NUT RI TI O N R ES E A RC H 3 6 (2 0 1 6) 14 1 51 42 2

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Original Research

Intake of bilberries (Vaccinium myrtillus L.) reduced

risk factors for cardiovascular disease by inducing
favorable changes in lipoprotein profiles

Marta Habanova a,, Jorge A. Saraiva b,, Miroslav Haban c , Marianna Schwarzova a ,
Peter Chlebo a , Lenka Predna a , Jan Gao d , Joanna Wyka e
a
Department of Human Nutrition, Slovak University of Agriculture in Nitra, 94976 Nitra-Chrenov, Slovakia
b
Department of Chemistry, University of Aveiro, Campus Universitrio de Santiago, Aveiro 3810-193, Portugal
c
Department of Pharmacognosy and Botany, Faculty of Pharmacy, Comenius University in Bratislava, 83232 Bratislava, Slovakia
d
Department of Genetics and Plant Breeding, Slovak University of Agriculture in Nitra, 94976 Nitra-Chrenov, Slovakia
e
Department of Human Nutrition, Wrocaw University of Environmental and Life Sciences, 51630 Wrocaw, Poland

ARTI CLE I NFO A BS TRACT

Article history: Cardiovascular diseases (CVDs) are the leading causes of death, and lifestyle modification,
Received 3 June 2016 including dietary changes, is recommended to improve this condition. In this study, regular
Revised 16 November 2016 consumption of bilberries was hypothesized to have beneficial effects on CVD risk reduction, by
Accepted 18 November 2016 changes in human health indicators such as decreasing low-density lipoprotein cholesterol (LDL-
C) and triglycerides (TGs) and increasing high-density lipoprotein cholesterol (HDL-C). The
Keywords: research involved women (n = 25) and men (n = 11) who consumed 150 g of frozen stored
Bilberries bilberries 3 times a week for 6 weeks. Anthropometric parameters, blood pressure, lipid profile,
Lipid profile glucose, liver enzymes, creatinine, albumin, magnesium, and antiradical activity were measured.
Cardiovascular diseases risk Except for the body mass index of women (P = .019), no significant changes were found for
Biochemical parameters anthropometric indicators. The consumption of bilberries led to a decrease in the following
Healthy participants parameters: total cholesterol (P = .017), LDL-C (P = .0347), TG (P = .001), glucose (P = .005), albumin
(P = .001), -glutamyltransferase (P = .046), and a positive increase in HDL-C (P = .044). In men,
additionally, favorable changes were observed in total cholesterol (P = .004), glucose (P = .015),
albumin (P = .028), aspartate aminotransferase (P = .012), -glutamyltransferase (P = .013), and
HDL-C (P = .009; in this group, LDL-C increased [P = .007]). Changes in other parameters were not
significant, for both women and men. Thus, the regular intake of bilberries can be important to
reduce CVDs risk, by decreasing LDL-C/TG and increasing HDL-C.
2016 Elsevier Inc. All rights reserved.

Abbreviations: AA, antiradical activity; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase;
CVD, cardiovascular diseases; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; GMT, -
glutamyltransferase; T-C, total cholesterol; TG, triglycerides.
Correspondence to: M. Habanova, Department of Human Nutrition, Slovak University of Agriculture in Nitra, Trieda Andreja Hlinku 2,
94976 Nitra, Slovakia. Tel.: +421 37 641 4467; fax: +421 37 6415599.
Correspondence to: J.M.A. Saraiva, Universidade de Aveiro, Campus Universitrio de Santiago, 3810-193 Aveiro, Portugal. Tel.: +351
234401513; fax: +351 234370084.
E-mail addresses: marta.habanova@uniag.sk, marta.habanova1@gmail.com (M. Habanova), jorgesaraiva@ua.pt (J.A. Saraiva),
miroslav.haban@gmail.com (M. Haban), Marianna.schwarzova@uniag.sk (M. Schwarzova), peter.chlebo@hotmail.com (P. Chlebo),
lenkapredna@gmail.com (L. Predna), jan.gazo@uniag.sk (J. Gao), joanna.wyka@up.wroc.pl (J. Wyka).

http://dx.doi.org/10.1016/j.nutres.2016.11.010
0271-5317/ 2016 Elsevier Inc. All rights reserved.
1416 N U T RI TI O N R ES E A RC H 3 6 ( 2 0 1 6) 14 1 51 42 2

energy and blood lipids, antioxidant status (AA), and other

1. Introduction
human health indicators, based on the possibility that the
intake of bilberries reduce CVD risk factors. This was a pre-
Bilberries (Vaccinium myrtillus L.) are recognized as a good source of
post intervention study [30], involving a total of 65 partici-
anthocyanins [1], chlorogenic acid, flavonoids, -linolenic acid,
pants from the staff of the Slovak Agriculture University who
pterostilbene, and vitamins [2,3]. Recent studies demonstrated the
were screened for eligibility to participate in the study.
benefit of consuming bilberries and blueberries to prevent age-
Twenty-two participants did not satisfy the inclusion criteria
related chronic diseases such as cardiovascular diseases (CVDs)
and 7 participants dropped out (Fig. 1). Twenty-five women
[4-6], cancer [7-11], diabetes [12-14], hyperlipidemia [15], hyper-
(n = 25) with a mean age (SD) of 47.7 5.23 years and 11 men
tension [16], obesity [17], metabolic syndrome [18], and
(n = 11) with a mean age (SD) of 49.00 6.48 years, all of
antiinflammation [19-21]. Bilberries have a high content of total
whom were apparently healthy, were included in the study.
polyphenols and anthocyanins, and their phenolic composition
The inclusion criteria were as follows: volunteers could not
has been often the subject of various studies [22-27]. The detailed
have been treated for cardiovascular, renal, and hepatic
analysis of phenolics in fresh bilberries and blueberries confirmed
diseases; could have neither high cholesterol nor high blood
that anthocyanins are the main phenolics in both bilberries and
pressure for the last 6 months; and could not have intake of
blueberries, as previously reported in 2 studies [28,29].
any nutritional supplements, namely, vitamins, minerals,
Although limited epidemiologic data inversely associate the
antioxidants, and flavonoids.
consumption of berries with inflammation and CVD, these
Volunteers were asked not to change neither their eating
conclusions need to be strengthened. Clinical studies in healthy
habits nor their lifestyle during the study. Anthropometric
human subjects with diabetes mellitus, dyslipidemia, metabolic
parameters (weight, height, and volume of the body param-
syndrome, or hypertension, or smokers show a significant
eters) and blood pressure were observed and recorded. The
decrease in CVD risk factors, especially glucose, lipids and lipid
following were monitored during the study: blood profiles (T-
peroxidation, and systolic blood pressure, after the consumption
C, LDL-C, HDL-C, TG) and ALT, AST, GMT, ALP, creatinine,
of berries. The principal mechanisms of action underlying the
albumin, glucose, Mg, and AA. The values obtained before
potential cardioprotective effects of berries include counteracting
(pre) the study were used as control.
free radical generation, attenuating inflammatory gene expres-
This study was conducted according to the guidelines of
sion, down-regulating foam cell formation, and up-regulating
the Declaration of Helsinki, and approved by the Slovak
endothelial nitric oxide synthase expression. Through these
Agriculture University in Nitra, Department of Human Nutri-
effects, the progression of atherosclerosis is slowed, and normal
tion, Slovakia, and by the Ethical Committee of the Special-
vascular function and blood pressure are preserved. Moreover, in
ized Hospital of St Nitra, Slovakia (Study No. 02/0906/2015). All
light of the possible decrease in nutritional and functional value
participants gave written, informed consent.
that occurs during food processing, including drying and
pasteurization, the consumption of fresh or minimally processed
2.2. Intervention
foods, such as frozen whole berries as part of a regular diet, may
be better than the intake of juices or extracts that need more
To test the biological efficiency of bilberries (V myrtillus L.),
elaborate processing for consumption [5].
wild bilberries originating from the region Kysuce and from
To test our hypothesis, the present study examined the
the northwestern part of Slovakia were used. The volunteers
effects of regular consumption of bilberries on CVD risk
were instructed to consume 150 g of frozen bilberries 3 times
reduction. Specifically, bilberries cause positive changes in
per week (Monday, Wednesday, and Friday, during the day,
human health indicators, such as decreasing low-density lipo-
e.g., as a snack with natural yogurt) for 6 weeks. The amount
protein cholesterol (LDL-C) and triglycerides (TGs) and increasing
(per dry weight) of some important bioactive compounds
high-density lipoprotein cholesterol (HDL-C). Volunteers (appar-
(total polyphenols and total anthocynins, quercetin,
ently healthy women and men) ingested whole frozen preserved
chlorogenic acid, and pterostilbene) as well as the AA of the
bilberries for 6 weeks and anthropometric (weight, height, and
bilberries was quantified. The total phenolic content was
volume of the body), blood pressure, and several blood health
determined according to the Folin-Ciocalteu method [40], the
indicators (lipid profile/(total cholesterol [T-C], LDL-C and HDL-C,
total anthocyanin content was analyzed as described by
and TG), as well as alanine aminotransferase [ALT], aspartate
Giusti and Wrolstad [41], and the quantification procedures
aminotransferase [AST], -glutamyltransferase [GMT], alkaline
for quercetin and chlorogenic acid were adapted from the
phosphatase [ALP], creatinine, albumin, glucose, magnesium
work of Justesen and Knuthsen [42]. The AA of the bilberries
[Mg], and antiradical activity [AA]). These parameters were
extract was measured using the stable radical 2.2-diphenyl-1-
monitored at the beginning and after 3 and 6 weeks of the
picrylhydrazyl [43,44], and the content of pterostilbene was
initiation of the ingestion of bilberries.
quantified according to Remsberg et al [45].

2.3. Anthropometric measurements

2. Methods and materials
Anthropometric measurements were performed by trained
2.1. Participants and study design personnel before (pre) at the beginning of the consumption of
the bilberries (week 0) and at the end (post) of the study (week
The objective of the present study was to evaluate the effect 6). Body weight (kg) and height (m) were measured on the
of regular consumption of bilberries on parameters related to outpatient electronic medical scales (Tanita WB-3000). The
 NUT RI TI O N R ES E A RC H 3 6 (2 0 1 6) 14 1 51 42 2 1417

Volunteers screened for eligibility

(n = 65)
Excluded (n = 22)
Not meeting
inclusion/exclusion
criteria (e.g.,
choleserol)

Women (n = 30) Men (n = 13) Pre

Pre (before) bilberries (before) bilberries
intervention analyses intervention analyses
(control) (control)

Drop out Drop out

(n = 5) (n = 2)

Post (after) bilberries Post (after) bilberries

intervention intervention
Data analyzed in Data analyzed in men
women group (n = 25) group (n = 11)

Fig. 1 Flowchart of participant selection in the pre-post intervention and those that completed the study.

body mass index (BMI) was calculated by dividing the body
weight in kilograms by the square of the height in meters.
Body fat (%) was determined using a bioelectric impedance
analyzer Bodystat Quadscan 4000 apparatus, and waist
circumference was quantified using Tanita ViScan 140.
Systolic and diastolic blood pressures were measured twice
in mm Hg using a Sphygmomanometer DM-3000, whereas
participants were seated at screening visits, and the means of
measurements were used for analysis. Participants rested for
at least 15 minutes before the measurements.
2.4. Clinical analyses
Blood samples were obtained as follows: the first samples
(before the start of bilberry consumption) were used as
control, the second blood sampling was performed after 3
weeks of bilberry consumption, and the third blood sampling
was performed after 6 weeks of bilberry consumption (at the
end of the study). Blood was collected in the morning after 8
hours of fasting in a standard manner using 2.5 mL of EDTA
solution and in a 7.5-mL serum gel tube. After the separation
of blood serum samples, they were stored at 80C until the
analyses were carried out. The lipid profile in blood serum (T-
C, LDL-C, HDL-C, and TG), ALT, AST, GMT, ALP, creatinine,
albumin, glucose, Mg, and total antioxidant status were
determined from thawed plasma samples using a biochem-
ical analyzer LISA 200, with commercially available kits
(Spinreact, Girona, Spain) from Ecomed (Slovakia) and Randox
(United Kingdom). Methods for the determination of bio-
chemical parameters were programmed using the application
protocol, provided by the supplier of reagents (Ecomed) and
adapted when necessary. The lipid profile was compared with
the reference values of The National Cholesterol Education
Program: Adult Treatment Panel III (NCEP ATP III) [46]. To
convert the concentration to mmol/L, values in mg/dL were
multiplied by 0.02586 for T-C, HDL, and LDL and multiplied by
0.01129 for TG. [47].
2.5. Statistical analyses
Data are expressed as means SDs (Tables 1 and 2) and as
median and interquartile ranges (Table 3). A power analysis
was conducted in G*Power to determine a sufficient sample
size using an value of .05, a power of 0.80, and a large effect
size [48]. The data were analyzed by t test and the Wilcoxon
signed rank test, depending on the data under analysis, using
Statistica CZ10 software. Differences were considered statis-
tically significant at P < .05.
3. Results
The amount of relevant bioactive compounds (total polyphe-
nols and total anthocynins, quercetin, chlorogenic acid, and
pterostilbene), as well as the AA of billberries, is shown in
Table 1 together with comparison with values reported in the
literature [8,31-39]. Generally the results obtained in the
present work were in the range of the results reported by
others [8,31-39].
The analyzed anthropometric parameters and blood pressure
of the participants before (pre) and after (post) the consumption
of bilberries are presented in Table 2. No significant differences
were observed for all monitored anthropometric indicators at the
start and at the end of the study, except for a slight increase in the
BMI of women (P = .019).
Concerning the parameters quantified in the blood sam-
ples, the results are presented in Table 3, where it can be seen
1418 N U T RI TI O N R ES E A RC H 3 6 ( 2 0 1 6) 14 1 51 42 2

Table 1 Amount (in dry weight) of bioactive substances in frozen bilberries used in the present study and comparison with
literature values
Parameter Units Values of the present study Values of references

Total polyphenols mg/kg 20 752.50 848.66 868.3-2950.9 [31]

Total anthocyanins mg/kg 3038.25 74.44 2651 781 [32]
2878.0 607.0 [33]
2050.0 566.0 [34]
3985.0 1290.0 [35]
2619.0-9296.2 [36]
Quercetin g/g 6.10 0.23 29-30 [37]
Chlorogenic acid g/g 25.86 0.48 23.1 1.0 [38]
Pterostilbene ng/g 190.00 22.5 99-520 [8]
AA, inhibition of DPPH % 76.937 1.474 73.54 1.94 [39]

The data are expressed as means SD (for each sample, n = 4).

DPPH, 2.2-diphenyl-1-picrylhydrazyl.

that T-C levels decreased from 5.63 mmol/L at the beginning

of the research to 5.29 mmol/L (P = .017) after bilberries
intervention. Similar observed beneficial effects were found
for LDL-C (3.39-3.14; P = .035), TG (1.46-1.06; P = .001), glucose
(5.54-5.14; P = .005), albumin (47.90-45.60; P = .001), and GMT
(0.54-0.49; P = .046), and there was a favorable increase in
HDL-C (1.57-1.67; P = .044). Similar advantageous changes
were observed for men for T-C levels that decreased from
5.38 to 4.96 mmol/L (P = .004), glucose (5.95-5.24; P = .012),
albumin (48.77-46.64; P = .028), AST (0.47-0.37; P = .012), and
GMT (0.71-0.58; P = .013), and there was a beneficial increase
in HDL-C (1.28-1.49; P = .009). In men, LDL-C levels increased
(3.26-3.78; P = .007). The changes observed for the other
parameters were not statistically significant.
The percentage change for the lipid profiles in the number
of participants (T-C, LDL-C, HDL-C, and TG) in plasma before
(pre) and after, following a 3-week and 6-week (post) intake of
bilberries according to NCEP ATP III, is presented in Table 4.
Generally, the lipid profiles for the number of volunteers
improved. The number of volunteers with optimal values,
near or above the optimal and borderline high values,
increased and the number of volunteers with high and very
high values decreased. This is evaluated as positive in terms
of reducing CVD risk factors.
4. Discussion
Results of the present study show that the short-term
inclusion of bilberries in the diet can contribute to improved
blood plasma lipid profiles of women and men. Although the
LDL-C levels increased in men, they were still below the
considered risk values (>4 mmol/L). Other studies also pointed
to the benefits of bioactive substances of bilberries for health
[22-24,49-51]. Wild bilberries (V myrtillus L.) are currently not
commercially available on the market, but only cultivated
species of blueberries can be purchased (Vaccinium corymbosum L.).
The composition of bioactive compounds of bilberries and
the comparison with that of blueberries were studied by
Giovanelli and Buratti [52] and Habanova et al [27]. Their
results indicate a high concentration of important bioactive
substances in bilberries, contributing to increasing refer-
ences to bilberries as a natural functional food [53], with
potential health benefits.
The amount of bilberries consumed by volunteers was
selected to be comparable to that of other studies, taking into
account the processed form used in other studies such as
fresh/frozen/dried, juice, smoothie, beverages, or capsules
[54]. The anthropometric parameters and blood pressure of

Table 2 Anthropometric parameters and blood pressure measurements of volunteers before (pre) and after (post)
bilberries intervention
Parameter Units Women (n = 25) Men (n = 11)

Pre Post Pre Post

Body weight kg 73.23 13.45 73.68 13.61 (0.062) 90.67 15.68 90.95 15.78 (0.556)
Waist circumference cm 87.24 12.19 88.24 12.56 (0.100) 99.00 11.84 98.91 11.37 (0.922)
Body fat % 34.06 6.39 33.31 7.60 (0.722) 23.64 5.58 22.98 5.58 (0.746)
Body fat mass kg 25.51 9.04 25.67 10.03 (0.215) 21.88 7.83 22.21 8.43 (0.217)
BMI kg/m2 26.3 4.73 26.5 4.78 (0.019) 29.04 3.94 29.15 4.03 (0.545)
Systolic blood pressure mm Hg 131.4 15.86 133.56 17.31 (0.430) 134.0 14.52 134.45 12.69 (0.881)
Diastolic blood pressure mm Hg 76.4 12.09 78.24 11.59 (0.250) 85.55 8.64 82.91 7.38 (0.355)

n, number of participants; pre, measurements taken before (pre) of the study; post, measurements taken at the end of the study. Data are
expressed as means SD; t test, P < .05.
Values in parentheses show the P value.
 NUT RI TI O N R ES E A RC H 3 6 (2 0 1 6) 14 1 51 42 2 1419

Table 3 Change in blood plasma parameters of volunteers before (pre) and after 6-week (post) bilberries intervention
Women (n = 25) Men (n = 11)

Units Pre Post P Pre Post P

1
T-C mmol/L 5.82 (4.74-6.39) 5.19 (4.85-5.74) .017 5.36 (4.77-6.07) 4.94 (4.47-5.43) .004
HDL-C mmol/L 1.52 (1.38-1.81) 1.70 (1.44-1.88) .044 1.22 (0.98-1.52) 1.49 (1.21-1.68) .009
LDL-C mmol/L 3.42 (2.60-4.15) 2.95 (2.75-3.68) .035 3,05 (2.95-3.78) 2.79 (2.50-3.12) .007
TG mmol/L 1.56 (0.83-2.02) 0.99 (0.84-1.24) .001 1.69 (0.92-2.82) 1.39 (0.66-2.06) .061
TAS mmol/L 1.69 (1.66-1.75) 1.72 (1.63-1.80) .553 1.85 (1.71-1.98) 1.89 (1.66-1.94) .789
Glucose mmol/L 5.43 (5.02-5.62) 4.92 (4.57-5.37) .005 5.97 (4.83-6.44) 5.05 (4.61-6.25) .012
Mg mmol/L 0.89 (0.82-0.94) 0.84 (0.82-0.92) .103 0.82 (0.77-0.96) 0.82 (0.75-0.87) .307
Creatinine mol L1 76.2 (71.8-9.90) 72.5 (66.7-79.1) .353 88.6 (79.1-103.2) 84.9 (74.7-98.1) .062
Albumin g/L1 46.8 (45.4-49.6) 46.0 (44.5-47.2) .001 49.2 (47.5-50.0) 46.9 (45.0-48.6) .028
ALT kat L1 0.15 (0.09-0.28) 0.16 (0.12-0.25) .976 0.29 (0.19-0,52) 0.25 (0.17-0.39) .059
AST kat L1 0.34 (0.29-0.44) 0.35 (29-0.39) .236 0.34 (0.30-0.59) 0.33 (0.27-0.49) .012
GMT kat L1 0.39 (0.29-0.68) 0.34 (0.25-0.48) .046 0.47 (0.31-0.98) 0.43 (0.32-0.83) .013
ALP kat L1 2.19 (1.88-2.64) 2.10 (1.85-2.47) .989 2.13 (1.50-2.62) 1.78 (1.53-2.79) .894

n, number of participants; pre, blood samples taken before (pre) the study; post, blood samples taken at the end of the study; TAS, total
antioxidant status.
Data are expressed as medians and interquartile ranges in parentheses. Statistical significant differences were verified by the Wilcoxon test,
P < .05.

volunteers before (pre) and after (post) bilberries consumption
that were obtained show that the consumption of bilberries
led to a significant change in the BMI of women (but still <1%
increase) and had no significant effect on the other studied
parameters (body weight, waist circumference, body fat, body
mass, and blood pressure). Basu et al [55] have previously
reported some health benefits of blueberries consumption. In
the study by these authors, 48 participants with metabolic
syndrome consumed freeze-dried blueberries, and the de-
crease in plasma oxidized LDL and serum malondialdehyde
and hydroxynonenal concentrations was higher in the group
ingesting blueberries (28% and 17%, respectively) than that in
the control group (9% and 9%; P = .01, respectively).
In our study, the positive effects of the consumption of
bilberries on CVD risk factors were also observed. Basu et al
[55] and Erlund et al [56] reported similar results, but our
findings are not directly comparable with the results of these
authors, because they used different doses of berries or a
different time for consumption, and additionally, the study
was carried out with volunteers with CVD risk (obese men and

Table 4 Distribution percentage changes (%) of blood lipids in participants before (pre), following a 3-week intervention of
bilberries, and after a 6-week. (post) intervention of bilberries according to NCEP ATP III
Reference values of NCEP ATP III Volunteers (%)

Women (n = 25) Men (n = 11)

Baseline 3-wk 6-wk Baseline 3-wk 6-wk

T-C
Desirable (<5.18 mmol/L) 38.9 50.0 50.0 42.8 42.8 85.7
Higher borderline (5.2-6.19 mmol/L) 33.3 27.7 33.3 42.8 28.6 14.3
High (6.2 mmol/L) 27.8 22.3 16.5 14.4 28.6 0
LDL-C
Optimal (<2.6 mmol/L) 0 11.2 0 0 0 14.3
Near or above optimal (2.6-3.3 mmol/L) 27.8 22.2 22.2 28.6 57.1 28.6
Higher borderline (3.4-4.1 mmol/L) 22.2 38.9 55.4 28.6 14.3 42.8
High (4.2-4.9 mmol/L) 27.8 22.2 11.2 42.8 28.6 14.3
Very high (>4.9 mmol/L) 22.2 5.5 11.2 0 0 0
HDL-C
High ( 1.55 mmol/L) 66.7 77.8 83.3 42.8 57.2 57.2
Higher borderline (1.04-1.54 mmol/L) 27.8 22.2 16.7 57.2 42.8 42.8
Low (<1.03 mmol/L) 5.5 0 0 0 0 0
TG
Optimal (<1.7 mmol/L) 66.8 77.9 83.4 42.8 42.8 57.1
Higher borderline (1.70-2.25 mmol/L) 27.7 16.6 11.1 14.3 28.6 28.6
High (2.26-5.64 mmol/L) 5.5 5.5 5.5 28.6 28.6 14.3
Very high (5.65 mmol/L) 0 0 0 14.3 0 0

Baseline, 3-wk, and 6-wk refer to the number of participants in % in each category before intervention, following a 3-week intervention, and
after 6-week (post) intervention. Values of T-C, HDL-C and LDL-C, and TG compared with the reference values of the NCEP ATP III [46].
1420 N U T RI TI O N R ES E A RC H 3 6 ( 2 0 1 6) 14 1 51 42 2
the metabolic syndrome in women). The results of the 2
teams of previously cited authors [55,56] showed significant
reductions of CVD risk factors linked to the consumption of
berries. The intake of bilberries in the present work was linked
to the normalization/improvement of several parameters in
blood related to CVD risk factors, similarly to results present-
ed by other authors [57-59]. Stamler et al [60] evaluated the
relationship between serum cholesterol and the risk of
premature death from coronary heart diseases in 356 222
men aged 35 to 57 years. This was the largest cohort with
standardized serum cholesterol measurements and long-
term mortality follow-up. Of all CVD deaths, 46% were
estimated to be deaths attributable to serum cholesterol
excess levels of 180 mg/dL or greater (4.65 mmol/L).
Approximately 1 mmol/L or lower T-C is associated with
approximately a one-half lowering of CHD mortality in both
sexes at ages 40-49, 50-69, and 70-89 years.
Moreover, beneficial changes (%) in the lipid profile of
volunteers before (pre) compared with a 3- and 6-week (post)
intervention of bilberries were found in the present work,
according to NCEP ATP III [46]. The results show that there
was a favorable shift of volunteers with risk values into more
favorable lipid profile categories. Results from intervention
studies aimed at monitoring the impact of fruits rich in
polyphenols to improve health status were reported by
several authors [56,57,61-65], indicating that regular con-
sumption of small fruits plays an important role in preventing
CVD. Clinical trials that use diet or drugs to lower serum
cholesterol levels have consistently shown a 2% reduction in
the incidence of coronary heart disease for every 1% reduction
in total serum cholesterol level [66].
Halliwell [67] reported that despite the results of several
intervention studies, some cause-effect relationships are still
unclear, because the relationships and mechanisms are
complex. For example, it is known that free radicals are
detrimental, because they contribute to aging and certain age-
related diseases, especially cancer and neurodegenerative
diseases, and therefore, antioxidants mechanism are difficult
to interpret. However, the intervention trials with antioxi-
dants often failed to demonstrate benefits in humans.
In addition, different results reported in the literature may
arise from the diversity of study designs, insufficient control,
relatively brief intervention periods, low doses of active
substances, and as a result of low amounts of consumption
of the tested foods, like berries. Also, some studies do not
indicate details on the content of the polyphenols of
consumed berries, making it difficult to compare data. To
confirm the observations verified in the present study, it is
recommended that well-organized intervention studies be
conducted to investigate a wider range of fruits and the long-
term impact of their consumption on the body [68,69].
In conclusion, the present study shows that even a short
period of regular consumption of whole wild bilberries,
preserved through minimal processing (frozen), is associated
with an improvement of the lipid profile in humans. The
results obtained in the present work support the hypothesis
designed to investigate the study that the regular consump-
tion of bilberries can contribute to beneficial effects on CVD
risk reduction, such as decreasing LDL-C and TG and
increasing HDL-C. Nonetheless, one has to be aware that the
current study was limited in a number of ways that deserve
careful attention: first, the study population was relatively
small, and this could be the main reason why significant
between-group differences for many of the biochemical
parameters assessed in this study were not detected; second,
the current study was subject to potential bias because of the
lack of blinding and placebo controlling; third, the intake
period in the current trial was 6 weeks, which may not have
been sufficient to detect significant differences for some of
the parameters studied; and lastly, it would be ideal to
standardize the intake of berries based on the content of
phytochemicals, such as anthocyanins and flavonoids, rather
than on crude fruit weight.
Acknowledgment
This work was supported by the Scientific Grant Agency of the
Ministry of Education, Science, Research and Sport of the Slovak
Republic (No. 1/0127/14) The determination of the capacity of
total polyphenols and antioxidants of the plant sources of natural
and agroecological conditions of the Slovak Republic and their
utilization in improving population health; project ITEBIO
Support and innovations of a special and organic products
Technologies for human healthy nutrition (ITMS 26220220115),
implemented under Operational Program Research and Devel-
opment; and FCT/MEC to the QOPNA research unit (FCT UID/QUI/
00062/2013), through national funds and where applicable co-
financed by the FEDER, within the PT2020 Partnership Agree-
ment. The authors also acknowledge editorial assistance to
improve the manuscript. The authors have no conflict of interest
to disclose.
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