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Green tea aroma fraction reduces β-amyloid peptide-induced toxicity in Caenorhabditis elegans transfected with human β-amyloid minigene Atsushi Takahashi
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, Tatsuro Watanabe , Takashi Fujita , Toshio Hasegawa , Michio Saito a
& Masami Suganuma a
Research Institute for Clinical Oncology, Saitama Cancer Center, Saitama, Japan
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Graduate School of Science and Engineering, Saitama University, Saitama, Japan
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Green Tea Laboratory, Saitama Prefectural Agriculture and Forestry Research Center, Saitama, Japan Published online: 12 Jun 2014.
Click for updates To cite this article: Atsushi Takahashi, Tatsuro Watanabe, Takashi Fujita, Toshio Hasegawa, Michio Saito & Masami Suganuma (2014) Green tea aroma fraction reduces β-amyloid peptide-induced toxicity in Caenorhabditis elegans transfected with human β-amyloid minigene, Bioscience, Biotechnology, and Biochemistry, 78:7, 1206-1211, DOI: 10.1080/09168451.2014.921553 To link to this article: http://dx.doi.org/10.1080/09168451.2014.921553
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Bioscience, Biotechnology, and Biochemistry, 2014 Vol. 78, No. 7, 1206–1211
Green tea aroma fraction reduces β-amyloid peptide-induced toxicity in Caenorhabditis elegans transfected with human β-amyloid minigene Atsushi Takahashi1,2,3, Tatsuro Watanabe1, Takashi Fujita2, Toshio Hasegawa2, Michio Saito3 and Masami Suganuma1,* 1
Research Institute for Clinical Oncology, Saitama Cancer Center, Saitama, Japan; 2Graduate School of Science and Engineering, Saitama University, Saitama, Japan; 3Green Tea Laboratory, Saitama Prefectural Agriculture and Forestry Research Center, Saitama, Japan
Received December 12, 2013; accepted February 27, 2014
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http://dx.doi.org/10.1080/09168451.2014.921553
Green tea is a popular world-wide beverage with health benefits that include preventive effects on cancer as well as cardiovascular, liver and Alzheimer’s diseases (AD). This study will examine the preventive effects on AD of a unique aroma of Japanese green tea. First, a transgenic Caenorhabditis elegans (C. elegans) CL4176 expressing human β-amyloid peptide (Aβ) was used as a model of AD. A hexane extract of processed green tea was further fractionated into volatile and non-volatile fractions, named roasty aroma and green tea aroma fractions depending on their aroma, by microscale distillation. Both hexane extract and green tea aroma fraction were found to inhibit Aβ-induced paralysis, while only green tea aroma fraction extended lifespan in CL4176. We also found that green tea aroma fraction has antioxidant activity. This paper indicates that the green tea aroma fraction is an additional component for prevention of AD. Key words:
Alzheimer’s disease; β-amyloid peptide; paralysis; lifespan; nematodes
Green tea is an acknowleged cancer preventive1) that is also associated with preventive effects on cardiovascular and liver diseases,2,3) resulting in longer lifespan.4) The active factors in these effects are (-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC), and (-)-epicatechin gallate (ECG), all found in the water-soluble fraction of green tea. Although (-)-epicatechin (EC) is an inactive catechin, it enhances the active catechin-induced activities, such as induction of apoptosis in lung cancer cells, PC-9, and inhibition of tumor necrosis factor release induced by tumor promoters.5) Thus, whole green tea infusion is the best mixture to gain health benefits.6) In addition to catechins, the aroma in the lipid-soluble fraction of green tea has recently attracted the attention of scien-
tists and doctors of aroma therapy, mental relaxation, and complementary and alternative medicine treatments. Using gas chromatography–mass spectrometry (GC–MS), scientists previously found over 630 volatile compounds in green tea.7,8) The odor-active compounds include various terpenes, such as geraniol and linalool,9,10) and the balance of numerous aroma compounds make up the essence of tea aroma. After reading some basic studies on aroma compounds, we became interested in the fractions of Japanese green tea (Camellia sinensis var. sinensis) obtained using microscale distillation under reduced pressure.11) The hexane extract from Japanese green tea was fractionated into volatile and non-volatile fractions using microscale distillation at 150 °C. The volatile fraction contains odoractive terpenes usually found in flowers and herbs, so we named it “roasty aroma fraction”; the non-volitile fraction has a unique green tea aroma that we named “green tea aroma fraction”.12) Dementia, including Alzheimer’s disease (AD) and vascular dementia, increases exponentially with aging: 1% of 60-year-olds have AD, while about 30% of 85year-olds have it.13) It is roughly estimated that there are 2,000,000 AD patients in Japan and 4,500,000 patients in the USA. Especially in developed countries, the number of AD patients is increasing with elongation of lifespan, so AD is becoming a widespread social dilemma. The pathogenesis of AD includes a number of different processes, including oxidative stress, inflammation, and neuronal dysfunction due to neuronal degeneration.14) Beta-amyloid (Aβ) plaques and hyperphosphorylated tau neurofibrillary tangles are common age-related lesions that are presumed to play primary roles in neuronal dysfuncitons of AD, but it is also understood that the correlation between these neuropathological lesions and dementia is not great. Recently, however, synaptic loss, which is the key neuropathological correlate of dementia severity in AD, has been linked to the accumulation of both Aβ and
*Corresponding author. Email: [email protected] Abbreviations: EGCG, (-)-epigallocatechin gallate; EGC, (-)-epigallocatechin; ECG, (-)-epicatechin gallate; AD, Alzheimer’s disease; Aβ, β-amyloid; DDS, 4-4’-diaminodiphenyl sulfone © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
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Green tea aroma fraction reduces β-amyloid peptide-toxicity
tau pathology. So, the amyloid hypothesis of AD is generally accepted.15) The amyloid hypothesis indicates that the primary pathological process of AD is the accumulation of normal Aβ from the β-amyloid precursor protein metabolism in the brain,16) resulting in the formation of senile plaques. The formation of neurofibrillary tangles containing tau protein probably results in an imbalance between Aβ production and Aβ clearance, leading to Aβ accumulation and toxicity.17) Several reports have indicated that EGCG has protective effects on the Aβ-induced apoptosis of cultured hippocampal neuronal cells, and that EGCG inhibits the fibrillogenesis of both α-synuclein and Aβ by directly binding to the native unfolded polypeptides18) and converting mature α-synuclein and Aβ fibrils into amorphous protein aggregates that are nontoxic to mammalian cells.19) Thus, the possibility of prevention of AD with EGCG is now being investigated in clinical trials.14) In order to determine the bioactivities in green tea aroma fraction, we looked at reports, especially a report about the protective effect of EGCG on a transgenic Caenorhabditis elegans (C. elegans) CL4176 (smg1ts[myo-3/Aβ1–42 long 3′-untranslated region (UTR)]) expressing human Aβ.20) CL4176 is a temperature-sensitive mutant C. elegans expressing human Aβ1–42, and expression of human Aβ1–42 transgene in its muscle cells was induced by raising the temperature from 16 to 23 °C at 22 h, resulting in paralysis.21,22) Although there is another transgenic C. elegans CL2355 that expresses human Aβ1–42 transgene in neurons, we used CL4176 because it allows simpler biological readout of Aβ toxicity as paralysis than CL2355 does. The green tea aroma fraction significantly delayed onset of paralysis, and it extended lifespan in transgenic C. elegans CL4176, partly due to the fraction’s antioxidant activity. All results clearly show that green tea aroma fraction has possible preventive effects on AD.
Materials and methods Green tea aroma fraction from green tea and reagents. Green tea leaves (C. sinensis var. sinensis, 100 g) grown and processed by our Green Tea Laboratory, Saitama Prefectural Agriculture and Forestry Research Center in 2012, were extracted with hexane (1 L) for 24 h at room temperature. The obtained solution was evaporated under reduced pressure, resulting in a viscous oil (hexane extract; 420 mg). The hexane extract was fractionated using the bulb-to-bulb microscale distillation method, gradually increasing temperature from 30 to 150 °C under 0.2 Torr.11) The result was a volatile “roasty aroma” fraction (5 mg) and a non-volatile “green tea aroma” fraction (349 mg). Hexane extract and the other fractions were dissolved in dimethyl sulfoxide (DMSO) and stored at −20 °C until use for experiments. EGCG (more than 99% purity) was obtained from Japanese green tea leaves. Curcumin was used as a positive control of paralysis assay,23) and 4–4′-diaminodiphenyl sulfone (DDS) was used as a positive control of lifespan assay.24) Both were purchased from Wako Chemical Pure Industries, Ltd. (Tokyo).
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C. elegans strains. C. elegans (CL4176 and CL802) was purchased from Caenorhabditis Genetics Center. CL4176 (smg-1ts[myo-3/Aβ1–42 long 3′-untranslated region (UTR)]) is a temperature-sensitive mutant that shows increasing human Aβ1–42 peptide in the muscle when the temperature is raised from 16 °C (permissive temperature for the smg-1ts mutation) to 25 °C (a non-permissive temperature).21) CL802 (smg-1ts) was control strain for CL4176. CL4176 and CL802 were maintained at 16 °C and propagated on solid nematode growth medium (NGM) agar plates (0.9 g NaCl, 7.5 g agar, 0.75 g bacto peptone) in 300 mL water. After autoclaving, 0.3 mL cholesterol (5 mg/mL), 0.3 mL MgSO4 (1 M), 0.3 mL CaCl2 (1 M), 7.5 mL potassium phosphate buffer (pH 6.0) (1 M) are added.)25) that were seeded with 1 mL of Escherichia coli (E. coli, OP50) for food every 7 days. Paralysis assay. Paralysis assay was performed as described previously.22,26) Fifty CL4176 nematodes eggs were laid on 9 cm NGM agar plate for 2 h at 16 °C. After removal of nematodes, the plate was incubated at 16 °C for 1 week. Ten synchronized young adult CL4176 nematodes were transferred to a 4 cm NGM agar plate that contained 1 × 107 cells/100 μL E. coli OP50 with test material—or 0.1% DMSO as vehicle—and the nematodes then laid eggs for 2 h at 16 °C. After removal of nematodes, the plate was incubated at 16 °C for 48 h. Twenty-five synchronized third larvae (L3) were transferred to a new 4 cm plate with test material, and the temperature was then raised to 25 °C (non-permissive temperature). To confirm the paralysis, each worm was gently touched with a platinum worm pick, and paralysis was considered if it moved its head only or did not move at all. Paralyzed nematodes were counted at 2 h intervals from 20 to 30 h after 25 °C. Results shown are the mean of three independent experiments, which were conducted with two plates. Lifespan assay. A thousand synchronized first larvae (L1) of CL4176 were transferred to a new 9 cm NGM agar plate containing 1 mL E. coli OP50, with or without test material, and kept at 16 °C. When nematodes grew to young adults, temperature was shifted to 25 °C, and the lifespan count was started as day 1.27) Test material dissolved in DMSO was given as a mixture in 1 x 107 cells E. coli (OP50)/mL 0.1% DMSO. Fifteen synchronized young adult nematodes were transferred to a new 4 cm NGM agar plate every 2 or 3 days, and the number of dead nematodes was scored at 25 °C. Fluorodeoxyuridine (FUdR) was used to prevent reproduction of progeny for the first 5 days. Nematodes were scored as dead if they did not respond to gentle touching with platinum worm pick. The results shown are the mean of three independent experiments, which were conducted with three plates. Antioxidant activity. Antioxidant activity was measured as the rate of discoloration of β-carotene coupled with the oxidation of linoleic acid.28) Briefly, first, linoleic acid—β-carotene emulsion was prepared as
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follows: a mixture of linoleic acid solution (11.1 μg/ 100 μL in chloroform), β-carotene solution (0.2 mg/ 250 μL chloroform), and Tween 40 (200 μg/100 μL chloroform) was dried under nitrogen gas and dissolved in 50 mL 20 mM phosphate buffer (pH 6.8). Twenty micro liter of test material was added to 980 μL linoleic acid—β-carotene emulsion at 50 °C, and changes of O.D. 470 nm were measured every 10 min for 70 min of incubation.
Statistical analysis. Statistical analysis was performed using Student’s t test and analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test comparing with a non-treated group.
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Results Inhibition of Aβ-induced paralysis of C. elegans (CL4176) transfected with human Aβ minigene by hexane extract and green tea aroma fraction C. elegans (CL4176) is a temperature-sensitive mutant that expresses human Aβ-peptide in muscle cells, resulting in induction of paralysis after upshifting the temperature to 25 °C at L3 stage.20) CL4176 nematodes at L3 stage induced paralysis 22 h after upshifting to 25 °C, and the paralysis time for 50% of nematodes (PT50) was 24.7 ± 1.1 h and 86.2 ± 8.1% nematodes showed paralysis at 28 h. Hexane extract at 100 μg/mL delayed the onset of paralysis, as shown in Fig. 1(A), and PT50 was delayed up to 26.5 ± 1.5 h a delay of 1.8 h, compared with that of non-treated group, while the paralysis ratio at 28 h was reduced from 86.2 ± 8.1% of non-treated group to 75.4 ± 17.0% (Table 1). Inhibitory effects of roasty aroma and green tea aroma fractions on induction of Aβ-induced paralysis were similarly determined. The roasty aroma fraction slightly delayed the onset of paralysis in CL4176 (Fig. 1(B)). While green tea aroma fraction showed a more significant delay (Fig. 1(C)), suggesting the inhibition of Aβ-induced toxicity. Specifically, the PT50 of green tea aroma fraction was delayed to 27.7 ± 0.4 h at 10 μg/mL and 28.7 ± 0.8 h at 100 μg/mL, compared with 26.3 ± 0.8 h in non-treated group. And the paralysis ratio at 28 h for green tea aroma fraction decreased from 75.6 ± 6.1% of non-treated group to 24.4 ± 22.0% at 100 μg/mL, a 51.2% reduction (Table 1). The activity of green tea aroma fraction was found to be very similar to curcumin 100 μM (36.8 μg/mL), which induces inhibitory activity of paralysis in CL4176.23) The results show that the green tea aroma fraction inhibited paralysis induced by toxicity of human Aβ1–42 peptide in muscle cells. Elongation of lifespan in transgenic C. elegans (CL4176) by green tea aroma fraction Unlike the paralysis experiment with L3, young adults of CL4176 did not show any paralysis as a result of upshifting temperature to 25 °C (non-permissive temperature), possibly because Aβ1–42 mRNA expression in young adults is lower than that in L3.21) However, the lifespan of CL4176 became shorter than that of CL802, a control strain for CL4176, at 25 °C: specifically, the
Fig. 1. Delayed onset of Aβ-induced paralysis in C. elegans (CL4176). Note: CL4176 transfected with human Aβ minigene were treated with hexane extract of green tea (A), roasty aroma fraction (B), and green tea aroma fraction (C) as described in Materials and Methods. The concentrations of hexane extract and green tea aroma fraction were 0 (×), 10 (○), 100 (●) μg/mL, and that of roasty aroma fraction was 0.2 (△) or 2 (▲) μg/mL. Nematodes at L3 were treated with upshift of temperature to 25 °C, and paralyzed nematodes were scored at 2 h intervals. Hexane extract (100 μg/mL) and green tea aroma fraction (10 and 100 μg/mL) significantly delayed onset of paralysis. The results show representative results of experiments. The F- and pvalues are hexane extract at 26 h: (F(2/6) = 1.3, p = 0.3); roasty aroma fraction at 26 h: (F(2/6) = 4.8, p = 0.1); and green tea aroma fraction at 26 h : (F(2/6) = 14.6, p < 0.01) and 28 h : (F(2/6) = 12.3, p < 0.01), * p < 0.05, **p < 0.01.
mean lifespan of CL4176 was 9.9 ± 1.3 days and that of CL802 was 10.7 ± 1.6 days (data not shown). We think that gradual expression of Aβ in muscle cells may be associated with shorter lifespan, so we next studied whether green tea aroma fraction could elongate lifespan of CL4176 at 25 °C by inhibition of Aβ toxicity: as expected, green tea aroma fraction at 100 μg/mL significantly extended mean lifespan to 12.0 ± 0.5 days (112.1%), from 10.7 ± 0.6 days in non-treated group, and maximal lifespan was 16.0 days compared with 13.7 days (Table 2), but green tea aroma fraction did not affect the mean lifespan of CL802. However, neither hexane extract (100 μg/mL) nor roasty aroma fraction (2 μg/mL) affected either the mean lifespan or the maximal lifespan (Table 2). All the results clearly showed that green tea aroma fraction prevents Aβ-induced toxicity.
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Table 1. Delayed onset of Aβ-induced paralysis in human Aβ-transgenic C. elegans CL4176 by hexane extract, roasty aroma and green tea aroma fractions. No. of nematodes Hexane extract
Curcumin Green tea aroma fraction
Roasty aroma fraction
0 μg/mL 10 μg/mL 100 μg/mL 100 μM 0 μg/mL 10 μg/mL 100 μg/mL 0.2 μg/mL 2.0 μg/mL
PT50 (h)
115 78 112 96 71 88 80 84 84
24.7 ± 1.1 26.1 ± 0.3 26.5 ± 1.5 26.7 ± 1.4 26.3 ± 0.8 27.7 ± 0.4 28.7 ± 0.8** 27.2 ± 0.0 27.7 ± 0.5
Delayed time (h)
28 h paralysis ratio (%)
– 1.4 1.8** 2.0a – 1.4* 2.4** 0.9 1.4
86.2 ± 8.1 87.1 ± 0.7 75.4 ± 17.0 72.1 ± 26.0 75.6 ± 6.1 60.5 ± 15.0 24.4 ± 22.0** 70.1 ± 6.1 57.5 ± 11.9
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Note: PT50 (paralysis time of 50% nematodes) and 28 h paralysis ratio are shown as mean ± S.D. of three independent experiments. Delayed time indicates the difference between PT50 of the test material and that of non-treated. Curcumin (100 μM = 36.8 μg/mL) is used as a positive control.23) Statistical analyses in hexane extract (0, 10, 100 μg/mL), in green tea aroma fraction (0, 10, 100 μg/mL), and in roasty aroma fraction (0, 0.2, 2.0 μg/mL) are followed by ANOVA. The F- and p-values are hexane extract (PT50: F(2/6) = 2.1, p = 0.2, delayed time: F(2/6) = 14.4, p < 0.01); green tea aroma fraction (PT50: F(2/6) = 12.3, p < 0.01, delayed time: F(2/6) = 10.6, p < 0.05, and 28 h paralysis ration: F(2/6) = 12.3, p < 0.01); and roasty aroma fraction (PT50: F(2/6) = 5.1, p = 0.1, and delayed time: F(2/6) = 4.4, p = 0.1). a Statistical analyses between non-treated (0 μg/mL) and curcumin are followed by Student’s t test (t(4) = 2.8, p < 0.05). * p < 0.05. ** p < 0.01.
Table 2.
Elongation of lifespan in transgenic C. elegans CL4176 by green tea aroma fraction.
Hexane extract
DDS Green tea aroma fraction
Roasty aroma fraction
0 μg/mL 10 μg/mL 100 μg/mL 2 mM 0 μg/mL 10 μg/mL 100 μg/mL 2.0 μg/mL
No. of nematodes
Mean lifespan (days)
Maximal lifespan (days)
130 130 130 131 132 80 130 130
9.9 ± 0.3 9.8 ± 0.8 10.3 ± 0.6 11.3 ± 0.5a 10.7 ± 0.6 10.7 ± 0.8 12.0 ± 0.5* 10.8 ± 0.4b
14.0 13.0 15.0 16.0 13.7 16.0 16.0 13.7
Note: The results shown are mean of three independent experiments. DDS, a positive control, was mixed in NGM at 2 mM (496 μg/mL) final concentration.24) Statistical analyses in hexane extract (0, 10, 100 μg/mL) and green tea aroma fraction (0, 10, 100 μg/mL) are followed by ANOVA. The F- and p-values are hexane extract: (F(2/6) = 0.5, p = 0.6); and green tea aroma fraction: (F(2/6) = 5.2, p < 0.05). * p < 0.05. a Statistical analyses between DDS and non-treated (0 μg/mL) are followed by Student’s t test (t(4) = 2.8, p < 0.05). b Statistical analyses between roasty aroma fraction and non-treated (0 μg/mL) are followed by Student’s t test (t(4) = 2.8, p = 0.9).
Antioxidant activity of green tea aroma fraction To determine the mechanism of action for inhibition of Aβ-induced toxicity by green tea aroma fraction, we examined antioxidant activity by measuring the rate of discoloration of β-carotene coupled with the oxidation of linoleic acid. In this experiment, O.D. at 470 nm was gradually reduced from 0.8 to 0.45 by 70 min incubation at 50 °C. Furthermore, the green tea aroma fraction at 20 μg/mL concentration completely inhibited discoloration, and at 2 μg/mL it showed inhibitory acitivity very similar to 2 μM (0.9 μg/mL) EGCG, indicating that green tea aroma fraction has antioxidant activity (Fig. 2). However, roasty aroma fraction at 4 μg/mL did not have any antioxidant activity (data not shown). These results show that the antioxidant acitivity of green tea aroma fraction partly contributes to preventing the activity of Aβ-induced toxicity in C. elegans.
Discussion C. elegans (CL4176) transfected with human Aβ minigene is a useful model for studying the inhibitory compounds found in AD. We first found that green tea aroma fraction inhibited the paralysis induced by
Fig. 2. Antioxidant activity of green tea aroma fraction. Note: Antioxidant activity was determined as: inhibition of discoloration of β-carotene observed at 470 nm absorbance from 0 to 70 min at 50 °C. The treated concentrations of green tea aroma fraction were 0 (×), 2 (●), 20 (○) μg/mL, and that of EGCG was 2 (△) μM. Green tea aroma fraction inhibited oxidation of β-carotene dose-dependently. These results are the average of two independent experiments. The F- and p-values are 20 min: F(2/6) = 1510.3, p < 0.01, 30 min: F(2/6) = 566.7, p < 0.01, 40 min: F(2/6) = 568.1, p < 0.01, 50 min: F(2/6) = 868.3, p < 0.01, 60 min: F(2/6) = 921.6, p < 0.01, and 70 min: F(2/6) = 1270.9, p < 0.01. **p < 0.01. aStatistical analyses between EGCG and non-treated are followed by Student’s t test (t(4) = 2.8, p < 0.01).
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human Aβ1–42 peptide production, and extended the lifespan in CL4176. The transgenic C. elegans (CL4176) accumulates Aβ1–42 peptide in muscle cells in non-permissive temperature conditions,21) and the upshift to non-permissive temperature at two different stages made it possible to induce different effects: at L3 stage, paralysis was induced, and at young adult stage, lifespan was shortened. It was interesting to note that hexane extract delayed the paralysis but did not extend the lifespan, while green tea aroma fraction both significantly delayed the paralysis and extended the lifespan dose-dependently. We think that green tea aroma fraction found in lipid-soluble fraction of green tea might strongly inhibit AD onset, similar to the effect of EGCG in water-soluble fraction.20) To confirm the inhibitory activity of green tea aroma fraction in Aβ-induced toxicity in neurons, we will use transgenic C. elegans (CL2355), which expresses Aβ1–42 peptide in neurons, in our next experiment. Green tea leaves contain numerous water-soluble and lipid-soluble compounds, and the catechins EGCG, EGC, and ECG are major water-soluble compounds that are biologically active. The antioxidant activity of EGCG is an important function in preventing AD. Like EGCG, α-tocopherol, which is contained in hexane extract of green tea, reduces the levels and plaque formation of Aβ in transgenic mice,29) and it also suppresses the development of tau pathology in transgenic mice,30) mediated through reducing oxidative stress in AD.31) We found that green tea aroma fraction has antioxidant activity and that its antioxidant activity at 20 μg/mL is equivalent to that of EGCG at 2 μM (0.9 μg/mL). The antioxidants in green tea aroma fraction may partly contribute to inhibition of Aβ toxicity through inhibition of reactive oxygen species generation. Since we previously determined that EGCG acts as a chemical chaperone because of its flexibility and mobility,32) we think that some chemical chaperone activity in EGCG helps to reduce Aβ toxicity by changing Aβ conformation. Interestingly, thioflavin has been reported to directly bind to Aβ, resulting in longer lifespan in CL4176,23) and we recently found that green tea aroma fraction showed changes of Aβ peptide bands similar to EGCG-induced change in Western blotting. Whether the compounds in green tea aroma fraction inhibit aggregation of Aβ through the action of a chemical chaperone32) remains to be investigated. Green tea aroma fraction consists of high-molecularweight compounds, while small amounts of roasty aroma fraction are mainly low-molecular-weight compounds: we found 37 compounds with molecular weights of 142–592 in green tea aroma fraction using GC–MS (Supplemental Fig. 1 and Table 1; see www. Biosci.Biotechnol.Biochem. Web site). It is interesting to note that anethole, one of the 37 compounds, is an aromatic compound with an antioxidant property,33) so it probably plays a role in inhibition of Aβ toxicity. Green tea aroma is composed of numerous aroma compounds in the lipid-soluble fraction, and the balance of the compounds contributes synergistically to green tea aroma.7,8) Thus, some compounds in green tea aroma fraction may contribute to inhibition of Aβ toxicity, delay onset of paralysis, and extend lifespan in CL4176. Alpha-tocopherol, a lipid-soluble compound,
and ascorbic acid, a water-soluble compound, both present in green tea, are reported to have synergistic effects on reducing the incidence of AD, but α-tocopherol alone was not effective in a cross-sectional and prospective study of dementia (The Cache Country Study).34) Thus, it is important to determine whether green tea aroma fraction also enhances activities of EGCG and other catechins in inhibition of AD. We think that green tea aroma fraction is worth further study in order to characterize the nature of the compounds for AD therapy, even if the compounds are in small quantities. We report here for the first time a new health benefit of green tea: green tea aroma fraction in lipid-soluble fraction is useful in reducing Aβ toxicity, in collaboration with catechins in water-soluble fraction. All results suggest that green tea aroma fraction in green tea is a candidate in complementary and alternative medicine for prevention and treatment of AD.
Acknowledgments We express sincere thanks to Dr. Naoaki Ishii and Dr. Kayo Yasuda, Department of Molecular Life Science Basic Medical Science and Molecular Medicine, Tokai University, for teaching us how to perform lifespan assay, and to Dr. Takashi Fujihara and Mr. Yasutsugu Tsukumo, Graduate School of Science and Engineering, Saitama University, for determination of GC–MS analysis. We greatly thank Dr. Hirota Fujiki for his critical reading of the manuscript, Dr. Kazue Imai for her helping statistical analyses, Mmes. Ikuko Shiotani, Kaori Suzuki, and Miki Kurusu-Kanno, Research Institute for Clinical Oncology, Saitama Cancer Center, for their technical assistance, and the members of the Green Tea Laboratory, Saitama Prefectural Agriculture and Forestry Research Center, for their generous collaborations.
Funding This work was supported by the Japan Society for the Promotion of Science; Scientific Research on Priority Areas for Cancer Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan; Selective Applied and Developed Research, and Green Tea Extracts Research Development for Cancer Prevention by the Department of Agriculture and Forests and the Department of Health and Human Services of Saitama Prefecture, Japan; Smoking Research Foundation, and the Urakami Foundation.
References [1] Fujiki H, Imai K, Nakachi K, Shimizu M, Moriwaki H, Suganuma M. Challenging the effectiveness of green tea in primary and tertiary cancer prevention. J. Cancer Res. Clin. Oncol. 2012;138:1259–1270. [2] Imai K, Nakachi K. Cross sectional study of effects of drinking green tea on cardiovascular and liver diseases. BMJ. 1995;310:693–696. [3] Nakachi K, Matsuyama S, Miyake S, Suganuma M, Imai K. Preventive effects of drinking green tea on cancer and cardiovascular disease: epidemiological evidence for multiple targeting prevention. Biofactors. 2000;13:49–54.
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Green tea aroma fraction reduces β-amyloid peptide-toxicity [4] Sueoka N, Suganuma M, Sueoka E, Okabe S, Matsuyama S, Imai K, Nakachi K, Fujiki H. A new function of green tea: prevention of lifestyle-related diseases. Ann. NY Acad. Sci. 2001;928:274–280. [5] Suganuma M, Okabe S, Sueoka N, Sueoka E, Matsuyama S, Imai K, Nakachi K, Fujiki H. Green tea and cancer chemoprevention. Mutation Res. 1999;428:339–344. [6] Suganuma M, Okabe S, Kai Y, Sueoka N, Sueoka E, Fujiki H. Synergistic effects of (-)-epigallocatechin gallate with (-)-epicatechin, sulindac, or tamoxifen on cancer-preventive activity in the human lung cancer cell line PC-9. Cancer Res. 1999;59:44–47. [7] Hattori S. Study of new approach for evaluation of Japanese green tea using OASIS method. Memoirs of the Faculty of Agricutlure Hokkaido University (in Japanese). 2006;28:85–120. [8] Yamanishi T. Flavor of tea. Food Rev. Int. 1995;11:477–525. [9] Prakash Chaturvedula VS, Prakash I. The aroma, taste, color and bioactive constituents of tea. J. Med. Plant Res. 2011;5:2110–2124. [10] Jumtee K, Komura H, Bamba T, Fukusaki E. Predication of Japanese green tea (Sen-cha) ranking by volatile profiling using gas chromatography mass spectrometry and multivariate analysis. J. Biosci. Bioeng. 2011;112:252–255. [11] Hasegawa T. Separation of odor constituents by microscale fractional bulb-to-bulb distillation. In: In distillation-advances from modeling to applications. S Zereshki, editor. Croatia: In Tech; 2012. p. 199–210. [12] Hasegawa T, Kishi Y, Kato Y, Akutsu K, Jingu D, Takahashi A, Tanaka E, Nakajima K, Matsumoto T. Comparison between aroma profiles of green tea leaves and black tea leaves derived from ‘Sayamakaori’. In proceedings of the 4th international conference on O-CHA (Tea) culture and science; 2010 October 26– 28; Shizuoka; 2011. p. 1–2. [13] Cummings JL. Alzheimer’s disease. New England J. Med. 2004;351:56–67. [14] Mähler A, Mandel S, Lorenz M, Ruegg U, Wanker EE, Boschmann M, Paul F. Epigallocatechin-3-gallate: a useful, effective and safe clinical approach for targeted prevention and individualised treatment of neurological diseases? EPMA J. 2013;4:5. [15] Arnold SE, Louneva N, Cao K, Wang LS, Han LY, Wolk DA, Negash S, Leurgans SE, Schneider JA, Buchman AS, Wilson RS, Bennett DA. Cellular, synaptic, and biochemical features of resilient cognition in Alzheimer’s disease. Neurobiol. Aging. 2013;34:157–168. [16] Hardy J, Chartier-Harlin MC, Mullan M. Alzheimer disease: the new agenda. Am. J. Human Genet. 1992;50:648–651. [17] Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science. 2002;297:353–356. [18] Ehrnhoefer DE, Bieschke J, Boeddrich A, Herbst M, Masino L, Lurz R, Engemann S, Pastore A, Wanker EE. EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers. Nat. Struct. Mol. Biol. 2008;15:558–566. [19] Bieschke J, Russ J, Friedrich RP, Ehrnhoefer DE, Wobst H, Neugebauer K, Wanker EE. EGCG remodels mature α-synuclein and amyloid-β fibrils and reduces cellular toxicity. Proc. Nat. Acad. Sci. 2010;107:7710–7715.
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[20] Brown MK, Evans JL, Luo Y. Beneficial effects of natural antioxidants EGCG and α-lipoic acid on life span and age-dependent behavioral declines in Caenorhabditis elegans. Pharmacol. Biochem. Behav. 2006;85:620–628. [21] Link CD, Taft A, Kapulkin V, Duke K, Kim S, Fei Q, Wood DE, Sahagan BG. Gene expression analysis in a transgenic Caenorhabditis elegans Alzheimer’s disease model. Neurobiol. Aging. 2003;24:397–413. [22] Wu Y, Wu Z, Butko P, Christen Y, Lambert MP, Klein WL, Link CD, Luo Y. Amyloid-β-induced pathological behaviors are suppressed by Ginkgo biloba extract EGb 761 and ginkgolides in transgenic Caenorhabditis elegans. J. Neurosci. 2006;26: 13102–13113. [23] Alavez S, Vantipalli MC, Zucker DJ, Klang IM, Lithgow GJ. Amyloid-binding compounds maintain protein homeostasis during ageing and extend lifespan. Nature. 2011;472:226–229. [24] Cho SC, Park MC, Keam B, Choi JM, Cho Y, Hyun S, Park SC, Lee J. DDS, 4,4′-diaminodiphenylsulfone, extends organismic lifespan. Proc. Nat. Acad. Sci. 2010;107:19326–19331. [25] Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974;77:71–94. [26] Dostal V, Link CD. Assaying β-amyloid toxicity using a transgenic C. elegans model. J. Vis. Exp. 2010;44. pii:2252, doi:10.3791/2252. [27] Wood JG, Rogina B, Lavu S, Howitz K, Helfand SL, Tatar M, Sinclair D. Sirtuin activators mimic caloric restriction and delay ageing in metazoans. Nature. 2004;430:686–689. [28] Tsushida T, Suzuki M, Kurogi M. Evaluation of antioxidant activity of vegetable extracts and determination of some active compounds. Nippon Shokuhin Kogyo Gakkaishi. (in Japanese). 1994;41:611–618. [29] Sung S, Yao Y, Uryu K, Yang H, Lee VM, Trojanowski JQ, Pratico D. Early vitamin E supplementation in young but not aged mice reduces Aβ levels and amyloid deposition in a transgenic model of Alzheimer’s disease. FASEB J. 2004;18:323– 325. [30] Nakashima H, Ishihara T, Yokota O, Terada S, Trojanowski JQ, Lee VM, Kuroda S. Effects of α-tocopherol on an animal model of tauopathies. Free Radical Biol. Med. 2004;37:176–186. [31] Nunomura A, Castellani RJ, Zhu X, Moreira PI, Perry G, Smith MA. Involvement of oxidative stress in Alzheimer disease. J. Neuropathol. Exp. Neurol. 2006;65:631–641. [32] Kuzuhara T, Suganuma M, Fujiki H. Green tea catechin as a chemical chaperone in cancer prevention. Cancer Lett. 2008;261:12–20. [33] Freire RS, Morais SM, Catunda-Junior FE, Pinheiro DC. Synthesis and antioxidant, anti-inflammatory and gastroprotector activities of anethole and related compounds. Bioorg. Med. Chem. 2005;13:4353–4358. [34] Zandi PP, Anthony JC, Khachaturian AS, Stone SV, Gustafson D, Tschanz JT, Norton MC, Welsh-Bohmer KA, Breitner JC. Reduced risk of Alzheimer disease in users of antioxidant vitamin supplements: the Cache County Study. Arch. Neurol. 2004;61:82–88.
Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Green tea aroma fraction reduces β-amyloid peptide-induced toxicity in Caenorhabditis elegans transfected with human β-amyloid minigene Atsushi Takahashi
abc
a
b
b
, Tatsuro Watanabe , Takashi Fujita , Toshio Hasegawa , Michio Saito a
& Masami Suganuma a
Research Institute for Clinical Oncology, Saitama Cancer Center, Saitama, Japan
b
Graduate School of Science and Engineering, Saitama University, Saitama, Japan
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Green Tea Laboratory, Saitama Prefectural Agriculture and Forestry Research Center, Saitama, Japan Published online: 12 Jun 2014.
Click for updates To cite this article: Atsushi Takahashi, Tatsuro Watanabe, Takashi Fujita, Toshio Hasegawa, Michio Saito & Masami Suganuma (2014) Green tea aroma fraction reduces β-amyloid peptide-induced toxicity in Caenorhabditis elegans transfected with human β-amyloid minigene, Bioscience, Biotechnology, and Biochemistry, 78:7, 1206-1211, DOI: 10.1080/09168451.2014.921553 To link to this article: http://dx.doi.org/10.1080/09168451.2014.921553
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Bioscience, Biotechnology, and Biochemistry, 2014 Vol. 78, No. 7, 1206–1211
Green tea aroma fraction reduces β-amyloid peptide-induced toxicity in Caenorhabditis elegans transfected with human β-amyloid minigene Atsushi Takahashi1,2,3, Tatsuro Watanabe1, Takashi Fujita2, Toshio Hasegawa2, Michio Saito3 and Masami Suganuma1,* 1
Research Institute for Clinical Oncology, Saitama Cancer Center, Saitama, Japan; 2Graduate School of Science and Engineering, Saitama University, Saitama, Japan; 3Green Tea Laboratory, Saitama Prefectural Agriculture and Forestry Research Center, Saitama, Japan
Received December 12, 2013; accepted February 27, 2014
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http://dx.doi.org/10.1080/09168451.2014.921553
Green tea is a popular world-wide beverage with health benefits that include preventive effects on cancer as well as cardiovascular, liver and Alzheimer’s diseases (AD). This study will examine the preventive effects on AD of a unique aroma of Japanese green tea. First, a transgenic Caenorhabditis elegans (C. elegans) CL4176 expressing human β-amyloid peptide (Aβ) was used as a model of AD. A hexane extract of processed green tea was further fractionated into volatile and non-volatile fractions, named roasty aroma and green tea aroma fractions depending on their aroma, by microscale distillation. Both hexane extract and green tea aroma fraction were found to inhibit Aβ-induced paralysis, while only green tea aroma fraction extended lifespan in CL4176. We also found that green tea aroma fraction has antioxidant activity. This paper indicates that the green tea aroma fraction is an additional component for prevention of AD. Key words:
Alzheimer’s disease; β-amyloid peptide; paralysis; lifespan; nematodes
Green tea is an acknowleged cancer preventive1) that is also associated with preventive effects on cardiovascular and liver diseases,2,3) resulting in longer lifespan.4) The active factors in these effects are (-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC), and (-)-epicatechin gallate (ECG), all found in the water-soluble fraction of green tea. Although (-)-epicatechin (EC) is an inactive catechin, it enhances the active catechin-induced activities, such as induction of apoptosis in lung cancer cells, PC-9, and inhibition of tumor necrosis factor release induced by tumor promoters.5) Thus, whole green tea infusion is the best mixture to gain health benefits.6) In addition to catechins, the aroma in the lipid-soluble fraction of green tea has recently attracted the attention of scien-
tists and doctors of aroma therapy, mental relaxation, and complementary and alternative medicine treatments. Using gas chromatography–mass spectrometry (GC–MS), scientists previously found over 630 volatile compounds in green tea.7,8) The odor-active compounds include various terpenes, such as geraniol and linalool,9,10) and the balance of numerous aroma compounds make up the essence of tea aroma. After reading some basic studies on aroma compounds, we became interested in the fractions of Japanese green tea (Camellia sinensis var. sinensis) obtained using microscale distillation under reduced pressure.11) The hexane extract from Japanese green tea was fractionated into volatile and non-volatile fractions using microscale distillation at 150 °C. The volatile fraction contains odoractive terpenes usually found in flowers and herbs, so we named it “roasty aroma fraction”; the non-volitile fraction has a unique green tea aroma that we named “green tea aroma fraction”.12) Dementia, including Alzheimer’s disease (AD) and vascular dementia, increases exponentially with aging: 1% of 60-year-olds have AD, while about 30% of 85year-olds have it.13) It is roughly estimated that there are 2,000,000 AD patients in Japan and 4,500,000 patients in the USA. Especially in developed countries, the number of AD patients is increasing with elongation of lifespan, so AD is becoming a widespread social dilemma. The pathogenesis of AD includes a number of different processes, including oxidative stress, inflammation, and neuronal dysfunction due to neuronal degeneration.14) Beta-amyloid (Aβ) plaques and hyperphosphorylated tau neurofibrillary tangles are common age-related lesions that are presumed to play primary roles in neuronal dysfuncitons of AD, but it is also understood that the correlation between these neuropathological lesions and dementia is not great. Recently, however, synaptic loss, which is the key neuropathological correlate of dementia severity in AD, has been linked to the accumulation of both Aβ and
*Corresponding author. Email: [email protected] Abbreviations: EGCG, (-)-epigallocatechin gallate; EGC, (-)-epigallocatechin; ECG, (-)-epicatechin gallate; AD, Alzheimer’s disease; Aβ, β-amyloid; DDS, 4-4’-diaminodiphenyl sulfone © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
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Green tea aroma fraction reduces β-amyloid peptide-toxicity
tau pathology. So, the amyloid hypothesis of AD is generally accepted.15) The amyloid hypothesis indicates that the primary pathological process of AD is the accumulation of normal Aβ from the β-amyloid precursor protein metabolism in the brain,16) resulting in the formation of senile plaques. The formation of neurofibrillary tangles containing tau protein probably results in an imbalance between Aβ production and Aβ clearance, leading to Aβ accumulation and toxicity.17) Several reports have indicated that EGCG has protective effects on the Aβ-induced apoptosis of cultured hippocampal neuronal cells, and that EGCG inhibits the fibrillogenesis of both α-synuclein and Aβ by directly binding to the native unfolded polypeptides18) and converting mature α-synuclein and Aβ fibrils into amorphous protein aggregates that are nontoxic to mammalian cells.19) Thus, the possibility of prevention of AD with EGCG is now being investigated in clinical trials.14) In order to determine the bioactivities in green tea aroma fraction, we looked at reports, especially a report about the protective effect of EGCG on a transgenic Caenorhabditis elegans (C. elegans) CL4176 (smg1ts[myo-3/Aβ1–42 long 3′-untranslated region (UTR)]) expressing human Aβ.20) CL4176 is a temperature-sensitive mutant C. elegans expressing human Aβ1–42, and expression of human Aβ1–42 transgene in its muscle cells was induced by raising the temperature from 16 to 23 °C at 22 h, resulting in paralysis.21,22) Although there is another transgenic C. elegans CL2355 that expresses human Aβ1–42 transgene in neurons, we used CL4176 because it allows simpler biological readout of Aβ toxicity as paralysis than CL2355 does. The green tea aroma fraction significantly delayed onset of paralysis, and it extended lifespan in transgenic C. elegans CL4176, partly due to the fraction’s antioxidant activity. All results clearly show that green tea aroma fraction has possible preventive effects on AD.
Materials and methods Green tea aroma fraction from green tea and reagents. Green tea leaves (C. sinensis var. sinensis, 100 g) grown and processed by our Green Tea Laboratory, Saitama Prefectural Agriculture and Forestry Research Center in 2012, were extracted with hexane (1 L) for 24 h at room temperature. The obtained solution was evaporated under reduced pressure, resulting in a viscous oil (hexane extract; 420 mg). The hexane extract was fractionated using the bulb-to-bulb microscale distillation method, gradually increasing temperature from 30 to 150 °C under 0.2 Torr.11) The result was a volatile “roasty aroma” fraction (5 mg) and a non-volatile “green tea aroma” fraction (349 mg). Hexane extract and the other fractions were dissolved in dimethyl sulfoxide (DMSO) and stored at −20 °C until use for experiments. EGCG (more than 99% purity) was obtained from Japanese green tea leaves. Curcumin was used as a positive control of paralysis assay,23) and 4–4′-diaminodiphenyl sulfone (DDS) was used as a positive control of lifespan assay.24) Both were purchased from Wako Chemical Pure Industries, Ltd. (Tokyo).
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C. elegans strains. C. elegans (CL4176 and CL802) was purchased from Caenorhabditis Genetics Center. CL4176 (smg-1ts[myo-3/Aβ1–42 long 3′-untranslated region (UTR)]) is a temperature-sensitive mutant that shows increasing human Aβ1–42 peptide in the muscle when the temperature is raised from 16 °C (permissive temperature for the smg-1ts mutation) to 25 °C (a non-permissive temperature).21) CL802 (smg-1ts) was control strain for CL4176. CL4176 and CL802 were maintained at 16 °C and propagated on solid nematode growth medium (NGM) agar plates (0.9 g NaCl, 7.5 g agar, 0.75 g bacto peptone) in 300 mL water. After autoclaving, 0.3 mL cholesterol (5 mg/mL), 0.3 mL MgSO4 (1 M), 0.3 mL CaCl2 (1 M), 7.5 mL potassium phosphate buffer (pH 6.0) (1 M) are added.)25) that were seeded with 1 mL of Escherichia coli (E. coli, OP50) for food every 7 days. Paralysis assay. Paralysis assay was performed as described previously.22,26) Fifty CL4176 nematodes eggs were laid on 9 cm NGM agar plate for 2 h at 16 °C. After removal of nematodes, the plate was incubated at 16 °C for 1 week. Ten synchronized young adult CL4176 nematodes were transferred to a 4 cm NGM agar plate that contained 1 × 107 cells/100 μL E. coli OP50 with test material—or 0.1% DMSO as vehicle—and the nematodes then laid eggs for 2 h at 16 °C. After removal of nematodes, the plate was incubated at 16 °C for 48 h. Twenty-five synchronized third larvae (L3) were transferred to a new 4 cm plate with test material, and the temperature was then raised to 25 °C (non-permissive temperature). To confirm the paralysis, each worm was gently touched with a platinum worm pick, and paralysis was considered if it moved its head only or did not move at all. Paralyzed nematodes were counted at 2 h intervals from 20 to 30 h after 25 °C. Results shown are the mean of three independent experiments, which were conducted with two plates. Lifespan assay. A thousand synchronized first larvae (L1) of CL4176 were transferred to a new 9 cm NGM agar plate containing 1 mL E. coli OP50, with or without test material, and kept at 16 °C. When nematodes grew to young adults, temperature was shifted to 25 °C, and the lifespan count was started as day 1.27) Test material dissolved in DMSO was given as a mixture in 1 x 107 cells E. coli (OP50)/mL 0.1% DMSO. Fifteen synchronized young adult nematodes were transferred to a new 4 cm NGM agar plate every 2 or 3 days, and the number of dead nematodes was scored at 25 °C. Fluorodeoxyuridine (FUdR) was used to prevent reproduction of progeny for the first 5 days. Nematodes were scored as dead if they did not respond to gentle touching with platinum worm pick. The results shown are the mean of three independent experiments, which were conducted with three plates. Antioxidant activity. Antioxidant activity was measured as the rate of discoloration of β-carotene coupled with the oxidation of linoleic acid.28) Briefly, first, linoleic acid—β-carotene emulsion was prepared as
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follows: a mixture of linoleic acid solution (11.1 μg/ 100 μL in chloroform), β-carotene solution (0.2 mg/ 250 μL chloroform), and Tween 40 (200 μg/100 μL chloroform) was dried under nitrogen gas and dissolved in 50 mL 20 mM phosphate buffer (pH 6.8). Twenty micro liter of test material was added to 980 μL linoleic acid—β-carotene emulsion at 50 °C, and changes of O.D. 470 nm were measured every 10 min for 70 min of incubation.
Statistical analysis. Statistical analysis was performed using Student’s t test and analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test comparing with a non-treated group.
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Results Inhibition of Aβ-induced paralysis of C. elegans (CL4176) transfected with human Aβ minigene by hexane extract and green tea aroma fraction C. elegans (CL4176) is a temperature-sensitive mutant that expresses human Aβ-peptide in muscle cells, resulting in induction of paralysis after upshifting the temperature to 25 °C at L3 stage.20) CL4176 nematodes at L3 stage induced paralysis 22 h after upshifting to 25 °C, and the paralysis time for 50% of nematodes (PT50) was 24.7 ± 1.1 h and 86.2 ± 8.1% nematodes showed paralysis at 28 h. Hexane extract at 100 μg/mL delayed the onset of paralysis, as shown in Fig. 1(A), and PT50 was delayed up to 26.5 ± 1.5 h a delay of 1.8 h, compared with that of non-treated group, while the paralysis ratio at 28 h was reduced from 86.2 ± 8.1% of non-treated group to 75.4 ± 17.0% (Table 1). Inhibitory effects of roasty aroma and green tea aroma fractions on induction of Aβ-induced paralysis were similarly determined. The roasty aroma fraction slightly delayed the onset of paralysis in CL4176 (Fig. 1(B)). While green tea aroma fraction showed a more significant delay (Fig. 1(C)), suggesting the inhibition of Aβ-induced toxicity. Specifically, the PT50 of green tea aroma fraction was delayed to 27.7 ± 0.4 h at 10 μg/mL and 28.7 ± 0.8 h at 100 μg/mL, compared with 26.3 ± 0.8 h in non-treated group. And the paralysis ratio at 28 h for green tea aroma fraction decreased from 75.6 ± 6.1% of non-treated group to 24.4 ± 22.0% at 100 μg/mL, a 51.2% reduction (Table 1). The activity of green tea aroma fraction was found to be very similar to curcumin 100 μM (36.8 μg/mL), which induces inhibitory activity of paralysis in CL4176.23) The results show that the green tea aroma fraction inhibited paralysis induced by toxicity of human Aβ1–42 peptide in muscle cells. Elongation of lifespan in transgenic C. elegans (CL4176) by green tea aroma fraction Unlike the paralysis experiment with L3, young adults of CL4176 did not show any paralysis as a result of upshifting temperature to 25 °C (non-permissive temperature), possibly because Aβ1–42 mRNA expression in young adults is lower than that in L3.21) However, the lifespan of CL4176 became shorter than that of CL802, a control strain for CL4176, at 25 °C: specifically, the
Fig. 1. Delayed onset of Aβ-induced paralysis in C. elegans (CL4176). Note: CL4176 transfected with human Aβ minigene were treated with hexane extract of green tea (A), roasty aroma fraction (B), and green tea aroma fraction (C) as described in Materials and Methods. The concentrations of hexane extract and green tea aroma fraction were 0 (×), 10 (○), 100 (●) μg/mL, and that of roasty aroma fraction was 0.2 (△) or 2 (▲) μg/mL. Nematodes at L3 were treated with upshift of temperature to 25 °C, and paralyzed nematodes were scored at 2 h intervals. Hexane extract (100 μg/mL) and green tea aroma fraction (10 and 100 μg/mL) significantly delayed onset of paralysis. The results show representative results of experiments. The F- and pvalues are hexane extract at 26 h: (F(2/6) = 1.3, p = 0.3); roasty aroma fraction at 26 h: (F(2/6) = 4.8, p = 0.1); and green tea aroma fraction at 26 h : (F(2/6) = 14.6, p < 0.01) and 28 h : (F(2/6) = 12.3, p < 0.01), * p < 0.05, **p < 0.01.
mean lifespan of CL4176 was 9.9 ± 1.3 days and that of CL802 was 10.7 ± 1.6 days (data not shown). We think that gradual expression of Aβ in muscle cells may be associated with shorter lifespan, so we next studied whether green tea aroma fraction could elongate lifespan of CL4176 at 25 °C by inhibition of Aβ toxicity: as expected, green tea aroma fraction at 100 μg/mL significantly extended mean lifespan to 12.0 ± 0.5 days (112.1%), from 10.7 ± 0.6 days in non-treated group, and maximal lifespan was 16.0 days compared with 13.7 days (Table 2), but green tea aroma fraction did not affect the mean lifespan of CL802. However, neither hexane extract (100 μg/mL) nor roasty aroma fraction (2 μg/mL) affected either the mean lifespan or the maximal lifespan (Table 2). All the results clearly showed that green tea aroma fraction prevents Aβ-induced toxicity.
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Table 1. Delayed onset of Aβ-induced paralysis in human Aβ-transgenic C. elegans CL4176 by hexane extract, roasty aroma and green tea aroma fractions. No. of nematodes Hexane extract
Curcumin Green tea aroma fraction
Roasty aroma fraction
0 μg/mL 10 μg/mL 100 μg/mL 100 μM 0 μg/mL 10 μg/mL 100 μg/mL 0.2 μg/mL 2.0 μg/mL
PT50 (h)
115 78 112 96 71 88 80 84 84
24.7 ± 1.1 26.1 ± 0.3 26.5 ± 1.5 26.7 ± 1.4 26.3 ± 0.8 27.7 ± 0.4 28.7 ± 0.8** 27.2 ± 0.0 27.7 ± 0.5
Delayed time (h)
28 h paralysis ratio (%)
– 1.4 1.8** 2.0a – 1.4* 2.4** 0.9 1.4
86.2 ± 8.1 87.1 ± 0.7 75.4 ± 17.0 72.1 ± 26.0 75.6 ± 6.1 60.5 ± 15.0 24.4 ± 22.0** 70.1 ± 6.1 57.5 ± 11.9
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Note: PT50 (paralysis time of 50% nematodes) and 28 h paralysis ratio are shown as mean ± S.D. of three independent experiments. Delayed time indicates the difference between PT50 of the test material and that of non-treated. Curcumin (100 μM = 36.8 μg/mL) is used as a positive control.23) Statistical analyses in hexane extract (0, 10, 100 μg/mL), in green tea aroma fraction (0, 10, 100 μg/mL), and in roasty aroma fraction (0, 0.2, 2.0 μg/mL) are followed by ANOVA. The F- and p-values are hexane extract (PT50: F(2/6) = 2.1, p = 0.2, delayed time: F(2/6) = 14.4, p < 0.01); green tea aroma fraction (PT50: F(2/6) = 12.3, p < 0.01, delayed time: F(2/6) = 10.6, p < 0.05, and 28 h paralysis ration: F(2/6) = 12.3, p < 0.01); and roasty aroma fraction (PT50: F(2/6) = 5.1, p = 0.1, and delayed time: F(2/6) = 4.4, p = 0.1). a Statistical analyses between non-treated (0 μg/mL) and curcumin are followed by Student’s t test (t(4) = 2.8, p < 0.05). * p < 0.05. ** p < 0.01.
Table 2.
Elongation of lifespan in transgenic C. elegans CL4176 by green tea aroma fraction.
Hexane extract
DDS Green tea aroma fraction
Roasty aroma fraction
0 μg/mL 10 μg/mL 100 μg/mL 2 mM 0 μg/mL 10 μg/mL 100 μg/mL 2.0 μg/mL
No. of nematodes
Mean lifespan (days)
Maximal lifespan (days)
130 130 130 131 132 80 130 130
9.9 ± 0.3 9.8 ± 0.8 10.3 ± 0.6 11.3 ± 0.5a 10.7 ± 0.6 10.7 ± 0.8 12.0 ± 0.5* 10.8 ± 0.4b
14.0 13.0 15.0 16.0 13.7 16.0 16.0 13.7
Note: The results shown are mean of three independent experiments. DDS, a positive control, was mixed in NGM at 2 mM (496 μg/mL) final concentration.24) Statistical analyses in hexane extract (0, 10, 100 μg/mL) and green tea aroma fraction (0, 10, 100 μg/mL) are followed by ANOVA. The F- and p-values are hexane extract: (F(2/6) = 0.5, p = 0.6); and green tea aroma fraction: (F(2/6) = 5.2, p < 0.05). * p < 0.05. a Statistical analyses between DDS and non-treated (0 μg/mL) are followed by Student’s t test (t(4) = 2.8, p < 0.05). b Statistical analyses between roasty aroma fraction and non-treated (0 μg/mL) are followed by Student’s t test (t(4) = 2.8, p = 0.9).
Antioxidant activity of green tea aroma fraction To determine the mechanism of action for inhibition of Aβ-induced toxicity by green tea aroma fraction, we examined antioxidant activity by measuring the rate of discoloration of β-carotene coupled with the oxidation of linoleic acid. In this experiment, O.D. at 470 nm was gradually reduced from 0.8 to 0.45 by 70 min incubation at 50 °C. Furthermore, the green tea aroma fraction at 20 μg/mL concentration completely inhibited discoloration, and at 2 μg/mL it showed inhibitory acitivity very similar to 2 μM (0.9 μg/mL) EGCG, indicating that green tea aroma fraction has antioxidant activity (Fig. 2). However, roasty aroma fraction at 4 μg/mL did not have any antioxidant activity (data not shown). These results show that the antioxidant acitivity of green tea aroma fraction partly contributes to preventing the activity of Aβ-induced toxicity in C. elegans.
Discussion C. elegans (CL4176) transfected with human Aβ minigene is a useful model for studying the inhibitory compounds found in AD. We first found that green tea aroma fraction inhibited the paralysis induced by
Fig. 2. Antioxidant activity of green tea aroma fraction. Note: Antioxidant activity was determined as: inhibition of discoloration of β-carotene observed at 470 nm absorbance from 0 to 70 min at 50 °C. The treated concentrations of green tea aroma fraction were 0 (×), 2 (●), 20 (○) μg/mL, and that of EGCG was 2 (△) μM. Green tea aroma fraction inhibited oxidation of β-carotene dose-dependently. These results are the average of two independent experiments. The F- and p-values are 20 min: F(2/6) = 1510.3, p < 0.01, 30 min: F(2/6) = 566.7, p < 0.01, 40 min: F(2/6) = 568.1, p < 0.01, 50 min: F(2/6) = 868.3, p < 0.01, 60 min: F(2/6) = 921.6, p < 0.01, and 70 min: F(2/6) = 1270.9, p < 0.01. **p < 0.01. aStatistical analyses between EGCG and non-treated are followed by Student’s t test (t(4) = 2.8, p < 0.01).
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human Aβ1–42 peptide production, and extended the lifespan in CL4176. The transgenic C. elegans (CL4176) accumulates Aβ1–42 peptide in muscle cells in non-permissive temperature conditions,21) and the upshift to non-permissive temperature at two different stages made it possible to induce different effects: at L3 stage, paralysis was induced, and at young adult stage, lifespan was shortened. It was interesting to note that hexane extract delayed the paralysis but did not extend the lifespan, while green tea aroma fraction both significantly delayed the paralysis and extended the lifespan dose-dependently. We think that green tea aroma fraction found in lipid-soluble fraction of green tea might strongly inhibit AD onset, similar to the effect of EGCG in water-soluble fraction.20) To confirm the inhibitory activity of green tea aroma fraction in Aβ-induced toxicity in neurons, we will use transgenic C. elegans (CL2355), which expresses Aβ1–42 peptide in neurons, in our next experiment. Green tea leaves contain numerous water-soluble and lipid-soluble compounds, and the catechins EGCG, EGC, and ECG are major water-soluble compounds that are biologically active. The antioxidant activity of EGCG is an important function in preventing AD. Like EGCG, α-tocopherol, which is contained in hexane extract of green tea, reduces the levels and plaque formation of Aβ in transgenic mice,29) and it also suppresses the development of tau pathology in transgenic mice,30) mediated through reducing oxidative stress in AD.31) We found that green tea aroma fraction has antioxidant activity and that its antioxidant activity at 20 μg/mL is equivalent to that of EGCG at 2 μM (0.9 μg/mL). The antioxidants in green tea aroma fraction may partly contribute to inhibition of Aβ toxicity through inhibition of reactive oxygen species generation. Since we previously determined that EGCG acts as a chemical chaperone because of its flexibility and mobility,32) we think that some chemical chaperone activity in EGCG helps to reduce Aβ toxicity by changing Aβ conformation. Interestingly, thioflavin has been reported to directly bind to Aβ, resulting in longer lifespan in CL4176,23) and we recently found that green tea aroma fraction showed changes of Aβ peptide bands similar to EGCG-induced change in Western blotting. Whether the compounds in green tea aroma fraction inhibit aggregation of Aβ through the action of a chemical chaperone32) remains to be investigated. Green tea aroma fraction consists of high-molecularweight compounds, while small amounts of roasty aroma fraction are mainly low-molecular-weight compounds: we found 37 compounds with molecular weights of 142–592 in green tea aroma fraction using GC–MS (Supplemental Fig. 1 and Table 1; see www. Biosci.Biotechnol.Biochem. Web site). It is interesting to note that anethole, one of the 37 compounds, is an aromatic compound with an antioxidant property,33) so it probably plays a role in inhibition of Aβ toxicity. Green tea aroma is composed of numerous aroma compounds in the lipid-soluble fraction, and the balance of the compounds contributes synergistically to green tea aroma.7,8) Thus, some compounds in green tea aroma fraction may contribute to inhibition of Aβ toxicity, delay onset of paralysis, and extend lifespan in CL4176. Alpha-tocopherol, a lipid-soluble compound,
and ascorbic acid, a water-soluble compound, both present in green tea, are reported to have synergistic effects on reducing the incidence of AD, but α-tocopherol alone was not effective in a cross-sectional and prospective study of dementia (The Cache Country Study).34) Thus, it is important to determine whether green tea aroma fraction also enhances activities of EGCG and other catechins in inhibition of AD. We think that green tea aroma fraction is worth further study in order to characterize the nature of the compounds for AD therapy, even if the compounds are in small quantities. We report here for the first time a new health benefit of green tea: green tea aroma fraction in lipid-soluble fraction is useful in reducing Aβ toxicity, in collaboration with catechins in water-soluble fraction. All results suggest that green tea aroma fraction in green tea is a candidate in complementary and alternative medicine for prevention and treatment of AD.
Acknowledgments We express sincere thanks to Dr. Naoaki Ishii and Dr. Kayo Yasuda, Department of Molecular Life Science Basic Medical Science and Molecular Medicine, Tokai University, for teaching us how to perform lifespan assay, and to Dr. Takashi Fujihara and Mr. Yasutsugu Tsukumo, Graduate School of Science and Engineering, Saitama University, for determination of GC–MS analysis. We greatly thank Dr. Hirota Fujiki for his critical reading of the manuscript, Dr. Kazue Imai for her helping statistical analyses, Mmes. Ikuko Shiotani, Kaori Suzuki, and Miki Kurusu-Kanno, Research Institute for Clinical Oncology, Saitama Cancer Center, for their technical assistance, and the members of the Green Tea Laboratory, Saitama Prefectural Agriculture and Forestry Research Center, for their generous collaborations.
Funding This work was supported by the Japan Society for the Promotion of Science; Scientific Research on Priority Areas for Cancer Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan; Selective Applied and Developed Research, and Green Tea Extracts Research Development for Cancer Prevention by the Department of Agriculture and Forests and the Department of Health and Human Services of Saitama Prefecture, Japan; Smoking Research Foundation, and the Urakami Foundation.
References [1] Fujiki H, Imai K, Nakachi K, Shimizu M, Moriwaki H, Suganuma M. Challenging the effectiveness of green tea in primary and tertiary cancer prevention. J. Cancer Res. Clin. Oncol. 2012;138:1259–1270. [2] Imai K, Nakachi K. Cross sectional study of effects of drinking green tea on cardiovascular and liver diseases. BMJ. 1995;310:693–696. [3] Nakachi K, Matsuyama S, Miyake S, Suganuma M, Imai K. Preventive effects of drinking green tea on cancer and cardiovascular disease: epidemiological evidence for multiple targeting prevention. Biofactors. 2000;13:49–54.
Downloaded by [University of Otago] at 20:36 27 December 2014
Green tea aroma fraction reduces β-amyloid peptide-toxicity [4] Sueoka N, Suganuma M, Sueoka E, Okabe S, Matsuyama S, Imai K, Nakachi K, Fujiki H. A new function of green tea: prevention of lifestyle-related diseases. Ann. NY Acad. Sci. 2001;928:274–280. [5] Suganuma M, Okabe S, Sueoka N, Sueoka E, Matsuyama S, Imai K, Nakachi K, Fujiki H. Green tea and cancer chemoprevention. Mutation Res. 1999;428:339–344. [6] Suganuma M, Okabe S, Kai Y, Sueoka N, Sueoka E, Fujiki H. Synergistic effects of (-)-epigallocatechin gallate with (-)-epicatechin, sulindac, or tamoxifen on cancer-preventive activity in the human lung cancer cell line PC-9. Cancer Res. 1999;59:44–47. [7] Hattori S. Study of new approach for evaluation of Japanese green tea using OASIS method. Memoirs of the Faculty of Agricutlure Hokkaido University (in Japanese). 2006;28:85–120. [8] Yamanishi T. Flavor of tea. Food Rev. Int. 1995;11:477–525. [9] Prakash Chaturvedula VS, Prakash I. The aroma, taste, color and bioactive constituents of tea. J. Med. Plant Res. 2011;5:2110–2124. [10] Jumtee K, Komura H, Bamba T, Fukusaki E. Predication of Japanese green tea (Sen-cha) ranking by volatile profiling using gas chromatography mass spectrometry and multivariate analysis. J. Biosci. Bioeng. 2011;112:252–255. [11] Hasegawa T. Separation of odor constituents by microscale fractional bulb-to-bulb distillation. In: In distillation-advances from modeling to applications. S Zereshki, editor. Croatia: In Tech; 2012. p. 199–210. [12] Hasegawa T, Kishi Y, Kato Y, Akutsu K, Jingu D, Takahashi A, Tanaka E, Nakajima K, Matsumoto T. Comparison between aroma profiles of green tea leaves and black tea leaves derived from ‘Sayamakaori’. In proceedings of the 4th international conference on O-CHA (Tea) culture and science; 2010 October 26– 28; Shizuoka; 2011. p. 1–2. [13] Cummings JL. Alzheimer’s disease. New England J. Med. 2004;351:56–67. [14] Mähler A, Mandel S, Lorenz M, Ruegg U, Wanker EE, Boschmann M, Paul F. Epigallocatechin-3-gallate: a useful, effective and safe clinical approach for targeted prevention and individualised treatment of neurological diseases? EPMA J. 2013;4:5. [15] Arnold SE, Louneva N, Cao K, Wang LS, Han LY, Wolk DA, Negash S, Leurgans SE, Schneider JA, Buchman AS, Wilson RS, Bennett DA. Cellular, synaptic, and biochemical features of resilient cognition in Alzheimer’s disease. Neurobiol. Aging. 2013;34:157–168. [16] Hardy J, Chartier-Harlin MC, Mullan M. Alzheimer disease: the new agenda. Am. J. Human Genet. 1992;50:648–651. [17] Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science. 2002;297:353–356. [18] Ehrnhoefer DE, Bieschke J, Boeddrich A, Herbst M, Masino L, Lurz R, Engemann S, Pastore A, Wanker EE. EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers. Nat. Struct. Mol. Biol. 2008;15:558–566. [19] Bieschke J, Russ J, Friedrich RP, Ehrnhoefer DE, Wobst H, Neugebauer K, Wanker EE. EGCG remodels mature α-synuclein and amyloid-β fibrils and reduces cellular toxicity. Proc. Nat. Acad. Sci. 2010;107:7710–7715.
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[20] Brown MK, Evans JL, Luo Y. Beneficial effects of natural antioxidants EGCG and α-lipoic acid on life span and age-dependent behavioral declines in Caenorhabditis elegans. Pharmacol. Biochem. Behav. 2006;85:620–628. [21] Link CD, Taft A, Kapulkin V, Duke K, Kim S, Fei Q, Wood DE, Sahagan BG. Gene expression analysis in a transgenic Caenorhabditis elegans Alzheimer’s disease model. Neurobiol. Aging. 2003;24:397–413. [22] Wu Y, Wu Z, Butko P, Christen Y, Lambert MP, Klein WL, Link CD, Luo Y. Amyloid-β-induced pathological behaviors are suppressed by Ginkgo biloba extract EGb 761 and ginkgolides in transgenic Caenorhabditis elegans. J. Neurosci. 2006;26: 13102–13113. [23] Alavez S, Vantipalli MC, Zucker DJ, Klang IM, Lithgow GJ. Amyloid-binding compounds maintain protein homeostasis during ageing and extend lifespan. Nature. 2011;472:226–229. [24] Cho SC, Park MC, Keam B, Choi JM, Cho Y, Hyun S, Park SC, Lee J. DDS, 4,4′-diaminodiphenylsulfone, extends organismic lifespan. Proc. Nat. Acad. Sci. 2010;107:19326–19331. [25] Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974;77:71–94. [26] Dostal V, Link CD. Assaying β-amyloid toxicity using a transgenic C. elegans model. J. Vis. Exp. 2010;44. pii:2252, doi:10.3791/2252. [27] Wood JG, Rogina B, Lavu S, Howitz K, Helfand SL, Tatar M, Sinclair D. Sirtuin activators mimic caloric restriction and delay ageing in metazoans. Nature. 2004;430:686–689. [28] Tsushida T, Suzuki M, Kurogi M. Evaluation of antioxidant activity of vegetable extracts and determination of some active compounds. Nippon Shokuhin Kogyo Gakkaishi. (in Japanese). 1994;41:611–618. [29] Sung S, Yao Y, Uryu K, Yang H, Lee VM, Trojanowski JQ, Pratico D. Early vitamin E supplementation in young but not aged mice reduces Aβ levels and amyloid deposition in a transgenic model of Alzheimer’s disease. FASEB J. 2004;18:323– 325. [30] Nakashima H, Ishihara T, Yokota O, Terada S, Trojanowski JQ, Lee VM, Kuroda S. Effects of α-tocopherol on an animal model of tauopathies. Free Radical Biol. Med. 2004;37:176–186. [31] Nunomura A, Castellani RJ, Zhu X, Moreira PI, Perry G, Smith MA. Involvement of oxidative stress in Alzheimer disease. J. Neuropathol. Exp. Neurol. 2006;65:631–641. [32] Kuzuhara T, Suganuma M, Fujiki H. Green tea catechin as a chemical chaperone in cancer prevention. Cancer Lett. 2008;261:12–20. [33] Freire RS, Morais SM, Catunda-Junior FE, Pinheiro DC. Synthesis and antioxidant, anti-inflammatory and gastroprotector activities of anethole and related compounds. Bioorg. Med. Chem. 2005;13:4353–4358. [34] Zandi PP, Anthony JC, Khachaturian AS, Stone SV, Gustafson D, Tschanz JT, Norton MC, Welsh-Bohmer KA, Breitner JC. Reduced risk of Alzheimer disease in users of antioxidant vitamin supplements: the Cache County Study. Arch. Neurol. 2004;61:82–88.
