One-month strawberry-rich anthocyanin supplementation ameliorates

cardiovascular risk, oxidative stress markers and platelet activation in humans

Jos M. Alvarez-Suarez
a
, Francesca Giampieri
a, b
, Sara Tulipani
c
, Tiziana Casoli
d
, Giuseppina Di Stefano
e
,
Ana M. Gonzlez-Params
f
, Celestino Santos-Buelga
f
, Franco Busco
g
, Jos L. Quiles
h
, Mario D. Cordero
i
,
Stefano Bompadre
j
, Bruno Mezzetti
b
, Maurizio Battino
a,

a
Dipartimento di Science Cliniche Specialistiche ed Odontostomatologiche, Facolt di Medicina, Universit Politecnica delle Marche, Ancona, Italy
b
Dipartimento di Scienze Agrarie, Alimentari ed Ambientali, Universit Politecnica delle Marche, Ancona, Italy
c
Research Laboratory, Virgen de la Victoria Clinical Hospital (IMABIS Foundation), Malaga, Spain
d
Neurobiology of Aging Laboratory, INRCA Scientific Technological Area, Ancona, Italy
e
Scientific Direction, INRCA Scientific Technological Area, Ancona, Italy
f
Grupo de Investigacin en Polifenoles (GIP-USAL), Universidad de Salamanca, Spain
g
Analysis Laboratory of INRCA Hospital, Ancona, Italy
h
Instituto de Nutricin y Tecnologa de los Alimentos Mataix Verd, Departamento de Fisiologa, Universidad de Granada, Granada, Spain
i
Departamento de Citologa e Histologa Normal y Patolgica, Facultad de Medicina, Universidad de Sevilla, Spain
j
Dipartimento di Scienze Biomediche e Sanit Pubblica, Facolt di Medicina, Universit Politecnica delle Marche, Ancona, Italy
Received 29 May 2013; received in revised form 19 August 2013; accepted 8 November 2013
Abstract
Strawberries are an important fruit in the Mediterranean diet because of their high content of essential nutrients and beneficial phytochemicals, which seem
to exert beneficial effects in human health. Healthy volunteers were supplemented daily with 500 g of strawberries for 1 month. Plasma lipid profile, circulating
and cellular markers of antioxidant status, oxidative stress and platelet function were evaluated at baseline, after 30 days of strawberry consumption and 15 days
after the end of the study. A high concentration of vitamin C and anthocyanins was found in the fruits. Strawberry consumption beneficially influenced the lipid
profile by significantly reducing total cholesterol, low-density lipoprotein cholesterol and triglycerides levels (8.78%, 13.72% and 20.80%, respectively;
Pb.05) compared with baseline period, while high-density lipoprotein cholesterol remained unchanged. Strawberry supplementation also significant decreased
serum malondialdehyde, urinary 8-OHdG and isoprostanes levels (31.40%, 29.67%, 27.90%, respectively; Pb.05). All the parameters returned to baseline
values after the washout period. A significant increase in plasma total antioxidant capacity measured by both ferric reducing ability of plasma and oxygen radical
absorbance capacity assays and vitamin C levels (+24.97%, +41.18%, +41.36%, respectively; Pb.05) was observed after strawberry consumption. Moreover, the
spontaneous and oxidative hemolysis were significant reduced (31.7% and 39.03%, respectively; Pb.05), compared to the baseline point, which remained
stable after the washout period. Finally, strawberry intake significant decrease (Pb.05) the number of activated platelets, compared to both baseline and washout
values. Strawberries consumption improves plasma lipids profile, biomarkers of antioxidant status, antihemolytic defenses and platelet function in healthy
subjects, encouraging further evaluation on a population with higher cardiovascular disease risk.
2014 Elsevier Inc. All rights reserved.
Keywords: Strawberry consumption; CVD risk; Platelet activation; LDL-C; Cholesterol; Triglycerides
1. Introduction
Cardiovascular diseases (CVD) are the worlds biggest killer and
major cause of death among non-transmittable diseases [1]. Substan-
tial evidence indicates that CVD is a life course disease that begins
with the development of subclinical atherosclerosis and the latent
increase of risk factors prior to culminating in the diagnosed
pathological state. Consequently, primary and secondary prevention
of CVD and early monitoring of CVD-related risk factors are
dramatically urgent public health priorities.
Diet plays a crucial role in the prevention of CVD [2], and dietary
patterns based on a high consumption of fruits and vegetables, such as
the Mediterranean diet [2], have been particularly associated with a
Available online at www.sciencedirect.com
ScienceDirect
Journal of Nutritional Biochemistry 25 (2014) 289294

Sources of Funding: The research was partially granted by EUBerry

Project: EU FP7 No. 265942. The GIP-USAL is financially supported by the
Spanish Government through the projects BFU2012-35228 and CSD2007-
00063 (Consolider-Ingenio 2010 Programme).

Corresponding author. Dipartimento di Science Cliniche Specialistiche

ed Odontostomatologiche, Sez. Biochimica, Facolt di Medicina, Universit
Politecnica delle Marche, Italy, Via Ranieri 65, 60100 Ancona, Italy. Tel.: +39
071 2204646; fax: +39 071 2204123.
E-mail address: m.a.battino@univpm.it (M. Battino).
0955-2863/$ - see front matter 2014 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jnutbio.2013.11.002
longer life expectancy and a significative decrease of incidence and
prevalence of CVD[3]. Since an imbalance between oxidative stress and
endogenous/exogenous body antioxidant defenses is involved in its
pathogenesis, antioxidant components of fruit and vegetables such as
polyphenols have been described to possess antiatherosclerotic
properties and play a role in protecting cellular macromolecules from
reactive oxygen species (ROS)/reactive nitrogen species (RNS)-induced
damage, and improving antioxidant status and endothelial function [3].
Among fruits, soft berries are of particular interest because of
their high antioxidant phytochemical content. Even if it is difficult
to prove categorically that certain foods can affect the decreased
risk of CVD, several studies carried out on berry supplementation
are encouraging [47]. A possible mechanism to explain the
decreased risk is related to the high content of antioxidants, i.e.,
polyphenols and vitamin C, in the fruit, but growing evidence
suggests that additional direct and indirect mechanisms of action
appear to be involved in the protective effects provided by the fruit
intake [3]. Among the mechanisms already proposed, a regular
consumption of berries/strawberries may positively affect risk
factors for CVD by improving the plasma lipid profile, increasing
plasma antioxidant activity [8] low-density lipoprotein (LDL)
resistance to oxidation [3] and improving endothelial function [9].
Significant increases in LDL peroxidation lag time [9], as well as
significant decreases of total and LDL cholesterol, small LDL
particles [10,11], and favourable changes in high-density lipopro-
tein (HDL) cholesterol and blood pressure [12,13] were reported
after relatively protracted periods of strawberry consumption.
Platelets deserve special attention: in part, activated blood
platelets are important in the development of CVD as they are
major components of thrombi that occlude arteries [14]; therefore,
favouring an optimal platelet function, via the reduction of diet-based
platelet hyperactivity, can be considered a feasible approach for the
maintenance of cardiovascular health. Impaired platelet reactivity can
be found in smokers and people under stress. Hence, favouring an
optimal platelet function can be considered a feasible approach for
the maintenance of cardiovascular health. However, current evidence
on the effects of the consumption of specific food items, such as
strawberries, on platelets activation is still scarce.
In the last few years, our group has been conducted several acute
and protracted strawberry consumption studies by selecting straw-
berry varieties particularly rich in phytochemical compounds [15
19]. Although our findings already suggested several health promot-
ing effects of strawberries, particularly improving plasma antioxidant
status and erythrocyte resistance to oxidative haemolysis in humans
[16], more information is clearly needed about the effects of
strawberry intake as a preventive factor of CVD. Moreover, at present
there are fewstudies to verify the possible maintenance of the healthy
effects following a nutritional intervention with strawberries (wash-
out period).
Since the increases of markers as cholesterol, LDL cholesterol
(LDL-C) and triglycerides are an alarm bell and represent risk factors
for the predisposition of CVD, a more detailed study of the effect of
strawberry on these markers could be an important target to
elucidate how the consumption of these fruits may be beneficial in
the prevention of such pathologies. Until now, our group has shown a
significant improvement of oxidative status in volunteers after the
consumption of strawberries, but more deep information is necessary
about the effect of their consumption on specific markers of CVD,
paying particular attention on lipid profile and platelet activity,
markers that have never been evaluated in depth in our studies.
Therefore, the aim of the present study was to assess in vivo the
possible beneficial effect of a protracted consumption of Alba
strawberry cultivar fruits on serum lipid profile, biomarkers of
antioxidant status and erythrocyte resistance to oxidative hemolysis,
as well as on platelet function in healthy subjects.
2. Methods
2.1. Strawberry fruit analysis
The commercial variety of "Alba" strawberry, from the strawberry breeding
program of the Marche Polytechnic University, Ancona, Italy, was selected for this
study. Compound extraction was carried out depending on the analysis to be
performed [19]. The total phenolic content (TPC) was determined by Folin-Ciocalteu
method [20], total flavonoid content (TFC) by the aluminium chloride spectrophoto-
metric method [19] while vitamin C was analyzed by reversed-phase high-
performance liquid chromatography (HPLC) [19]. Anthocyanins (ACYs) extraction
and HPLC-MS/MS analysis were performed as previously described [21]. Total
antioxidant capacity (TAC) was determined using Trolox Equivalent Antioxidant
Capacity [22] and Oxygen Radical Absorbance Capacity (ORAC) assays [23].
All results were expressed per total fresh weight of strawberries consumed daily (500 g
fresh weight [FW]) by each subject during the test as grams of gallic acid (TPC), catechin
(TFC), vitamin C (Vit C) and milligrams of pelargonidin-3-glucoside (Pg-3-glc) or cyanidin-
3-glucoside equivalents, while TAC values were expressed as mM of Trolox equivalents.
2.2. Subjects and study design
Twenty-three healthy volunteers (11 men and 12 women, age 273.2, weight 63.5
12.7 kg and body mass index 21.742.5 kg/m
2
) were included in the study. The study
was performed in accordance with the principles of the Declaration of Helsinki as
revised in 2000; the protocol was approved by the Medicine School of Marche
Polytechnic University Ethical Committee and informed written consent was obtained
from each participant. Smokers and subjects declaring vitamin or other dietary
supplementation, case history of allergy to strawberries or other berry fruits, history of
any chronic disease possibly linked to oxidative stress and current symptoms of any
acute disease were excluded from the study.
Fig. 1 shows a scheme of the study design. To standardize baseline data and to
prevent dietary changes during the study, two weeks before the experiment (pre-study
period), subjects began a strawberry-free and low-in-polyphenolics diet. A list of daily
recommended foods, limited foods and beverages and forbidden consumption
(strawberries) was provided to the subjects. At baseline, subjects started a 30-day
period of consumption of 500 g of fresh strawberries per day
1
(in-study period),
preferably at mid-morning and mid-afternoon between meals. Participants were
advised not to change their habitual diet until the end of the study (washout) and filled
out a detailed daily diet diary from the pre-study period until the end of the 30 days of
strawberry supplementation. During the washout period 15 days, subjects were invited
to avoid the consumption of strawberries. Energy, macronutrient and micronutrient
intake of the subjects before and during the study was calculated using Funiber
Nutriber software (version 1.1.1.R4). Table 1 shows the details of the energy, macro
and micronutrient intake of the subjects through the baseline diet and contribution of
strawberries daily dose.
2.3. Samples
Blood samples (10 mL) were collected by antecubital venipuncture into a sodium
citrate vacutainer (BD Vacutainer CPTTM) from overnight fasting subjects at baseline,
after a 30-day supplementation period (time 30d), and 15 days after the end of the
study (washout). Plasma was isolated and stored at 80C for biochemical studies.
Morning urine samples were also collected at each time points and frozen at 80C
until analysis.
2.4. Biochemical analysis and biomarker determination of antioxidant status and
oxidative stress
The enzymatic test kits were used for the determination of plasma glucose
(GOD-PAP), total cholesterol (CHOD-PAD), triglycerides (GPO-PAP), high-density
lipoprotein cholesterol (HDL-C) and LDL-C (HDL-C plus 3rd generation and LDL-C
plus 2nd generation, respectively), while albumin was determined by colorimetric
test kit (BCG). All commercial kits were purchased from Roche Diagnostics GmbH,
Mannheim, Germany, and the analyses were conducted using automated clinical
chemistry analyzers Roche/Hitachi. Plasma TAC was estimated by evaluating the
plasma antioxidant capacity by ORAC [20] and by ferric reducing ability of plasma
(FRAP) assays [24]. The serum concentrations of ascorbic and uric acids were
measured by HPLC coupled to electrochemical detection [15] while the extent of
plasma lipid peroxidation was estimated by HPLC-UV quantification of malondial-
dehyde (MDA) [25].
Evaluation of urinary 8-hydroxy-2-deoxyguanosine (8-OHdG) was determined
using a competitive in vitro enzyme-linked immunosorbent assay (ELISA) kit (JaICA,
Japan). Determination of urinary isoprostanes level was carried out using an enzyme
1
The daily dose of strawberries corresponded to the amount given to a
hypothetical 63 kg individual. A few extra strawberries were respectively given
to subjects with a body weight above the mean value (63 kg), in order to
standardize the dose/body weight ratio.
290 J.M. Alvarez-Suarez et al. / Journal of Nutritional Biochemistry 25 (2014) 289294
immunoassay (ELISA) kit (Neogen, Lexington, KY, USA). The analyses were performed
within 3 months of postcollection.
2.5. Haemolysis test
Erythrocyte resistance to oxidative hemolysis induced ex vivo was evaluated as
previously described [16]. Briefly, red blood cell (RBC) pellets were washed once with
0.9% NaCl solution, then three times with phosphate-buffered saline (PBS) at room
temperature and resuspended (4% haematocrit) in PBS buffer (spontaneous haemo-
lysis) or in a 12.5 mM AAPH (2,2'-azobis-2-methyl-propanimidamide, dihydrochlor-
ide) buffered solution (AAPH-induced haemolysis) at pH 7.4, and incubated for up to 5
h at 37C with gentle shaking. The spontaneous and AAPH-induced haemolysis were
recorded after 1, 3 and 5 h of RBCs incubation and spectrophotometrically evaluated at
540 nm as haemoglobin (Hb) released from cells in the supernatant [26]. Results are
expressed as % haemolysis.
2.6. Determination of platelet function by electron microscopy
For the platelet study ten of the twenty-three volunteers were randomly chosen (5
men and 5 women). To obtain platelet-rich-plasma blood samples were centrifuged for
10 min at 200g and platelet pellets were separated by centrifugation at 2000g for
20 min, fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) and
washed twice with the same buffer. After 1% buffered osmium tetroxide post-fixation,
platelets were dehydrated in a graded series of ethanol, treated with propilenoxide and
embedded in Durcupan ACM resin. Ultrathin sections were mounted on copper grids
and stained with uranyl acetate and lead citrate. An average of 130 platelets were
randomly chosen from each sample at the Zeiss EM 109 transmission electron
microscope (TEM) and classified, according to their ultrastructure, into three
categories: resting, central clustered and degranulated that represent platelets in the
basal state, during activation and in the post activation phase, respectively [27].
2.7. Statistical analysis
Statistical analyses were performed using STATISTICA software package (Statsoft,
Tulsa, OK, USA). Plasma, serum and erythrocyte data were subjected to the Wilcoxon
paired samples test. The mean of three analyses was used and the results reported as
meanS.E.M. and as % changes from the baseline values. To analyze whether the
strawberry supplementation had influenced platelet function, repeated measures
analysis of variance was applied within each class of platelets and data were reported
as percentage meanS.E.M. Differences at Pb.05 and at Pb.01 were considered
statistically significant and highly significant, respectively.
3. Results
3.1. Biomarkers of antioxidant status and oxidative damage
Table 2 shows the values of general biochemical parameters,
biomarkers of antioxidant status and oxidative stress of the subjects
during the study. The consumption of strawberries positively affected
the plasma lipid profile. Serum levels of total cholesterol at time 30d
were significantly lower (8.78%, Pb.05) than those of the baseline
period. Similar findings for LDL-C (13.72%, Pb.05) and triglyceride
levels (20.80%, Pb.05) were found, although they both returned to
Fig. 1. General scheme of the intervention study.
Table 1
Energy, macronutrient and micronutrient intake of the subjects through the baseline
diet and contribution of strawberries daily dose
Parameter Baseline diet
b
Contribution of 500 g/day of
fruits to daily overall intake
c
Energy (kcal) 1938.27192.03 1600.02
Protein (g) 86.697.47 3.350.01
Total lipid (g) 51.736.81 1.50.01
Saturated (g) 12.531.68 0.0750.01
Monounsatured (g) 23.642.65 0.2150.01
Polyunsaturated (g) 9.280.54 0.7750.01
Total carbohydrate (g) 268.1828.50 48.350.03
Dietary fiber (g) 11.511.91 100.02
Folate (g) 144.8628.55 1200.01
Vit C (g/day)
b
0.0890.0 0.170.01
Vitamin E, - tocopherol (mg) 3.320.73 1.450.01
Total phenolic (g/day)
d
- 1.130.02
Total flavonoid (g/day)
d
- 0.470.01
Total anthocyanins (mg/day)
d
- 307.590.01
Cy-3-glucoside - 9.510.02
Pg 3-glucoside - 253.760.01
Pg 3-rutinoside - 10.440.01
Cy 3-malonylglucoside - 0.580.01
Pg 3-malonylglucoside - 32.740.02
Pg 3-acetylglucoside - 0.560.00
TAC (mM TE/day)
d
-
FRAP - 5.530.7
ORAC - 24.81.7
Nutritional values correspond to the daily dose (500 g) consumed by each subject
during the test.
a
a
The total daily intakes are expressed as meansstandard error (SEM).
b
Intake were determined using Funiber Nutriber software (version 1.1.1.R4).
c
Intakes were estimated by using the database of US Department of Agriculture,
Agriculture Research Service. USDA National Nutrient for Standard References, Fruits
and fruit juices (http://www.ars.usda.gov/Services/docs).
d
Intake were based on chemical analyses of strawberry fruit.
Table 2
Biochemical parameters and biomarkers of antioxidant status and oxidative stress of
the volunteers during the study
Parameters (reference values) Baseline Time 30d Washout
General biochemical analysis
Glucose (g/L) 4.710.07
a
4.760.09
a
4.710.01
a
Albumin (g/L) 50.50.3
a
49.30.2
a
49.30.5
a
Total cholesterol (mmol/L) 4.580.13
a
4.180.12
b
4.500.12
a
HDL-C (mmol/L) 1.540.07
a
1.570.07
a
1.520.06
a
LDL-C (mmol/L) 2.540.10
a
2.190.09
b
2.520.10
a
Triglycerides (mmol/L) 0.850.09
a
0.670.06
b
0.820.06
a
Antioxidant status
FRAP (mol TE/L) 46.125.16
a
57.644.18
b
47.546.54
a
ORAC (mol TE/L) 16 636105
a
23 487137
b
15 013121
a
Vit C (mol/L) 39.842.94
a
56.322.39
b
41. 792.67
a
Uric acid (mol/L) 248.125.65
a
2467.12
a
249.548.32
a
Biomarkers of oxidative stress
MDA (nM/mL) 1.210.13
a
0.830.01
b
1.110.02
a
8-OHdG (ng/mL) 5.930.06
a
4.070.11
b
5.240.10
a
Isoprostanes (ng/mL) 0.860.04
a
0.620.01
b
0.790.03
a
Data are presented as meansstandard error (SEM). Different superscript letters in the
same row are significantly different at Pb.05.
291 J.M. Alvarez-Suarez et al. / Journal of Nutritional Biochemistry 25 (2014) 289294
baseline concentrations after the washout period. HDL-C remained
unchanged throughout the in-study period.
Similar improvements were observed in several markers of
antioxidant status and plasma urinary oxidative stress. Following 30
days of strawberries consumption, TAC values of plasma significantly
increased by both FRAP (24.97%, Pb.05) and ORAC (41.18%, Pb.05)
assays, together with a highly significant increase of the serum
concentration of ascorbic acid (41.36 %, Pb.01). No significant changes
were observed in the serum levels of uric acid throughout the in-
study period.
Concomitantly, a significant decrease was observed in plasma
MDA (31.40%, Pb.05) and the urinary 8-OHdG and isoprostanes
(29.67%, Pb.05 and 27.90%, Pb.05) following the strawberry interven-
tion, compared to baseline. All the parameters returned to baseline
values after the washout period.
3.2. Erythrocyte susceptibility to hemolysis
Fig. 2 depicts the % erythrocyte hemolysis after 1 h, 3 h and 5 h
incubation in PBS (% spontaneous hemolysis, Fig. 2A) and in AAPH
solution (% AAPH-induced hemolysis, Fig. 2B). An increased resistance
to hemolysis was observed at time 30d for both the spontaneous and
AAPH-induced hemolysis, compared to baseline. As shown in Fig. 2A a
significant reduction (31.70%, Pb.05) of the percentage of spontane-
ous hemolysis after 5 hours of incubation was observed in time 30d
compared to baseline, remaining below (22.40%, Pb.05) the baseline
values also after the washout period. Similarly, in the AAPH-treated
erythrocytes at time 30d (Fig. 2B) a better response to oxidative
damage was found after strawberry consumption, compared to the
baseline point, showing a significant decrease in the % of AAPH-
induced hemolysis (39.03%, Pb.05). The erythrocyte resistance to
hemolysis remained higher compared to baseline also after the 15-
day washout period with a significantly lower percentage of
hemolysis (21.51%, Pb.05), indicating a possible long-lasting effect
in the synergism between the compounds of strawberries and
erythrocyte membranes.
3.3. Determination of platelet activation
Table 3 shows the study of platelets at each time point according
to their ultrastructural characteristics and level of activation classified
as: resting (A), central clustered (B) and degranulated platelets (C), as
observed at TEM. These categories correspond to three different
physiological conditions of platelets: resting is referred to unstimu-
lated platelets, central clustered to activated platelets, and degranu-
lated to the final phase of activation (Fig. 3). At time 30d a significant
decrease (Pb.05) in the number of central clustered platelets was
observed compared to both baseline and washout values. Moreover,
no significant differences were found in the two other groups
investigated, namely resting and degranulated platelets, between
the three times of the study, suggesting that strawberry supplemen-
tation had a favorable effect on platelet function.
4. Discussion
The strawberry cultivar Alba was chosen as fruit material for the
present study due to its high nutritional attributes [1519]. The
concomitant increase of plasma TAC and vitamin C levels agrees with
our previous results [15,16]. Moreover, the parallel return to baseline
concentrations after washout period strengthens our previous reports
[15,16] and the hypothesis that the main contribution of strawberry
consumption to the rise of plasma TAC is the transient increase of
vitamin C intake and bioavailability. Certainly, it should be taken into
account that, in the recent past, other strawberries phytochemicals
have attracted the attention for their potential contribution to the
increase of plasma TAC, in synergy with vitamin C, such as several
classes of polyphenols (including flavonols, some flavanols such as
anthocyanins and ellagitannins) or flavor constituents (i.e., the
recently considered 2,5-dimethyl-4-hydroxy-3-[
2
H] furanone) [4].
However, the effective in vivo bioavailability and antioxidant
bioefficacy of these non-nutrient compounds still lack of sufficient
scientific support [28]. In the present study, plasma MDA, urine 8-
OHdG and isoprostane concentrations were measured along the
study, to evaluate the extent of lipids and DNA oxidative damage, and
the possible changes following strawberry intake. The findings
supported the hypothesis that a strawberry-enriched diet may
significantly improve the markers of oxidative stress, by decreasing
lipid peroxidation oxidation [3] and protecting cells against DNA [29].
A B
Fig. 2. Percentage of spontaneous (A) and AAPH-induced hemolysis (B) at baseline, time 30d of strawberry intake and after the washout period. Data are presented as % hemolysis
evaluated after 1, 3 and 5 h of incubation in control (PBS) or AAPH-containing solution. Asterisks indicate a significant difference compared to the corresponding column of the baseline
time point. *Significant difference at Pb.05; **highly significant difference at Pb.001.
Table 3
Percentage of platelets in the different functional categories at each time point
Platelet classification Baseline Time 30d Washout
Resting 99.050.86 99.341.25 98.611.57
Central clustered 0.290.03 0.090.02

0.420.03
Degranulated 0.660.02 0.570.03 0.970.28
Data are expressed as meanS.E.M. (n=10).
Significant difference at Pb.05 vs. central clustered platelets at baseline and
washout.
292 J.M. Alvarez-Suarez et al. / Journal of Nutritional Biochemistry 25 (2014) 289294
Interestingly, the consumption of strawberries was also associated
with a general improvement of the serum lipid profile of the subjects,
through a reduction of total cholesterol, LDL-C and triglyceride levels,
as previously reported [30,31], indicating that some of the constitu-
ents present in the fruit, alone or in combination, favorably affect the
plasma lipid profile.
From the fruit analysis it was evidenced the high content of
vitamin C and that among polyphenols, ACYs were quantitatively the
most representative class of flavonoids, representing approximately
65.31% of total flavonoid contents, with Pg-3-glc as the major
anthocyanin. The contribution of 500 g/day of strawberry to daily
overall intake provides approximately 0.17 g/day and 307.59 mg/day
of vitamin C and ACYs, respectively (Table 1). There is convincing
evidence that vitamin C is a strong inhibitor of LDL oxidation [3], a
recognized factor in the pathogenesis and progression of human
atherosclerosis, by scavenging free radicals and other reactive species,
and preventing their interaction to oxidize LDL [3]. Also ACYs have
been found to exert beneficial effects against LDL oxidation. In vitro
studies have revealed that elderberry anthocyanins protect endothe-
lial cells against several oxidative stressors [32], suggesting the
possibility that endothelial cells can incorporate anthocyanins into
the membrane and cytosol, and supporting the theory that strawberry
antioxidants can preserve endothelial function and prevent the
initiation of endothelial cell changes associated with CVD. The
supplementation of the diet with bilberry anthocyanin-rich extracts
led to a significant inhibition of atherosclerotic plaque development
in Apo E-deficient mice, whereas no effect on total antioxidant
capacity was seen [10]. After bilberry anthocyanin-rich extracts
supplementation in Apo E-deficient mice the nutrigenomic analysis
identified 1261 genes implicated in several cellular processes, such as
oxidative stress, angiogenesis and adhesion molecules, whose
expression was modulated by bilberry anthocyanin in the aorta
[11], suggesting that the cardiovascular effect of ACYs are related to
their modulating properties at gene expression level than their
antioxidant properties. Although between bilberries and strawberry
exist differences in their respective ACYs composition, some of these
are common to both, as the cyanidin derivatives, which were the
second most abundant ACYs found in strawberries used for this study.
Therefore, the above results are encouraging and provide a starting
point for further studies in strawberries, because at present there
are few studies available considering strawberry intake under
this approach.
Even if it is difficult to analyze the effect of a single compound of
diet on health, recent studies on the effect of dietary anthocyanins on
human health are encouraging, leading to support the results here
exposed. In an study conducted by Qin et al. [33] in a dyslipidemic
subjects supplemented with 160 mg anthocyanins (a mix of
glycosylated anthocyanins as 3-O-b-glucosides, 3-O-b-galactosides
and 3-O-b-arabinosides of cyanidin, delphinidin, petunidin, peonidin,
malvidin and delphinidin) twice daily or placebo for 12 weeks in a
double-blind, randomized, placebo-controlled trial was found that
anthocyanin consumption increased HDL-cholesterol concentrations
(13.7% and 2.8%) and decreased LDL-cholesterol concentrations
(13.6% and 20.6%) in the anthocyanin and placebo groups, respec-
tively. More recently the results of a meta-analysis by Cassidy et al.
[34], following 93.600 women from 25 to 42 years of age,
demonstrated that high anthocyanin intake was associated with a
reduction of risk of myocardial infarction (MI) in young and middle-
aged women. In this study an inverse association between higher
intake of anthocyanins and risk of MI was observed. Combined intake
of 2 anthocyanin-rich foods, blueberries and strawberries, tended to
be also associated with a decreased risk of MI when compared to
those consuming N3 servings a week and those with lower intake. For
every 15 mg increase in anthocyanins intake, the relative risk of MI
decreased by 17%. Moreover, the intakes of other flavonoid subclasses
were not significantly associated with MI risk. Therefore, according to
the data obtained in this study and in several others cited above, the
polyphenols and vitamin C in the fruit seem to be the most likely
constituents, which exert in vivo effects in the prevention of the
CVD risk.
As far as the improvement in cholesterol level is concerned also
fiber is well known to have a significant effect on reducing cholesterol
[35]. The reduction of total cholesterol found in our study may have
been due, at least partly, to the increased intake of fiber during time
30d. Strawberries provide approximately 2 g fiber/100 g fresh fruit;
therefore, each volunteer received about 10 g of extra fiber per day
during the 4 weeks of the study, and this fact might have contributed
to the cholesterol-lowering effect of the strawberry.
Oxidative damage of RBCs membrane involves lipid peroxidation,
which may cause its malfunctioning by altering its fluidity and the
membrane-bound enzyme and receptor functions. This has been
proposed as a general mechanism leading to RBCs hemolysis [36]. We
recently reported a significant increase in erythrocyte resistance to
the hemolysis maintained after washout period [16]. It was
hypothesized that potentially bioactive compounds ingested through
strawberry intake, once absorbed and metabolized, could accumulate
within the cell membrane by partitioning into its lipophilic core,
altering membrane composition, fluidity and functionality [36,37].
More data are necessary to support the hypothesis that a phyto-
chemical-rich diet causes a change in the RBC membrane structure
and functionality; however, the beneficial effect of strawberries
against oxidative damage in RBC was clearly evidenced.
Several studies have been performed to understand the effects of
strawberry supplementation on platelet function but, to date, no
Fig. 3. Ultrastructural features of resting, central clustered and degranulated platelets. (A) A platelet in the resting state: the shape is discoidal, numerous granules and some cisternae
of the open canalicular system can be observed inside the cell. (B) A central clustered platelet corresponding to the activation phase: granules are fused centrally in an electron-dense
mass and the open canalicular system is fully dilated. (C) A completely degranulated platelet at the end of activation process. Bar=0.25 m.
293 J.M. Alvarez-Suarez et al. / Journal of Nutritional Biochemistry 25 (2014) 289294
definite conclusion has been drawn, because they show important
differences regarding health status of the study population, chronic
vs. acute intake and assessment of platelet function [12,38,39]. Our
approach was to analyze platelet basal activation in healthy subjects
after strawberry consumption without external manipulations, as a
preventive factor against CVD through a diet rich in antioxidants.
Although the majority of platelets remained in the resting state after
treatment, as expected, we found a significant change during the
supplementation period with a significant decrease in the number of
activated platelet compared to baseline and washout values. This
finding indicates that probably the constituents in fruit exert a
favorable effect on platelet function, as these cells could result less
responsive to activation stimuli. Even if the change is small it is
indicative of a specific effect of the treatment. The significant
decrement of central clustered platelets observed after strawberry
supplementation did not take place concomitantly with a decrease
of degranulated platelets in the same period. Degranulated platelets
represent the next phase to central clustering [27]. In fact, after
activation, which consists in the formation of the central dark mass
inside platelets, there is the loss of granules. These two phases do
not have the same duration, the step of central clustering being very
rapid, while the phase of degranulated platelets can be longer.
Degranulated platelets rapidly lose surface P-selectin, but continue
to circulate and function [39]; therefore, it is plausible that a
decrease in activated platelets does not correspond to a decrease in
degranulated platelets.
Through the present study we added new favorable evidence of
the effects of strawberries after 30-days consumption on the overall
improvement of the plasma antioxidant status, highlighting a
potential beneficial role on biomarkers of antioxidant status, lipid
profile and platelet function. Moreover, the potential effect of
strawberry intake in improving the RBC antioxidant status and
protection against oxidation was confirmed. The findings presented
here are interesting, because they may partly explain the protective
role of a diet rich in fruit and vegetables in preventing CVD and other
chronic diseases mediated by oxidative stress.
Acknowledgments
We are indebted to Prof. Jos Manuel Villalba (Universidad de
Crdoba) for critically reading the manuscript. The authors would like
to thank Mrs. Belinda Giorgetti for performing sample preparation for
the electron microscopy studies and Ms. Monica Glebocki for
extensive editing of the manuscript.
References
[1] Butler D. UN targets top killers. Nature 2011;477:2601.
[2] Bach-Faig A, Berry EM, Lairon D, Reguant J, Trichopoulou A, Dernini S, et al,
Mediterranean Diet Foundation Expert Group. Mediterranean diet pyramid today.
Science and cultural updates. Public Health Nutr 2011;14:227484.
[3] Kaliora AC, Dedoussis GVZ, Schmidt H. Dietary antioxidants in preventing
atherogenesis. Atherosclerosis 2006;187:117.
[4] Giampieri F, Tulipani S, Alvarez-Suarez JM, Quiles JL, Mezzetti B, Battino M. The
strawberry: composition, nutritional quality, and impact on human health.
Nutrition 2012;28:919.
[5] Kalea AZ, Clark K, Schuschke DA, Kristo AS, Klimis-Zacas DJ. Dietary enrichment with
wildblueberries (Vacciniumangustifolium) affects thevascular reactivityintheaorta
of young spontaneously hypertensive rats. J Nutr Biochem 2010;21:1422.
[6] Youdim KA, McDonald J, Kalt W, Joseph JA. Potential role of dietary flavonoids in
reducing microvascular endothelium vulnerability to oxidative and inflammatory
insults. J Nutr Biochem 2002;13:2828.
[7] Vendrame S, Daugherty A, Kristo AS, Riso P, Klimis-Zacas D. Wild blueberry
(Vaccinium angustifolium) consumption improves inflammatory status in the
obese Zucker rat model of the metabolic syndrome. J Nutr Biochem 2013. http://
dx.doi.org/10.1016/j.jnutbio.2012.12.010 [Epub ahead of print].
[8] Henning SM, Seeram NP, Zhang Y, Li L, Gao K, Lee RP, et al. Strawberry
consumption is associated with increased antioxidant capacity in serum. J Med
Food 2010;13:11622.
[9] Franzini L, Ardig D, Valtuea S, Pellegrini N, Del Rio D, Bianchi MA, et al. Food
selection basedonhightotal antioxidant capacity improves endothelial function in
a low cardiovascular risk population. Nutr Metab Cardiovasc Dis 2012;22:507.
[10] Mauray A, Milenkovic D, Besson C, Caccia N, Morand C, Michel F, et al.
Atheroprotective effects of bilberry extracts in apo E-deficient mice. J Agric
Food Chem 2009;57:1110611.
[11] Mauray A, Felgines C, Morand A, Mazur A, Scalbert A, Milenkovic D. Bilberry
anthocyanin-rich extract alters expression of genes related to atherosclerosis
development in aorta of apo E-deficient mice. Nutr Metab Cardiovasc Dis
2012;22:7280.
[12] Erlund I, Koli R, Alfthan G, Marniemi J, Puukka P, Mustonen P, et al. Favorable
effects of berry consumption on platelet function, blood pressure, and HDL
cholesterol. Am J Clin Nutr 2008;7:32331.
[13] Jenkins DJA, Nguyen TH, Kendall CWC, et al. The effect of strawberries in a
cholesterol-lowering dietary portfolio. Metabolism 2008;57:163644.
[14] Vorchheimer DA, Becker R. Platelets in atherothrombosis. Clin Proc 2006;8:5968.
[15] Tulipani S, Romandini S, Busco F, Bompadre S, Mezzetti B, Battino M. Ascorbate,
not urate, modulates the plasma antioxidant capacity after strawberry intake.
Food Chem 2009;117:1818.
[16] Tulipani S, Alvarez-Suarez JM, Busco F, Bompadre S, Quiles JL, Mezzetti B, et al.
Strawberry consumption improves plasma antioxidant status and erythrocyte
resistance to oxidative haemolysis in humans. Food Chem 2011;128:1806.
[17] Tulipani S, Romandini S, Alvarez-Suareza JM, Capocasa F, Mezzetti B, Busco F, et al.
Folate content in different strawberry genotypes and folate status in healthy
subjects after strawberry consumption. BioFactors 2008;34:4755.
[18] Tulipani S, Marzban G, Herndl A, Laimer M, Mezzetti B, Battino M. Influence of
environmental and genetic factors on health-related compounds in strawberry.
Food Chem 2011;124:90613.
[19] Tulipani S, Mezzetti B, Capocasa F, Bompadre S, Beekwilder J, de Vos CH, et al.
Antioxidants, phenolic compounds, and nutritional quality of different strawberry
genotypes. J Agric Food Chem 2008;56:696704.
[20] Slinkard K, Singleton VL. Total phenol analysis: automation and comparison with
manual methods. Am J Enol Vitic 1977;28:4955.
[21] Lopes da Silva F, Escribano-Bailon MT, Perez Alonso JJ, Rivas-Gonzalo J, Santos-
Buelga C. Anthocyanin pigments in strawberry. LWT Food Sci Technol 2007;40:
37482.
[22] Bompadre S, Leone L, Politi A, Battino MA. Improved FIA-ABTS method for
antioxidant capacity determination in different biological samples. Free Radic Res
2004;38:8318.
[23] Gillespie KM, Chae JM, Ainsworth EA. Rapid measurement of total antioxidant
capacity in plant. Nat Protoc 2007;2:86770.
[24] Benzie IFF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of
antioxidant power: The FRAP assay. Anal Biochem 1996;239:706.
[25] Karatas F, Karatepe M, Baysar A. Determination of free malondialdehyde in human
serumby high-performance liquid chromatography. Anal Biochem2002;311:769.
[26] Cesquini M, Torsoni MA, Stoppa GR, Ogo SH. T-BOOH-induced oxidative damage
in sickle red blood cells and the role of flavonoids. Biomed Pharmacother 2003;57:
1249.
[27] Olcay L, Erdemli E, Kesimer M, Bykasik Y, Okur H, Kalkanoglu HS, et al. High
cystine in platelets from patients with nephropathic cystinosis: a chemical,
ultrastructural, and functional evaluation. J Clin Pathol 2005;58:93945.
[28] Bast A, Haenen GR. Ten misconceptions about antioxidants. Trends Pharmacol Sci
2013;34:4306.
[29] Collins AR, Azqueta A, Langie SA. Effects of micronutrients on DNA repair. Eur J
Nutr 2012;51:26179.
[30] Zunino SJ, Parelman MA, Freytag TL, Stephensen CB, Kelley DS, Mackey BE, et al.
Effects of dietary strawberry powder on blood lipids and inflammatory markers in
obese human subjects. Br J Nutr 2011;9:110.
[31] Basu A, Fu DX, Wilkinson M, Simmons B, Wu M, Betts NM, et al. Strawberries
decrease atherosclerotic markers in subjects with metabolic syndrome. Nutr Res
2010;30:4629.
[32] Youdim KA, Martin A, Joseph JA. Incorporation of the elderberry anthocyanins by
endothelial cells increases protection against oxidative stress. Free Radic Biol Med
2000;29:5160.
[33] Qin Y, Xia M, Ma J, Hao YT, Liu J, Mou HY, et al. Anthocyanin supplementation
improves serum LDL- and HDL-cholesterol concentrations associated with the
inhibition of cholesteryl ester transfer protein in dyslipidemic subjects. Am J Clin
Nutr 2009;90:48592.
[34] Cassidy A, Mukamal KJ, Liu L, Franz M, Eliassen AH, Rimm EB. High anthocyanin
intake is associated with a reduced risk of myocardial infarction in young and
middle-aged women. Circulation 2013;127:18896.
[35] Erkkila AT, Lichtenstein AH. Fiber and cardiovascular disease risk: how strong is
the evidence? J Cardiovasc Nurs 2006;21:38.
[36] Youdim KA, Shukitt-Hale B, MacKinnon S, Kalt W, Joseph JA. Polyphenolics
enhance red blood cell resistance to oxidative stress: in vitro and in vivo. Biochim
Biophys Acta 2000;1523:11722.
[37] Tedesco I, Russo M, Russo P, Iacomino G, Russo GL, et al. Antioxidant effect of red
wine polyphenols on red blood cells. J Nutr Biochem 2000;11:1149.
[38] Ostertag LM, O'Kennedy N, Kroon PA, Duthie GG, de Roos B. Impact of dietary
polyphenols on human platelet functiona critical review of controlled dietary
intervention studies. Mol Nutr Food Res 2010;54:6081.
[39] Ellis CL, Edirisinghe I, Kappagoda T, Burton-Freeman B. Attenuation of meal-
induced inflammatory and thrombotic responses in overweight men and women
after 6-week daily strawberry (Fragaria) intake. A randomized placebo-controlled
trial. J Atheroscler Thromb 2011;18:31827.
294 J.M. Alvarez-Suarez et al. / Journal of Nutritional Biochemistry 25 (2014) 289294