Dietary Supplementation with Polyphenol-Rich Chokeberry Juice Improves Skin Morphology in Cellulite

Abstract

Abstract The aim of our study was to investigate possible beneficial effects of organic chokeberry juice (OCJ) consumption in the treatment of cellulite. Twenty-nine women aged 25-48 with a cellulite grade 2 according to the Nurnberger-Muller scale were included. Anthropometric and biochemical parameters were measured. Skin structure was analyzed by ultrasonography. All subjects consumed 100 mL of OCJ per day, during 90 days. Measurements of investigated parameters were performed at 0, 45, and 90 days of the study. A marked reduction in the subcutaneous tissue thickness was observed in all subjects, with the average reduction of 1.9 mm. The length of subcutaneous tissue fascicles (ScTFL) was reduced in 97% (28 out of 29) of subjects, with the average value of 1.18 mm. After 45 days of chokeberry juice consumption, reduction of edema was observed in 55.2% of the subjects with edema at the baseline, while at the endpoint of the study, edemas were not observed in any of the subjects involved in the study. OCJ could have beneficial effects on the cellulite condition, including the length of ScTFL, subcutaneous tissue, and dermis thickness as well as on edema reduction.

Full text

FULL COMMUNICATION
Dietary
AU1 cSupplementation with Polyphenol-Rich Chokeberry Juice
Improves Skin Morphology in Cellulite
Katarina
AU2 cS
ˇavikin,
1
Nemanja Menkovic
´,
2
Gordana Zdunic
´,
1
Dejan Pljevljakus
ˇic
´,
1
Slavica Spasic
´,
3
Nevena Kardum,
4
and Aleksandra Konic
´-Ristic
´
4
AU3 c
1
Institute for Medicinal Plants Research, Belgrade, Serbia.
2
Clinical Center of Serbia, Center for Radiology and Magnetic Resonance, Belgrade, Serbia.
3
Department of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia.
4
Department of Nutrition and Metabolism, Institute for Medical Research, University of Belgrade, Belgrade, Serbia.
ABSTRACT The aim of our study was to investigate possible beneficial effects of organic chokeberry juice (OCJ)
consumption in the treatment of cellulite. Twenty-nine women aged 25–48 with a cellulite grade 2 according to the
Nurnberger–Muller scale were included. Anthropometric and biochemical parameters were measured. Skin structure was
analyzed by ultrasonography. All subjects consumed 100 mL of OCJ per day, during 90 days. Measurements of investigated
parameters were performed at 0, 45, and 90 days of the study. A marked reduction in the subcutaneous tissue thickness was
observed in all subjects, with the average reduction of 1.9 mm. The length of subcutaneous tissue fascicles (ScTFL) was
reduced in 97% (28 out of 29) of subjects, with the average value of 1.18 mm. After 45 days of chokeberry juice consumption,
reduction of edema was observed in 55.2% of the subjects with edema at the baseline, while at the endpoint of the study,
edemas were not observed in any of the subjects involved in the study. OCJ could have beneficial effects on the cellulite
condition, including the length of ScTFL, subcutaneous tissue, and dermis thickness as well as on edema reduction.
KEY WORDS: anthropometric biochemistry cellulite chokeberry fascicles subcutaneous tissue
INTRODUCTION
Cellulite is a condition that occurs in over 85% of
women and has also been diagnosed in men.
1
Although
it is not recognized as a medical disorder, it is of special
importance in esthetic medicine.
2,3
Cellulite is characterized
by an irregular, dimpled appearance of the skin, mainly found
on the thighs, buttocks, and abdomen.
4
Multifactorial etiology
is assumed, including structural, genetic, and endocrine ab-
normalities. It was shown that weakened connective tissues,
enlarged fat cells, and diminished microcirculation, play key
roles in the pathophysiology of cellulite.
2,5–7
Cellulite progression or reduction is evaluated using dif-
ferent techniques, including imprecise palpation and de-
scribing using various scales,
8
as well as instrumental methods
such as termography,
9
macrophotography,
10
magnetic reso-
nance, computed tomography, and ultrasonography.
11–15
A variety of topical preparations, massage-based thera-
pies, and surgical techniques, as well as herbal preparations
for oral applications, functional foods, or changes in dietary
habits have been used to improve cellulite conditions.
2,3,17,18
There is increasing demand for functional foods and in this
category, functional beverages are the fastest growing seg-
ment.
19
A lot of them are made from berries, among them
chokeberry products are very frequent.
Chokeberry (Aronia melanocarpa) is a rich source of
proanthocyanidins, anthocyanins, flavonols, and phenolic
acids and due to this diverse chemical profile it possesses
numerous beneficial effects in humans and animals.
20–22
It is
shown that chokeberry is among the plant species with the
highest antioxidant activity.
20,23
A large number of inter-
vention studies demonstrated the beneficial effects of
chokeberry ingredients on various risk factors for cardio-
vascular diseases, the levels of total cholesterol, LDL, ox-
LDL, triglycerides, glucose, HbA1c and systolic and diastolic
blood pressure, body mass index, improving vasodilatation
dependent on endothelial function as well as vasoactive and
vasoprotective properties.
22,24–26
In addition, it was shown
that anthocyanins play a role in the redistribution of micro-
vascular blood
27
and together with other phenolics have
beneficial effects on small blood vessel permeability.
28
Ac-
cording to our knowledge, there is no information in the lit-
erature about the effects of chokeberry juice on the condition
of skin and subcutaneous tissue region affected by cellulite.
Taking into account all these facts, the aim of this study
was to investigate the possible beneficial effects of organic
chokeberry juice (OCJ) consumption in the treatment of
cellulite in a female subject with cellulite type 2 according
Manuscript received 12 April 2013. Revision accepted 6 November 2013.
Address correspondence to: Dejan Pljevljakus
ˇic
´
AU4 c, Institute for Medicinal Plants Research,
Tadeus
ˇa Kos
ˇc
´us
ˇka 1, Belgrade 11000, Serbia, E-mail: dpljevljakusic@mocbilja.rs
JOURNAL OF MEDICINAL FOOD
J Med Food 00 (0) 2013, 1–6
#Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2013.0102
JMF-2013-0102-ver9-Savikin_1P
Type: research-article
1
JMF-2013-0102-ver9-Savikin_1P.3d 12/14/13 9:34am Page 1

to the Nurnberger–Muller scale. Different parameters were
monitored, including body mass index, percent of fat, the
abdominal, thigh, knee, and hip circumference, blood tests,
and also ultrasound examinations of the skin.
MATERIALS AND METHODS
Organic chokeberry juice
OCJ (A. melanocarpa) used in this study was imported and
donated from Conimex Trade d.o.o. (Belgrade, Serbia).
Producer of OCJ is Tlocznia Rembowscy (Popowko, Poland).
Total phenolics
The total concentration of phenols was estimated by the
Folin–Ciocalteu method with slight modifications.
29
Two
hundred microliters of properly diluted OCJ was added to
1 mL of 1:10 diluted Folin–Ciocalteu reagent. After 4 min,
800 lL of sodium carbonate (75 g/L) was added. After 2h of
incubation at room temperature, the absorbance at 765 nm
was measured. Gallic acid (0–100 mg/L) was used for cal-
ibration of a standard curve. The results were expressed as
milligrams of gallic acid equivalents per 100 g of juice (mg
GAE/100 g FW). Triplicate measurements were taken and
mean values were calculated.
Total anthocyanins
The total anthocyanin content was investigated according
to the procedure described in European Pharmacopoeia
6.0,
30
with slight modifications. A 50-fold dilution of OCJ in
a 0.1% v/v solution of hydrochloric acid in methanol was
prepared. The absorbance of the solution was measured at
528 nm, using a 0.1% v/v solution of hydrochloric acid in
methanol as the compensation liquid.
The percentage content of anthocyanins, expressed as
cyanidin-3-glucoside chloride, was calculated from the ex-
pression: A·5000/718 ·m(A=absorbance at 528 nm;
718 =specific absorbance of cyanidin-3-glucoside chloride at
528 nm; m=mass of the substance to be examined in grams).
1,1-Diphenyl-2-picrylhydrazyl radical scavenging activity
The free radical scavenging activity of OCJ on the stable
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was carried out
according to the procedure described previously,
31
with slight
modifications. The antiradical capacity of OCJ was evaluated
using a dilution series, to obtain a large spectrum of sample
concentrations. The samples (100 lL) were mixed with
1400 lLof80lM methanolic solution of DPPH. Absorbance
at 517 nm was measured after 20 min. The percentage of in-
hibition was calculated using the following equation:
Inhibition ¼[(A0Ai)=A0]·100;
where A
0
is absorbance of the control and A
i
is absorbance of
the samples. IC
50
values were estimated using a nonlinear
regression algorithm. All test analyses were run in triplicate.
Trolox was used as a positive control.
Subjects
Twenty-nine women aged 25–48 (mean age 34.9) par-
ticipated in the study. Cellulite grade 2 (orange peel ap-
pearance spontaneously visible and disappearing in layered
position), according to the Nurnberger–Muller scale,
32
was
the main inclusion criterion together with a regular monthly
menstrual cycle. Exclusion criteria included age under 18
and over 50 years; body mass index over 30; significant
changes in dietary habits, life style, or exercise program in
the past 12 months; cardiovascular diseases; pregnancy or
lactation; history of malignancy; any type of cellulite
treatment applied within 12 months before the study; sys-
temic lymphedema; menopause and premenopause.
Protocol
The study protocol was approved by the Ethics Com-
mittee of Faculty of Pharmacy, University of Belgrade,
Serbia. The study was conducted in accordance with the
revised Declaration of Helsinki. All participants provided
written informed consent. The study was performed from
February till May 2012. Subjects were instructed to con-
sume 100 mL of OCJ per day, during 90 days, 30 min before
the main meal and to retain their regular diet, exercise
program, and life style. Analyzed parameters included pa-
rameters of epidermal, dermal, and subcutaneous structure
measured by ultrasound (subcutaneous tissue thickness
[ScTT], thickness of subcutaneous tissue and dermis,
thickness of dermis and epidermis [DET], the length of
subcutaneous tissue fascicles [ScTFL], the presence of
edemas within the dermis), biochemical parameters (total
cholesterol, triglycerides, HDL, LDL, glucose, urea, ALT,
AST, iron and hemoglobin levels, leukocytes and erythro-
cytes counts, sedimentation), and anthropometric parame-
ters (weight, body mass index, total body fat, arterial
pressure, abdominal, thigh, knee, and hip circumference).
Biochemical parameters were determined with a clinical
chemistry analyzer (Cobas e411; Roche Diagnostics, Basel,
Switzerland) and Roche Diagnostics kits according to the
manufacturer’s instructions, while full blood count was
performed using a hematology analyzer (Coulter Ac.T Diff;
Beckman Coulter, Miami, FL, USA).
Measurements of investigated parameters were per-
formed at three time points, at 0, 45, and 90 days of the study
for all parameters with the exception of biochemical pa-
rameters measured at the beginning and the end of the study.
The upper back and front part of the thigh were photo-
graphed at the beginning and the end of the study by a digital
camera (Nikon D90, Nikor lens 18–105 mm) at standardized
angles and positions.
At day 90, subjects were asked to fulfill a questionnaire on
self perception of the treatment effects.
High-frequency ultrasound measurements
The high-frequency ultrasound examinations were carried
out using SA 9900 Multi-beam 3D Ultrasound, system Kretz
5–12 MHz, and linear probe PB–99L5-12IR. The ultrasound
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images obtained were saved on a personal computer, and
then analyzed using experimental software. The examina-
tions were performed on the posterior part of the thigh in
triplicate, always at the same location.
Statistical analysis
Data are presented as mean and standard deviations for
all 29 participants. All measurements were performed in
triplicate. The paired Student’s t-test and one-way ANOVA
for repeated measures (with Bonferroni adjustment for
multiple comparisons) were used to compare the data in two
or three time points, respectively, where P<.05 was con-
sidered statistically significant. SPSS program, version 19
(SPSS, Inc., Chicago, IL, USA) was used for the analysis.
RESULTS
Chemical characteristics and radical scavenging
activity of OCJ
Active ingredients were analyzed in our laboratories and
characteristics of OCJ were as follows: soluble solids
10.94% (Mettler Toledao HB43-S); pH 3.35 (pH meter
Testo 206); total phenolics 386 mg GAE/100 g of juice; total
anthocyanins 25 mg/100 g of juice. The radical scavenging
activity was investigated against synthetic DPPH radical
and the obtained IC
50
value for OCJ was 1.74 –0.04 mg/mL.
Effects of chokeberry juice consumption on epidermis,
dermis, and subcutaneous tissue characteristics
The most significant changes that have been observed
during this study were recorded by ultrasonography. In this
study, statistically significant changes in all investigated
characteristics of epidermis, dermis, and subcutaneous tis-
sue, measured by ultrasound, were observed after the in-
tervention and compared with the baseline values (
T1 cTable 1).
ScTT and the thickness of dermal and subcutaneous tissue
(DScTT) were reduced in all subjects after 45 days of in-
tervention, with further reduction until the end of the study.
The average reductions of ScTT and DScTT at the end of the
study were 1.9 and 2.1 mm (9.5% and 9.6%), respectively,
compared with the baseline values. DET was reduced in
65% (19 out of 29) of subjects after 45 days of chokeberry
juice consumption and in 90% (26 out of 29) of subjects, at the
end of the study. An average reduction in DET for the whole
group was 0.3 mm (10.2%), compared with the baseline val-
ues. The growth of the ScTFL into the dermis is a typical
feature of cellulite
15,33
and in our study, the length of ScTFL
was reduced in 97% (28 out of 29) of subjects after 45 days of
the study, with further reduction at the end of the study ob-
served in the same percentage of subjects. An average value
of ScTFL reduction was observed at the end of the study.
Chokeberry juice consumption also showed positive ef-
fects on edema reduction as shown in bT2
Table 2. At the be-
ginning of the study, edema was observed in 60% (16 out of
29) of subjects. At the second time point (45 days after juice
consumption), reduction of edema severity was observed in
37% (6 out of 16) of subjects, and the absence of edema in
63% (10 out of 16) of subjects in whom edema was diag-
nosed at the beginning of the study. At the end of the study,
edema was not observed in any of the subjects included in
the study.
Digital photographs of the upper back and front part of the
thigh, taken at the beginning and at the end of the study,
were evaluated for visual changes by 10 independent eval-
uators. Five of them noticed an improvement in cellulite
appearance after the study was finished.
Biochemical and anthropometric measurements
Anthropometric and biochemical parameters of partici-
pants (29 women aged 25–48, with a height range from 156
to 181.5 cm and weight 49.3–93.8 kg) measured at the be-
ginning and the end of the study are shown in bT3
Table 3.
Although statistically significant difference of the results
obtained before and after the study was not shown for any of
analyzed parameters, beneficial changes were observed in
certain number of participants. Regarding anthropometric
parameters, at the end of the study, decrease in body weight
was observed in 16 of 29 women, reduction in body fat (%)
was noticed in the greatest number of subjects (n=20), re-
presenting 69% of the study group, with the maximum value
of 17%. Reduction in the abdominal circumference was
observed in 62% (18 out of 29) of subjects. Thigh circum-
ference reduction was not significant and was observed in
only 12 subjects, which was inconsistent to the results of
ultrasound measurements in the same region. Since only
healthy subjects were included in the study, most of them
were within the reference ranges for most of the biochemical
parameters before entering the study and at the final point of
control. Although not statistically significant for the whole
study population, changes in biochemical parameters after
the intervention were observed in more than 50% of the
Table 1. Characteristics of Epidermis, Dermis,
and Subcutaneous Tissue Measured by Ultrasound
Time
point
Dermis and
subcutaneous
tissue thickness
(mm)
Subcutaneous
tissue
thickness
(mm)
Dermis and
epidermis
thickness
(mm)
Length of
subcutaneous
fascicles
(mm)
Day 0 22.23 –3.28
a
20.42 –3.26
a
2.69 –0.48
a
2.96 –0.72
a
Day 45 21.10 –3.23
b
19.44 –3.07
b
2.52 –0.51
ab
2.11 –0.40
b
Day 90 20.09 –3.33
c
18.52 –3.24
c
2.38 –0.39
b
1.61 –0.35
c
Values are mean –SD (n=29). All measurements were performed in
triplicate. Data were analyzed by one-way repeated measures ANOVA, with
Bonferroni confidence interval adjustment for multiple comparisons of main
effects, whereas means followed by different letters differ significantly P<.05.
Table 2. Effect of Intervention on Subcutaneous Edema
Day of the study Subjects with edema (n,%)
0 16/29 (55.2%)
45 6/29 (20.7%)
90 0
CHOKEBERRY JUICE IMPROVES SKIN MORPHOLOGY 3
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subjects. In addition, investigated parameters, which are
indicators of liver and kidney function (urea, ALT, AST),
remained in reference values thus indicating safety of
chokeberry juice consumption.
Questionnaire analysis
Analysis of the answers from the questionnaire fulfilled
by all subjects at the end of the study showed that 41.4% of
subjects observed visual improvement in the condition of
cellulite, 48.3% did not observed any change, while 10.3%
of investigated subjects were ambivalent. The positive
overall effect of chokeberry juice consumption, which was
mainly referred on skin tightness, was reported by 69% of
subjects. Side effects, related to the gastrointestinal system
function (motility, constipation, diarrhea), were reported by
13.8% of subjects.
DISCUSSION
Considering that cellulite involves both the subcutaneous
tissue and the dermis, different methods for the analysis of
structure and characteristics of these tissues, including high-
frequency ultrasonography, are applied in both the diagnosis
and treatment of cellulite.
10,13,15
It was shown that some of
the key roles in pathophysiology of cellulite play enlarged
fat cells, weakened connective tissues, and diminished mi-
crocirculation.
6,7
It was shown that anthocyanins from bil-
berries could be effective in promoting and enhancing
arteriolar rhythmic diameter changes that play a role in the
redistribution of microvascular blood flow and interstitial
fluid formation,
27
while phenolics from bilberries, cranber-
ries, and grape seed extracts facilitate the repair of vessel
damage responsible for small blood vessel permeability.
28
Positive effects of polyphenols that reach OCJ consumma-
tion in the treatment of cellulite could be connected with
improvements in microcirculation as well as with protection
and restoration of endothelial cells and consequently their
function. It was shown previously that the flavonoid-rich
extract from chokeberry might preserve the function of the
endothelium and contributes to the prevention of athero-
sclerosis progression,
34
while the anthocyanin-rich choke-
berry extract inhibited the releasing of PGE2 in normal
human endothelial cells.
35
In addition, positive effects of
chokeberry on fat metabolism could contribute to beneficial
effects in cellulite conditions.
20,36
Ultrasonography was also used in the study of Mlosek et
al.
15
who investigated the effects of anticellulite cream
which, among other components, contained cranberry ex-
tracts rich in polyphenols. Although two different ap-
proaches to the treatment were applied, similar results were
obtained. They also noticed a decrease in all measured pa-
rameters, including thickness of subcutaneous tissue, length
of ScTFL, and the number of women with dermal edemas.
One of the mechanisms assumed was improvement in
microcirculation.
Recent investigations on gene expression levels provided
more insight in the pathophysiology of cellulite, and pos-
tulated new targets in the prevention and therapy of this
cosmetic problem. Emanuele et al.
37,38
showed that adipo-
nectin mRNA expression in the subcutaneous adipose tissue
of the gluteal region was significantly lower in areas with
cellulite compared with areas with normal skin and subcu-
taneous tissue architecture, without any change in plasma
Table 3. Anthropometric and Biochemical Characteristics
of Subjects (n=29) Before and After the Study
Before After P
BMI (%) 22.9 –4.0 22.9 –4.1 NS
Fat (%) 30.0 –6.5 29.6 –6.9 NS
Abdominal circumference (cm) 75.1 –10.4 74.0 –9.8 NS
Hip circumference (cm) 100.7 –7.6 101.3 –7.9 NS
Thigh circumference (cm) 58.5 –4.8 58.6 –4.9 NS
Knee circumference (cm) 42.1 –4.7 42.1 –4.2 NS
Glucose (mM) 4.5 –0.4 4.4 –0.5 NS
Triglycerides (mM) 0.9 –0.4 0.8 –0.4 NS
Total cholesterol (mM) 5.1 –1.2 5.0 –1.0 NS
HDL cholesterol (mM) 1.5 –0.3 1.5 –0.3 NS
LDL cholesterol (mM) 3.2 –1.1 3.1 –0.9 NS
Urea (mM) 3.9 –0.8 4.1 –1.2 NS
ALT (U/L) 17.8 –9.4 17.0 –8.0 NS
AST (U/L) 19.9 –4.4 19.2 –5.5 NS
Values are mean –standard deviation (n=29). Data were analyzed by a
paired Student’s t-test.
NS, statistically not significant if P‡.05.
FIG. 1. Ultrasound images at the beginning (A)
and end (B) of the study, indicating the reduction
in the length fascicle. bAU7
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adiponectin levels.
37
Based on the gene expression analysis,
they also highlighted the independent role of angiotenzin-
converting enzyme and hypoxia-inducible factor 1A in
predisposing to cellulite.
38
At the same time, anthocyanins
have shown a beneficial influence on gene expression of
several adipocytokines, including adiponectine
39,40
in ani-
mal in vivo models. The inhibitory potential of anthocyanins
and their metabolites on ACE activity in vitro was also re-
ported.
41
These findings could hypothetically explain the
observed beneficial effects of anthocyanin-rich chokeberry
juice on morphological features of cellulite, measured by
ultrasound.
On the other side, low sensitivity and reproducibility of
circumference measurements could contribute to the obtained
inconsistency, postulating the advantage of the direct analysis
of skin and subcutaneous tissues thus indicating that cellulite
disturbed architecture of these tissues could be the optimal
target rather than fat tissue alone. Sasaki et al.
18
investigated
the effectiveness of anticellulite gel combined with LED light
and although they noticed positive changes using ultrasound
imaging, significant changes in body weight, BMI, body fat
indices, and thigh circumferences were not observed.
Results obtained in this study showed marked potential of
anthocyanin-rich chokeberry juice to improve the mor-
phology of skin and subcutaneous tissue in the regions af-
fected by cellulite under in vivo conditions. These findings
add new evidence in the dossier of beneficial effects of
anthocyanins and postulate a new research strategy for both
prevention and treatment of cellulite.
ACKNOWLEDGMENTS
The authors acknowledge their gratitude to the Ministry
of Education and Science of Serbia for financial support,
project number 46013. We heartily thank the Conimex
Trade d.o.o., Belgrade, Serbia.
AUTHOR DISCLOSURE STATEMENT
The authors disclose any commercial associations that
might create a conflict of interest in connection with sub-
mitted manuscripts.
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AUTHOR QUERY FOR JMF-2013-0102-VER9-SAVIKIN_1P
AU1: Please note that gene symbols in any article should be formatted as per the gene nomenclature. Thus, please make sure
that gene symbols, if any in this article, are italicized.
AU2: Please review all authors’ surnames for accurate indexing citations.
AU3: Please confirm the authors’ affiliations.
AU4: Please mention the degrees of the corresponding author.
AU5: Please cite Ref. 16 in the text.
AU6: Please provide title of the work in Ref. 30.
AU7: Please cite Figure 1 in the text.
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