hong Kong, Chinac School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, China
a r t i c l e i n f o
Article history:Received 2 June 2009Received in revised form 18 July 2009Accepted 15 September 2009Available online 19 September 2009
Keywords:CatalaseBlack teaLifespanFliesLipid peroxidationSuperoxide dismutaseMethuselah
0531-5565/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.exger.2009.09.004
Abbreviations: CAT, catalase; CuZnSOD or SODsuperoxide dismutase; BTE, black tea extract; LPOmethuselah; MnSOD or SOD2, manganese containingsuperoxide dismutase; ROS, reactive oxygen species.
* Corresponding author. Tel.: +852 2609 6382; fax:E-mail address: [email protected] (Z.Y. Ch
a b s t r a c t
Black tea extract (BTE) is a mixture of epicatechins and theaflavins. The present study investigated theeffect of BTE on the lifespan of Drosophila melanogaster. Results showed the mean lifespan was signifi-cantly extended from 51 to 56 days upon BTE treatment. Gene expression of superoxide dismutase(SOD1 and SOD2), catalase (CAT), and methuselah (MTH) was characterized by an increase in youngand then a decrease in aged fruit flies. Higher gene expression of SOD1 and CAT was observed in theBTE-treated group than the control flies. However, BTE exerted a minimal effect on the expression ofSOD2 and MTH genes. Dietary fat could induce oxidative stress and shorten the maximum lifespan to15 days, while addition of 10 mg/ml BTE into diet extended it to 28 days. Paraquat and H2O2 challengetests demonstrated that BTE prolonged the survival time only for Oregon-R wild type flies but not forSODn108 or Catn1 mutants. This suggests that the lifespan-prolonging activity of BTE is mediated at leastin part through SOD and CAT.
� 2009 Elsevier Inc. All rights reserved.
1. Introduction
Interest in the relationship between diet and ageing is growing.Research has shown that a moderate reduction of nutrient intakeand/or dietary calorie restriction extends the lifespans of rodents(McCay et al., 1935), fruit flies (Partridge et al., 2005), nematodeworms (Lee et al., 2006), and yeast (Lin et al., 2002). The proposedunderlying mechanisms responsible for lifespan extension are thatdietary restriction may retard growth, reduce body fat, decreasethe metabolic rate, and attenuate oxidative stress (Masoro, 2009).Some earlier studies have also suggested that besides total energyintake, the composition of nutrients in the diet also affects the life-span and ageing of an organism (Piper and Bartke, 2008).
Dietary antioxidants have become popular supplements in pre-vention of ageing (Willis et al., 2009). Oxygen is essential to aerobicorganisms because it functions as a final electron acceptor. How-ever, oxygen can continuously generate reactive oxygen species(ROS), which are believed to be one of the causes of an organism’sageing (Gutteridge and Halliwell, 2000). Aerobic organisms possess
ll rights reserved.
1, copper–zinc containing, lipid hydroperoxide; MTH,
superoxide dismutase; SOD,
+852 2603 7246.en).
an antioxidant enzyme system, which includes superoxide dismu-tase (SOD), catalase (CAT), and glutathione peroxidase, to removethe ROS in cells (Cutler, 1991). In addition, dietary antioxidants,including ascorbic acid, vitamin A, vitamin C, a-tocopherol andplant flavonoids, are also responsible for scavenging the ROS incells (Ames et al., 1993). Both endogenous antioxidant enzymesand exogenous dietary antioxidants build a defense base to termi-nate the propagation of free radical reactions, limit the formationof new free radicals and slow down the ageing process.
Fruit fly, Drosophila melanogaster, is one of the most commonlyused models to investigate the genetic determinants of ageing(Minois, 2006). The Methuselah (MTH) gene has been shown to beinvolved in longevity in fruit flies, although its underlying mecha-nism remains poorly understood (Lin et al., 1998). Green tea is anexcellent source of dietary antioxidants as it contains fourepicatechin derivatives: (�)-epigallocatechin gallate (EGCG), (�)-epigallocatechin (EGC), (�)-epicatechin gallate (ECG), and (�)-epi-catechin (EC). We have previously demonstrated that green teaextracts are able to prolong the lifespan of fruit flies (Li et al.,2007). Black tea is also rich in antioxidants. In addition to EGCG,EGC, ECG, and EC, black tea also contains four theaflavin deriva-tives: theaflavin-1 (TF1), theaflavin-3-gallate (TF2A), theaflavin-30-gallate (TF2B), and theaflavin-3,30-digallate (TF3). In the presentstudy we investigated whether black tea extract (BTE) has thecapacity to scavenge free radicals and prolong the lifespan ofD. melanogaster. In particular, we focused on the interaction
774 C. Peng et al. / Experimental Gerontology 44 (2009) 773–783
between BTE and gene expression of the endogenous antioxidantenzymes SOD and CAT in D. melanogaster.
2. Materials and methods
2.1. BTE and lard fatty acids
BTE was purchased from Siming Natural Plant Co., Zhejiang,China. According to the supplier, BTE (theaflavins-enriched) wasprepared by enzymatic oxidation of green tea polyphenols (Tuand Xia, 2004). Tea epicatechin and theaflavin were quantifiedusing a Shimadzu LC-10AD HPLC (Tokyo, Japan) as we describedpreviously (Su et al., 2003). In brief, BTE mixture (10 ll, 0.5 mg/ml) was injected onto column (Hypersil ODS, 250 � 4.6 mm, 5 m,Alltech, Deerfield, IL, USA) via a Rheodyne valve (Shimadzu, Tokyo,Japan). The mobile phase consisted of 2% acetic acid in water (vol/vol) (Solvent A) and acetonitrile (Solvent B). After the injection ofthe sample, Solvent B was increased from 8% to 15% over 28 min,to 31% over additional 52 min and then back to the starting ratioover an additional 5 min. The flow rate was maintained at1.0 ml/min. The individual catechin and TF were monitored at280 nm and quantified using (+)-catechin as an internal standard.
Lard fatty acids were prepared according to the method previ-ously described (Li et al., 2008). In brief, 35 g potassium hydroxidewas ground into power and dissolved into 2 L of methanol solutioncontaining 130 g of lard. The mixture was heated for 2 h in a waterbath under a gentle stream of nitrogen gas to prevent oxidation.The methanol was removed in a rotary evaporator. The resultantsaponified mixture was then acidified using 10% sulfuric acid toprecipitate the free fatty acids followed by washing five times withdistilled water. The free fatty acids were then stored at �20 �C.
2.2. Fly strains
The fly strains used in this study included Oregon-R-C (OR),SODn108/TM3 (SODn108), and OE�/SM5 x Catn1/TM3 (Catn1) (Bloom-ington Drosophila Stock Center, Department of Biology, IndianaUniversity, Bloomington, IN, USA). OR is a wild type fly whichwas used in all experiments unless specified otherwise. SODn108
is a mutant with one pair of single SOD gene on 3L chromosomeknocked out, while Catn1 is a mutant with the CAT gene on chromo-some 3L knocked out by a point mutation.
2.3. Diet
A standard diet was prepared according to a previouslydescribed formulation (Li et al., 2007; Roberts and Standen,1998). In brief, 1000 ml diet contained 105 g cornmeal, 21 g yeast,105 g glucose, and 13 g agar. Ethyl-4-hydroxybenzoate (0.4%) wasadded into the diet to prevent mold growth. BTE was added intothe basal diet at 5 and 10 mg/ml, respectively. For the fat-inducedmortality experiments, the fatty acids derived from lard wereadded into the basal diet at 10% on a weight basis. The mixturewas cooked and poured into each vial (5 ml each). For rearingthe stocks, 15 ml of the basal diet was poured and set into a vial.For the experimental flies, 5 ml of the basal or experimental dietswas prepared per vial. BTE was obtained from Professor You-YingTu, Tea Research Institute of Zhejiang University, Hangzhou, China,while the lard fatty acids were prepared according to a previouslydescribed method (Li et al., 2008).
2.4. Effect of BTE on longevity of OR flies fed the basal diet
Male flies (2-day-old) developed from eggs were divided intothree groups, with 200 flies in each group, and were reared in 10
vials (20 flies per vial). The first group was maintained on the basaldiet, while the other two groups were fed one of the two dietscontaining 5 or 10 mg BTE/ml. The dead flies were counted every2–3 days and the remaining flies were transferred to a new vialcontaining the same diet. The feeding lasted 76 days. The two setsof experiments described above were similarly repeated and thefruit flies were killed at various time points to quantify the expres-sion of SOD, CAT, and MTH.
2.5. Effect of BTE on food intake and body weight of OR flies fed onstandard diet
Quantification of food intake by a fruit fly is technically difficultbecause they are small insects and evaporation of moisture fromfoods cannot be avoided during incubation. Therefore, the changein average body weight per fly was used as an indication ofwhether BTE could affect the food intake of fruit flies. In brief, fliesin each vial were anesthetized by carbon dioxide and then weighedin a balance (Mettler Toledo AG285, Switzerland) at day 0, 15, 25,35, 45, and 55. The average body weight per fly in each group wasrecorded.
An alternative method to measure food intake was to use thegustatory assay as previously described (Bahadorani and Hilliker,2008; Bahadorani et al., 2008). In brief, 60 newly eclosed male flieswere collected (20 per vial) and reared on a standard diet for 5 daysand then starved for 24 h on Kimwipes paper soaked with distilledwater. Afterward, flies were maintained on the standard or BTE-supplemented diet containing 0.2% sulforhodamine B sodium salt(Acid-Red) for 2 h. The flies were anesthetized by carbon dioxide,and the degree of abdomen redness was blind-scored using a grad-ing scale ranging from grade 0 (colorless abdomen) to grade 5(fully red abdomen). Food intake was compared on the basis ofthe difference in the degree of abdomen redness between the con-trol and BTE-fed group.
2.6. Paraquat treatment
Paraquat (1,10-dimethyl-4,40-bi-pyridinium dichloride; Pq2+)(Sigma, St. Louis, MO, USA) is able to generate superoxide anion rad-icals (Michaelis and Hill, 1933). To examine the resistance of flies tosuperoxide-induced stress, both OR flies (n = 400 in 20 vials) andSODn108 mutant flies (n = 400 in 20 vials) were maintained on eitherthe standard control diet or an experimental diet containing 10 mgBTE/ml. All the flies were raised at 25 �C. At day 25, the fruit flies inthe two groups were first starved for 2 h, and then transferred tonew vials containing a filter paper saturated with 1 ml of 20 mMparaquat diluted in a 6% glucose solution. The number of dead flieswas counted every 4–6 h until all the flies were dead.
2.7. Hydrogen peroxide (H2O2) treatment
H2O2 is able to generate a hydroxyl radical in the presence ofsome metal ions, and was therefore also used to examine the resis-tance of flies against OH-induced oxidative stress. OR flies (n = 400)and Catn1 mutant flies (n = 400) were maintained on either thestandard control diet or an experimental diet containing 10 mgBTE/ml and incubated at 25 �C. Similarly, the fruit flies in the twogroups were first starved for 2 h, and then were transferred tonew vials containing a filter paper saturated with 1 ml of 30%H2O2 diluted in a 6% glucose solution at day 25. The number ofdead flies was counted every 4–6 h until all the flies were dead.
2.8. Effect of BTE on longevity of wild type flies fed on a high-fat diet
Two-day-old male flies were divided into three groups with 200flies in each group, and reared in 10 vials (20 flies per vial). The first
0.50
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y W
eigh
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0
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Fig. 2. Effect of black tea extract (BTE) supplementation on body weight (Top) andstomach redness index in wild type flies (OR) fed on the control (CTL) and the BTEdiet. Data are expressed as means ± SD.
C. Peng et al. / Experimental Gerontology 44 (2009) 773–783 775
group was maintained on the standard control diet containing nolard fatty acids (CTL), while the two experimental groups werefed on diets containing 10% lard fatty acid only [CTL(lard)] or withthe addition of 10 mg BTE/ml [BTE(lard)]. The dead flies werecounted every 2–3 days and the flies that remained alive were thentransferred to a new vial containing the same diet. The feedinglasted 32 days. The fruit flies were killed at day 0, 5, and 10 toquantify the gene expression of SOD, CAT and MTH.
2.9. Measurement of lipid hydroperoxides (LPO)
An LPO assay kit was used to quantify the lipid oxidation in fruitflies (Cayman Chemical, Michigan, USA). Hydroperoxides react withferrous ion to produce ferric ion, which can be detected using thio-cyanate ion as a chromogen. Two hundred fruit flies were homoge-nized in 2 ml of HPLC grade water and centrifuged at a speed of1500g for 5 min at 0 �C. The supernatant (800 ll) was aliquoted intriplicates followed by deproteinization and extraction using a mix-ture of methanol and chloroform (1:2, vol/vol) saturated with nitro-gen gas. The sample was then centrifuged at 1500g for 5 min at 0 �C,and the chloroform layer was collected in a test tube on ice. To ini-tiate the reaction, 500 ll of chloroform extract together with 450 llof methanol/chloroform solvent (1:2, vol/vol) was mixed with thechromogen, which contained 50 ll of FTS Reagent 1 (4.5 mM fer-rous sulfate in 0.2 M hydrochloric acid) and 50 ll FTS Reagent 2(3% methanolic solution of ammonium thiocyanate). After incuba-tion at room temperature for 5 min, the absorbance of each samplewas measured at 500 nm using a quartz cuvette.
2.10. SOD activity
An assay kit was used to quantify the SOD activity in fruit flies(Cayman Chemical, Michigan, USA). The principle is that a tetrazo-
Fig. 1. HPLC chromatogram of black tea extract (BTE). Peak identification: 1, epigallocagallate (ECG); 5, theaflavin-1 (TF1); 6, theaflavin-3-gallate (TF2A); 7, theaflavin-30-gallat
lium salt can detect superoxide anion generated by xanthine oxi-dase and hypoxanthine while SOD is able to remove thesuperoxide anion. In general, one unit of SOD is defined as theamount of enzyme needed to exhibit 50% dismutation of the super-oxide anions. The fruit flies (n = 100 in 5 vials) were homogenized
techin (EGC); 2, epicatechin (EC); 3, epigallocatechin gallate (EGCG); 4, epicatechine (TF2B); 8, theaflavin-3,30-digallate (TF3).
0%
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100%
0 30 40 50 60 70 80Days
Su
rviv
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ate
CTLBTE5BTE10
P<0.01, BTE5 Vs CTLP<0.01, BTE10 Vs CTL
Fig. 3. Lifespan curve of wild type flies (OR) fed on diets containing 0 mg/ml(control), 5 mg/ml and 10/ml mg black tea extract (BTE5 and BTE10). Data wereexpressed as the maximum lifespan of last fly, 50% survival time and mean lifespan(n = 200 flies) for each group (Table 1). The Kaplan–Meier test showed both BTE5and BTE10 could significantly extend the mean lifespan of fruit flies (p < 0.01).
Table 1Lifespan of OR wild type flies fed the control diet and the two experimental dietscontaining 5 and 10 mg/ml BTE.
Maximum lifespan of lastfly (day)
50% survival(day)
Mean lifespan(day)
Control 74 50 51 ± 2a
5 mg/mlBTE
77 55 55 ± 2b
10 mg/mlBTE
77 56 56 ± 2b
a,bMeans with different letters in the same column differ significantly at p < 0.05.
776 C. Peng et al. / Experimental Gerontology 44 (2009) 773–783
in 1 ml of cold 20 mM HEPES buffer (pH 7.2, with 1 mM EGTA,210 mM mannitol, and 70 mM sucrose) and then centrifuged at aspeed of 1500g for 5 min at 4 �C. The supernatant was transferredto a new tube on ice and then subjected to centrifugation at 10,000
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Fig. 4. Effect of black tea extract (BTE) supplementation (10 mg/ml diet) on the whole bosuperoxide dismutase (SOD1), manganese containing superoxide dismutase (SOD2) and cgroup, n = 20/vial) were incubated at 25 �C for 0, 25, and 45 days. Data are expressed as
g for 15 min at 4 �C. The supernatant contained the cytosolic cop-per–zinc containing SOD (CuZn SOD or SOD1), and the pelletcontained mitochondrial manganese containing SOD (Mn SOD orSOD2). The supernatant was transferred to a new tube and themitochondrial pellet was resuspended in 0.5 ml cold HEPES buffer.The sample (10 ll) in triplicates was used for each test. The dilutedradical detector containing tetrazolium salt (200 ll) was addedonto 96-well plates together with 10 ll sample. The reaction wasinitiated by adding 20 ll of diluted xanthine oxidase and thenshaking the plate for 20 min at room temperature. After incuba-tion, the absorbance was recorded at 450 nm using a micro-platereader.
SOD2
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dy lipid hydroperoxide (LPO) level, and enzymatic activity of copper–zinc containingatalase (CAT) compared with the control diet (CTL). The wild type (OR) flies (n = 300/mean ± SD. *p < 0.05; **p < 0.01 compared with the control value.
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Fig. 5. Effect of black tea extract (BTE) supplementation (10 mg/ml diet) on mRNA of copper–zinc containing superoxide dismutase (SOD1), manganese containingsuperoxide dismutase (SOD2), catalase (CAT), and methuselah (MTH) compared with the control diet (CTL). The wild type (OR) flies (n = 300/group, n = 20/vial) wereincubated at 25 �C for 0, 15, 25, 35, 45, and 55 days. Data are expressed as mean ± SD. *p < 0.05; **p < 0.01 compared with the control value.
C. Peng et al. / Experimental Gerontology 44 (2009) 773–783 777
2.11. CAT activity
CAT was measured using a catalase assay kit (Sigma, St. Louis,MO, USA). The principle is based on the measurement of the hydro-gen peroxide substrate remaining after the action of CAT present inthe sample. The flies (n = 100) were homogenized in 1 ml enzymedilution buffer and then centrifuged at a speed of 1500g for 5 minat 4 �C. The supernatant was moved into a new tube and diluted 15times by 1� assay buffer (5 mM potassium phosphate buffer, pH7.0) in triplicates. The resultant sample (10 ll) was diluted againwith 65 ll of 1� assay buffer. Then, 25 ll of 200 mM hydrogen per-oxide solution was added to initiate the reaction. At exactly 1 min,900 ll of stop solution (15 mM sodium azide) was added. The reac-tion mixture (10 ll) was mixed with 1 ml color reagent containing0.25 mM 4-aminoantipyrine, 2 mM 3,5-dichloro-2-hydroxyben-zenesulfonic acid, and freshly added peroxidase (0.8–1.2 U/mg).After incubation at room temperature for 15 min, absorbance ofeach sample was measured using a spectrometer at 520 nm.
2.12. Real-time PCR
Total RNA was extracted using the commercial extraction agentTRIzol (Invitrogen, Carslbad, CA, USA). Fruit flies (n = 15) werehomogenized in 800 ll of TRIzol solution and centrifuged at12,000 g at 4 �C for 10 min. The supernatant was transferred intoa new tube containing 160 ll chloroform. The mixture was thensubjected to centrifugation at 12,000 g at 4 �C for 15 min. Theupper layer was mixed with 400 ll isopropanol. After incubationat room temperature for 10 min, the samples were centrifuged at12,000 g at 4 �C for 10 min, and the pellet was saved and washedin 1 ml of 75% ethanol and re-centrifuged. Finally, 25 ll DEPCwater was employed to resuspend the RNA pellet. The concentra-tion and purity of the RNA obtained were checked by measuring
its absorbance at 260 and 280 nm. High Capacity cDNA ReverseTranscription Kit (Applied Biosystems, Foster City, CA, USA) wasused to construct cDNA. RNA (2 lg) was used for each reaction to-gether with MgCl2, 10� RT buffer, dNTPs, random hexamers, RNaseinhibitor, and MultiScribe Transcriptase. The final volume was ad-justed to 10 ll. cDNA was synthesized in the thermocycler Gene-Amp PCR system 9700 (Applied Biosystems, Foster City, CA, USA)and stored at �20 �C.
Real-time PCR amplification was carried out on a Fast Real-timePCR System 7500 (Applied Biosystems, Foster City, CA, USA). Thefour target genes were SOD1 (NCBI Reference SequenceNM_057387.3), SOD2 (NCBI Reference Sequence NM_057577.2),CAT (NCBI Reference Sequence NM_080483.2), and MTH (NCBI Ref-erence Sequence NM_079147.2). The expression of the target geneswas normalized with that of rp49 (NCBI Reference SequenceNM_079843.2), and a housekeeping gene used as an internal con-trol. Gene expression was calculated on the basis of the compara-tive threshold cycle (CT) value. Levels of gene expression in allgroups were shown as a ratio of the day 0 control group value.
2.13. Western blot Analysis
Total proteins were extracted and subjected to Western blotanalysis. In brief, 50 flies were homogenized in a 1.5 ml tube con-taining 500 ll homogenizing buffer (20 mM Tris–HCl, 2 mM MgCl2,and 0.2 M sucrose) and protease inhibitor cocktail (Roche, Mann-heim, Germany). The extracts were centrifuged at 13,000 g for5 min at 4 �C and the supernatant was collected. Protein concentra-tion was determined using a protein concentration assay kit inaccordance with the manufacturer’s instructions (Bio-Rad, Hercu-les, CA, USA). After adding 6� loading dye and homogenizing bufferto adjust the volume, the protein was boiled at 95 �C for 5 min andthen stored at �80 �C. For the measurement of CAT and b-actin,
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* *
Fig. 6. Effect of black tea extract (BTE) supplementation (10 mg/ml diet) on the relative immunoreactive mass of copper–zinc containing superoxide dismutase (SOD1),manganese containing superoxide dismutase (SOD2), and catalase (CAT) compared with the control diet (CTL). The wild type (OR) flies (n = 300/group, n = 20/vial) wereincubated at 25 �C for 0, 15, 25, 35, 45, and 55 days. Data are expressed as mean ± SD. *p < 0.05 compared with the control value.
778 C. Peng et al. / Experimental Gerontology 44 (2009) 773–783
20 lg total protein was size-fractionated on a 7% SDS–PAGE gel at130 V for 70 min. For the measurement of SOD1 and SOD2, the
same amount of total protein was loaded and size-fractionatedon a 15% SDS–PAGE gel at 130 V for 180 min. The protein was then
Wild Type Flies
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P<0.05, BTE Vs CTL
Fig. 7. Effect of paraquat treatment or hydrogen peroxide treatment on the survival time of (a) mutant flies (SODn108) and (b) mutant flies (Catn1) fed on diets containing 0 mg/ml (CTL) or 10 mg/ml black tea extract (BTE) compared with that of the wild type (OR) flies. The Kaplan–Meier test found both that BTE-fed OR group survived better than itscorresponding OR control (p < 0.05) while the survival rate of the BTE-fed SODn108 and Catn1 groups was not different from their corresponding control groups.
C. Peng et al. / Experimental Gerontology 44 (2009) 773–783 779
transferred onto a Hybond-P PVDF membrane (Millipore, Billerica,MA, USA). The membrane was incubated for one hour in blockingsolution (5% non-fat milk) at room temperature and then in the
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a
bba
bb
Fig. 8. Lifespan of OR fruit flies fed either a diet containing no lard fatty acid[CTL(no lard)] or a diet containing 10% lard fatty acid [CTL(lard)] with addition of10 mg black tea extract [BTE(lard) at 25 �C. The Kaplan–Meier test found both thatBTE(lard) group survived better than the CTL(lard) (p < 0.01). (B) Effect of BTE on thewhole body lipid hydroperoxide (LPO) level in CTL(no lard), CTL(lard), and BTE(lard)groups. a,bMeans at the same time point differ significantly at p < 0.05.
same solution containing diluted anti-catalase/anti-actin/anti-SOD1/anti-SOD2 antibodies, respectively, at 4 �C overnight. Themembrane was then washed in 1� TBST and incubated for onehour at 4 �C in diluted horseradish peroxidase-linked goat anti-rabbit IgG (Santa Cruz Biotechnology, Inc., California, USA) oranti-mouse IgG (Santa Cruz Biotechnology, Inc., California, USA).The washes were repeated before the membranes were developedwith ECL enhanced chemiluminescence agent (Santa Cruz Biotech-nology, Inc., California, USA) and subjected to autoradiography forone second to five minutes on SuperRX medical X-ray film (Fuji,Tokyo, Japan). Densitometry was quantified using the computersoftware Quantity one (Bio-Rad, CA, USA).
2.14. Statistics
Data were expressed as means ± standard deviation. The Kap-lan–Meier test was employed to compare the difference betweenthe survival curves using SPSS 15.0 (Statistical Package for the So-cial Sciences software, SPSS Inc., Chicago, USA). The significance ofdifference between means was assessed using T-test and one wayANOVA. Differences were considered significant when p < 0.05.
3. Results
3.1. Composition of BTE
HPLC analysis showed that BTE used in the present study was amixture of epicatechins and theaflavins (Fig. 1). Results revealedthat BTE contained 11% TF1, 12% TF2A, 13% TF2B, 24% TF3, 12% EC,12% EGC, 10% EGCG, and 6% ECG.
3.2. Effect of BTE on food intake and body weight of OR flies fed onstandard control diet
No significant difference in average body weight was observedbetween the control and BTE-fed flies. Similarly, the stomach red-
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Fig. 9. The relative immunoreactive mass of copper–zinc containing superoxidedismutase (SOD1), manganese containing superoxide dismutase (SOD2), andcatalase (CAT) in OR fruit flies fed either a diet containing no lard fatty acid[CTL(no lard)] or a diet containing 10% lard fatty acid [CTL(lard)] with addition of10 mg black tea extract [BTE(lard) at 25 �C.
780 C. Peng et al. / Experimental Gerontology 44 (2009) 773–783
ness index in the BTE-fed flies was not significantly different fromthat of the control group (Fig. 2).
3.3. Effect of BTE on longevity of OR flies fed on standard control diet
Both 5 and 10 mg/ml BTE treatment showed a lifespan exten-sion effect on OR wild type male flies. The maximum life spanincreased more than 4% in BTE-treated groups compared withthe control group. Meanwhile, the 50% survival time was ex-tended from 50 days to 55 (5 mg BTE/ml) and 56 (10 mg BTE/ml) days. The mean lifespan for the control and the two BTE-treated groups were 55 (5 mg BTE/ml) and 56 (10 mg BTE/ml)days, as compared with 51 days for flies fed on the standardcontrol diet (Fig. 3 and Table 1). The Kaplan–Meier test demon-strated that both 5 and 10 mg BTE/ml treatment could signifi-cantly extend the mean lifespan of fruit flies (p < 0.05).However, no significant difference was found between the twoconcentrations of BTE used.
The present study also investigated the effect of 10 mg BTE/ml-containing diet on the activity of SOD1, SOD2, and CAT in OR wildtype male flies at days 0, 25, and 45 (Fig. 4). No significant differencein SOD1 and SOD2 activity was observed between the control andthe BTE-treated group. In contrast, CAT activity decreased with age-ing, and the BTE-treated group showed a greater CAT activity thanthe standard control diet group at day 25. The total body LPO levelincreased with ageing. Supplementation of 10 mg BTE/ml in the dietcould reduce the LPO formation significantly at day 45 (p < 0.05).
Gene expression of SOD1, SOD2, CAT, and MTH in wild type flieswas studied, and we found that the expression of these genes first in-creased and then decreased with age in both the control and 10 mg/ml BTE-treated groups (Fig. 5). The expression level of SOD1 wasgreater in the BTE-treated group than in the control at days 35 and55, while gene expression of CAT in the BTE-treated group was great-er than in the control at day 15 and 25. Otherwise, no significant dif-ference in gene expression of SOD1, SOD2, CAT, and MTH wasobserved between the control and the BTE-treated group.
Western blot analyses found that the changes in protein abun-dance of SOD1, SOD2, and CAT were consistent with their corre-sponding patterns of gene expression in both the control andBTE-treated groups (Fig. 6). We also found that the CAT protein le-vel, but not the SOD1 and SOD2 protein levels, was greater in theBTE-treated group than in the control at days 15 and 25.
3.4. Effect of BTE on paraquat and H2O2 resistance in OR, Catn1, andSODn108 flies
Results from the paraquat challenge test showed that the10 mg/ml BTE diet could prolong the survival time of OR wild type(p < 0.05) flies, but not that of SODn108 flies (Fig. 7). When fed BTE-containing diet, OR wild type flies showed a maximum survivaltime of 74 h while the standard control diet OR flies only survivedfor 70 h. The 50% survival time was prolonged by 18% in the BTE-treated OR flies compared with the control flies (p < 0.05). In con-trast, the maximum and the 50% survival time of SODn108 flies fedwith the BTE-containing diet did not differ significantly from thoseof the control diet group.
Similar results were also observed in the H2O2 challenge test.The maximum survival time and the 50% survival time were allprolonged in the BTE-treated OR wild type but not in the BTE-trea-ted Catn1 flies (Fig. 7).
3.5. Effect of BTE on longevity of OR flies fed a high-fat diet
Addition of 10% fatty acid derived from lard could induce oxida-tive stress and significantly shorten the maximum lifespan of ORwild type flies to 15 days (Fig. 8A). Supplementation of 10 mg/ml
of BTE in the diet could partially reverse such fat-induced mortal-ity. We showed that the maximum lifespan was prolonged from15 days in the control group to 28 days in the BTE-treated group(an 87% increase). Addition of fatty acids to the diet increased
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Fig. 10. mRNA of copper–zinc containing superoxide dismutase (SOD1), manganese containing superoxide dismutase (SOD2), and catalase (CAT) in OR fruit flies fed either adiet containing no lard fatty acid [CTL(no lard)] or a diet containing 10% lard fatty acid [CTL(lard)] with addition of 10 mg black tea extract [BTE(lard) at 25 �C. a,bMeans at thesame time point differ significantly at p < 0.05.
C. Peng et al. / Experimental Gerontology 44 (2009) 773–783 781
the LPO values. However, no statistical difference was observed be-tween the control [lard] and the BTE-treated group [lard] (Fig. 8B).
Western blot analyses did not show any changes in proteinabundance of SOD1, SOD2, and CAT between the control andBTE-treated OR flies (Fig. 9). SOD1 gene expression however in-creased during the same period of study. At day 5, the control(lard) showed a significant decrease in SOD gene expression whileBTE treatment increased the SOD gene expression to a level similarto that of the normal diet control (no lard) (Fig. 10). At day 10, lardfatty acid treatment down-regulated SOD2 gene expression butBTE exerted no modulatory effect on SOD2 expression. At day 10,both CAT and MTH gene expression were down-regulated signifi-cantly with the addition of lard fatty acids to the diet, while BTEsupplementation could up-regulate expression of these genes tothe levels of the normal diet control (no lard) (Fig. 10).
As far as enzyme activity was concerned, no difference in SOD1and SOD2 activity was found between the control and treatmentgroups (Fig. 11). However, addition of lard fatty acid decreasedCAT activity while addition of BTE completely restored it (Fig. 11).
4. Discussion
The present study is the first investigation of the effect of BTE onthe lifespan of D. melanogaster. Results clearly demonstrated thatBTE could prolong the mean lifespan and 50% survival time of fruitflies. This observation was in agreement with the findings by Cuiet al. (1999) and Li et al. (2007, 2008) that green tea extract couldextend the lifespan of fruit flies. An EGCG-containing diet was alsoreported to extend the lifespan of Caenorhabditis elegans by 10–170% (Abbas and Wink, 2009; Zhang et al., 2009). Finally, whenmale mice were given a water solution containing 80 mg/l of teapolyphenols at the age of 13 months until death, their average life-span increased by 6.4% (Kitani et al., 2007). Although there is no di-rect evidence to show that drinking tea increases human lifespans,it has been reported that daily consumption of green tea in suffi-cient amounts could prolong life by averting premature death, par-ticularly death caused by cancer (Nakachi et al., 2003).
The underlying mechanisms by which BTE extends the meanlifespan of fruit flies remain poorly understood. One possiblemechanism is probably related to the free radical scavengingactivity of BTE. It is generally believed that the free radical speciescan cause deterioration of an organism while the antioxidant candelay the process of ageing (Gutteridge and Halliwell, 2000).HPLC analysis showed that BTE used in the present study con-tained 60% theaflavins and 40% epicatechins. It is known thatboth theaflavins in black tea and epicatechins in green tea areequally effective antioxidants (Leung et al., 2001). In fact, thepresent study demonstrated that BTE treatment reduced totalbody LPO level (Figs. 4 and 8). In HPF-1 cells, theaflavins generallyexhibit a greater antioxidant activity than EGCG (Yang et al.,2008). In this regard, the genetically manipulated long lived strainof D. melanogaster (La strain) has been shown to produce a lowerLPO level with a greater activity of SOD and CAT activities at alltime points during its life than the short lived strain, suggestingthat oxidative stress is at least one of the factors leading to ageing(Arking et al., 2000).
The second mechanism by which BTE extents the lifespan offruit flies may be mediated by up-regulation of endogenous anti-oxidants enzymes of SOD1 and CAT at both transcriptional andtranslation levels. In wild type flies, expression of these enzymeswas characterized by an increase in lifespan of up to 15 days fol-lowed by a decrease thereafter (Figs. 5 and 6), suggesting that theability of scavenging the free radicals in fruit flies decreases withageing. We showed that BTE supplementation could up-regulateSOD1 and CAT more than in fruit flies fed with a control diet.In this regard, the following two explanations are offered. First,both Western blot and RT-PCR analyses are semi-quantitativeand insufficiently sensitive to detect the difference in geneexpression of these enzymes associated with BTE supplementa-tion. Second, three vials of fruit flies per time point might notgenerate enough power to conduct the statistical analyses. Therehas been no report to date regarding the effect of BTE on lifespanof fruit flies, so we were only able to make comparisons with thedata from published studies of green tea epicatechins. We have
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Fig. 11. Enzymatic activity of copper–zinc containing superoxide dismutase(SOD1), manganese containing superoxide dismutase (SOD2), and catalase (CAT)in OR fruit flies fed either a diet containing no lard fatty acid [CTL(no lard)] or a dietcontaining 10% lard fatty acid [CTL(lard)] with addition of 10 mg black tea extract[BTE(lard) at 25 �C. a,bMeans at the same time point differ significantly at p < 0.05.
782 C. Peng et al. / Experimental Gerontology 44 (2009) 773–783
previously demonstrated that green tea epicatechins could up-regulate the expression of both SOD and CAT in flies maintainedon a diet containing 10 mg/ml green tea epicatechins (Li et al.,2007, 2008). Sohal et al. (1995) found that flies had increasedSOD activity by 26% and CAT activity by 73% in response to a34% increase in lifespan. In our previous report (Li et al., 2007),it was found that supplementation of green tea epicatechins indiet increased the mean lifespan by 16% in response to 32% activ-ity enhancement in SOD1, 40% in SOD2 and 19% in CAT. However,Orr and Sohal (1993) showed that over-expression of SOD1 alonedid not increase the lifespan of the flies. In another study, over-expression of CAT gene alone did not increase the longevity ofthe flies (Mockett et al., 2003). This suggests that longer lifespan
of the flies is associated with up-regulation of gene expression ofboth SOD and CAT.
Additional evidence supporting the lifespan-prolonging effect ofBTE in fruit flies is associated with the up-regulation of genes forSOD and CAT. Paraquat and H2O2 challenge tests demonstratedthat BTE prolonged the survival time only in OR wild type fliesbut did not affect the survival of SODn108 or Catn1 mutants, in whichthe gene of either SOD or CAT was knocked out (Fig. 7). Results im-plied that the lifespan-prolonging activity of BTE was at least med-iated in part by the interaction of BTE with the SOD and CAT genes.In this regard, BTE has been shown to significantly elevate theactivity and gene expression of SOD in various models (Khan,2006; Das et al., 2002). Similarly, green tea epicatechins have beenalso shown to up-regulate the gene expression of SOD and CAT(Chow et al., 2002; Das et al., 2002; Mori and Hasegawa, 2003;Yamamoto et al., 2003; Ying et al., 2004).
Experiments in various models have shown that caloric restric-tion or reduction in food intake extends lifespan (Lee et al., 2006;Lin et al., 2002; McCay et al., 1935; Partridge et al., 2005). One con-cern with our data was that BTE might make the food less appeal-ing to the flies and therefore reduce food intake. In this regard, it isimpossible to directly measure the food consumption by each flybecause its food intake is too small to be quantified and the mois-ture lost from diet during incubation makes the measurement evenmore difficult. To rule out the possibility that BTE might affect thefood intake, we opted to use the gustatory assay and measure thechange in body weight as an indirect measure. It was assumed thatthe food intakes were the same if there was no difference in theaverage body weight and the stomach redness index between thecontrol and the BTE-fed groups. We found that BTE did not affectthe body weight and the stomach redness index, implying it wasunlikely that the lifespan-prolonging activity of BTE in the fruitflies was associated with any changes in food intake.
It was noteworthy that gene expression of SOD1, SOD2, and CATdecreased after day 15 (Fig. 5), suggesting that the oxidative de-fense system becomes weaker in aged fruit flies compared withtheir younger counterparts. In this regard, over-expression of thesegenes in Saccharomyces cereviseae and D. melanogaster has beenshown to reduce oxidative damage and extend lifespan (Landisand Tower, 2005). Similarly, a gradual decrease in gene expressionof MTH was also observed after day 15 in aged fruit flies (Fig. 5).The MTH gene in D. melanogaster has been a major target of inter-est in the biology of aging (Lin et al., 1998; Paaby and Schmidt,2008). MTH encodes the G-protein coupled receptor and MTH mu-tants have extended longevity. However, we did not find any sig-nificant effect of BTE on the gene expression of MTH.
In conclusion, gene expression of SOD1, SOD2, CAT, and MTHdecreased with ageing. The present study showed that BTE supple-mentation could increase the mean lifespan and survival time of D.melanogaster under various oxidative stress with reduction of theLPO level. The effect of BTE on prolonging the lifespan of fruit flieswas associated partially with up-regulation of the expression ofendogenous SOD1 and CAT but probably not with that of SOD2and MTH. It should be pointed out that the bioavailability of teaepicatechins and theaflavins is very low in both humans and rats(Wiseman et al., 2001). Future studies should address the issueof bioavailability and metabolism of tea epicatechins and theaflav-ins in fruit flies and their interaction with SOD1 and CAT at cellularlevel.
Acknowledgements
We are grateful to Professor John P. Phillips for SODn108/TM3strains the Bloomington Stock Center for Oregon-R-C and OE�/SM5 x Catn1/TM3 strains. We would like to thank Doctor David Wil-mshurst for comments on a draft of this paper.
C. Peng et al. / Experimental Gerontology 44 (2009) 773–783 783
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