Food Research International 106 (2018) 335–343

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Food Research International

journal homepage: www.elsevier.com/locate/foodres

Portulaca oleracea extracts and their active compounds ameliorate T

inflammatory bowel diseases in vitro and in vivo by modulating TNF-α, IL-6
and IL-1β signalling
Yesol Kima,1, Hyung Jin Lima,b,1, Hyun-Jae Janga, Soyoung Leea, Kyungsook Junga,
⁎ ⁎
Seung Woong Leea, Seung-Jae Leea, , Mun-Chual Rhoa,
a
Immunoregulatory Materials Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Jeongeup, Jeonbuk 56212, Republic of Korea
b
Department of Bioactive Material Sciences, Chonbuk National University, Jeonju, Jeonbuk 54896, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Keywords: Portulaca oleracea L. (P. oleracea) is an herb that is widely used in traditional medicine to treat various diseases.
Portulaca oleracea However, its effects on inflammatory diseases, such as inflammatory bowel disease (IBD), are not yet well
Inflammatory bowel diseases characterized. Here, we investigated the impact of the ethyl acetate (EtOAc) and ethanol (EtOH) extracts of P.
cis-N-Feruloyl-3′-methoxytyramine oleracea on lipopolysaccharide (LPS)-induced inflammatory responses and phosphorylation of ERK, JNK, and
Dextran sulphate sodium
p38 expression in RAW264.7 macrophages. In addition, the inhibitory effects of these extracts and fractions on
Mitogen-activated protein kinases
3% dextran sulphate sodium (DSS)-induced ulcerative colitis were examined using an ICR mouse model. DSS-
induced colitis, including body weight loss, reduced colon length, and histological colon injury, was significantly
ameliorated in mice fed the P. oleracea extracts (200 and 500 mg/kg). In particular, P. oleracea extracts also
inhibited pro-inflammatory cytokine (TNF-α, IL-6, and 1L-1β) production in mice with DSS-induced colitis; the
P. oleracea extracts displayed higher and/or similar inhibitory activity to sulfasalazine at high concentrations.
Furthermore, the chemical structures of active compounds separated from the EtOAc extract of P. oleracea were
elucidated using nuclear magnetic resonance (NMR) spectroscopy (see Figure in supplementary materials), re-
sulting in the identification of three known compounds. Among these active compounds, cis-N-feruloyl-3′-
methoxytyramine (2) exhibited the strongest effects on preventing DSS-induced IBD in animal models. Thus,
extract of P. oleracea and their active compounds represents a new therapeutic approach for patients with in-
flammatory bowel diseases.

1. Introduction tract that are pathologically characterized by intestinal inflammation

Cytokine secretion may either induce or relieve inflammation
(Cavaillon, 2001). Levels of inflammatory cytokines such as interleukin-
6 (IL-6) and -1β (IL-1β), and tumour necrosis factor-α (TNF-α) have
been reported to be elevated in patients with inflammatory bowel dis-
ease (Grimm, Elsbury, Pavli, & Doe, 1996; Sanchez-Muñoz, Dominguez-
Lopez, & Yamamoto-Furusho, 2008). In addition, inflammatory bowel
disease not only increases the levels of nitric oxide (NO) produced by
inducible nitric oxide synthase (iNOS) but also activates the production
of cyclooxygenase-2 (COX-2), leading to the expression of pros-
taglandin E2 (PGE2) and inflammatory factors (Cross & Wilson, 2003;
Wang & Dubois, 2010).
Inflammatory bowel diseases (IBD), such as Crohn's disease and
ulcerative colitis, are chronic relapsing disorders of the gastrointestinal
and epithelial injury (Baumgart & Sandborn, 2012; Danese & Fiocchi,
2011). The exact pathogenesis of IBD is poorly understood. However,
infection, environmental factors, heredity and immunological ab-
normalities have often been proposed as possible causes (Abraham,
Cho, & H., 2009). Most of the current therapies for IBD involve treat-
ment with glucocorticosteroids, 5-aminosalicylic acid and im-
munosuppressive drugs. Although all of these treatments have shown
efficacy in patients with these intestinal conditions, the frequency and
severity of adverse effects, inconvenient dosing regimen and somewhat
prohibitive price limit their long-term use (Siegel, 2011). For this
reason, the development of new therapies that combine efficacy, con-
venient dosing and fewer side effects is an important goal in human IBD
therapy.
P. oleracea has been used as a folk medicine in many countries

⁎
Corresponding authors.
E-mail addresses: seung99@kribb.re.kr (S.-J. Lee), rho-m@kribb.re.kr (M.-C. Rho).
1
These authors contributed equally to this study.

https://doi.org/10.1016/j.foodres.2017.12.058
Received 14 September 2017; Received in revised form 28 November 2017; Accepted 20 December 2017
Available online 21 December 2017
0963-9969/ © 2017 Elsevier Ltd. All rights reserved.
Y. Kim et al.
because it acts as a febrifuge, antiseptic, and vermifuge, among others
(Lee, Kim, Lee, Kang, & Lee, 2012). It exhibits a wide range of phar-
macological effects, including antibacterial (Liu et al., 2015), anti-
ulcerogenic (Karimi, Hosseinzadeh, & Ettehad, 2004), anti-in-
flammatory (Chan et al., 2000), antioxidant (Chen et al., 2012), wound-
healing (Rashed, Afifi, & Disi, 2003), ameliorate acute intestinal in-
flammation (Obeng, Schwartz & Plahar, 2015) and protective effect of
ulcerative colitis (Yang et al., 2016). P. oleracea has high potential for
use as human and animal food and as a pharmacological agent in
medicine. However, little information have described IBD associated
with inflammation.
In the present study, we have isolated compounds of extracts from P.
oleracea. We further identified its structure, and evaluated its anti-in-
flammation and analysed using a dextran sulphate sodium (DSS)-in-
duced intestinal colitis model in ICR mice.
2. Materials and methods
2.1. Reagents and chemicals
Cell culture reagents, including Dulbecco's modified Eagle's medium
(DMEM), fetal bovine serum (FBS) and penicillin/streptomycin, were
purchased from Gibco BRL (Grand Island, NY, USA).
Lipopolysaccharide (LPS), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyltetrazolium bromide) powder, genistein, and sulfasalazine were
purchased from Sigma (St. Louis, MO, USA). Primary antibodies against
iNOS, COX-2, phosphorylated p38 (p-p38), p-Jun N-terminal kinase
(JNK), p-extracellular signal-regulated kinase (ERK), β-actin and sec-
ondary antibodies were obtained from Cell Signaling Technology
(Boston, MA, USA). DSS was purchased from MP Biomedicals (MW:
36,000–50,000, MP Biomedicals, Solon, OH). The enzyme im-
munoassay kit was obtained from R&D Systems (Minneapolis, MN,
USA). All other reagents were of the highest commercially available
grade.
2.2. Preparation of the extract and purification of compounds from
Portulaca oleracea
The dry P. oleracea (12 kg) powder was extracted three times with
95% ethanol (EtOH; 3 × 36 L) at room temperature, and the EtOH
solution was evaporated under reduced pressure. The EtOH extract
(612 g) was suspended in H2O and partitioned with ethyl acetate
(EtOAc; 3 × 4 L). Chromatography was performed, and the EtOAc
fraction (381 g) was separated on a silica gel column (10 × 100 cm,
200–400 mesh) and eluted with a hexane/ethyl acetate gradient system
to generate twelve fractions (POE1–POE12). POE7 (12.6 g) was dis-
solved in methanol (MeOH) and partitioned with hexane solvent. The
MeOH-soluble POE7A fraction (1.7 g) was evaporated under reduced
pressure and then subjected to chromatography on a C18 medium-
pressure liquid chromatography (MPLC) column with a gradient solvent
composed of H2O and MeOH (19:1–0:1, v/v) to generate nine fractions
(POE7A1–POE7A9). Compound (1), Portulacanone C (16.5 mg) was
purified from POE7A5 (76.2 mg) by semi-preparative HPLC
(Phenomenex Luna 5 μm, C18, 150 × 21.2 mm, acetonitrile
(MeCN):H2O = 40:60, flow rate 6 mL/min).
POE9 (12 g) was suspended in MeOH and partitioned with hexane.
The MeOH-soluble POE9A fraction (8.6 g) was chromatographed on a
C18 MPLC column to generate fractions (POE9A1–POE9A6) with a
gradient solvent system containing H2O and MeOH (9:1–0:1). The
POE9A2 fraction (2.7 g) was again separated on a C18 MPLC column to
generate fractions (POE9A2A–POE9A2G) using a gradient solvent
system containing H2O and MeOH (9:1–1:1). Compounds (2), trans-n-
feruloyltyramine (17.8 mg) and cis-n-feruloyl-3′-methoxytyramine (3)
(48.1 mg) were purified from the POE9A2D fraction (320 mg) by semi-
preparative HPLC (Phenomenex Luna 5 μm, C18, 150 × 21.2 mm,
MeCN:H2O = 19:81, flow rate 6 mL/min). The purity of each
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compound was verified as ≥ 95% by HPLC (see Fig. S10 in
Supplementary materials).
2.3. Cell culture
RAW264.7 cells, a mouse macrophage cell line obtained from
American Type Culture Collection (ATCC TIB71, Rockville, MD, USA),
were cultured in DMEM supplemented with 10% heat-inactivated FBS
and antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin) in a
humidified, 5% CO2 atmosphere at 37 °C.
2.4. Cell viability assay
Cells were seeded in 96-well culture plates and cultured for 24 h.
After 24 h, the culture medium was replaced with new complete
medium, and the cells were treated with the indicated concentrations of
extracts or fractions for 48 h. Cell viability was determined using the
MTT assay according to the manufacturer's protocol (Sigma Chemical
Co., St. Louis, MO).
2.5. NO assay
RAW264.7 cells were seeded in a 24-well plate and incubated with
various concentrations of extracts or fractions from P. oleracea in the
presence or absence of LPS (1.0 μg/mL) for 18 h. The culture super-
natant (100 μL) was mixed with Griess reagent (100 μL, 1% sulphani-
lamide and 0.1% N-1-naphthyl ethylenediamine) for 10 min, and the
absorbance was measured at 550 nm. A standard curve was constructed
using known concentrations of sodium nitrite.
2.6. Western blot analysis
The total protein was extracted with cell lysis buffer (Cell Signaling
Technology) supplemented with a protease inhibitor cocktail (Thermo
Scientific, Cheshire, UK). Protein concentrations in the samples were
measured using a bicinchoninic acid (BCA) protein assay kit (Sigma-
Aldrich Co.). Equal protein concentrations were loaded on 4–12% gels,
separated by sodium dodecyl sulphate-polyacrylamide gel electro-
phoresis (SDS-PAGE) and transferred to polyvinylidene difluoride
(PVDF) membranes (Amersham Bioscience, Freiburg, Germany). After
blocking nonspecific sites with 5% non-fat milk for 1 h, the membrane
was incubated with a specific primary antibody overnight at 4 °C and
then incubated with a secondary antibody for 1 h at room temperature.
Signals from the immunoblots were visualized with an enhanced che-
miluminescence (ECL) kit (Intron Biotechnology, Seongnam, Korea),
and the membranes were exposed to X-ray film.
2.7. Experimental animals
Male ICR mice weighing 25–30 g were purchased from Orient Bio
(Jeonju, Korea). Animals were provided food and water ad libitum. The
animals were maintained on a 12 h dark-light cycle and allowed free
access to purified feed diet and tap water at controlled temperatures
(25 ± 2 °C). Animals were allowed to habituate for 7 days prior to the
initiation of the experimental protocol. All animal experiments were
conducted under KRIBB guidelines and the animal protocol was ap-
proved by the Ethical Committee for Animal Care and Use at the KRIBB
(KRIBB-AEC-17059).
2.8. Induction of colitis
Experimental colitis was induced by providing mice drinking water
containing 3% (w/v) DSS for 14 days ad libitum. Mice in each of the
groups were carefully monitored each day to confirm that they con-
sumed an approximately equal volume of water containing DSS. For
each experiment, the mice were divided into experimental groups. The
Y. Kim et al.
first group was used as a vehicle-treated control and administered PBS
via the same route as P. oleracea, and the second group was only pro-
vided drinking water containing DSS throughout the experimental
period. The other 7 groups comprised mice that received 3% DSS and
were administered sulfasalazine (50 mg/kg/day p.o.) or P. oleracea
extracts (100, 300, and 500 mg/kg/day p.o.) daily for 14 days ac-
cording to the experimental design. All materials were dissolved in
vehicle (PBS). Control groups were administered vehicle daily for
14 days, as appropriate. The administration of each drug was initiated
simultaneously with the DSS treatment. The experiments were con-
ducted two times with five mice in each group per experiment.
2.9. Histopathology
The resected large intestine was grossly examined for mucosal de-
fects, haemorrhage, or ulcerative lesions and then immediately fixed
with 4% neutral formalin. For the histopathological analysis, tissue
sections were generated from a representative region of the large in-
testine using conventional tissue preparation methods, stained with
haematoxylin and eosin (H&E) and viewed under a light microscope
(10–1000×).
2.10. Measurement of cytokine levels
Cardiac puncture was performed and blood was collected in en-
dotoxin-free silicone coated tubes without additives. Blood samples
were allowed to clot at room temperature for 30 min before cen-
trifugation (2200 × g, 4 °C, 10 min), and the serum was collected and
stored at − 80 °C until analysis. The Quantikine quantitative sandwich
enzyme immunoassay technique was used to analyse cytokine levels
(ELISA; R&D Systems, Minneapolis, MN, USA).
2.11. Statistical analysis
Statistical analyses were performed 3 times for all experiments. All
quantitative results are presented as means ± standard deviations
(SD). Statistical analyses were performed using Prism 5 software
(GraphPad Software, San Diego, CA) and p values were analysed using
Student's t-test. The results for comparisons of mean values of triplicate
samples in all experiments were considered statistically significant at
*p < 0.05, **p < 0.01, and ***p < 0.001.
3. Results
3.1. P. oleracea extracts inhibit LPS-induced NO production and cell
viability
The Griess test was used to determine the inhibitory effects of the
extracts on NO production, which is known to be an important factor in
inflammation. RAW264.7 cells were treated with various concentra-
tions of the two different P. oleracea extracts (10, 30, 60, or 100 μg/mL
EtOAc or EtOH extracts) and/or LPS (1.0 μg/mL), and triplicate MTT
assays were conducted to determine the potential toxic effects of the
treatments on the cells (Fig. 1A and B). P. oleracea extracts and LPS did
not display significant cytotoxicity toward RAW264.7 cells that were
pre-treated with different concentrations of these compounds for 4 h
(Fig. 1C and D). Furthermore, P. oleracea extracts alone did not exert
toxic effects on other macrophage lines, such as J774.A1 cells (data not
shown), proving the non-toxic nature of the extracts. RAW264.7 mac-
rophage cells were pre-treated with different concentrations of P. oler-
acea extracts (10, 30, 60, or 100 μg/mL) for 30 min, followed by an LPS
(1.0 μg/mL) treatment for 24 h, and the levels of NO in the culture
media were determined using the Griess assay to evaluate the effects of
the P. oleracea extracts on LPS-induced NO production. As shown in
Fig. 1C and D, unstimulated macrophages produced a background level
of NO (2.97 ± 0.2 μM) in the culture medium. When RAW264.7 cells
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were stimulated with LPS (1.0 μg/mL) for 24 h, the levels of nitrite, a
stable oxidized product of NO, were markedly increased to
23.63 ± 0.6 μM in the culture medium, while the percentages of the
EtOAc and EtOH extracts-treated cells were 4.75 ± 0.2 and
3.19 ± 2.1 μM at 100 μg/mL, respectively. P. oleracea extracts po-
tently inhibited the increased NO production in LPS-stimulated mouse
macrophages. Treatment with the indicated doses of the P. oleracea
extracts (100 μg/mL of extracts) alone did not yield any changes in the
overall production of NO in RAW264.7 cells.
3.2. Effects of P. oleracea extracts on LPS-induced inflammatory cytokine
production in RAW264.7 cells
Pro-inflammatory cytokines play important roles in regulating the
inflammatory response. Our in vitro ELISAs revealed significant in-
creases in levels of the inflammatory cytokines TNF-α, IL-6 and IL-1β in
macrophages stimulated with LPS (1.0 μg/mL for 24 h) compared to
control macrophages. The levels of inflammatory mediators such as
TNF-α (235.91 ± 10.8 pg/mL), IL-6 (323.82 ± 16.6 pg/mL), and IL-
1β (29.24 ± 2.4 pg/mL) detected in the LPS group were significantly
decreased (*p < 0.05, **p < 0.01) in groups that were also treated
with the EtOAc P. oleracea extracts (30 and 60 μg/mL); the levels of
TNF-α, IL-6, and IL-1β were 37.17 ± 3.0 and 12.92 ± 0.3 pg/mL,
232.32 ± 6.1 and 130.47 ± 4.6 pg/mL, 23.13 ± 0.3 and
12.95 ± 0.5 pg/mL, respectively. In addition, the levels of these cy-
tokines in the EtOH extract-treated groups were 94.23 ± 0.1 and
17.60 ± 0.1 pg/mL, 110.99 ± 8.50 and 37.37 ± 0.1 pg/mL,
17.33 ± 1.4 and 12.29 ± 0.4 pg/mL, respectively. The P. oleracea
extracts decreased the levels of TNF-α, IL-6, and IL-1β in a dose-de-
pendent manner, as shown in Fig. 2.
3.3. Effects of P. oleracea extracts on MAPK activation in macrophages
MAPKs play important roles in cell growth and division, cytokine
production and stress control and act through signalling pathways such
as the ERK, JNK, and p38 pathways. The mechanism by which P.
oleracea extracts inhibit the inflammatory response and the potential
MAPK pathways involved were identified by western blotting.
According to the western blots, LPS-induced ERK phosphorylation was
reduced in RAW264.7 cells treated with 10, 30, and 60 μg/mL P. oler-
acea extracts (Fig. 3A). In addition, we measured the levels of p-JNK
(Fig. 3B) and p-p38 (Fig. 3C). P. oleracea extracts decreased the levels of
phosphorylated proteins in a dose-dependent manner.
3.4. P. oleracea ameliorated disease progression and pathological changes
associated with DSS-induced colitis in mice
In the present study, we used a mouse model of DSS-induced IBD to
evaluate the therapeutic effect of P. oleracea extracts. Mice were chal-
lenged with 3% DSS for 14 days, which directly induced colonic mu-
cosal injury and thus led to inflammatory conditions in the intestine.
Beginning on day 7, mice started to show increasingly severe symp-
toms, including dramatic body weight loss, evident rectal bleeding and
diarrhoea. Compared to the control group, daily administration of 200
and 500 mg/kg P. oleracea extracts markedly attenuated the body
weight loss of DSS-challenged mice (Fig. 4A). Sulfasalazine (SS) was
used as a positive control because it is efficacious in treating IBD. The P.
oleracea extracts exhibited similar therapeutic effects to SS. A reduced
colon length is an important feature of colitis, and DSS-treated mice
displayed a prominently reduced colon length compared to the control
group. Treatment with 500 mg/kg P. oleracea alleviated the colon
shortening (Fig. 4B and C). H&E staining revealed severe pathological
changes, including mucosal damage and necrosis, as well as infiltration
of inflammatory cells, such as neutrophils and monocytes, in colon
samples from DSS-treated mice. P. oleracea extracts exerted protective
effects on this histological damage (Fig. 4D).
Y. Kim et al. Food Research International 106 (2018) 335–343

Fig. 1. EtOAc and EtOH P. oleracea extracts inhibit nitrite production in LPS-stimulated RAW264.7 cells. (A and B) RAW264.7 cells were pre-treated with various concentrations of P.
oleracea extracts (0, 10, 30, 60 or 100 μg/mL) for 4 h. Cell viability was measured using the MTT assay. (C and D) RAW264.7 cells were pre-treated with different concentrations of P.
oleracea extracts for 4 h and then treated with LPS (1.0 μg/mL) for 18 h. The concentration of NO in the culture medium was determined using the Griess assay. The results are expressed
as the means ± SD from three independent experiments. *p < 0.05, **p < 0.01 compared to the group treated with LPS alone.

3.5. Inhibitory effects of P. oleracea extracts on inflammatory cytokines in
DSS-treated mouse serum
In vivo tests were performed to validate the in vitro results. The in-
hibitory effects of the extracts on inflammatory cytokine secretion were
measured using ELISA. As shown Fig. 5 the levels of TNF-α, IL-6, and IL-
1β in the DSS group value were 39.32 ± 0.64, 51.95 ± 2.12, and
61.49 ± 10.97 pg/mL, respectively. In the EtOAc and EtOH extracts-
treated groups (500 mg/kg), the levels of TNF-α value was
13.57 ± 0.40, and 7.20 ± 3.19 pg/mL, respectively. Also, IL-6 values
was 3.61 ± 1.01 and 8.47 ± 3.50 pg/mL, respectively. In addition,
IL-1β values were 8.93 ± 2.69 and 10.45 ± 1.14 pg/mL, respec-
tively. Specifically, as the EtOAc and EtOH extracts had higher and/or
similar inhibitory effects of TNF-α, IL-6, and IL-1β expression than
positive control SS (50 mg/kg).
3.6. Purified active compounds slow disease progression and ameliorate
pathological changes in a mouse model of DSS-induced IBD
Portulacanone C (1), trans-n-feruloyltyramine (2) and cis-n-feruloyl-
3′-methoxytyramine (3) were purified EtOAc fraction (Fig. 6A). The
chemical structures (Fig. 6B–D) of these compounds were verified by
1D (1H and 13C NMR) and 2D (heteronuclear multiple quantum co-
herence (HMQC) and heteronuclear multiple bond coherence (HMBC))
spectroscopic analyses (see Fig. S1–9 in Supplementary materials).
Portulacanone C (1), trans-n-feruloyltyramine (2) and cis-n-feruloyl-3′-
methoxytyramine (3) were orally administered at doses of 3 mg/kg
daily for 5 days after the mice were administered 3% DSS. The body
weight of the DSS group was significantly lower than that of the control
group, and all treated groups showed less weight loss (Fig. 7A). As
shown in Fig. 7B and C, the length of the colon was shorter in mice
administered DSS compared to treated (or control) mice. Among all
compounds, feruloyltyramine (2) exerted the strongest protective ef-
fects on the animal model of DSS-induced IBD. In particular, changes in
the length of the colon indirectly confirm the progression of in-
flammation caused by IBD. The trans-n-feruloyltyramine (2)-treated
group, unlike the group treated with DSS alone, did not display ex-
tensive reductions in colon length. Thus, trans-n-feruloyltyramine (2)
significantly prevented DSS-induced IBD. The colonic tissues from each
group were stained with H&E to verify the damaging effect of DSS-
induced ulcerative colitis on colon tissue. The epithelial cells of the
control group were normal and invasion of immune cells was not ob-
served. In the DSS group, the crypts of epithelial cells and in the sub-
mucosal layer disappeared, and immune cell invasion was confirmed.
On the other hand, trans-n-feruloyltyramine (2) and SS showed similar
protective effects on IBD compared to the non-treated group (Fig. 7D).
3.7. Inhibitory effects of bioactive compounds on inflammatory cytokine
production in DSS-treated mice
Next, we investigated the effect of portulacanone C (1), trans-n-
feruloyltyramine (2) and cis-n-feruloyl-3′-methoxytyramine (3) on in-
flammatory cytokine levels. The following levels of the inflammatory
mediators were observed in the DSS group: TNF-α (41.27 ± 2.4 pg/
mL), IL-6 (225.65 ± 5.1 pg/mL), and IL-1β (165.35 ± 3.7 pg/mL).
The levels of these cytokines were decreased in all groups compared to
the DSS group. Groups treated with portulacanone C (1), trans-n-fer-
uloyltyramine (2) and cis-n-feruloyl-3′-methoxytyramine (3) were
evaluated and showed significantly decreased levels of TNF-α
(21.33 ± 1.62, 18.56 ± 0.08 and 15.57 ± 0.06 pg/mL), IL-6
(80.42 ± 0.09, 65.19 ± 0.02 and 110.91 ± 0.16 pg/mL), and IL-1β
(79.38 ± 3.12, 31.26 ± 1.02 and 27.29 ± 2.54 pg/mL), respec-
tively. In addition, levels of other cytokines, such as interferon-γ (IFN-
γ), IL-4, and IL-17, were also significantly decreased. Portulacanone C

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Fig. 2. EtOAc and EtOH P. oleracea extracts in-

hibit TNF-α (A and B), IL-6 (C and D), and IL-1β
(E and F) production in RAW264.7 cells, as de-
termined using ELISAs. RAW264.7 cells were pre-
treated with 30 or 60 μg/mL of EtOAc or EtOH
extracts and then exposed to LPS (1.0 μg/mL) for
12 h. Data are presented as the mean
values ± SD from at least three independent
experiments. *p < 0.05, **p < 0.01 compared
to the group treated with LPS alone.

Fig. 3. Inhibitory effects of EtOAc and EtOH P. oleracea extracts on p-ERK (A), p-JNK (B) and p-p38 (C) levels in LPS-stimulated RAW264.7 cells, as measured by western blot analyses.
Total ERK, JNK, and p38 proteins were used as internal controls. RAW264.7 cells were pre-treated with 10, 30 or 60 μg/mL P. oleracea extracts and then exposed to LPS (1.0 μg/mL) for
12 h. Total cell lysates (20 μg) were separated on 10% SDS-PAGE gels. *p < 0.05, **p < 0.01 compared to the group treated with LPS alone. These experiments were repeated three
times with similar results.

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Y. Kim et al.
Fig. 4. Analysis of body weight and colon length in a DSS-induced mouse model treated with P. oleracea extracts. Changes in body weight (A), colon length (B), images of the colon (C),
and H&E-stained colon tissues (D) during the experimental period. Mice were orally administered P. oleracea extracts and 3% DSS in the drinking water for 14 days (n = 7). *p < 0.01, as
compared with control group. #p < 0.05, vs. DSS-treated animals. SS (50 mg/kg/day) was used as a positive control.
(1), trans-n-feruloyltyramine (2) and cis-n-feruloyl-3′-methoxytyramine
(3) decreased the levels of the inflammatory cytokines TNF-α, IL-6, IL-
1β, IFN-γ, IL-4, and IL-17 in DSS-treated mice, as shown in Fig. 8.
4. Discussion
Ulcerative colitis and Crohn's disease are forms of IBD with mild
symptoms including gradual loosening of the stool, abdominal cramps
and diarrhoea. As the disease progresses, patients may experience
weight loss, fatigue, anorexia, malnutrition, mucus in the stool, severe
rectal bleeding, fever and anaemia (Cuffari, 2009; Panaccione, 2013).
Sulfasalazine, 5-ASA, and steroids are mainly used to treat IBD
(Vermeire et al., 2012). However, the long-term use of these drugs re-
sults in negative side effects, toxicity, and resistance to the dose of the
drug (Probert, 2013).
Interest in plant extract-based medicines as treatments for in-
flammation has increased in recent years. P. oleracea is an herb com-
monly used for medicine and food in Asia. It has pharmacological ef-
fects on various diseases, such as rheumatism, aphtha, gastritis, liver
inflammation and intestinal ulcers (Iranshahy et al., 2017; Joshi &
Joshi, 2000; Chan et al., 2000; Hu, Xu, & Wang, 2003; Kim et al., 2009;
Xiang et al., 2005; Yang et al., 2016). In this study, an animal model
was established and tested to evaluate the inhibitory effects of P. oler-
acea extracts on IBD. Inflammatory cytokines, such as TNF-α, IL-6 and
IL-1β, are known to be major factors contributing to IBD. The innate
immune response plays a crucial role in IBD, and activated immune
cells release inflammatory cytokines such as TNF-α, IL-6 and IL-1β that
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regulate the differentiation of T cells and the inflammatory response.
Dysregulation of T cells or over-production of effector T cells is in-
volved in the progression and exacerbation of IBD (Leon, Smythies,
Smith, & Kelsall, 2006; Sanchez-Muñoz et al., 2008). Tissue analyses of
patients with ulcerative colitis have reported significant increases in the
secretion of pro-inflammatory cytokines, such as TNF-α, IL-6 and IL-1β
(Holub et al., 1998; Komatsu et al., 2001; Louis et al., 1997). Thus,
finding a substance that modulates the production of inflammatory
cytokines is thought to be important for the treatment of IBD. Specifi-
cally, TNF-α is an immune-related cytokine that plays an important role
in IBD by inhibiting tumours or viruses, but its over-production causes
continuous inflammatory reactions. In this study, the effects of P.
oleracea extracts and their active compounds on IL-6 and TNF-α se-
cretion in mice with DSS-induced IBD. In the DSS-induced mouse
model, the inhibitory effect of the P. oleracea EtOAc fraction and its
active compounds on TNF-α secretion was superior to SS, a commonly
used therapeutic agent. IL-6 and IL-1β levels in animals treated with the
EtOAc extract and the active compounds were similar to the control
group. Thus, P. oleracea inhibits IBD by controlling the levels of the
inflammatory cytokines TNF-α, IL-6 and IL-1β in mice with DSS-in-
duced IBD.
These results provide a new lead for the development of IBD agent.
Accordingly, further detailed mechanisms studies will be needed by a
derivative of compound and synthesis of new structures in anti-in-
flammatory activity.
Y. Kim et al. Food Research International 106 (2018) 335–343

Fig. 5. EtOAc and EtOH P. oleracea extracts inhibit TNF-α (A and B), IL-6 (C and D), and IL-1β (E and F) production in the mouse intestine, as determined using ELISAs. Mice were pre-
treated with 200 or 500 mg/kg EtOAc and EtOH extracts and then exposed to DSS (3%). The results from all experiments are shown as the mean values ± SD of at least three
experiments. *p < 0.05, **p < 0.01 compared to the group treated with DSS alone; SS (50 mg/kg/day) was used as a positive control.

5. Conclusions
In this study, we evaluated cytotoxicity, the inhibition of NO pro-
duction and inflammatory cytokines by ELISA of P. oleracea extracts on
macrophage cells. In addition, extracts were presumed to be involved in
inhibiting the expression of genes involved in various inflammation-
related signalling pathways, such as ERK, JNK, and p38. Next, we iso-
lated a potent active compounds from P. oleracea extracts. Finally, we
found that this compounds has Portulacanone C, trans-n-feruloyltyr-
amine and cis-n-feruloyl-3′-methoxytyramine were identified by HPLC,
NMR and electrospray ionization-mass spectrometry (ESI-MS).
Consequentially, extracts and compounds of P. oleracea display a strong
natural inhibition of IBD on in vitro and in vivo model. Overall, this
extracts and compounds has the potential to be developed into new
biomaterials.
Ministry of Agriculture, Food and Rural Affairs (MAFRA) (Grant No.
116082-3) and High-Value Food Technology Development Program
funded by the Ministry of Agriculture, Food and Rural Affairs through
KOREA Pharmaceutical Co., Ltd. (Grant No. 116038-03). This research
was supported by a grant from the KRIBB Research Initiative Program
(KGM2221723).
Authors' contribution
Y.K. and H.J.L. designed and performed the various experiments.
H.-J.J., and S.L. performed additional in vitro experiments. K.J. ana-
lysed the data. S.W.L. contributed reagents, materials, and analytical
tools. S.-J.L. and M.-C.R. wrote the manuscript and supervised the
overall project.

Acknowledgements Additional information

This work was supported by Korea Institute of Planning and Competing financial interests: The authors have no competing fi-
Evaluation for Technology in Food Agriculture, Forestry (IPET) through nancial interests to declare.
Agri-Bio industry Technology Development Program funded by

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Y. Kim et al. Food Research International 106 (2018) 335–343

Fig. 6. Schematic of the isolation of bioactive compounds from the EtOAc fraction of P. oleracea EtOH extracts (1–3): portulacanone C (1), trans-n-feruloyltyramine (2) and cis-n-feruloyl-
3′-methoxytyramine (3).

Fig. 7. Changes in body weight and colon lengths in a DSS-induced mouse model upon treatment with bioactive compounds (1–3). Change in body weight (A), colon length (B), images of
the colon (C), and H&E-stained colon sections (D) during the experiment period. Mice were orally administered bioactive compounds (1–3) and 3% DSS in drinking water for 14 days
(n = 7). *p < 0.01, as compared with control group. #p < 0.05, vs. DSS-treated animals. The positive control (SS) was omitted.

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Y. Kim et al.
Fig. 8. Purified bioactive compounds (1–3) from P. oleracea inhibit TNF-α (A), IL-6 (B), and IL-1β (C) production in the mouse intestine, as determined using ELISAs. Mice were pre-
treated with 3 mg/kg bioactive compounds (1–3) and then exposed to DSS (3%). The results from all experiments are shown as the mean values ± SD of experiments that were repeated
more than three times. *p < 0.05, **p < 0.01 compared to the group treated with DSS alone.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodres.2017.12.058.
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