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The effects of raspberry, strawberry and bilberry juices and green and black tea on early atherosclerosis in hamsters were investigated. They received an atherogenic diet and at the same time either a juice or a tea at a daily dose corresponding to the consumption of 275 ml by a 70 kg human. After 12 weeks berry juices and teas inhibited aortic lipid deposition by 79–96% and triggered reduced activity of hepatic antioxidant enzymes, not accompanied by lowered plasma cholesterol. These findings suggest that moderate consumption of berry juices and teas can help prevent the development of early atherosclerosis. There were substantial differences between the five beverages in terms of composition and concentration of individual phenolic compounds that were present. This indicates that anti-atherosclerotic effects can be induced by a diversity of phenolic compounds rather than a few specific components. The possible mechanisms by which this is brought about are discussed.
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Berry juices, teas, antioxidants and the prevention of atherosclerosis in hamsters
Jean-Max Rouanet
a,*
, Kelly Décordé
a
, Daniele Del Rio
b
, Cyril Auger
c
, Gina Borges
c
, Jean-Paul Cristol
a
,
Michael E.J. Lean
d
, Alan Crozier
c
a
Unité Mixte de Recherche 204-Prévention des Malnutritions et des Pathologies Associées, CC 023, Université Montpellier, 2, Place Eugène Bataillon, 34095 Montpellier, France
b
Department of Public Health, University of Parma, Via Volturno 39, 43100 Parma and National Institute of Biostructures and Biosystems (INBB), Italy
c
Division of Environmental and Evolutionary Biology, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom
d
Human Nutrition Section, University of Glasgow Division of Developmental Medicine, Queen Elizabeth Building, Royal Infirmary, Glasgow G31 2ER, United Kingdom
article info
Article history:
Received 20 February 2009
Received in revised form 17 March 2009
Accepted 28 April 2009
Keywords:
Atherosclerosis
Nutrition
Phenolic compounds
Antioxidants
Berry juices
Teas
abstract
The effects of raspberry, strawberry and bilberry juices and green and black tea on early atherosclerosis in
hamsters were investigated. They received an atherogenic diet and at the same time either a juice or a tea
at a daily dose corresponding to the consumption of 275 ml by a 70 kg human. After 12 weeks berry
juices and teas inhibited aortic lipid deposition by 79–96% and triggered reduced activity of hepatic anti-
oxidant enzymes, not accompanied by lowered plasma cholesterol. These findings suggest that moderate
consumption of berry juices and teas can help prevent the development of early atherosclerosis. There
were substantial differences between the five beverages in terms of composition and concentration of
individual phenolic compounds that were present. This indicates that anti-atherosclerotic effects can
be induced by a diversity of phenolic compounds rather than a few specific components. The possible
mechanisms by which this is brought about are discussed.
Ó2009 Elsevier Ltd. All rights reserved.
1. Introduction
The postulated involvement of lipid peroxidation in atherogen-
esis invoked intensive research on antioxidants. Consumption of
fruits and vegetables has been linked with lower prevalence of cor-
onary heart disease (Bazzano, 2006; Dauchet, Amouyel, Hercberg,
& Dallongeville, 2006; Feldman, 2001; Liu et al., 2000). Drinking
tea has also been linked with reduced mortality arising from
cardiovascular disease (Kuriyama et al., 2006), although some
epidemiological data are inconclusive (Yang & Landau, 2000).
Fruits, vegetables and teas contain a wide range of antioxidant
compounds, including phenolic compounds and vitamins. Phenolic
compounds, such as anthocyanins, flavan-3-ols, flavonols,
hydroxycinnamates and tannins, are widespread in fruits and veg-
etables, with especially high quantities being found in berries and
teas. Berries are rich in anthocyanins and can also contain substan-
tial quantities of ellagitannins, while flavan-3-ols and their related
derivatives predominate in teas (Crozier, Jaganath, Marks, Salt-
marsh, & Clifford, 2006).
Golden Syrian hamsters represent an useful test system because
when fed a fat-rich diet they develop dyslipidemia and atheroscle-
rotic plaques, similar in many respects to human atheroma (Auger
et al., 2002).
A relatively straight-forward way to evaluate influences on ath-
erosclerosis progression in animal models is to measure the extent
of fatty streak development, the continuous accumulation of lipids
(due mainly to large accumulations of macrophages) in the sub-
endothelial space. Using this approach, we have evaluated the ef-
fects of raspberry, strawberry and bilberry juices and green and
black tea, sources of potentially anti-atherogenic phenolic com-
pounds, by feeding the beverages to golden Syrian hamsters on a
high fat diet for a 12-week period.
2. Materials and methods
2.1. Chemicals
5-O-Caffeoylquinic acid, procyanidin B2, (–)-epicatechin (+)-
catechin, (–)-gallocatechin, (–)-epicatechin, (–)-epicatechin gallate,
(–)-epigallocatechin, (–)-epigallocatechin gallate, gallic acid,
caffeine, theobromine, theaflavins and ellagic acid were purchased
from Sigma–Aldrich (Poole, UK). Quercetin, myricetin, quercetin-3-
O-rutinoside, quercetin-3-O-glucoside, quercetin-3-O-arabinoside,
quercetin-3-O-galactoside, kaemperol-3-O-rutinoside, kaemperol-
3-O-glucoside, caffeic acid and p-coumaric acid were obtained
from AASC Ltd. (Southampton, UK). Cyanidin-3-O-glucoside was
purchased from Extrasynthese (Genay, France). Methanol and ace-
tonitrile were obtained from Rathburn Chemicals (Walkerburn,
Peebleshire, UK). Formic acid was obtained from Fisher Scientific
(Loughborough, UK).
0308-8146/$ - see front matter Ó2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2009.04.116
*Corresponding author. Fax: +33 0467143521.
E-mail address: jm.rouanet@univ-montp2.fr (J.-M. Rouanet).
Food Chemistry 118 (2010) 266–271
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
2.2. Berries juices and teas
Bouvrage raspberry (Rubus idaeus L.;1 ml = 0.6 g berries), bil-
berry (Vaccinium myrtillus; 1 ml = 0.32 g berries) and strawberry
juices (Fragaria ananassa; 1 ml = 0.22 g berries) drinks were ob-
tained from Ella Drinks Ltd. (Alloa, Clackmannanshire, UK). The
green and black teas (The Tetley Group, Greenford, Middlesex,
UK) were prepared by adding 300 ml of boiling water to 3 g of
leaves. After brewing for 3 min with continuous stirring, tea solids
were removed by filtration through a sieve and the resulting tea
was allow to cool before preparation of aliquots which were stored
at 20 °C prior to use.
2.3. HPLC–PDA–MS
2
analysis of berry juices and teas
Berry juices and teas were analysed on a Surveyor HPLC system
comprising of a HPLC pump, photodiode array detector (PDA) scan-
ning from 250 to 700 nm, and an autosampler set at 4 °C (Thermo
Finnigan, San Jose, USA) with the separation carried out using a
250 4.6 mm i.d. 4
l
m Synergi RP-Max column (Phenomenex,
Macclesfield, UK) eluted at a flow rate of 1 ml/min. A mobile phase
consisting of a 5–40% gradient over 60 min of acetonitrile in 0.1%
formic acid was used for the analysis of all samples. After passing
through the flow cell of the diode array detector the column eluate
was split and 0.3 ml was directed to a LCQ Deca XP ion trap mass
spectrometer fitted with an electrospray interface (Thermo Finni-
gan, San Jose, USA). Analysis was carried out using full scan mode
from 100 to 2000 amu, with data dependent tandem MS (MS
2
)
scanning, in both negative and positive ion mode.
A combination of co-chromatography with authentic standards,
where available, absorbance spectra and mass spectra, using MS
2
,
were used to confirm the identity of compounds previously re-
ported in the literature (Stewart, Mullen, & Crozier, 2005). Quanti-
tative estimates are based on calibrations generated by the PDA
detector using the compound under study when a standard was
available – see Chemicals. When this was not possible, a closely re-
lated derivative was used instead. For instance, all anthocyanins,
were quantified by reference to cyanidin-3-O-glucoside, while
chlorogenic acids, such as 3-O-p-coumarylquinic acid were quanti-
fied by reference to the appropriate aglycone. In all instances the
standard curve of reference compounds ranged from 2 to 500 ng.
The thearubigin content of the black tea was estimated as de-
scribed by Stewart et al. (2005).
2.4. Animals, diets and experimental design
Sixty weanling male Syrian golden hamsters (Elevage Janvier, Le
Genest-St-Isle, France) weighing ca.100 g were maintained in plas-
tic cages in a temperature controlled environment (23 ± 1 °C) sub-
jected to a 12-h light/dark cycle and allowed free access to both
food and water. Hamsters were handled according to the guide-
lines of the Committee on Animal Care at the University of Mont-
pellier and NIH guidelines (National Research Council, 1985).
They were randomly assigned to six groups of 10 not statisti-
cally different for weight. For 12 weeks all the hamsters were fed
a semi-purified atherogenic diet (Scientific Animal Food and Engi-
neering, Augy, France) consisting of casein (200 g/kg),
L
-methio-
nine (3 g/kg), corn starch (393 g/kg), sucrose (154 g/kg), cellulose
(50 g/kg), lard (150 g/kg), and cholesterol (5 g/kg) (Auger et al.,
2002). The diet also contained vitamin (10 g/kg) and mineral mixes
(35 g/kg). It was formulated according to AIN-93 guidelines (Re-
eves, Nielsen, & Fahey, 1993) and was devoid of selenium, vitamin
C and vitamin E. During the 12-week period, the hamsters received
either water (control), raspberry juice, strawberry juice, bilberry
juice, green tea or black tea daily by gavage. The volume of solu-
tions fed was adjusted daily to the weight of hamsters. The calcu-
lation is based on a consumption of 275 ml/day for a 70 kg human
as based on the US Food and Drug Administration Center for Drug
Evaluation and Research dose calculator (http://www.fda.gov/
cder/cancer/animalframe.htm).
2.5. Analytical procedures
At the end of the 12-week experimental period the hamsters
were fasted overnight and blood was drawn by cardiac puncture
under anesthesia. Plasma was prepared by centrifugation at
2000gfor 10 min at 4 °C, and then stored at 80 °C before analysis.
The liver was perfused with saline to remove residual blood, rap-
idly excised, rinsed in ice cold saline, blotted dry, weighted, sec-
tioned for analyses and stored in liquid nitrogen. The aortic
tissues were then processed as described below.
Following blood collection and liver removal, the intact aorta
was first perfused with phosphate buffered saline containing
1 mmol/l CaCl
2
and 15 mmol/l glucose for 5 min, then with
0.1 mmol/l sodium cacodylate buffer pH 7.4 containing
2.5 mmol/l CaCl
2
, 2.5% paraformaldehyde and 1.5% glutaraldehyde
for the fixation of the vasculature. The aorta was carefully dis-
sected and processed as previously described (Auger et al., 2002),
lipids being stained in Oil red O. An image acquisition and analysis
system (ImageJ, Scion Corporation, Frederick, MD) incorporated in
an Olympus microscope was used to capture and analyse the total
Oil Red O stained area of each aortic arch. The area covered by
foam cells (aortic fatty streak area or AFSA) was expressed as a per-
centage of the total area.
Plasma total cholesterol (TC) and HDL cholesterol (HDL-C) were
determined by commercially available enzymatic methods
(respectively Nos. CH 200 and CH 203, Randox Laboratories Ltd.,
Crumlin, UK) on a Pentra 400 automated analyser (HORIBA ABX,
Montpellier, France). Plasma very low- + low-density lipoprotein
cholesterol (referred to as « non-HDL-C in the data tables) was pre-
cipitated with phosphotungstate reagent and HDL-C was measured
in the supernatant.
The liver was homogenised in 5 vol of 0.15 M KCl buffer (pH 7.4)
and the homogenate was spun at 13,000gfor 15 min at 4 °C. The
supernatant was stored at 80 °C prior to the assay of glutathione
peroxidase (GSHPx) and superoxide dismutase (SOD) activity on a
Pentra 400 analyser. GSHPx activity was measured by the method
of Randox (Randox Laboratories Ltd., Crumlin, UK) using a com-
mercial kit (Ransel, No. RS505). Superoxide dismutase (SOD) activ-
ity was determined using a Randox kit (Ransod, No. SD 125).
To extract and analyse livers by HPLC–PDA–MS
2
, the livers from
two hamsters from each feeding group were combined and
homogenised in 2 ml of methanol/water (v/v) containing 5% formic
acid and 20 mM sodium diethyldithiocarbamate using an Ultratur-
rax homogeniser. The resultant homogenate was shaken continu-
ously for 30 min before being centrifuged at 2000gfor 20 min.
The supernatant was decanted and the pellet re-extracted twice.
The three supernatants were combined and reduced to dryness in
vacuo. The extract was dissolved in 25
l
l methanol in 475
l
l aque-
ous 1% formic acid and loaded onto an activated 2 g Sep–Pak C
18
cartridge (Waters, Milford, MA, USA) which was washed with
4 ml acidified water (pH 3.0) before elution with 4 ml methanol
containing 1% formic acid. The methanolic eluates were reduced
to dryness and resuspended in 50
l
l methanol in 950
l
l aqueous
1% formic acid before analysis for anthocyanin and flavan-3-ol
metabolites by HPLC–PDA–MS
2
using single and selected ion
monitoring.
2.6. Statistical analyses
Data are shown as the means ± SEM, n= 10 measurements/
group. Tea and berry juices samples were analysed in triplicate.
J.-M. Rouanet et al. / Food Chemistry 118 (2010) 266–271 267
Statistical analysis of the data was carried out using the Stat View
IV software (Abacus Concepts, Berkeley, CA, USA) by one-way AN-
OVA followed by Fisher’s Protected Least Significant Difference
test. Differences were considered significant at p< 0.05.
3. Results
3.1. Phenolic compounds in berry juices and teas
Twenty seven phenolic compounds were detected in the bil-
berry juice (Table 1) with the 13 anthocyanins comprising
599 nmoles/ml of a total flavonoid and phenolic content of
744 nmoles/ml. The juice also contained 76 nmoles/ml of gallic
acid and smaller quantities of flavan-3-ols and a number of flavo-
nols in low concentrations. The major components in the raspberry
juice were anthocyanins (164 nmoles/ml), principally cyanidin-3-
sophoroside and cyanidin-3–2
G
-glucosylrutinoside, and the ellagit-
annins, lambertianin C and sanguiin H-6 (Table 2). The strawberry
juice contained much lower overall levels of flavonoids and phen-
olics, 181 nmoles/ml (Table 3), than the bilberry and raspberry
juices. The main constituents were pelargonidin-3-glucoside
(91 nmoles/l) and a p-coumaric acid hexose conjugate (46 nmo-
les/l) (Table 3).
The compositions of the two teas are summarised in Table 4.
Both green and black tea contained higher amounts of phenolic
compounds than the juices, with 2894 and 2285 nmoles/ml,
respectively for green and black tea. The main green tea constitu-
ents were catechins which comprised a group of eight flavan-3-
ols, accounting for 2414 nmoles/ml with the major component
being (–)-epigallocatechin (921 nmoles/ml). Black tea contained
much lower concentrations of catechins (52 nmoles/ml) than green
tea, but a large amount of theaflavins and thearubigins (1839 nmo-
les/ml) that were not present in green tea. Both teas also contained
broadly similar levels of chlorogenic acids and a diverse array of
flavonols.
3.2. Effects of berry juices and teas on fatty steak deposits
Fig. 1A shows the effects of bilberry, raspberry and strawberry
juice consumption on aortic fatty streak deposits in hamsters fed
a high-fat diet for 12 weeks. In the control animals, which ingested
water rather than juice, fatty streaks covered 21.2 ± 2.7% of the aor-
tic wall. The extent of these deposits when juices or teas were
administered to hamsters, were dramatically and significantly low-
er with respect to controls (all p< 0.001 when compared to water
control). The aortic fatty streak area was 4.5 ± 0.5% for the group
fed with bilberry juice, 2.4 ± 0.5% with strawberry juice,
1.1 ± 0.2% with raspberry juice and finally 0.75 ± 0.13% and
1.40 ± 0.31% for green and black tea, respectively. Representative
pictures of fatty streak deposits are presented in Fig. 2.
Table 1
Concentration of phenolic compounds in bilberry juice. Data expressed as nmoles/
ml ± SEM (n= 3).
Compound Concentration
Gallic acid 76 ± 1
5-Caffeoylquinic acid 22 ± 1
Caffeic acid hexoside 12 ± 0
Total gallic and caffeic acid derivatives 110
Delphinidin-3-galactoside 38 ± 2
Delphinidin-3-glucoside 75 ± 2
Delphinidin-3-arabinoside 45 ± 0
Cyanidin-3-galactoside 32 ± 1
Cyanidin-3-glucoside 120 ± 1
Cyanidin-3-arabinoside 46 ± 1
Petunidin-3-glucoside 64 ± 1
Petunidin-3-arabinoside 12 ± 0
Peonidin-3-galactoside 4.8 ± 0.1
Peonidin-3-glucoside 45 ± 1
Malvidin-3-galactoside 31 ± 1
Malvidin-3-glucoside 74 ± 1
Malvidin-3-arabinoside 12 ± 1
Total anthocyanins 599
(–)-Epicatechin 8.1 ± 0.2
Procyanidin dimer 7.4 ± 0.5
Procyanidin trimer 3.2 ± 0.1
Total flavan-3-ols 19
Myricetin-3-galactoside 1.6 ± 0.0
Myricetin-3-glucoside 2.6 ± 0.1
Myricetin-3-glucuronide 1.1 ± 0.0
Quercetin-3-galactoside 2.9 ± 0.0
Quercetin-3-glucoside 1.5 ± 0.0
Quercetin-3-glucuronide 3.6 ± 0.1
Myricetin 1.2 ± 0.0
Quercetin 1.2 ± 0.0
Total flavonols 16
Total phenolics and flavonoids 744
Table 2
Concentration of phenolic compounds in raspberry juice. Data expressed as nmoles/
ml ± SEM (n= 3).
Compound Concentration
Cyanidin-3-sophoroside 108 ± 1
Cyanidin-3-(2
G
-glucosylrutinoside) 32 ± 1
Cyanidin-3-glucoside 12 ± 0
Pelargonidin-3-sophoroside 4.1 ± 0.1
Cyanidin-3-rutinoside 5.6 ± 0.1
Pelargonidin-3-(2
G
-glucosylrutinoside) 2.0 ± 0.1
Total anthocyanins 164
Procyanidin dimer B4 1.5 ± 0.1
Total flavan-3-ols 1.5
Sanguiin H-10 46 ± 1
Lambertianin C 59 ± 2
Sanguiin H-6 235 ± 6
Ellagic acid 16 ± 0.1
Ellagic acid-4-acetylpentose 0.9 ± 0.0
Total hydrolysable tannins and ellagic acid derivatives 357
Quercetin-3-hexosyl-rhamnoside 0.2 ± 0.1
Quercetin-3-galactosylrhamnoside 0.6 ± 0.0
Quercetin-3-rutinoside 0.2 ± 0.0
Quercetin-3-galactoside 2.2 ± 0.1
Quercetin-3-glucoside 0.7 ± 0.0
Quercetin-3-glucuronide 2.4 ± 0.1
Quercetin 0.5 ± 0.0
Total flavonols 7.7
Total phenolics and flavonoids 530
Table 3
Concentration of phenolic compounds in strawberry juice. Data expressed as nmoles/
ml ± SEM (n= 3).
Compound Concentration
p-Coumaric acid-hexose 46 ± 1
Total hydroxycinnamates 46
Pelargonidin-3-glucoside 91 ± 1
Pelargonidin-3-(6-malonylglucoside) 22 ± 0
Total anthocyanins 113
Procyanidin dimer B1 0.9 ± 0.0
Procyanidin dimer B3 4.9 ± 0.1
Procyanidin trimer 2.8 ± 0.0
Total flavan-3-ols 8.6
Sanguiin 9.9 ± 0.2
Ellagic acid rhamnoside 2.0 ± 0.0
Total hydrolysable tannins and ellagic acid derivatives 12
Quercetin-3-glucuronide 0.9 ± 0.1
Kaempferol-3-glucoside 0.3 ± 0.0
Kaempferol-malonyl-hexoside 0.7 ± 0.0
Total flavonols 1.9
Total phenolics and flavonoids 181
268 J.-M. Rouanet et al. / Food Chemistry 118 (2010) 266–271
3.3. Effects of berry juices and teas on circulating cholesterol and
hepatic antioxidant enzymes
The lower fatty streak deposition in juice and tea hamster
groups was not accompanied by lower circulating cholesterol lev-
els (total cholesterol, HDL-cholesterol and non-HDL-cholesterol
were not significantly different between all groups; not shown
here) but was associated to a reduced activity of liver antioxidant
defense system in hamsters fed antioxidant rich beverages in com-
parison to controls (Fig. 1B and C). Teas induced a greater inhibi-
tion in the antioxidant enzymes compared to berry juices.
Analysis of liver extracts by HPLC-mass spectrometry operating
in selected ion and selected reaction monitoring mode did not de-
tect the presence of any flavonoids or phenolic compounds derived
from the teas or berry juices despite the hamsters being fed the
supplements for a period of 12 weeks.
4. Discussion
Daily consumption of each of the test beverages for a 12-week
period resulted in a substantially lower fatty streak deposition in
the arteries of the hamsters compared to water-treated controls,
with stronger effects for green tea and raspberry juice (Figs. 1A
and 2). This marked limitation of the onset of atherosclerosis was
not associated with any significant change in plasma cholesterol
profile.
The observation that the plasma cholesterol profile did not
change among groups of hamsters (Table 5) is in keeping with
the work of Andrews et al. (1995) that demonstrates that absolute
cholesterolaemia is not pivotal in determining the aortic fatty
streak deposition. These results thus strengthen the hypothesis
that oxidation of LDL, more than their plasma level, must be impli-
cated in the pathogenesis of atherosclerosis (Breinholt, Lauridsen,
& Dragsted, 1999). This can explain, at least in part, the effects ob-
served on aortic atherosclerosis after antioxidant juices and tea
consumption.
Table 4
Concentration of phenolic compounds, and purine alkaloids in green and black tea
infusions. Data expressed as nmoles/ml ± SEM (n= 3).
Compound Green tea Black tea
Gallic acid 6.3 ± 0.2 132 ± 7
5-Galloylquinic acid 64 ± 1 77 ± 1
Total gallic acid derivatives 70 209
(–)-Gallocatechin 225 ± 2 n.d.
(–)-Epigallocatechin 921 ± 11 19 ± 1
(+)-Catechin 168 ± 6 7.4 ± 0.2
(–)-Epicatechin 459 ± 10 6.8 ± 0.1
(–)-Epigallocatechin-3-gallate 494 ± 25 7.4 ± 0
(–)-Epicatechin-3-gallate 147 ± 5 11 ± 0
Total flavan-3-ols 2,414 52
3-Caffeoylquinic acid 30 ± 1 5.0 ± 0.2
5-Caffeoylquinic acid 118 ± 1 32 ± 0.1
4-p-Coumaroylquinic acid 85 ± 2 76 ± 1
Total caffeic and coumaric acid derivatives 233 113
Quercetin-rhamnosylgalactoside 4.5 ± 0.2 3.6 ± 0.1
Quercetin-3-rutinoside 39 ± 1 29 ± 1
Quercetin-3-galactoside 46 ± 1 29 ± 1
Quercetin-rhamnose-hexose-rhamnose 7.2 ± 0.2 5.9 ± 0
Quercetin-3-glucoside 72 ± 1 46 ± 1
Kaempferol-rhamnose-hexose-rhamnose 7.7 ± 0 7.4 ± 0.2
Kaempferol-3-galactoside 17 ± 1 12 ± 1
Kaempferol-3-rutinoside 21 ± 1 18 ± 1
Kaempferol-3-glucoside 41 ± 0 28 ± 2
Kaempferol-3-arabinoside 2.0 ± 0.1 n.d.
Unknown quercetin conjugate 0.8 ± 0.1 0.9 ± 0.1
Unknown quercetin conjugate 6.7 ± 0.2 4.9 ± 0.2
Unknown kaempferol conjugate 2.0 ± 0 n.d
Unknown kaempferol conjugate 0.4 ± 0.0 10 ± 0.0
Total flavonols 267 185
Theaflavin n.d. 20 ± 1
Theaflavin-3-gallate n.d. 16 ± 1
Theaflavin-3-gallate n.d. 8.8 ± 0.1
Theaflavin-3,3-digallate n.d. 13 ± 0
Thearubigins n.d. 1781
Total theaflavins and thearubigins n.d. 1839
Total phenolics and flavonoids 2984 2285
Theobromine 57 ± 1 25 ± 1
Caffeine 804 ± 15 503 ± 3
Total purine alkaloids 861 528
n.d. – not detected.
0
5
10
15
20
25
Control Bilberry Raspberry Strawberry Green tea Black tea
% of aortic arch area
a
dc
dcd
b
0
100
200
300
400
500
600
Control Bilberry Raspberry Strawberry Green tea Black tea
SOD activity (U/mg protein)
a
bbb
c
c
0
5
10
15
20
25
30
Control Bilberry Raspberry Strawberry Green tea Black tea
GSHPx activity (U/mg protein)
a
bbb
cc
0
A
B
C
Fig. 1. Effects of dietary treatments on aortic fatty streak area and hepatic
antioxidant enzymes activities. Mean values expressed as (A) a percent of aortic
area ± SEM (n= 10) for aortic arch area, and as units per mg of hepatic pro-
teins ± SEM (n= 10) for (B) SOD and (C) GSHPx activity. Bars with different letters
are significantly different (p< 0.05).
J.-M. Rouanet et al. / Food Chemistry 118 (2010) 266–271 269
There are reports that consumption of fruit juice and green tea
both increase the activity of hepatic antioxidant enzymes (Lin
et al., 1998; Young et al., 1999). This contrasts with the findings
of the present study where berry juice and tea consumption low-
ered hepatic GSHPx and SOD activities (Fig. 1B and C), and agree
with previous results in hamsters receiving either pure catechin,
quercetin or resveratrol (Auger et al., 2005) or phenolics from
purple grape, apple, purple grape juice and apple juice (Décordé,
Teissèdre, Auger, Cristol, & Rouanet, 2008). One explanation for
this down regulation is that it is a consequence of dietary antiox-
idants being able to scavenge oxygen radicals and thus reduce the
need for enzymatic endogenous antioxidants.
The prevention of fatty streak deposition by berry juice and tea
consumption does seemingly involve mechanisms allowing the
possibility of phenolic compounds to induce local antioxidant ef-
fects which cannot be ruled out. Recent data suggests that dietary
phenolics can modulate inflammatory pathways, hence reducing
the severity of local inflammation (Rahman, Biswas, & Kirkham,
2006). The pathogenesis of atherosclerosis has been linked to the
occurrence of inflammatory processes inside the arterial wall dur-
ing the initiation of lesions (Call, Deliargyris, & Newby, 2004). Com-
pounds derived from the ingested phenolics could, therefore, delay
the progression of atherosclerosis by inhibition of arterial wall
inflammation.
Endothelial dysfunction is also associated with the increased
production of the vasoconstrictive peptide endothelin-1, which
has been linked with chronic inflammation of the arterial wall
(Feletou & Vanhoutte, 2006; Schiffrin, 2005). Endothelin is also
related with the onset and development of atherosclerosis, with
atherosclerotic plaques containing an increased endothelin con-
centration (Bacon, Cary, & Davenport, 1996). Moreover, endothe-
lin-1 production is induced by oxidised LDL, which in turn can
recruit macrophages and monocytes in the arterial wall (Schiffrin,
2001). Endothelin, thus, plays a pivotal role in development of dis-
eases related to vascular function. As demonstrated by Corder et al.
(2006), phenolic compounds, principally procyanidins, are able to
reduce the production of endothelin-1 by endothelial cells. The
green tea polyphenol (–)-epigallocatechin gallate is also reported
to reduce endothelin expression (Spinella et al., 2006). Inhibition
of endothelin-1 over-expression is, therefore, a further potential
mechanism for the observed protective effects of juice and tea
consumption.
Tea antioxidants, in particular catechins, act either as activators
or inhibitors of signal transduction kinases interfering with multi-
ple pathways of signal transduction in cardiovascular relevant cells
(Stangl, Dreger, Stangl, & Lorenz, 2007). Arguably, this could ex-
plain the strong effect of green tea with respect to the other bever-
ages in this study.
The fact that the five beverages, which reduced the onset of ath-
erosclerosis, contained a very different spectrum of phenolics im-
plies that a wide variety of compounds may be bioactive and
that the observed preventive effects may be due to the influence
of several constituents working either independently or in tandem.
What compounds enter the circulatory system, reduce the arterial
fatty streaks deposition and the activity of hepatic SOD and GSHPx
remains to be determined. Analysis of the livers of hamsters after
12 weeks plus an overnight treatment between last feed and sacri-
fice showed no trace of either the parent compounds from the bev-
erages or their glucuronyl-, methyl- or sulfo-metabolites,
indicating that they do not accumulate in these tissues, at least
in detectable quantities.
In conclusion, we have demonstrated that berry juices and teas
fed to hamsters under atherogenic diet are able to facilitate a very
strong inhibition of aortic fatty streaks deposition. These effects are
physiologically relevant as they were induced by a daily supple-
ment equivalent to 275 ml of beverage consumed on a daily base
by a 70 kg human. The features and progression of the lesions ob-
served in the hamster model of atherosclerosis are morphologically
similar to atheromatous lesions observed in humans. The hamster
is therefore considered to be a good animal model to study the for-
mation of atheromas in humans (Yamanouchi et al., 2000). It is also
of interest to note while both bilberry and strawberry juices pre-
vent aortic lipid deposition in hamsters, blueberry, a close relative
of bilberry, and strawberry extracts both bring about improve-
ments in neuronal function and behaviour in a rodent model of
accelerated aging (Shukitt-Hale, Carey, Jenkins, Rabin, & Joseph,
2007).
Carotid atherosclerosis is associated with aortic atherosclerosis
(Shimizu et al., 2003) and the intima-media thickness of the com-
mon carotid artery has been shown to predict coronary events and
Fig. 2. Photomicrographs of hamster aortic arches after 12 weeks on an atherogenic diet (control) and 12 weeks on an atherogenic diet supplemented with either strawberry
juice, bilberry juice, raspberry juice, green tea or black tea. The micrographs are examples of the aortic arch surface covered by lipid inclusion in the intima with lipids
coloured red using Oil Red O stain. Quantifications of fatty streaks are summarised in and Fig. 1A. All micrographs have the same scale.
270 J.-M. Rouanet et al. / Food Chemistry 118 (2010) 266–271
is, therefore, a non-invasive predictor of future ischemic stroke
incidence (Chambless, Folsom, Clegg, Sharrett, & Shahar, 2000).
Thus, polyphenol-rich berry juices and green and black tea intake
may be of significant relevance to clinical and public health.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
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Oxidation of low density lipoprotein (LDL) is implicated in the pathogenesis of atherosclerosis. In this study the susceptibility to oxidation of LDL (from patients with atherosclerosis) is related to the progression of the disease. LDL were isolated from 37 patients with demonstrable atherosclerotic plaques. The susceptibility of LDL to oxidation (induced by an exogenous oxidative stress) was assessed by measuring the breakdown products of lipid peroxidation, the increased formation of conjugated dienes, and changes in surface charge of the apolipoprotein B (apo B). Progression of the atherosclerotic plaque was assessed by measuring the maximum velocity of blood through the narrowest portion of the vessel at inclusion and after one year. Twenty-nine of the 37 samples taken were found to have LDL that were partially oxidised, whereas 8 samples showed LDL whose state of oxidation was within the normal range. Progression of the atherosclerotic plaque occurred in 19 (66%) of the 29 patients whose lipoproteins were partially oxidised compared with only 2 (25%) of the 8 patients with normal lipoproteins (P = 0.055, Fisher's exact test). These data support an association between the progression of atherosclerotic plaques in carotid and femoral vessels and the susceptibility to oxidation of LDL.
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Combining data from two large-scale prospective observational studies in women and men, investigators link the consumption of fruits and vegetables to the risk of ischemic stroke. Of interest is their breakdown of these foods into cruciferous vegetables and citrus fruit juice, which confer the greatest protection.
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In this animal study, Wistar rats were fed 2.5% green tea (longjing) leaves, for 27 and 63 weeks; the changes of GOT, GPT, γ-GT, and creatinine were not significant in the treated group as compared with the control. These results suggested that long-term feeding of green tea leaves was not toxic to the liver or kidney. Serum total cholesterol, triglyceride, and LDL-C were decreased in the tested group. Interestingly, the dietary intakes of the two groups were approximately the same, but the body weights of the tea-fed group were decreased 10−18% compared with those of the control. The activities of antioxidant enzymes (SOD and catalase) and phase II enzyme (GST) and glutathione concentration in the liver of Wistar rats were significantly higher in the treated group. The biological significance of these results can be implicated in relation to the hypolipidemic effect as well as the cancer chemopreventive action of green tea. Keywords: Longjing tea; GST; HDL-C; hypolipidemic; LDL-C; phase II enzyme; SOD
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