Published January 23, 2015

Extracts of brown seaweeds can attenuate the bacterial lipopolysaccharide-induced

pro-inflammatory response in the porcine colon ex vivo1

B. Bahar,*† J. V. O’Doherty,*2 M. Hayes, ‡ and T. Sweeney†

*School of Agriculture and Food Science; †School of Veterinary Medicine, University College Dublin, Belfield, Dublin 4,
Ireland; and ‡Ashtown Food Research Centre, Teagasc, Ashtown, Dublin 15, Ireland

ABSTRACT: Bioactive compound-rich brown
seaweeds are demonstrated to have numerous
health benefits including anti-microbial and
immunomodulatory bioactivities in the pig intestine.
In this study, the immunomodulating effects of
extracts of brown seaweed (Ascophyllum nodosum
and Fucus serratus) were evaluated on the porcine
colon using a bacterial lipopolysaccharide (LPS) ex
vivo model. Approximately 1.5 × 1.5 cm of pig colon
(n = 6) was stripped of its overlying muscle layer and
incubated in 1 mL Dulbecco’s Modified Eagle Medium
containing bacterial LPS (10 μg) and seaweed extracts
(1 mg). Gene expression of interleukin-8 (IL-8) and
interleukin-6 (IL-6) and tumor necrosis factor-alpha
(TNFA) were measured using quantitative real time
PCR. In contrast to the low level of expression of IL-8,
IL-6, and TNFA genes in the colonic tissue at 0 h, LPS
treatment increased (P < 0.05) the expression of IL-8,
IL-6, and TNFA genes to 2.38 ± 0.86, 1.90 ± 0.66, and
1.90 ± 0.57 fold, respectively. This pro-inflammatory
response induced by the LPS was suppressed by the
extracts of Ascophyllum. Ascophyllum extract reduced
(P < 0.05) the expression of IL-8, IL-6, and TNFA genes
to 0.99 ± 0.53, 0.75 ± 0.33, and 1.01 ± 0.17 fold, and
Fucus extract reduced (P < 0.05) the expression of
the corresponding genes to 0.70 ± 0.32, 0.69 ± 0.38,
and 1.15 ± 0.25 fold, respectively. It is concluded that
the extracts of Ascophyllum and Fucus seaweeds have
potential to suppress the pro-inflammatory response
induced by the bacterial LPS in the pig colon.

Key words: anti-inflammatory, cytokines, gene expression, interleukin-6, interleukin-8,

tumor necrosis factor-alpha
© 2012 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2012.90:46–48
doi:10.2527/jas53944

INTRODUCTION and secondary metabolites such as phlorotannin, have

not yet been extensively explored.
Addition of seaweed extracts to animal diet has
Recently, ex vivo tissue challenges with bacterial
been considered as an alternative to in-feed antibiotics.
lipopolysaccharide (LPS) to porcine colon and ileum
Brown seaweeds are a rich source of bioactive
tissues have uncovered differential responses in these
compounds, which have previously been demonstrated
tissues following the dietary inclusion of seaweed
to favorably alter the gastrointestinal microbiota in pigs
extracts (Smith et al., 2011). Hence, we hypothesized
(Gardiner et al., 2008). Dietary provision of brown
that the expression of pro-inflammatory cytokine genes
seaweed extract laminarin was found to induce cytokine
induced by an LPS treatment could be modulated by the
response in the porcine gut (Smith et al., 2011). To date,
seaweed extracts in vitro in the porcine colonic tissue.
only a limited number of brown seaweed species have
This experiment was conducted to evaluate the effects
been characterized for their immune stimulating effects
of extracts of brown seaweeds (A. nodosum and F.
on the pig gut. Brown seaweeds Ascophyllum and
serratus) in suppressing the pro-inflammatory response
Fucus, which contain sulfated polysaccharide fucose
induced by bacterial LPS treatment in the pig colonic
1
This work is carried out under the Sea Change Strategy with the tissue ex vivo.
support of the Marine Institute and the Department of Agriculture,
Food and the Marine, funded under the National Development Plan
2007–2013.
2
Corresponding author: john.vodoherty@ucd.ie
46
 Brown seaweeds can attenuate pro-inflammation 47
MATERIALS AND METHODS (Fermentas, GmbH, St Leone-Rot, Germany) following
the manufacturer’s instructions. The expression of
Oven-dried samples of two brown seaweeds (A.
cytokines interleukin-8 (IL-8), interleukin-6 (IL-6), and
nodosum and F. serratus) were solvent extracted as
tumor necrosis factor-alpha (TNFA) and 4 reference
previously described (Herrero et al., 2005) using an
genes [glyceraldehyde 3-phosphate dehydrogenase
automated Dionex 200 accelerated solvent extraction
(GAPDH), beta actin (ACTB), beta 2-microglobulin
(ASE) system (Dionex Corporation, Salt Lake City,
(B2M) and peptidylprolyl isomerase A (PPIA)] were
UT). Dried algal samples (2.5 g) were mixed with 30
measured and analyzed as described previously (Leonard
g of silica gel and diatomaceous earth prior to loading
et al., 2012).
into the sample cells. Extraction conditions for the ASE
The data were analyzed as a split plot design using the
were preheating for 5 min, 6,895 kPa pressure, 100°C
GLM procedure of SAS (SAS Institute, Cary, NC). The
temperature, 5 min heat time, flush volume 75% of the
statistical model included effect of treatment (seaweed
cell volume, 200 s purge time, and 4 static cycles. The
type) and effect of pig within treatment. All the data were
solvent used for A. nodosum extraction was ethanol:water
checked initially for outliers and normality using the
(80:20 v/v). The solvent used for F. serratus extraction
PROC Univariate procedure of SAS.
was water (100%).
Animals and Tissue Sample Collection and Treatment RESULTS
of Colon In contrast to the low level of expression of IL-8, IL-
6, and TNFA genes in the colonic tissue at 0 h (Figure
All experimental procedures were conducted under
1), LPS treatment increased the expression of IL-8, IL-6,
experimental license from the Irish Department of Health
and TNFA genes to 2.38 ± 0.86 (P < 0.01), 1.90 ± 0.66
in accordance with the Cruelty to Animals Act 1876 and
(P < 0.05), and 1.90 ± 0.57 (P < 0.01) fold, respectively.
the European Communities (Amendments of the Cruelty
All of the 3 pro-inflammatory cytokine responses were
to Animals Act, 1876) Regulation, 1994. Pigs (n = 6;
inhibited by dexamethasone (Figure 1). The extract
Large White × Landrace; age 32 d) were euthanized by
of Ascophyllum had an inhibitory effect on the LPS-
a lethal injection of Euthatal (Vortech Pharmaceuticals,
induced expression of IL-8 (P < 0.05), IL-6 (P < 0.05),
Dearborn, MI) and a portion of the colon was surgically
and TNFA (P < 0.05) genes in the pig colonic tissue ex
removed immediately postslaughter. The fecal material
vivo (Figure 1), which inhibited the expression of IL-8,
was removed and the tissue was washed with Kreb’s
IL-6, and TNFA genes to 0.99 ± 0.53, 0.75 ± 0.33 and
solution. Five (corresponding to the 5 ex vivo conditions)
1.01 ± 0.17 fold, respectively. The extract of Fucus also
consecutive pieces of colon (each approx 1.5 cm × 1.5
had an inhibitory effect on the LPS-induced expression
cm) were collected from each of 6 animals.
of IL-8 (P < 0.01), IL-6 (P < 0.05), and TNFA (P < 0.05)
The overlying muscle layer of the colonic section was
genes, which inhibited the expression of IL-8, IL-6, and
removed and a section of approximately 1.5 × 1.5 cm of
TNFA genes to 0.70 ± 0.32, 0.69 ± 0.38, and 1.15 ± 0.25
the colon was transferred into 1 mL Dulbecco’s Modified
fold, respectively.
Eagle Medium in a 12-well cell culture plate containing
10 μg/mL bacterial LPS (Escherichia coli serotype
DISCUSSION
O111: B4, Sigma Aldrich, St. Louis, MO) in presence or
absence of seaweed extract (1 mg/mL) and incubated for In this experiment, challenging the porcine colonic
3 h in a humidified cell culture incubator with 5% CO2 at tissue ex vivo with bacterial LPS induced the expression
37°C. Steroidal compound dexamethasone (10 ng/mL) of pro-inflammatory cytokine genes IL-8, IL-6, and TNFA.
was used as positive anti-inflammatory control. Colonic The LPS-induced pro-inflammatory cytokine response
tissue section after ex vivo treatment was collected in was attenuated by the extracts of Ascophyllum and
RNAlater (Ambion, Austin, TX) and processed for RNA Fucus indicating that these two extracts have potential as
extraction. anti-inflammatory dietary supplements in pig. This pro-
inflammatory response was also inhibited by the steroidal
RNA Extraction, Quantitative Real-Time PCR
anti-inflammatory compound dexamethasone. Induction
Analysis, and Statistical Analysis
of pro-inflammatory cytokine genes IL-8 and IL-6 was
Total RNA was prepared from 25 mg tissue samples previously reported in the porcine colonic tissue ex vivo
using GenElute Mammalian Total RNA Miniprep after treatment with bacterial LPS (Smith et al., 2011).
Kit (Sigma-Aldrich) following the manufacturer’s
Brown seaweeds are a rich source of bioactive
instructions and stored at –80°C. Synthesis of cDNA was
polysaccharides (laminarin, fucans, and alginic acids),
performed with 1 μg of total RNA and oligo dT primer
polyunsaturated fatty acids, vitamins, antioxidants, and
using RevertAid H minus First Strand cDNA synthesis kit
secondary metabolites such as phlorotannin, which may
48
Figure 1. The expression of inflammatory cytokine interleukin-8 (IL-8) (a), interleukin-6 (IL-6) (b), and TNFΑ (c) genes in the porcine colonic tissue
ex vivo due to bacterial lipopolysaccharide (LPS) treatment and attenuation of the pro-inflammatory cytokine response by dexamethasone and Ascophyllum
nodosum and Fucus serratus extracts.
have potential immunomodulatory activities (Holdt
and Kraan, 2011). Whereas the previous report on
dietary inclusion of whole Ascophyllum demonstrated
no improvement on the intestinal health in pig
(Turner et al., 2002), suppression of LPS-induced pro-
inflammatory cytokines IL-8, IL-6, and TNFA genes by
both Ascophyllum and Fucus extracts in the present study
indicates a potentially anti-inflammatory effect, which
may be useful in reducing the intestinal inflammation
of weaning piglets. It is concluded that the extracts of
Ascophyllum and Fucus seaweeds have potential to
suppress the pro-inflammatory response induced by the
bacterial LPS in the pig colon.
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