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The Immunomodulation Effect of Aronia Extract Lacks Association with Its

Antioxidant Anthocyanins

Article in Journal of Medicinal Food · April 2013

DOI: 10.1089/jmf.2012.0151 · Source: PubMed

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JOURNAL OF MEDICINAL FOOD
J Med Food 16 (4) 2013, 334–342
# Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2012.0151

The Immunomodulation Effect of Aronia Extract

Lacks Association with Its Antioxidant Anthocyanins
Jin Xu,1,2 and Biljana Mojsoska1
1
Department of Science, Systems, and Models, Roskilde University, Roskilde, Denmark.
2
Department of Medicine, Copenhagen University Hospital, Herlev, Denmark.

ABSTRACT Polyphenols comprise a diverse group of molecules with antioxidative and anti-inflammatory activities. To
compare the antioxidative and anti-inflammatory capacity of Aronia melanocarpa berries (chokeberries), recognized for their
high content of anthocyanins, a noncytotoxic isolation method was developed to obtain high-purity anthocyanins in the
extract. The antioxidative activity of the extract, the anthocyanin-rich fraction (AF) was determined by 1,1-diphenyl-2-
picrylhydrazyl radical and ferric-reducing ability of plasma along with resveratrol as a reference. The immunomodulation
properties were assessed in lipopolysaccharide (LPS)-stimulated human monocytes mono mac 6. The isolated AF, containing
six different anthocyanins, exhibited a stronger antioxidative capacity compared to resveratrol. Resveratrol enhanced tumor
necrosis factor-a and reduced interleukin-10 (IL-10) production by LPS, whereas AF only had a slight effect in reducing IL-
10. These results demonstrated that there was no major relationship between the antioxidative effect and immunomodulation
capacities of AF and resveratrol. The immunomodulatory activity of the extract is associated with bioactive compounds in
Aronia other than its anthocyanins.

KEY WORDS:  anthocyanins  anti-inflammatory  antioxidative  Aronia  resveratrol

INTRODUCTION by cyanidin-3-O-b-glucoside.11 The anti-inflammatory ca-

pacity of anthocyanins on oxLDL-induced endothelial injury
B erries and red-colored fruits are important dietary
sources of phenolic antioxidants that are known to have
significant health benefits. Aronia melanocarpa (chokeberry)
were found to be significantly correlated with different sub-
stituents at C30 , C40 , and C3 positions.12 Thus, a beneficial
effect of anthocyanins on endothelial cells seems well estab-
is widely used as a raw material in natural coloring for the
lished. In contrast, a single study found that cyanidin-3-O-b-
food industry. Aronia berries have preventive potential to-
glucoside upregulated TNF-a production in lipopolysaccharide
ward various diseases, including uveitis,1,2 gastric mucosal
(LPS)/interferon-c-stimulated murine macrophage-like RAW
damage,3 and colon cancer.4 Extract of Aronia berries has
264.7 cells (RAW) macrophages.13 To our knowledge, this
been shown to inhibit platelet adhesion, reduce aggregation,
is the only study that has addressed the anti-inflammatory/
and generation of O2- , as well as reduce the production of
immunomodulatory activity of the anthocyanin-rich fraction
inflammatory markers.5,6
(AF) in immune cells that are often involved in proin-
Anthocyanins represent one of the most potent groups of
flammatory processes, for example, monocytes or other
polyphenolic antioxidants7 and be an abundant compound
phagocytosing cells.
in Aronia berries.8 It has been proposed that anthocyanins
Many other plant antioxidants have been shown to pos-
in the extracts of many berries and fruits are the compo-
sess an anti-inflammatory activity in their pure form, for
nents responsible for a large proportion of their anti-
example, flavonoids and stilbenes14 and curcuminoids.15
inflammatory activity.9 Studies on endothelial cell lines
One molecule, trans-3,5,40 -trihydroxystilbene, also known
have shown that the anthocyanins cyanidin-3-O-b-glucoside
as resveratrol, has been shown to hold anti-inflammatory
and peonidin-3-O-b-glucoside inhibit CD40-induced acti-
activities in many different cells, including monocytes,
vation of the cells,10 while tumor necrosis factor alpha
macrophages, and dendritic cells.14,16–19 The antioxidative
(TNF-a)–induced endothelial dysfunctions were abrogated
activity of resveratrol is well known, although it is moderate
compared to antioxidants in Aronia berries.20 Resveratrol is
Manuscript received 3 June 2012. Revision accepted 30 September 2012. abundant in the skin of grapes, in peanuts, and in many berry
species, but it is not found in Aronia berries.21
Address correspondence to: Jin Xu, MSc, Department of Science, Systems, and Models,
Roskilde University, Universitetsvej 1, 18.2, Roskilde 4000, Denmark, E-mail: jxu@
For many years, it was believed that the antioxida-
ruc.dk tive effect of plant polyphenols was also the cause of their

334
 THE IMMUNOMODULATION EFFECT OF ARONIA EXTRACT
anti-inflammatory activities. In recent years, polyphenols,
however, were found to be involved in the proinflammatory
signaling pathways (e.g., the Janus kinases 1 and 2),22,23 re-
sulted in inhibition of the activation of transcription factors
such as nuclear factor kappa-B (NF-jB) and activator
protein–1 (AP-1).24 Accordingly, strong antioxidative activ-
ity may not necessarily imply strong anti-inflammatory ac-
tivity of a polyphenolic compound, and vice versa.
With this in mind, the study aimed at characterizing the
anthocyanin fraction of Aronia berries in regard to their an-
tioxidant and anti-inflammatory capabilities, respectively, as
compared to resveratrol, a well-described reference com-
pound as a therapeutic agent for inflammatory diseases.14,16
MATERIALS AND METHODS
Resveratrol, piceid, chlorogenic acid, neochlorogenic
acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid (Trolox), 1,1-diphenyl-2-picrylhydrazyl radical
(DPPH), 2,4,6-tripyridil-s-triazine (TPTZ), and LPS from
Escherichia coli (026:B6) were purchased from Sigma
Aldrich (St. Louis, MO, USA). The organic Aronia crude
extract was from Aronia melanocarpa (purchased from
Berrifine ApS., Slagelse, Denmark). The extract was stored
at -18C. Anthocyanin standards were obtained from
Polyphenols Laboratories A/S (Sandnes, Norway).
Characterization of anthocyanins
The purified anthocyanins and the anthocyanins present in
Aronia crude extract were identified and quantified by high-
performance liquid chromatography (HPLC; TSP Spectra
System) with an UV 6000 LP diode array detector with a
5-cm flow cell with a mass spectrometry (MS) detector.
Ten-microliter aliquots of Aronia crude extract were applied
to a Phenomenex Luna C 18 column (Torrance, CA, USA;
150 mm · 2.0 mm, 3 lm particle size) and eluted with a
linear eluent gradient program of 5% formic acid in water
(A) and methanol (B) with a flow rate 0.17 mL/min:
0–15 min, 8%–20% B; 15–35 min, 20%–27% B; 35–50 min,
27%–45% B; 50–60 min, decreasing from 45% to 8% B;
60–70 min, constant 8% B. The mass spectrometry was a
LCQ-Deca ion-trap instrument from ThermoFinnigan (San
Jose, CA, USA) equipped with an electrospray ionization
interface run in a positive mode. A positive potential
(+ 3 kV) was applied to the silica needle, discharge current
80 mA, capillary voltage 23 V, capillary temperature 225C,
tube lens offset - 5 V, sheath gas (N2) and auxiliary gas (N2)
84 and 41 arbitrary units, respectively. The diode array
detector (DAD) absorption spectra were recorded at wave-
lengths between 190 and 800 nm or more narrow intervals,
with a fixed filter. The ion trap was run in the data-dependent
tandem mass spectrometry (MS/MS) scanning mode and the
first scan event was obtained in the m/z interval 250–1200.
This was followed by a second scan event of the most in-
tensive ion from the first scan in which a high-intensity MS2
fragment of the MS/MS spectrum was obtained. Isolation
m/z width = 1, normalized collision energy 28, activation
time 30 ms, minimum signal required 105 counts.
335
Purification of anthocyanins
Aronia crude extract (1 mL) was diluted with an aqueous
solution of 1% formic acid (60 mL). The solution was then
eluted through a strong cation-exchange column (Varian
MEGA-BE-SCX, 1 g, 6 mL; AA, Walnut Creek, CA, USA)
binding the positively charged anthocyanins, while neutral
molecules were removed by washing with water. The strong
cation-exchange column based on –SO3H groups had a
capacity of 1–3 mg of anthocyanins (2–6 lmol/g) corre-
sponding to 1%–3% of the available –SO3H groups on the
stationary phase may exchange their proton for a positive
anthocyanin molecule. The anthocyanins were eluted from
the column by concentrated hydrochloric acid/water/ethanol
(8:12:80), diluted 10 times with water, and applied to a
reverse-phase C18 sep-pack column (Varian MEGA-BE-
C18, 1 g, 6 mL; Walnut Creek, CA, USA). The C18 column
was carefully washed with water and the anthocyanins were
eluted with 0.1 M formic acid in ethanol. Finally, the ethanol
was removed by rotary evaporation and the product dried in
a vacuum oven at 40C.
Determination of total anthocyanin content
The total anthocyanin content of Aronia 25 crude extract
was determined by the pH differential method25 and HPLC
(Supplementary Fig. S1; Supplementary Data are available
online at www.liebertpub.com/jmf). The total anthocyanin
isolated from the extract was determined by dissolving
0.8 mg dry matter in 8 mL 0.1 M aqueous formic acid, which
was then analyzed by the pH differential method and HPLC.
Anthocyanins were quantified using the pelargonidin-3-O-
glucoside calibration curve at 520 nm on HPLC. Commer-
cially available pelargonidin-3-O-glucoside standard was
dissolved in 0.1 M aqueous formic acid and thereafter di-
luted (0.04–0.6 mM) to make a standard curve (R2 ‡ 0.99).
The results were expressed as cyanidin-3-O-glucoside
equivalents. The determination was performed in triplets.
Determination of antioxidative capacity using
the DPPH assay
The DPPH method was performed according to the
protocol described previously.27 In brief, the antioxidant
solution (100 lL) was transferred to a UV/Vis-cuvette
containing 3 mL DPPH in methanol (1 · 10 - 4 mol/L), in-
cubated for 30–300 min at room temperature, sealed in the
dark, and monitored at 515 nm. The antiradical power
(ARP = nDPPH/n50) was obtained from a plot of % remaining
DPPH (nDPPH) versus n50, the amount of antioxidant nec-
essary to remove 50% DPPH (Supplementary Fig. S2).26
Determination of antioxidative capacity using
the ferric-reducing ability of plasma assay
The ferric-reducing ability of plasma (FRAP) reagent was
prepared by a 10:1:1 mixture of acetate buffer (300 mM, pH
3.6), 2,4,6-TPTZ (10 mM) dissolved in HCl (40 mM), and
FeCl3$6H2O (20 mM). The measurements were performed
according to Benzie and Strain.27 The antioxidant solution
336 XU AND MOJSOSKA
(100 lL) was added to the FRAP reagent (3 mL) and the
absorbance obtained after 4 min at 593 nm. FeSO4$7H2O
solutions (5–20 mM) were used as standard antioxidant
solutions (Supplementary Fig. S3).
Cell culture conditions
Mono Mac 6 cells (MM6) were obtained from German
Collection of Microorganisms and Cell Cultures (DSMZ).
The MM6 cells were cultured in the growth medium RPM1
1640 (Cambrex, East Rutherford, NJ, USA) with 10% fetal
calf serum (BioWhittaker, Walkersville, MD, USA), 1%
penicillin/streptomycin (Cambrex), 2 mM glutamine (Cam-
brex), 2 mM nonessential amino acids (Cambrex), 1 mM
sodium pyruvate (InvitrogenTM; Gibco, Auckland, NZ), and
9 lg/mL bovine insulin (Sigma Aldrich, St. Louis, MO,
USA). Twice weekly, the cells were harvested, counted, and
diluted to 2 · 105 cells/mL. The cells were incubated in a
humidified atmosphere with 5% CO2 at 37C.
Stimulation of MM6 cells with plant extracts
and resveratrol
Three-day old cultures of MM6 cells were harvested and
cell density adjusted to 0.67 · 106 cells/mL. Resveratrol
dissolved in dimethyl sulfoxide (DMSO), Aronia crude
extract, and the AF in 70% ethanol were diluted in Dul-
becco’s phosphate-buffered saline (DPBS; Biowhittaker),
and added to MM6 cells for 30 min, and then LPS (final
concentration 1 lg/mL) was added and the cells were in-
cubated for 18 h in a humidified atmosphere with 5% CO2 at
37C. For the combinatory stimulations, Aronia crude ex-
tract or the AF diluted with resveratrol or DMSO/DPBS was
added to the cells incubated under the conditions described
above. The supernatants were harvested and the cytokine
production (interleukin [IL]-6, IL-10, and TNF-a) was an-
alyzed by enzyme-linked immunosorbent assay (DuoSet
ELISA Development System, R&D Systems, Inc., Min-
neapolis, MN, USA; Supplementary Figs. S4–S6) according
to the manufacturer’s instructions.
Statistical analysis
Unless otherwise stated, all experiments were performed
three times, and the results are given as mean values
– standard deviation. No outliers were rejected from the data
sets. One-way analysis of variance (ANOVA) followed with
Tukey’s multiple comparison test was performed to deter-
mine the different immunomodulation ability among single
compounds or in combined. One-way ANOVA with Dun-
nett’s test was used to determine whether the effect by single
compounds is significantly different from the controls. For
nonparametric analysis, ANOVA (Kruskal–Wallis test) was
used, followed by Dunnett’s multiple comparison test; anal-
ysis was performed using a significance level of P < .05.
Graphpad Prism 5.0 for Windows (Graphpad Prism Software,
San Diego, CA, USA) was used. Microsoft Excel 2010 was
used for antioxidative capacity data statistics according to the
protocol.28 No outliers were removed from any data sets.
RESULTS AND DISSUSION
Characterization and isolation of the anthocyanins
from Aronia crude extract
To characterize the anthocyanin content in Aronia crude
extract as well as in the AF isolated from Aronia crude
extract, reversed-phase separation analysis was performed.
Figure 1A and B show the HPLC-DAD chromatograms of
aliquots from a single batch of Aronia crude extract obtained
in the wavelength intervals 190–800 nm and in the maxi-
mum absorption mode 500–540 nm, respectively. In total,
six anthocyanin derivatives (peaks 3–8, in Fig. 1) were
present in Aronia crude extract, but comparison of the two
chromatograms revealed two additional major non-
anthocyanin components (peaks 1, 2, Fig. 1A). These two
nonanthocyanin compounds were identified as neo-
chlorogenic acid (peak 1) and chlorogenic acid (peak 2) based
on retention time match with authentic reference samples. The
six anthocyanin compounds were identified from their UV/Vis
and electrospray molecular ion of [M + ] and fragment ion of
MS/MS spectra and comparisons with literature data29 are
summarized in Table 1 and their chemical structures in Figure
2. The four major anthocyanins (liquid chromatography peaks
3–6, Fig. 1B), corresponding to cyanidin-3-O-galactoside, cy-
anidin-3-O-glucoside, cyanidin-3-O-arabinoside, and cyanidin-
3-O-xyloside, were all identified and extensively characterized
in previous studies.30 The remaining two species were identified
as cyanidin pyruvates, commonly found in the skins of some
deep frozen stored grapes and some red wines.31 Based on
retention times and MS fragment patterns, the crude extract did
not contain any resveratrol (fragment m/z 227), its glucoside,
piceid (fragment m/z 389), or other resveratrol derivatives.32
Anthocyanins are positively charged at low pH (< 2–3) and
may therefore be conveniently isolated and separated from
other neutral molecules in Aronia crude extract by means of a
strong cation-exchange column based on –SO3H groups on
the stationary phase, which may exchange their proton for a
positive anthocyanin molecule. To our knowledge, the same
noncytotoxic separation method that is competent for the
immunomodulation study has not been reported previously.
Figure 1C and D show the HPLC-DAD chromatograms of
cation-exchange-purified Aronia AF obtained in the maxi-
mum absorption mode in the wavelength intervals 280–
350 nm and 500–540 nm, respectively. Comparison of the
chromatograms of AF with the anthocyanin chromatogram
of Aronia crude extract (Fig. 1B) revealed almost identical
profiles, indicating that our developed strong cation-
exchange purification protocol for AF is capable of extract-
ing the entire content of anthocyanins from Aronia crude
extract. Furthermore, the analyzed cytokines TNF-a, IL-6,
and IL-10 in the nonstimulated monocytes were not affected
by the addition of AF (data not shown). This demonstrated
the noncytotoxic quality of the purification methods and
made it possible to evaluate immunomodulatory effects of
various plant extracts with or without anthocyanins.
The efficiency of cation-exchange chromatography is
shown to be comparable to the commonly used C18 solid-
phase extraction. Compared to C18 extraction, strong
 THE IMMUNOMODULATION EFFECT OF ARONIA EXTRACT 337

FIG. 1. High-performance liquid

chromatography–diode array detector
(HPLC-DAD) chromatogram of Ar-
onia extract and anthocyanin fraction
purified from Aronia. (A) Aronia ex-
tract HPLC-DAD chromatogram ob-
tained UV/Vis and mass spectrometry
data 190–800 nm. (B) Aronia extract
(kmax mode 500–540 nm). (C) Cation-
exchange purified anthocyanin-rich
fraction (AF; kmax mode 280–350 nm).
(D) Like (C) with a wavelength inter-
val 500–540 nm. Peaks 1–8 correspond
to the compounds listed in Table 1.

cation-exchange chromatography is rapid, easy to modify,
and low cost to obtain high-purity anthocyanins. Based on
the color observed in the experiments, the chemical forms of
anthocyanins are pH dependent and easily reduced at pH
> 6. To obtain high purity, the acidic solvent is favorable to
keep anthocyanins in its flavylium cation form during the
isolation process. The hydrophilic condition is more suitable
for retaining anthocyanin species. However, strong cation-
exchange chromatography does not eliminate the aqueous
solvent, and rotary evaporation would need to be applied
subsequently.
Aronia crude extract and AF were dehydrated and dry
weights (DWs) were determined, three independent exper-
iments were repeated. DW of Aronia crude extract was
found to be 600 – 100 g/L.

Table 1. Retention Times, Ultraviolet/Visible

and Mass Spectrometry Data of Compounds 1–8

MS1 MS2
UV/Vis [M + ] [M + ] Mole
(nm) (m/z) (m/z) Identification percenta
1 324 355 163 Neochlorogenic acid 22.0 – 1.0
2 326 355 163 Chlorogenic acid 18.0 – 1.0
3 514 449 288 Cyanidin-3-galactosideb 39.0 – 3.0
4 515 449 287 Cyanidin-3-glucosideb 2.33 – 0.19
5 515 419 287 Cyanidin-3-arabinosideb 17.3 – 1.9
6 518 419 287 Cyanidin-3-xylosideb 0.86 – 0.31
7 503 517 355 Cyanidin-3-O-hexo- 0.56 – 0.050
pyruvate
8 509 487 355 Cyanidin-3-O-pento- 3.14 – 0.94
pyruvate
a
Total anthocyanin content in Aronia extract was 4.49 – 0.87 g/L determined
by pH differential method and the high-performance liquid chromatography FIG. 2. Structures of compounds, which are part of this investigation
analysis. Mean values – SD, n = 6. and have been previously identified in Aronia.29 In this study, cyanidin
b
The exact hexose/pentose structures were obtained from Wu et al.29 glycosides, 5-carboxypyranocyanidin glycosides, neochlorogenic acid,
MS, mass spectrometry; SD, standard deviation. and chlorogenic acid were identified in the extract used.
338 XU AND MOJSOSKA

Table 2. Radical Scavenging Parameters of Resveratrol and
Anthocyanin-Rich Fraction Determined by DPPH and FRAP
DPPH FRAP
Antioxidant stoic. ratioa relative to FeSO4
Resveratrol 1.255 – 0.090 (1)b 0.706 – 0.040
Trolox 2.10 – 0.10 (2.2)b 1.53 – 0.16 (2.0)c
Anthocyanin-rich fractiond 6.085 – 0.035 1.72 – 0.16
Mean value – SD, n = 3.
a
Number of DPPH radicals, which can be removed by one molecule of
antioxidant. Stoichiometric ratio = antiradical power/2.35
b
Value from Villano et al.36
c
Value from Benzie and Strain.27
d
Anthocyanin-rich fraction isolated from Aronia.
DPPH, 1,1-diphenyl-2-picrylhydrazyl radical; FRAP, ferric-reducing ability
of plasma; stoic., stoichiometric.
The anthocyanin content was quantified by the pH dif-
ferential method and HPLC analysis. The results obtained
from these two methods were comparable. Using the pH
differential method, the anthocyanin content in Aronia crude
extract was found to be 4.0 – 1.0 g/L, whereas the HPLC
analysis gave a concentration of 4.98 – 0.43 g/L. The two
methods also showed that the 0.8 – 0.2 mg dehydrated iso-
lation product contained 0.87 – 0.32 mg anthocyanins as
measured by pH differential and 0.73 – 0.15 mg as measured
by HPLC. Together, this showed that our purification pro-
tocol is capable of obtaining high-purity products. The total
anthocyanins content in Aronia crude extract was deter-
mined to be 7.5 mg/g DW, which is consistent with previ-
ously reported numbers for extract from black chokeberry.2
In other studies, the total content of anthocyanins from
Aronia raw extract was reported to vary from 6.5 to 10.4 mg/g
DW.33,34 The difference may be explained by different plant
origins.
Antioxidative capacity of AF and kinetic behavior
The antioxidative capacities of the extract, AF, and re-
sveratrol were determined by DPPH and FRAP assay. The
stock solutions used in cell experiments were measured and
expressed as Trolox equivalent in lmol/mL for both DPPH
and FRAP. The kinetic behavior was assessed according to
the previously established protocol.26,27
Table 2 shows that resveratrol had approximately half the
molar antioxidative capacity of the vitamin E analog Trolox,
while the AF exhibited 1.1–2.9 times the antioxidative ac-
tivity as Trolox.27,35,36 Table 3 shows the antioxidative ca-
pacities of the stock solutions used in cell experiments;
Aronia crude extract, AF, and 44 lM (10 mg/L) resveratrol,
expressed as Trolox equivalent (TE) in lmol/mL. Samples
of 44 lM resveratrol exhibited an antioxidative capacity in
the order of a 600 · dilution of AF or to a 2000 · dilution of
Aronia crude extract as used in cell experiments.
The DPPH analysis determined the antioxidative ca-
pacity of Aronia crude extract to be 241–369 lmol TE/g
DW (Table 3). This result is consistent with the values re-
cently reported30,37 when normalized to the same DW
(600 g/L). However, the antioxidative capacity of AF as
measured by DPPH was 1.7-fold higher than the value
measured by FRAP. Similar observations have been made in
previous studies.38 The DPPH method measures the ca-
pacity of antioxidants to transfer an H-atom to the DPPH
radical, whereas the FRAP method expresses the reductive
capacity of the antioxidant.27,35 This may explain the dis-
crepancies between values obtained in the two different
assays. The percent contribution of the anthocyanin fraction
to the total antioxidative capacity of Aronia crude extract, as
determined by DPPH and FRAP were 81% and 33%, re-
spectively. In contrast, Zheng and Wang found30 53.1% for
the anthocyanin fraction in Aronia berries by the oxygen
radical absorbance capacity assay. Thus, other antioxidative
compounds, in addition to anthocyanins, are present in Ar-
onia crude extract. The phenolic composition of the
chokeberry analyzed by Zheng and Wang were similar to
what we have observed.30 He reported that two chlorogenic
acids represented high proportions of antioxidative acitiv-
ities found in Aronia.30 The combination of anthocyanins
and chlorogenic acids was hypothesized to contribute sig-
nificantly to the total antioxidative capacity of Aronia crude
extract. The presence of other compounds with antioxidant
activities cannot be fully excluded based on the findings of
this study or Zheng and Wang.30

Table 3. Antioxidative Capacity of Aronia Extract, Anthocyanin-Rich Fraction,

and Resveratrol Stock Solutions Used in Cell Experiments

DPPH (Trolox eq. FRAP (Trolox eq.


DPPH (Trolox lmol/mL for the solutions FRAP (Trolox eq. lmol/mL for the solutions
eq. lmol/g DW) used in cell experiments)a lmol/g DW) used in cell experiments)a
Aronia extract 241 – 82.4 145b – 49.6 369 – 67.8 221 – 40.6b
Anthocyanin-rich fraction 29,435 – 12,510 34c – 14.5 10,892 – 971.5 12c – 1.1
Resveratrol 3199 – 1223 0.6d – 0.23 2025 – 106.44 0.4d – 0.02

Mean values – SD, n = 3.

a
Antioxidative capacity expressed in Trolox eq. lmol/mL = amount of test compound stocks in g/L · Trolox eq. lmol/g DW by DPPH or FRAP.
b
600 – 100 g/L Aronia extract stock used in cell experiments.
c
1.14 – 0.29 g/L anthocyanin-rich fraction stock used in cell experiments.
d
0.2 g/L resveratrol used before loading in cell wells.
DW, dry weight.
 THE IMMUNOMODULATION EFFECT OF ARONIA EXTRACT 339

Characterization of the immunomodulatory effect of Aronia
crude extract, anthocyanidin-rich fraction, and resveratrol
in LPS-stimulated human monocytes
To test the immunomodulatory effect of Aronia crude
extract, AF, and resveratrol, these samples were added to
cultures of the human monocyte cell line, MM6, before
stimulation with LPS. They were evaluated by measuring
the production of proinflammatory cytokines TNF-a, IL-6,
and the anti-inflammatory cytokine IL-10.
We first compared the immunomodulatory activity of re-
sveratrol and Aronia crude extract added to cells in compa-
rable antioxidant concentrations as determined in Figure 3
and Table 3. Resveratrol increased the LPS-induced TNF-a
production and reduced the IL-10 production at the highest
tested concentration (44 lM). The IL-6 production was not
affected. Addition of Aronia crude extract in 600 · and
2000 · dilution led to a dose-dependent reduction in LPS-
induced IL-10 and TNF-a production, and increase in IL-6.
Thus, although the antioxidant concentration measured in the
diluted extract and in the resveratrol sample were compara-
ble, the modulatory effects on the LPS-stimulated monocytes
were rather disparate: Aronia crude extract affected the pro-
duction of each cytokine analyzed, led to an increase in the
IL-6 production and, in contrast to resveratrol, decreased the
TNF-a production.
The dose-dependent downregulation of the TNF-a and
IL-10 production in the monocytes by Aronia crude extract
is consistent with previous studies in RAW cells.1 Similar
results were also obtained from an extract of raspberries,39
whereas increased TNF-a levels were observed when ex-
posing RAW cells to the extracts of blue berries, black
currants, blackberries, and Saskatoon berries in an experi-
mental setup very similar to the present experiments.13
The results obtained from resveratrol are consistent with
previous studies. Resveratrol has previously been shown to
downregulate IL-10 and to upregulate TNF-a in macro-
phages and monocytes.16,40 It should be noted, however,
that resveratrol seems to affect cytokine production ac-
cording to the cell type studied; In studies of, for example,
dendritic cells, resveratrol led to an increase in LPS-
induced TNF-a production.39,41 Furthermore, resveratrol
was reported to regulate transcription factors: For example,
regulation of NF-jB in a cell-type-specific manner, dis-
crepancy may be explained by that resveratrol regulates
individual NF-jB subunits differently. For example, dif-
ferent roles of p50–p50 homodimers and p50–p65 hetero-
dimers may consequently result in different cytokine
production.42 Therefore, it will be interesting to study the
effect of test groups on certain NF-jB subunits in future
studies. The lack of immunomodulatory effects of antho-
cyanins corresponds well with earlier observations of
Ohgami et al.,1 while others found increased TNF-a levels
when the RAW cells were exposed to increasing concen-
trations of pure anthocyanins such as cyanidin and cyani-
din glycosides.13

FIG. 3. The modulation of cytokine expression in Mono Mac 6 (MM6) cells by resveratrol. MM6 cells were exposed to increasing concen-
trations of pure compounds resveratrol (0.4–44 lM) (A), or Aronia extract (diluted in 2000 · , 600 · times) (B), 30 min before the exposure to
1 lg/mL lipopolysaccharide (LPS). Cells were incubated for additional 18 h and the culture supernatants were harvested, the levels of cytokines
were determined by enzyme linked immunosorbent assay (ELISA). The levels of tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), and IL-10
are shown as means – standard deviation (SD) of three, four, or six experiments. Compared with LPS: **P < .01, ***P < .001.
340 XU AND MOJSOSKA

As Aronia crude extracts may contain many other com-
pounds with or without the antioxidative activity that might
modulate the cytokine response, for example, chlorogenic
acids or quercetin derivatives,13 it was important to assess
the contribution of the AF in Aronia crude extract per se to
the immunomodulation exerted by Aronia crude extract and
furthermore, to assess the combined immunomodulatory
effects of resveratrol and either Aronia crude extract or AF
(Fig. 4). If the antioxidative activity in a sample relates
directly to the immunomodulatory activity, the combined
action of the two samples holding the antioxidative activity
should enhance the immunomodulatory activity induced by
the individual samples.43 The cell experiment was repeated
as indicated in figure captions. An average value of the
cytokine expression was then calculated. In contrast to
the addition of Aronia crude extract, addition of AF to the
monocytes did not affect the LPS-induced TNF-a and IL-6
production, but it did reduce the IL-10 production dose
dependently in basal cytokine measurements. Resveratrol or
the AF alone did not induce cytokine production above
background. The addition of Aronia crude extract alone led
to the responses in TNF-a and IL-10 that did not exceed
200 pg/mL. Only Aronia crude extract induced a slight re-
sponse in IL-6, which increased by fivefold when resveratrol
was added to a 600 · dilution of the extract (data not
shown). Taken together, this demonstrated that AF only
exhibits a weak reducing effect in IL-10 production in LPS-
stimulated monocytes, and that Aronia crude extract
contains bioactive compounds other than anthocyanidins.
Furthermore, these other bioactive compounds affect the
inflammatory response of the cells differently as compared
to resveratrol.
When resveratrol was added together with the extract, the
enhanced TNF-a production caused by resveratrol was re-
duced dose dependently by the extract (Fig. 4A). Thus, as
might be expected from the results obtained by the indi-
vidual samples, the two samples conferred contrasting ef-
fects. The resveratrol-induced reduction of LPS-stimulated
IL-10 production was further reduced dose dependently by
Aronia crude extract (Fig. 4E). Interestingly, the IL-6 pro-
duction, which was not affected by resveratrol and increased
by Aronia crude extract, exhibited a dose-dependent de-
crease upon addition of both resveratrol and Aronia crude
extract (Fig. 4C). In contrast, addition of AF, which only

FIG. 4. The modulation of cytokine expression in MM6 cells by Aronia crude extract, anthocyanidin-rich fraction, resveratrol, and their
combination. To investigate the contribution of the antioxidant effect of AF from Aronia extract in cytokine expression. MM6 cells were exposed
to the Aronia crude extract (A, C, E) or anthocyanidin (B, D, F) at increasing concentrations with or without 44 lM resveratrol 30 min before the
addition of 1 lg/mL LPS. After an additional 18 h of incubation, cytokine levels TNF-a (A, B), IL-6 (C, D), or IL-10 (E, F) in culture supernatants
were determined by ELISA. The levels are means – SD of three end up to nine independent determinations. Compared with LPS: *P < .05,
**P < .01, ***P < .001; Aronia extract/AF versus corresponding Aronia/AF + resveratrol: #P < .05, ##P < .01, ###P < .001; compared with resver-
atrol: R1P < .05, R2P < .01, R3P < .001.
 THE IMMUNOMODULATION EFFECT OF ARONIA EXTRACT
weakly decreased the IL-10 production, did not give rise to
alterations in the TNF-a, IL-6, and IL-10 responses induced
by resveratrol and LPS together (Fig. 4B, D, F).
The observed effect on the LPS-induced IL-6 by Aronia
crude extract together with resveratrol (Fig. 4C) may be
explained by the fact that the two compounds regulate LPS-
induced IL-6 signaling through different modes of action.
As this effect was not observed by combining resveratrol
and AF, the role of the AF on the IL-6 production can be
ruled out. TNF-a and IL-10 expression followed the same
pattern as Aronia crude extract regardless of the presence of
resveratrol (Fig. 4A, E), especially in the TNF-a responses,
the reducing effect of Aronia crude extract became more
pronounced when combined with resveratrol. This inter-
estingly explains the French paradox,44 as red wine is rich in
both resveratrol and flavonoid. In contrast, the AF slightly
increased the resveratrol-induced increase in TNF-a. No
previous combinatory treatment by these two compounds
has been studied; however, more studies would be necessary
to further narrow the conclusions of the concentrations of
Aronia crude extract to show whether the effect is dose
dependent. Additional cytokine species as well as other
models for the assessment of inflammation will be necessary
for analysis to further confirm the results of this study.
Aronia berries are known to be a rich source of antioxi-
dants, anthocyanins in particular;45 it has been speculated
that this property is the root cause behind many beneficial
health effects reported for Aronia.46,47 Recently, increasing
evidence of polyphenols directly interfering with intracel-
lular proinflammatory enzymes has been reported.22,23 By
using the noncytotoxic cation-exchange-purified method, it
was possible to assess the immunomodulatory effects of
adding resveratrol and AF or reservatrol and Aronia crude
extract to stimulated monocytes. This study demonstrated
no major relationship between the antioxidative activity and
immunomodulatory activity. In addition, our results re-
vealed that the combined immunomodulatory effects of
distinct polyphenols cannot be predicted from the action of
the individual compounds. These findings underlined the
extra caution that should be given when interpreting the
individual classes of polyphenols because the characteriza-
tion of the major classes of polyphenols cannot be predicted
based on testing complex extracts. Resveratrol and Aronia
crude extract in combination, as contrasted to them as single
compounds may to some extent have more health promotive
effects in reducing the risk of diseases such as diabetes,
cardiovascular, or inflammatory diseases5 or cancer.
ACKNOWLEDGMENTS
The authors thank Dr. Hanne Frøkiær (BioCentrum,
Technical University of Denmark, Kongens Lyngby, Den-
mark), Dr. Torben Lund (Department of Chemistry, Ros-
kilde University), Dr. Jens E.T. Andersen (Department of
Chemistry, Technical University of Denmark), and Dr.
Bangjun Wang (Department of Biology, University of Fri-
bourg, Fribourg, Switzerland) for providing us with critical
comments in the process of manuscript writing; Dr. Ole
341
Vang (Department of Science, Systems, and Models, Ros-
kilde University) for providing us project coordination and
testing materials; for excellent technical assistance, Ms.
Louise Henningsen and Ms. Marianne K. Petersen (both
from Technical University of Denmark) in the cellular ex-
periments, and Mr. Jacob Krake (Roskilde University) for
HPLC techniques; and Miss Katherine Chiang (Waterloo,
ON, Canada) and Ms. Lisa Hughes (Roskilde, Denmark, and
Greensboro, NC, USA) for helping with language corrections.
AUTHOR DISCLOSURE STATEMENT
No competing financial interests exist.
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