JOURNAL OF MEDICINAL FOOD

J Med Food 20 (7) 2017, 667675

# Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2016.3822

Aronia Berry Extract Ameliorates the Severity of Dextran Sodium

Sulfate-Induced Ulcerative Colitis in Mice
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Sa-Haeng Kang,1,2,* Yong-Deok Jeon,2,* Kwang-Hyun Moon,3 Jeong-Ho Lee,3 Dae-Geun Kim,3
Wook Kim,3 Hyun Myung,4 Jong-Sung Kim,5 Hyun-Ju Kim,6 Keuk-Soo Bang,2 and Jong-Sik Jin2
1
Department of Oriental Pharmacy, College of Pharmacy, Wongkwang-Oriental
Medicine Research Institute, Wongkwang University, Iksan, South Korea.
2
Department of Oriental Medicine Resources, Chonbuk National University, Iksan, South Korea.
3
Sunchang Research Institute of Health and Longevity, Sunchang, South Korea.
4
Department of Ecology Landscape Architecture-Design, College of Environmental
and Bioresource Sciences, Chonbuk National University, Iksan, South Korea.
5
Department of Hotel and Restaurant Culinary Art, Kunjang University, Gunsan, Korea.
6
Industrial Technology Research Group, Research and Development Division,
World Institute of Kimchi, Gwangju, South Korea.

ABSTRACT Inflammatory bowel disease, including Crohns disease and ulcerative colitis (UC), is a group of inflammatory
conditions of the colon and small intestine. UC is a chronic inflammatory disorder of the colon and rectum that includes
intervals of acute exacerbation. Although recent studies have suggested that proinflammatory cytokines might have initiated
the inflammatory responses in UC, its etiology remains unclear. Aronia berries are rich in dietary polyphenols such as phenolic
acids, anthocyanins, flavonoids, and proanthocyanidins with various health benefits, including antioxidant, anti-inflammatory,
and antiaging activities. The objective of this study was to determine whether Aronia berry can be an effective intervention for
the treatment of UC. BALB/c mice were administered 5% dextran sulfate sodium (DSS) to induce UC. They were then given
Aronia berry extracts at concentrations of 10 or 100 mg/kg. During the induction of UC, the expression levels of nuclear
factor-kappa B were increased in colonic epithelial cells and immune cells, leading to increased proinflammatory cytokine
levels. Aronia berry extract significantly improved the clinical signs of DSS-induced UC, including body weight loss, colon
length shortening, and disease activity index increase, with histological markers of colon injury. Furthermore, oral admin-
istration of Aronia berry extract inhibited prostaglandin E2 production in DSS-induced colitis and decreased the levels of nitric
oxide, interleukin-6, and tumor necrosis factor-a in lipopolysaccharide-stimulated macrophages. These results suggest that
Aronia berry extract could efficiently ameliorate clinical signs and inflammatory mediators of UC. Therefore, Aronia berry
might be a promising natural treatment for UC.

KEYWORDS:  Aronia berry  colitis  inflammation  macrophage

INTRODUCTION with associated histopathological findings, it is possible to

I nflammatory bowel diseases (IBD), including ulcer-
ative colitis (UC) and Crohns disease, are serious chronic
inflammatory disorders of the intestine. UC involves ulcers
or inflammation in the large intestine. The main symptoms of
UC are abdominal pain and diarrhea. In addition, inflam-
mation and ulcers can appear in the intestinal mucosa. Along
*The first two authors contributed equally to this article.
Manuscript received 21 August 2016. Revision accepted 1 May 2017.
Address correspondence to: Keuk-Soo Bang, PhD, Department
Resources, Chonbuk National University, Iksan, Jeollabuk-do
E-mail: ksbang@jbnu.ac.kr or Jong-Sik Jin, PhD, Department
Resources, Chonbuk National University, Iksan, Jeollabuk-do
E-mail: jongsik.jin@jbnu.ac.kr
confirm the phenomenon of inflammatory cell infiltration
and decrease in the length of the rectum.1,2 Recently, the
prevalence of UC has increased in different countries be-
cause people have adopted a western lifestyle.3 There is no
current treatment for UC. Medications that are commonly
administered include 5-aminosalicylicacid (5-ASA) agents,
steroids, and immunomodulatory drugs. However, long-
term administration of these medications has side effects
such as nausea, loss of appetite, and fever.4 Therefore, it is
necessary to find natural treatments with fewer side effects.
Aronia berries (chokeberries) in the Rosaceae family are
of Oriental Medicine perennial herbs native to North America.5 Recently, Aronia
54538, South Korea, extracts have gained a lot of attention in Korea and farmers
of Oriental Medicine
54538, South Korea,
have increased production of Aronia species. Chokeberries
have three species: red chokeberry (Aronia arbutifolia),

667
 668 KANG ET AL.
black chokeberry (A. melanocarpa), and purple chokeberry
(A. prunifolia). Black chokeberries have been used as food
and medicine.6 Aronia berries have several chemical com-
ponents, including phenolic acids, anthocyanins, flavonoids,
and proanthocyanidins. In particular, Aronia berries are
known to have a higher content of anthocyanin than other
fruits.7 Aronia berries also contain many tannins. Therefore,
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it is highly astringent when eaten raw. Aronia berries are
usually taken as a powder or extract. Anthocyanins in Ar-
onia berries have remarkable antioxidant and anticancer
effects.8 Some studies have reported that anthocyanins can
be used to treat cardiovascular diseases such as stroke and
heart disease.9
Berries have shown promise in the treatment of chronic
inflammatory diseases. For example, black chokeberry can
decrease the expression levels of nuclear factor-kappa B
(NF-jB) p65 in dextran sulfate sodium (DSS)-administered
mice as an anti-inflammatory agent.10 Aronia berry extract
can also inhibit the secretion of interleukin-6 (IL-6) in li-
popolysaccharide (LPS)-induced murine splenocyte, but
induce the secretion of IL-10.11
Although the etiology of UC remains unknown, recent
studies have suggested that an inflammatory response initiated
by the interaction between the immune system (including
macrophages and dendritic cells) and antigens is involved.12
The inflammatory response in UC is induced by the activation
of macrophages by bacterial products such as LPS. Activated
macrophages will release proinflammatory cytokines such as
IL-1b, tumor necrosis factor-a (TNF-a), and IL-6. Patients
with UC have been reported to express high levels of inflam-
matory cytokines such as IL-6 and TNF-a.13
Although Aronia berries are known to have various health
benefits, whether they have beneficial effect on UC remains
unclear. Therefore, the objective of this study was to de-
termine whether Aronia berry could inhibit clinical signs
and inflammatory mediators in DSS-induced UC.
MATERIALS AND METHODS
Reagents
DSS (mol wt: 36,00050,000) was purchased from MP
Biochemicals (Solon, OH, USA). Anti-phospho-JNK, anti-p38,
and anti-NF-jB antibodies were purchased from Cell Signaling
Technology (Woodland, CA, USA). Anti-mouse IL-6 and
TNF-a antibodies were purchased from BD Pharmingen (San
Diego, CA, USA). Anti-JNK and anti-NF-jB antibodies were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA,
USA). Anti-GAPDH antibody was purchased from Thermo
Scientific (Pittsburgh, PA, USA). Hematoxylin was purchased
from Muto Pure Chemicals Co. (Bunkyo-ku, Tokyo, Japan).
Eosin was purchased from Sigma-Aldrich (St. Louis, MO,
USA). Prostaglandin E2 (PGE2) assay kits were purchased from
Enzo Bioscience (Farmingdale, NY, USA).
Experimental animals
All experimental protocols (CBNU2016-0016) were ap-
proved by the Committee on the Care of Laboratory Animal
Resources, Chonbuk National University. All experiments
were conducted in accordance with the Guide for the Care
and Use of Laboratory Animals. Male BALB/c mice (8
weeks old, 2125 g) and C57BL/6 mice (5 weeks old, 19
20 g) were purchased from Samtako (Osan, Korea). These
mice were group-housed under a controlled temperature
(2527C) with a 12 h lightdark cycle. They were provided
free access to a standard diet and tap water (or specified
drinking solution). All mice were acclimated under these
conditions for at least 7 days before inclusion in experi-
ments. Mice were sacrificed on day 10, and their colons
were removed. After length measurement, the colons were
cut into small pieces (about 23 cm in length) and kept in
15 mL tubes. The tips of colons were fixed in formalin for
histological analysis.
Extraction of Aronia
Aronia berries were purchased from cooperative farmers
market in Sunchang village ( Jeonbuk, Korea). They were
washed twice with distilled water and dried. Aronia berry
extract was prepared by decocting with 70% ethanol for 1 h
30 min at 80C. The solution in ethanol was then filtered and
allowed to evaporate using a rotary evaporator at a tem-
perature of 4045C. The extract was diluted in 0.9% saline
and filtered through a 0.22 lm syringe filter (HYUNDAI
Micro, Seoul, Korea).
Induction of colitis with DSS
Mice were separated into five groups: CON (normal),
DSS (5% w/v DSS), AR10 (5% DSS with oral administra-
tion of Aronia 10 mg/kg), AR100 (5% DSS with oral ad-
ministration of Aronia 100 mg/kg), and ASA (5% DSS with
oral administration of 5-ASA). The DSS groups were given
5% DSS (MP Biochemicals) in drinking water for 7 days.
Body weight was monitored daily.
Disease activity index
Diarrhea with blood and mucus are used to clinically
identify UC.14 Disease activity index (DAI) scores were
used to calculate intestinal disease activity in mice as de-
scribed in Table 1.15
DAI fWeight loss score diarrhea score
rectal bleeding scoreg=4:
Table 1. Criteria for Disease Activity Index
Stool Bloodstain or gross
Score Weight loss (%) consistency bleeding
0 None Normal Negative
1 15 Loose stool Negative
2 510 Loose stool Positive
3 1015 Diarrhea Positive
4 >15 Diarrhea Gross bleeding
 ARONIA INHIBIT ULCERATIVE COLITIS
Enzyme-linked immunosorbent assay
At the end of experiment, serum samples were prepared
and subjected to enzyme-linked immunosorbent assay
(ELISA) to determine serum levels of IL-6 and TNF-a using
ELISA kits from BD Pharmingen. In brief, microwells were
coated with 100 lL per well of capture antibodies and in-
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cubated at 4C overnight. After aspiration of the media,
100 lL of each standard or sample was added into each well
and the plates were incubated at room temperature (RT) for
3 h. Plates were then washed and 100 lL of Working
Detector (Detection Ab + SAv-HRP) was added to each
well. Plates were incubated at RT for 1 h. Then, 100 lL of
substrate solution was added to each well, and plates were
incubated at RT for 30 min in the dark. Absorbance at
450 nm was measured using a VersaMax microplate reader
(Molecular Devices, Sunnyvale, CA, USA).
Western blot analysis
Colons were homogenized in lysis buffer (iNtRON Bio-
tech, Seongnam-Si, Republic of Korea) and centrifuged by
high speed centrifuge 1248R (Labogene, Daejeon, Republic
of Korea) at 16,582 g for 10 min at 4C. The supernatants
were collected and transferred to fresh tubes. Protein con-
centrations were quantified using the BCA protein assay
reagent (Sigma-Aldrich). Lysates (50 lg of protein) were
separated by 10% sodium dodecyl sulfate-polyacrylamide
gel electrophoresis and transferred to polyvinylidene di-
fluoride membranes (Roche Diagnostics, Indianapolis, IL,
USA). After blocking with 5% skim milk in phosphate-
buffered saline-Tween-20 (PBST) for 1 h at RT, membranes
were incubated with primary antibodies against mitogen-
activated protein kinases (MAPK), NF-jB, and phospho-
IjB at 4C overnight. After washing three times with PBST,
membranes were then incubated with secondary antibodies
at RT for 1 h. After washing again with PBST three times,
the protein-antibody complexes were then developed with
ECL western blotting luminol reagent (Santa Cruz Bio-
technology) and recorded with an LAS-4000 Image Reader
(Fujifilm Life Sciences, Tokyo, Japan).
Histological analysis
Cross sections of the distal colons (2 cm) were fixed in
10% paraformaldehyde solution. They were then cut into
small fragments, dehydrated in an ethanol series (70100%),
washed with xylene, and embedded in paraffin. The frag-
ments were sliced into 5 lM-thick sections and stained with
hematoxylin-eosin (H&E) stain. These sections were visu-
alized with an optical microscope equipped with a camera to
capture images at 100 magnification.
PGE2 assay
Whole mouse colons were washed with cold PBS and
immediately stored at -80C until use. Colon tissues were
homogenized in 1 mL ice-cold lysis buffer (10 mM Tris-HCI
[pH 7.4], 1 mM ethylenediaminetetraacetic acid, 10 lM in-
domethacin, and 1% protease and phosphatase inhibitor
669
cocktail [Sigma-Aldrich]) using a homogenizer. Tissue ho-
mogenates were transferred to microcentrifuge tubes, vor-
texed, and centrifuged at 4C for 10 min at 16,582 g. The
supernatant (5 lL) from each sample was used to determine
protein concentration using a DC Protein Assay Kit (Bio-
Rad, Hercules, CA, USA) according to the manufacturers
protocol. Determination of PGE2 levels was carried out
using a PGE2 Monoclonal Enzyme Immunoassay Kit (Enzo
Bioscience). All samples were diluted by Assay Buffer in
1:100. Experiments were run in triplicates and a standard
curve was created at the same time. Absorbance at 405 nm
was measured using the VersaMax microplate reader.
Isolation and culture of peritoneal macrophages
Mice were given an intraperitoneal injection of 4%
thioglycolate solution (3 mL) and sacrificed on the third day
after injection. Peritoneal cavities were rinsed with Dul-
beccos Modified Eagle Medium (DMEM) (5 mL). The
peritoneal lavage fluid was centrifuged at 4000 rpm for
5 min. Cells were then resuspended in DMEM and seeded
into 24-well microplates. These cells were cultured in 24-
well plates at 37C for 24 or 48 h in DMEM plus 10% fetal
bovine serum.
Cell viability assay
To determine cell viability, thiazolyl blue tetrazolium
bromide (MTT) was used. Mice peritoneal macrophages
were pretreated with Aronia berry extract (0.001, 0.01, 0.1,
or 1 mg/mL) for 24 h. MTT solution at final concentrations
of 0.5 mg/mL was then added to each well and incubated for
4 h. After the incubation, the supernatant was removed, and
the formazan crystals were dissolved in 500 lL of dimethyl
sulfoxide. The absorbance of each well at wavelength of
540 nm was then measured using the VersaMax microplate
reader.
Measurement of nitric oxide level
Mice peritoneal macrophages were pretreated with Ar-
onia berry extract (0.001, 0.01, 0.1, or 1 mg/mL) for 1 h.
After that, they were treated with 1 lg/mL of LPS and in-
cubated for 48 h. After the incubation, the supernatants were
collected for nitric oxide (NO) determination. To measure
nitrite, an equal volume of Griess reagent (1% sulfanil-
amide/0.1% naphtylethyenediamine dihydrochloride in
2.5% H3PO4) was mixed with cell culture supernatant at RT
for 10 min. Nitrite concentration was determined by mea-
suring the absorbance at wavelength of 540 nm using the
VersaMax microplate reader. NaNO2 was used to obtain the
standard curve.
Statistical analysis
Results are shown as a summary of data from at least
three experiments. Data are presented as means standard
errors of the mean. Statistical evaluation of results was
performed by independent t-test. For all experiments, sta-
tistical significance was defined when P-value was <.05.
 670 KANG ET AL.
RESULTS
Effect of Aronia berry extract on clinical signs
of UC induced by DSS
To measure the effect of Aronia berry extract on UC,
Aronia berry extract was given to an animal model of UC
induced by DSS, and changes in body weight were mea-
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sured. Mice were administered DSS followed by Aronia
berry extract at a concentration of 10 or 100 mg/kg for
10 days. 5-ASA was used as a positive control. As shown in
Figure 1, the greatest weight loss was observed in the group
administered with DSS alone. In the AR10 and AR100
groups, body weight loss was significantly (P < .05) sup-
pressed compared with that in the group administered with
DSS alone. Mice given Aronia berry extract experienced
less weight loss than those given 5-ASA, the positive control
(Fig. 1A). DAI scores were also significantly lower in the
AR100 group compared with those of the DSS group. The
effect of Aronia berry extract on DAI in DSS-induced UC
model was better than 5-ASA administered group. (Fig. 1B).
Effect of Aronia berry extract on colon length
shortening in UC induced by DSS
In DSS-induced animal models, various physical symp-
toms will appear, including changes in the length of the large
intestine. The length of the large intestine can be used to
indirectly assess the degree of inflammation.14 In this study,
the average colon length of the control group colon was
10.2 0.52 cm. The length of the colon in the DSS-induced
group was significantly (P < .05) decreased to 6.21 0.53 cm
compared with that in the control. Colons from the AR10 and
AR100 groups were significantly (P < .05) increased to 7.38
0.45 cm and 8.35 0.46 cm, respectively (Fig. 2A, B), com-
pared with that in the DSS alone group.
Effect of Aronia berry extract on serum
levels of inflammatory cytokines
Inflammatory cytokines appear early in the inflammatory
response of UC.16 To determine the effect of Aronia berry
extract on serum levels of TNF-a and IL-6, ELISA was
conducted. Serum IL-6 levels were found to be significantly
(P < .05) lower in the AR100 group (49.41 7.94 pg/mL)
than that in the DSS group (85.40 10.71 pg/mL) (Fig. 3A).
Serum TNF-a levels were also significantly (P < .05) de-
creased in the AR100 group (601.52 54.98 pg/mL) com-
pared with those in the DSS group (828.62 110.38 pg/mL)
(Fig. 3B).
Effect of Aronia berry extract on PGE2
production in colon tissue
The levels of PGE2 have been reported to be elevated in
the intestines of UC patients.17 We used ELISA to determine
the effect of Aronia berry extract on inflammatory mediator
FIG. 1. Effects of Aronia berry ex-
tract on body weight and DAI in DSS-
induced colitis mice. Experimental
colitis in mice was induced by treating
animals with 5% DSS for 10 days. 5-
ASA (50 mg/kg/day) was administered
orally as a reference drug. Aronia berry
extract was administered orally at do-
ses of 10, 100 mg/kg once a day for
10 days before 5% DSS supplement.
(A) Body weight was measured through
the experimental duration. (B) Disease
activity index scores in the five study
groups. Disease activity index = {(weight
loss score) + (diarrhea score) + (rectal
bleeding score)}/4 was calculated.
(#P < .05 vs. control group, *P < .05 vs.
DSS group). 5-ASA, 5-aminosalicylicacid;
DSS, dextran sulfate sodium; DAI,
disease activity index.
 ARONIA INHIBIT ULCERATIVE COLITIS
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FIG. 2. Effect of Aronia berry extract on colon length in DSS-
induced colitis mice. Experimental colitis in mice was induced by
treating animals with 5% DSS for 10 days. Aronia berry extract was
administered orally at 10 and 100 mg/kg per day during the experi-
ment. (A) Colons harvested on day 10. (B) Colon lengths in the five
study groups. Values are mean SEM. (#P < .05 vs. control group,
*P < .05 vs. DSS group). SEM, standard error of the mean.
PGE2 in DSS-induced UC mouse colon tissue. PGE2 levels
in the DSS group (20615.5 2403.7 pg/mL) were found to
be significantly (P < .05) higher compared with those of the
CON group (10806.1 1131.6 pg/mL). However, PGE2
levels in the AR100 group (11082.4 2686.7 pg/mL) were
significantly (P < .05) lower than those in the DSS group (Fig. 4).
Effect of Aronia berry extract on epithelial
injury in DSS-induced colitis
For histological examination of the colon, tissues were
stained with H&E and observed at 40 and 100 magnifica-
tion using an optical microscope. The epithelium is composed
of an epithelial cell layer and lamina propria. DSS treatment
caused epithelial injury. Results of H&E staining of colon
tissue revealed that the epithelial cells of the CON group were
normal. No inflammation was observed. However, the epi-
thelial cell and crypt structures were destroyed in the DSS
group. On the contrary, 100 mg/kg Aronia berry extract or 5-
ASA treatment attenuated these injuries (Fig. 5A, B).
Effects of Aronia berry extract on inflammatory cytokine
production in LPS-challenged peritoneal macrophages
MTT assay was used to examine the effect of Aronia
berry extract on inflammatory cytokine production in LPS-
671
FIG. 3. The effect of Aronia berry extract on production of proin-
flammatory cytokines in serum of mice with DSS-induced colitis.
Ulcerative colitis was promoted in male BALB/c mice by adminis-
tering 5% DSS for 10 days. Cytokine production was determined by
ELISA. (A) IL-6; (B) TNF-a production in colon tissue at day 10.
Values are mean SEM. Data were analyzed by Students t-test.
(#P < .05 vs. control group, *P < .05 vs. DSS group). ELISA, enzyme-
linked immunosorbent assay; IL-6, interleukin-6; TNF-a, tumor ne-
crosis factor-a.
challenged peritoneal macrophages. Cells were treated with
Aronia berry extract (0.001, 0.01, 0.1, or 1 mg/mL) for 24 h,
and cell viability was measured. Although cell viability was
decreased slightly, to 96% compared with that of the un-
treated group (100 0.49%), there was no significant dif-
ference in cell viability (Fig. 6A). Macrophages challenged
with LPS after pretreatment with Aronia berry extract did
not show significant decreases in cell viability either
(Fig. 6B). Inflammatory cytokine is an important indicator
of inflammation. Therefore, we investigated the effect of
Aronia berry extract on the production of inflammatory
cytokines in LPS-treated macrophages. IL-6 level was sig-
nificantly reduced by Aronia berry extract treatment at a
concentration of 1 mg/mL (1.43 0.39 ng/mL) (Fig. 6C). In
addition, TNF-a level in the group treated with Aronia
berry extract 1 mg/mL (1.53 0.14 ng/mL) was significantly
(P < .05) lower compared with those in groups treated with
other concentrations (0.001, 0.01, or 0.1 mg/mL) of Aronia
berry extract (Fig. 6D). These results showed that Aronia
 672 KANG ET AL.

extract significantly suppressed the generation of LPS-induced

inflammatory cytokines in a concentration-dependent manner.

Effect of Aronia berry extract on NO production

in LPS-stimulated peritoneal macrophages
NO has been considered as an important proinflammatory
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mediator that plays a key role in the pathogenesis of IBD.18

After macrophages were pretreated with Aronia berry ex-
tract (0.01, 0.1, or 1 mg/mL) for 4 h, the production of LPS-
induced NO was measured. LPS at a concentration of 1 lg/
mL was used to stimulate microphage for 48 h. Using Griess
reagent, the NO production was dose-dependently decreased
by Aronia berry extract treatment (Fig. 7).

FIG. 4. Effect of Aronia berry extract on PGE2 production in colon
tissues. PGE2 levels were measured using PGE2 assay kits. PGE2
production in colon tissues. Data were representatives of three in-
dependent experiments. Values are mean SEM. Data were analyzed
by Students t-test. (#P < .05 vs. control group, *P < .05 vs. DSS group).
PGE2, prostaglandin E2.
Effect of Aronia berry extract on phosphorylation
of MAPK in peritoneal macrophages
We hypothesized that MAPKs could mediate the anti-
inflammatory effects in the colon. To determinate the po-
tential implication of MAPKs in the anti-inflammatory effects

FIG. 5. Effect of Aronia berry extract on epithelial injury in colon tissues of DSS-induced colitis mice. Over the same period, Aronia (10,
100 mg/kg) and the positive control 5-ASA (50 mg/kg) were orally administered once daily. (A) Paraffin sections of colon tissue were stained with
hematoxylin and eosin. (B) Histological score was measured from the colon tissues on day 10. Values are mean SEM. (#P < .05 vs. control group,
*P < .05 vs. DSS group).
 ARONIA INHIBIT ULCERATIVE COLITIS 673
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FIG. 6. Effects of Aronia berry extract on inflammatory cytokines production on LPS-stimulated peritoneal macrophages. Macrophage were
treated with various concentrations (0, 100 lg/mL, 1, 10, 100 mg/mL) of Aronia berry extracts for 4 h before being incubated with LPS (1 lg/mL)
for 24 h. Cell viability was measured by MTT assay. (A) Only Aronia berry extract. (B) LPS-induced macrophage. Cytokines production was
determined by ELISA. (C) IL-6 production; (D) TNF-a production. Values are mean SEM. (#P < .05 vs. Blank group, *P < .05 vs. LPS-induced
group). LPS, lipopolysaccharide.

observed in macrophages after Aronia berry extract treat-
ment, phosphorylation levels of ERK, JNK, and p38 were
examined by western blot analysis using ERK, JNK, and
p38 phosphor-specific MAPKs antibodies. Results are shown
in Figure 8A. Treatment with LPS significantly activated
ERK, JNK, and p38 proteins, indicating that MAPK proteins
FIG. 7. Inhibition of LPS-induced NO production by Aronia berry
extract. Macrophage were preincubated with 0.01, 0.1, 1 mg/mL of
Aronia berry extracts for 4 h and treated with 1 lg/mL of LPS for
48 h. The NO production was measured by Griess reagent system.
Values are mean SEM. (#P < .05 vs. blank group, *P < .05 vs. LPS-
induced group). NO, nitric oxide.
are activated at acute stage of colonic lesion. However, after
treatment with AR at 1 mg/mL, phosphor-ERK and JNK
activations were significantly lower (Fig. 8B).
DISCUSSION
UC presents with symptoms such as weight loss, abdom-
inal pain, and bloody stools. It is a form of IBD that causes
inflammation and ulceration of the colon.19,20 Treatment of
UC entails the use of glucocorticoids, sulfasalazine, and im-
munosuppressive drugs.21 However, these drugs are associ-
ated with severe side effects. There is a need for treatment
options that do not have serious side effects. Recently, there is
an increasing interest in the use of traditional herbal medi-
cines to inhibit inflammation. Herbal medicines used to treat
UC include Scutellaria baicalensis and Ixeris dentata.
However, these herbs have failed to show clear benefits in
clinical trials.22,23 We hypothesized that anthocyanin-rich
Aronia could decrease the symptoms associated with UC. We
therefore administered Aronia berry extract orally to BALB/
C mice with DSS-induced colitis and observed histological
and immunohistological changes as well as macroscopic
morphological changes that might indicate benefits. A pre-
vious study on the effect of an Aronia species (A. melano-
carpa) on inflammation has reported that Aronia extract can
inhibit the inflammatory response in macrophages by directly
 674
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FIG. 8. Effect of Aronia berry extract on activation of MAPK in macrophage. (A) Protein expression of MAPKs. (B) Densitometry was
normalized to the LPS group. Values are mean SEM. (n = 6). Data were analyzed by Students t-test. (#P < .05 vs. blank group, *P < .05 vs. LPS-
induced group). MAPK, mitogen-activated protein kinase.
blocking the expression of iNOS and COX-2, thereby sup-
pressing uveitis induced in the RAW264.7 macrophage cell
line.8 Aronia extract can also ameliorate inflammation caused
by histamine and serotonin treatment.24 It has also been
shown that orally administered Aronia extract protects the
liver following acute liver damage caused by carbon tetra-
chloride in mice.25 In this study, we established an animal
model of UC to evaluate the effect of Aronia berry extract.
Inflammatory cytokines such as TNF-a and IL-6 are
known factors that can mediate the inflammatory response
in models of UC.26 Excessive secretion of these inflamma-
tory cytokines causes sepsis and a variety of IBDs.12,27 In
particular, TNF-a levels have been reported to be signifi-
cantly increased in patients with UC.28 Thus, modulators of
these inflammatory cytokines may provide important clues
to the treatment of UC. TNF-a plays an essential part in
suppressing tumors and viruses. However, when TNF-a is
overexpressed, a continuous inflammatory reaction oc-
curs.16 Therefore, suppressing the level of TNF-a might
ameliorate chronic inflammatory diseases. In this study, we
determined the effect of Aronia berry extract on IL-6 and
TNF-a expression in the serum and colon tissue in a DSS-
induced UC mouse model. Aronia berry extract inhibited
IL-6 and TNF-a production more than 5-ASA administra-
tion, a drug currently used to modulate secretion of IL-6 and
TNF-a in patients with UC. Based on these results, we
conclude that Aronia berry extract can ameliorate DSS-
induced UC by modulating the production of inflammatory
cytokines.
Various inflammatory mediators are increased in colon
tissue from patients with UC. PGE2 produced by COX-2 is
an important inflammatory factor that causes inflammatory
reactions and vasodilation.29 When overexpressed, PGE2
may cause tumors.30 Inhibition of PGE2 can reduce the
symptoms of colitis in vivo and in vitro in a DSS-induced
mouse model.3133 We showed that PGE2 was reduced in
DSS-induced UC following Aronia extract administration.
We also confirmed that Aronia berry extract exerted a
KANG ET AL.
modulating effect on the MAPK pathway and the secretion
of inflammatory cytokines in murine macrophages. Our re-
sults indicated that Aronia berry extract might be useful
herbal medicine in IBD.
In summary, we found that Aronia berry extract suppressed
the symptoms of UC in a DSS-induced mouse model. We
also examined inflammation-related activities of Aronia
berry extract using mouse peritoneal macrophages. Aronia
berry extract was found to be more effective than the anti-
inflammatory drug 5-ASA, which is currently used to treat
IBD. In addition, Aronia extract reduced the expression of
COX-2 in peritoneal macrophages. Given these results,
Aronia extract might be a safe and effective treatment to
suppress inflammation by adjusting the levels of inflam-
matory mediators. Therefore, Aronia may have potential as
a natural treatment for colitis that can replace current drug
therapies. Further studies are needed to identify the active
ingredients in Aronia berry extract using component anal-
ysis and to confirm the results in human clinical trials.
ACKNOWLEDGMENTS
This research was supported by Basic Science Research
Program through the National Research Foundation of
Korea (NRF) funded by the Ministry of Science, ICT and
Future Planning (2015R1C1A1A01054675) and the In-
dustrial Technology Research Infrastructure Program
(N0000004) funded by the Ministry of Trade, Industry and
Energy (Sejong, Korea) and Sunchang Research Institute of
Health and Longevity.
AUTHOR DISCLOSURE STATEMENT
No competing financial interests exist.
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