Studies on isolation, characterization of fucoidan from brown algae Turbinaria decurrens and evaluation of it's in vivo and in vitro anti-inflammatory activities

Article abstract of DOI:10.1016/j.ijbiomac.2020.05.152

In the present study, the anti-nociception and anti-inflammatory activity of fucoidan isolated from T. decurrens on formalin induced paw-edema in mice model were investigated. The extracted fucoidan contain 54.86% of total sugar, 23.51% of sulfate and 3.4% of protein. The monosaccharide composition analysis revealed that fucoidan encompassed of fucose (59.3%), galactose (12.6%), mannose (9.6%), rhamnose (6.4%) and xylose (11.4%). Further, the structural characterization was done by UV–visible spectroscopy, X-ray diffraction, FT-IR and ¹HNMR analysis. The fucoidan reduced the licking time thereby suggesting anti-nociceptive effect and decreased the size of paw swelling in the formalin induced inflammatory edema condition. The isolated fucoidan could significantly decreased the MDA and also increase the SOD, CAT, GPx, GST and GSH activity in paw edema tissue of formalin injected mice. Furthermore, fucoidan administration retained p65/NF-κB transcription factor in the cytosol thereby showing down regulation of the gene expression of pro-inflammatory mediators such as IL-1β, COX-2 and MMP-9 in fucoidan treated mice. The anti-inflammatory effect of fucoidan was attributed to its capacity on modulating the levels of enzymatic antioxidants, master regulator NF-κB and pro-inflammatory cytokines. The fucoidan has reduced LPS induced cytotoxicity in IC-21 macrophage at a dose depended on manner.

Full text of DOI:10.1016/j.ijbiomac.2020.05.152

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Studies on isolation, characterization of fucoidan from brown
algae Turbinaria decurrens and evaluation of it's in vivo and in
vitro anti-inflammatory activities
Ramar Manikandan, D. Parimalanandhini, K. Mahalakshmi,
Manikandan Beulaja, Munuswamy Arumugam, Sundaram
Janarthanan, Subramanian Palanisamy, SangGuan You,
Narayanasamy Marimuthu Prabhu
PII:
S0141-8130(20)33306-7
DOI:
https://doi.org/10.1016/j.ijbiomac.2020.05.152
BIOMAC 15635
Reference:
To appear in:
International Journal of Biological Macromolecules
Received date:
Revised date:
Accepted date:
3 April 2020
13 May 2020
18 May 2020
Please cite this article as: R. Manikandan, D. Parimalanandhini, K. Mahalakshmi, et al.,
Studies on isolation, characterization of fucoidan from brown algae Turbinaria decurrens
and evaluation of it's in vivo and in vitro anti-inflammatory activities, International
Journal
of
Biological
Macromolecules
(2020),
https://doi.org/10.1016/
j.ijbiomac.2020.05.152
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Studies on isolation, characterization of fucoidan from brown algae Turbinaria decurrens
and evaluation of it's in vivo and in vitro anti-inflammatory activities
1
*
1
1
2
Ramar Manikandan , D. Parimalanandhini , K. Mahalakshmi , Manikandan Beulaja ,
1
1
3,4
3,4
Munuswamy Arumugam , Sundaram Janarthanan , Subramanian Palanisamy , SangGuan You ,
5
Narayanasamy Marimuthu Prabhu
1
Department of Zoology, University of Madras, Guindy Campus, Chennai-600 025.
2
Department of Biochemistry, Annai Veilankanni’s College for Women, Saidapet, Chennai-
6
00015.
3
East Coast Life Sciences Institute, Gangneung-Wonju National University, 120 Gangneung,
Gangwon, 210-720, Republic of Korea
4
Department of Marine Food Science and Technology, Gangneung-Wonju National University,
1
20 Gangneungdaehangno, Gangneung, Gangwon, 210-702, Republic of Korea.
5
Disease Control and Prevention Lab, Department of Animal Health and Management, Alagappa
University, Karaikudi, 630 003, Tamil Nadu, India.
*
Corresponding author
Dr. R. Manikandan
Assistant Professor
Department of Zoology
University of Madras, Guindy Campus, Chennai-600 025
E. Mail: manikandanramar@yahoo.co.in
1

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Abstract
In the present study, the anti-nociception and anti-inflammatory activity of fucoidan
isolated from T. Decurrens on formalin induced paw-edema in mice model were investigated. The
extracted fucoidan contain 54.86% of total sugar, 23.51% of sulfate and 3.4% of protein. The
monosaccharide composition analysis revealed that fucoidan encompassed of fucose (59.3%),
galactose (12.6%), mannose (9.6%), rhamnose (6.4%) and xylose (11.4%). Further, the structural
1
characterization was done by UV-visible spectroscopy, X-ray diffraction, FT-IR and HNMR
analysis. The fucoidan reduced the licking time thereby suggesting anti-nociceptive effect and
decreased the size of paw swelling in the formalin induced inflammatory edema condition. The
isolated fucoidan could significantly decreased the MDA and also increase the SOD, CAT, GPx,
GST and GSH activity in paw edema tissue of formalin injected mice. Furthermore, fucoidan
administration retained p65/NF-κB transcription factor in the cytosol thereby showing down
regulation of the gene expression of pro-inflammatory mediators such as IL-1β, COX-2and MMP-
9
in fucoidan treated mice. The anti-inflammatory effect of fucoidan was attributed to its capacity
on modulating the levels of enzymatic antioxidants, master regulator NF-κB and pro-inflammatory
cytokines. The fucoidan has reduced LPS induced cytotoxicity in IC-21 macrophage at a dose
depended on manner.
Keywords: Fucoidan; Turbinaria decurrens; Anti-inflammatory.
2

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1
. Introduction
Inflammation is the main driving force of an adaptive immune response produced by
complex interactions between cells and soluble factors during infection as well as tissue injury. A
multitude of factors results in inflammation releasing large number of inflammatory mediators
such as cytokines, chemokines and reactive oxygen/nitrogen intermediates in its vicinity. The
same beneficial immune response sometimes converted into dysregulated inflammatory process
leads to diverse disease states and cause many chronic as well as degenerative diseases such as
rheumatoid arthritis, Crohn’s disease, type II diabetes and Alzheimer’s disease etc., [1].In chronic
condition, increased production of reactive oxygen/nitrogen intermediates can also damage DNA
that leads to increase in the chances of mutation and cancer risk [2].
Pain is a symptomatic sensation of the complex sensory phase of inflammatory reaction,
occurring during tissue or nerve damage that begins in the activation of nociceptors [3].
Nociception is different from pain, wherein it refers to the detection of noxious stimuli by
nociceptors and passing them to brain thereby it serves as an alarming signal to living organisms
[
4]. Millions of people worldwide suffering from acute and chronic pain due to trauma, cancer and
arthritis represent 20% of general population [5]. Nowadays, pain management comes with high
socio-economic costs, requires more trained health care workers and major policy decision from
the government, since in the case of Europe3-10% of GDP was spent on pain management[6]. In
India, the large elderly population suffering from arthritis will be 60 million in 2025 and 1 million
new cancer patients are added every year to the existing patients’ lists that further alleviate the
crisis of pain management. In addition, to the poor accessibility of opioid by deserving cancer
patients and high cost of knee replacement surgery for example has kept the poor Indian patients
in dilemma with life-long chronic pain [7].The management of pain during various disease
conditions is still a challenging problem. The most prescribed drugs for pain management are
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opioids and non-steroidal anti-inflammatory drugs (NSAIDs). However, the adverse side effects
of these medications range from opioid deaths to liver and kidney damage by NSAIDs [8,9].The
current scenario necessitates a need for safer and effective alternate anti-inflammatory drugs other
than the currently available opioids and NSAIDs. The search for naturally occurring biomolecules
with potent anti-inflammatory activity reported in our traditional medicinal systems such as
Ayurveda and Siddha has been gaining momentum. Unfortunately, most of the plant-based drugs
are reported as crude extract and their anti-inflammatory mechanisms are still not elaborately
reported.
The brown seaweed possesses rich source of anti-inflammatory compounds with different
combinations in chemical variations. Such bioactive compounds range from phenols,
polysaccharides, fatty acids, halogenated compounds to carotenoids [10].Among them sulfated
polysaccharides such as fucoidan that represents 20 to 60% of fructose having alternating α (1→3)
and α (1→4) glycosidic linkages dominates as potent and widely used biomolecule [10–12].
Fucoidan are increasingly recognized source of bioactive metabolites that are utilized in
cosmetics, functional foods, nutritional supplements, animal feeds and pharmaceuticals including
the incorporation of fucoidan nano composites in 3D scaffold [13,14]. However, the much
celebrated fucoidan do have some limitations; small variations in monosaccharide content leads to
alteration of the structure, different extraction method gives different results, species to species
variation as well as seasonal variation. All of these have affected the commercialization of
fucoidan based drugs[13,15]. Therefore, the bioactive fucoidan have to be separately isolated from
each species of brown seaweed and tested for its anti-inflammatory activity. This ensures isolation
of large amount of fucoidan with unambiguous pharmaceutical effects, which further enables the
selling of standardized drug in the commercial market.
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The brown seaweeds Turbinaria ornata and Turbinaria decurrens are commonly found
and cultivated in the Gulf of Mannar area. The anti-inflammatory activity of T. ornata is well
established. Recently, it has been reported about the anti-inflammatory effect of T. ornate on
cotton pellet induced granuloma model in rats[16]. However, the anti-inflammatory activity of
related species T. decurrens has not been investigated although it possesses anti-coagulant [17],
anticancer [18], hepatoprotective [19] and neuroprotective [20] effects. Therefore, the present
study was aimed at determination of the anti-inflammatory activity of fucoidan isolated from the
T. decurrens and its antioxidant as well as anti-nociceptive effect on formalin induced
inflammatory condition in mice. Further, this study also provides bio prospective information
regarding medicinal applications of cultivable seaweeds in the east coast region of India.
Since information on the mechanism of action of isolated bioactive metabolites is essential
for the future pharmaceutical exploitation of the drug. In this study, we have also aimed to
investigate the anti-inflammatory effect of isolated fucoidan molecule from T. decurrens and
studied the molecular mechanisms using transcription factor and pro-inflammatory genes in
formalin-induced paw edema in mice model as well as anti-inflammatory activities in in vitro
analysis of IC-21 macrophages.
2
. Materials and methods
2
.1. Materials
Formalin, dexamethasone, superoxide dismutase, epinephrine, 3,3′-diaminobenzidine
tetrahydrochloride (DAB), thiobarbituric acid (TBA), 1-chloro-2,4-dinitrobenzene (CDNB),
reduced glutathione, 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and diacetylmonoxime (DAM),
were purchased from Sigma Chemical Company (St. Louis, USA).Primary antibody p65/NF-κB
polyclonal rabbit antibodies were purchased from BD Bioscience, USA. Secondary antibody goat
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anti-rabbit IgG-HRP conjugated, trizol, diethyl pyrocarbonate and primers were obtained from
Genei, Bangalore, India.
2
.2. Isolation of fucoidan from T. decurrens
Brown seaweed, T. decurrens was collected from Gulf of mannar region, Tamil nadu,
India and authenticated with the help of a botanist. Collected seaweeds were thoroughly washed in
running tap water followed by distilled water to remove the adhering sand, epiphytes, extraneous
matter and necrotic parts. After this, the seaweeds were shade dried at room temperature (37 ºC).
The dried seaweeds were pulverized to powder using an electronic blender and stored in an
airtight container. The extraction of fucoidan was reported by Yang [21]. Briefly, 50 g of seaweed
powder was extracted using one litre of 85% ethanol, stirred with mechanical stirrer for 12 h at
room temperature and centrifuged at 15,000 rpm for 10 min to remove proteins and pigments.
Then the residue was allowed to dry at room temperature. 5 g of powder was taken, mixed in100
ml of distilled water stirred for 1h at 65ºC and centrifuged at 15,000 rpm for 10 min. Then the
supernatant was collected and mixed with 1% CaCl . The solution was maintained overnight at 4
2
ºC to precipitate alginic acid. Subsequently, the solution was centrifuged again at 15,000 rpm for
1
0 min. The collected supernatant was mixed with 99% ethanol to obtain final ethanol
concentration of 30% and kept at 4 ºC. After 4 h incubation, solution was centrifuged at 15,000
rpm for 10 min and the supernatant was collected. Then, 99% ethanol was added to arrive at a
final concentration of 70% and this solution was stored overnight at 4 ºC. Subsequent to overnight
incubation, this solution was filtered with a nylon membrane (0.45 μm size) to obtain fucoidan. To
estimate the yield of fucoidan, the obtained sample was treated with 85% ethanol as a percentage
of algal dry weight (% dry weight). The dried fucoidan was packed in an airtight container until
further use.
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2
.3 Purification of fucoidan
About, 250 mg of extracted fucoidan was dissolved in 25 ml of distilled water heated with
.0 M HCl for 3hrs. Then, the reaction mixture was cool down to room temperature followed by
3
the centrifugation at 5000 rpm for 10 min. The collected supernatant was neutralized and
precipitated with ethanol. Finally, the fucoidan washed with distilled water and freeze dried.
2
.4 Determination of chemical composition
The presence of total sugar content was determined by phenol-sulphuric acid method using
D-glucose as a standard [22]. The sulphate content was quantified by BaCl –gelatine method
2
described by Dodgson and Price using potassium sulfate (K SO ) as a standard [23]. In this study,
2
4
the uronic acid content was determined by carbazole reaction using glucuronic acid as a standard
24]. Amount of protein was measured by folin-phenol reagent as per the method prescribed by
Lowry et al [25] and in this assay, bovine serum albumin (BSA) was used as a standard.
[
2
.5 CHN analysis
The presence of elements such as carbon, hydrogen and nitrogen content were measured
by CHN analysis (Perkin-Elmer 2400 Series CHNS/O Analyzer). In this analysis, 2, 5-Bis (5′
tertbutyl-2-benzoxazol-2-yl) thiophene (BBOT) and aspartic acid were considered as reference.
2
.6 Determination of monosaccharides composition
The monosaccharides composition was determined by High-performance liquid
chromatography (HPLC) with 1 – phenyl – 3-methyl-5 –pyrazolone [26]. Briefly, the fucoidan
sample (6 mg) was hydrolyzed by 2 M trifluoro acetic acid (0.3 ml) at 121°C for 1 hr. Then, it was
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turned into PMP derivatives and eluted by thermo hypersil ODS -2 HPLC columns (250 mm x 4.6
-1
o
mm; flow rate of 1 ml min ) at 30 C.
2
.7 UV-visible spectroscopy analysis
About, 1mg of fucoidan was completely dissolved in 1 ml of distilled water and the
spectrum was recorded in the range of 200–400 nm using UV–vis spectrophotometer (UV-1800
spectrophotometer, Shimadzu, Japan)
2
.8 XRD analysis
The XRD analysis was carried out using X’ Pert PRO, PAN analytical, Netherland
instrument. In this analysis, 40 kV of voltage and 30 mA current was applied and the spectrum
was measured in the 2θ range of 30–80°.
2
.9 FT-IR and NMR analysis
The structural characterization of fucoidan was determined by FT-IR and NMR analysis.
The fucoidan sample was ground with potassium bromide and pressed to form a hard pellet using
spectra lab pelletizer. The FT-IR spectrum was measured using a Perkin Elmer model-983/ G
1
detector double beam spectrophotometer. The H NMR spectral analysis was carried out using an
advance spectrometer (400MHz Bruker model). Approximately, 10 mg of fucoidan was dissolved
in deuterium oxide and the spectrum was measured at 27 ºC.
2
.10 Collection and maintenance of experimental animals
Healthy Swiss albino mice weighing about 20 g were procured from Biogen laboratory
animal facility, Hosur. They were maintained in polypropylene cages with distilled water and fed
with standard mice feed pellets ad libitum (Hindustan Lever Ltd.). A 12 h light and dark cycle was
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maintained throughout the experimental period. The animals were allowed to acclimatize to
laboratory condition for four days before conducting each experiment. All experiments were
conducted according to the ethical norms approved by Government of India and Institutional
Animal Ethics Committee guidelines.
2
.11 Experimental design
The Swiss albino mice were divided into five Groups of five animals each for the
experiment at both 24 and 48 h, respectively.
Group I
:
:
Control (saline injected)
Group II
Formalin (2%) injected sub-cutaneously in mice hind paw and orally gavaged
with physiological saline.
Group III
Group IV
Group V
:
:
:
Mice injected with formalin (as in Group II) and gavaged orally with T.
decurrens derived fucoidan (50 mg/kg body weight).
Mice injected with saline and gavaged orally with T. decurrens derived
fucoidan (50 mg/kg body weight).
Mice injected with formalin that received dexamethasone intraperitoneally
(dosed once at 2.5 mg/kg body weight) 1 h before formalin paw injection.
2
.12Toxicity of isolated fucoidan on mice
In this experiment, the animals divided into three Groups such as control, and experimental
Groups. The experimental Groups of animals were orally gavaged with 25 and 50 mg/kg body
weight for seven consecutive days respectively. Body weights and animal behaviour were
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monitored continuously until the end of study. Mice in all Groups were finally weighed 24 h after
o
last dose, sacrificed and paw was harvested and stored in -80 C.
2
.13Methodology of formalin and fucoidan administration
a) Assessment of anti-nociceptive effect
(
Mice across the five experimental Groups were utilized in this study. The drug, T.
decurrens derived fucoidan substance (50 mg/kg body weight) was dissolved in distilled water
and gavaged orally to the mice for 5 days. One hour after the last treatment edema was induced in
mice by 20µl formalin administration (2%in saline v/v) at plantar region of the right hind paw.
The anti-nociception activity of isolated fucoidan against formalin injected mice was carefully
observed immediately after injection, 5 and 30 min later. During this observation phase, the
control and treated mice were individually placed in a separate glass chamber. The licking of
injected paw area by mice indicated pain. Therefore, licking time was visually observed and
recorded at initial 5 min (neurogenic phase) and 30 min later (inflammatory phase) to know the
action of fucoidan on two different phases of inflammation. The positive control Group were
treated with dexamethasone (2.5 mg/kg body weight dissolved in 10% ethanol) and it was
administered intraperitoneally one hour prior to formalin injection.
(b) Assessment of acute anti-inflammatory effect
The acute anti-inflammatory effect of isolated fucoidan was tested in formalin induced
mice paw edema method as described by Domiati [27] with slight modifications. Two different
concentrations of T. decurrens derived fucoidan substance (25 and 50 mg/kg body weight) were
dissolved in distilled water and gavaged orally to the mice for 5 days. One hour after the last
treatment acute inflammation was induced in mice by administration of 20µl of formalin (2%in
saline v/v) at the plantar region of the right hind paw. Control mice received equal volume of
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saline solution. The positive control Group were treated with dexamethasone (2.5 mg/kg body
weight dissolved in 10% ethanol) and it was administered intraperitoneally one hour prior to
formalin injection. Percentage reduction of paw edema volume was measured using
plethysmometer in the control and treated mice before, 1, 3 and 5 h after injection of formalin.
2
.14 Collection of samples for biochemical analysis
In order to study the biochemical and molecular mechanisms behind the anti-inflammatory
action of isolated fucoidan on formalin induced paw inflammation. Animals were sacrificed and
paws were dissected out immediately and stored at-80 ºC. 200 mg of paw tissue sample was taken
and homogenized in tris-HCl buffer (0.1 M; pH 7.4) in ice cold condition. The tissue homogenates
were centrifuged at 11,000 rpm for 30 min, clear supernatants were collected and kept at -80°C
until its use for further biochemical analysis.
2
.15Estimation of malondialdehyde (MDA)
The level of lipid peroxide was estimated by the method of Ohkawa [28]. Briefly, 0.2 ml
of paw tissue homogenates, 0.2 ml of 20% SDS, 1.5 ml of 20% acetic acid and 1.5 ml of 0.8%
thiobarbituric acid were added. The mixture was made up to 4 ml with double distilled water and
then heated in a water bath at 95 °C or 60 min using glass balls as a conductor. After cooling, 1 ml
of double distilled water and 5 ml of n-butanol/pyridine mixture (v/v) were added, shaken
vigorously and centrifuged at 11,000 rpm, for 10 min at 4 °C. Absorbance of the organic layer was
read at 532 nm in a UV spectrophotometer. 1’, 1’,3’,3’-tetramethoxy propane was used as a
standard and lipid peroxide levels were expressed in n mol of MDA/g wet weight.
2
.16 Antioxidant assay
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Antioxidant enzymes such as SOD [29], CAT [30], GPx [31], GST [32], GSH [33] were
estimated using paw edema tissue homogenates. To calculate the specific enzyme activity, protein
in each sample was estimated by the method of Lowry et al,[25].
2
.17 Isolation of RNA, RT-PCR and target sequence amplification
a) Isolation of total cellular RNA from mice paw tissues
Mouse paw tissue from each group was weighed separately. Briefly, the paw tissue was
(
pulverized in a mortar and pestle using liquid nitrogen until powdered. Subsequently, Trizol (200
µl for 20 mg tissue) was added to the ground tissue and transferred to an Eppendorf tube.
Thereafter, an equal volume of ice-cold chloroform was added and incubated for 10 min at room
temperature. Following this, the sample was vortexed and centrifuged at 12,000 rpm for 20 min at
4
°C.The cellular RNA that preferentially separates into aqueous layer on top was carefully
removed to a new Eppendorf tube. Subsequently, the ice-cold isopropanol (1:2.5) was added to the
aqueous layer. This mixture was again let to stand at room temperature for about 10 min and
centrifuged at 12,000 rpm for 20 min at 4°C.The resulting pellet was washed twice in ice cold
7
5% ethanol and re-suspended in 0.1% DEPC treated double distilled water until complete
solubilisation.
(b) Reverse transcriptase polymerase chain reaction (RT-PCR)
RNA was quantified and reverse transcribed to cDNA using the Thermo Scientific First
strand cDNA synthesis kit. In brief, 1 µg of isolated RNA from all experimental Groups was made
in a reaction volume of 20 µl containing oligo (dT) primer, dNTPs, RNase inhibitor and MuLV
Reverse Transcriptase. The forward and reverse primer sequences were designed for chosen genes
from the NCBI site using Primer BLAST programme. Sequences were designed keeping in mind
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the isolation of total cellular RNA as opposed to mRNA. Hence, primers spanning an exon-exon
boundary were selected for our experiment. The primer sequences are presented in Table. 1.
About 100 ng of cDNAs was utilized from each Group and amplified using a thermocycler
with the following conditions:Initial denaturation 95°C for 4 min; denaturation 95°C for 1min;
annealing 55°C for 1min (Tm for both primers were at least 4-5 degrees higher than the set
annealing temperature); extension 72°C for 1 min; final extension 72°C for 5 min; Hold at 4°C.
Steps 2, 3 and 4 were run for a total of 35 steps each. The PCR amplified products were then run
and visualized under UV light for target gene amplification.
2
.18 Western blot analysis
Animals from each Group were sacrificed; paw tissue was weighed separately and snap-
frozen immediately in liquid nitrogen. Prior to homogenization, the frozen tissue was thawed and
ground in a mortar and pestle on ice with approximately two and a half times the weight of tissue
in detergent free buffer (50 mM tris, pH 7.4; 100 mM NaCl, 10% glycerol) on ice supplemented
with 1 mM PMSF added fresh. The homogenates were centrifuged and resulting clear
supernatants were used for further analysis.
After electrophoretic separation, the gel was blotted onto a nitrocellulose membrane,
blocked and incubated with primary antibody, p65/NF-κB polyclonal rabbit antibodies, at a
dilution of 1:500 followed by incubation with secondary antibody, goat anti-rabbit IgG-HRP
conjugate at a dilution of 1:2000. The blot was then developed using ECL kit (Ultra Cruz, Santa
Cruz) following the manufacturer’s instruction.
2
.19 Cell Culture
The IC-21 cell line was derived by transforming peritoneal macrophages from C57BL/6
mouse with simian virus 40 and it was purchased from NCCS Pune. The cell line was maintained
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in RPMI 1640 medium containing 10% heat inactivated foetal bovine serum and 2mM L-
2
˚
glutamine at 37 ºC, 5% CO . The cells were cultured in 25-cm culture flasks at 37 C in a
2
humidified atmosphere of 5% CO and was sub cultured when they reached 90-100% confluence
2
using PBS.
2
.20 LPS induced cytotoxicity assay
4
The macrophage cells were seeded in a flat bottomed 96-well plate at a density of 1x 10
cells per well and allowed to reach confluence provided with 10% FBS containing RPMI-1640
medium. Once confluence is reached the 10% FBS containing medium is removed and the cells
were maintained in 0.5% containing RPMI-1640 medium for overnight. The toxicity effect was
determined by incubating the IC-21 cells with different concentration of fucoidan (32 to 500
µg/ml) or LPS (0.25-4 µg/ml) for 24 h separately. The percentage of cell viability was calculated
using alamar blue assay. Briefly, the cells were treated with 0.5 μg/ml of LPS for 5h and different
concentrations of fucoidan (32 to 500 µg/ml) were added to the LPS treated wells and incubated
for 24 h. After incubation 0.1% of 10 µl of alamar blue was added wells and extended the
incubation period for 5 h. The absorbance was measured at 570 and 600 nm using (Power waves
XS). Alamar blue reduction was calculated and the percentage viability was obtained after
normalizing the data by untreated control cells. Untreated control cells served as 100% cell
viability.
2
.21 Statistical analysis
The statistical significance was assessed by SPSS/10.0 software. The comparison between
groups was determined by student’s t-test. Difference in values between control and treated group
were considered to be significant atp≤0.05.
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3
. Results and Discussion
3
.1. Chemical characterization of fucoidan
Fucoidan are sulfated polysaccharides derived from marine brown algae and essentially
contain fucose and sulphate groups along with molecules such as galactose, xylose, mannose and
uronic acids. Fucoidan were reported to possess various biological effects in vitro and in vivo such
as anti-inflammatory, anticoagulant, antithrombotic, antiviral including anti-HIV,
immunomodulatory, antioxidant, antitumor activities and also inhibit transcription factors (NF-
κB), pro-inflammatory mediators such as IL-1β and more importantly cyclooxygenases [34].In
this study, 5.32±0.31% of fucoidan was extracted from the T. decurrens using hot-water extraction
and ethanol precipitation method. As shown in the Table 1, the extracted fucoidan mainly consist
of total sugar (54.86±0.23%) sulfate (23.51±0.32), uronic acid (3.4±0.7) and protein (2.7±0.13).
The monosaccharide composition revealed that the existence of fucose (59.3%), galactose
(12.6%), mannose (9.6%), rhamnose (6.4%) and xylose (11.4%). A similar trend was observed in
the previous studies reported by Synytsya et al. [35] and Vishchuk et al. [36] where the
polysaccharides of Saccharina japonica and Undaria pinnatifida had shown the higher level of
fucose content than galactose. Further, the presence of carbon (21.03±0.21), hydrogen (3.16±0.17)
and nitrogen (2.06±0.02) elements were confirmed by CHN analysis (Table 1b).The composition
of sugar mostly varied between the species due to the period of sample collection, treatment of
sample and extraction procedure [26].
3
.2 Structural characterization of fucoidan
The structural characterization of fucoidan was assessed by UV-Visible spectroscopy,
1
XRD, FT-IR and HNMR analysis. The isolated fucoidan showed the maximum absorbance peak
at 260 nm in the UV-Visible spectral analysis which denoted the covalent bonding of aromatic
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compounds with the polysaccharides (Fig. 1a) and also, the absorption value of these fucoidan
confirmed as fucose enriched sulphated polysaccharides [37].The microstructural changes were
determined by XRD analysis. The expression of a major peak at 32.30º confirms the semi-
crystalline nature of polymer (Fig. 1b). It is well known that the structural arrangement of
polysaccharide is directly affecting the various properties of the polymer such as tensile strength,
solubility, flexibility and swelling [38].
-1
As shown in Fig 2a, the FT-IR spectrum of fucoidan revealed a peak at 1252 cm due to-
S-O anti-symmetric stretching vibration of the sulphate group. The peaks arising at 1136 and 1054
-1
cm is due to presence of C-C and C-O stretching vibrations in the pyramid ring and also by C-O-
-1
C stretches of the glycosidic bonds. A peak at 862 cm characterize sulfation at the equatorial
position where sulfate ester binds to that of fucose to form sulphate fucose. A strong absorption
-1
band was observed at 1255 cm which was attributed to O=S=O stretching of sulfates and a good
marker of sulfated polysaccharides [39]. Further, NMR spectroscopy is performed as an excellent
tool to investigate the anomeric configuration and the sulfation pattern of the polysaccharides. In
1
the present study, the HNMR spectrum of fucoidan was displayed in Fig. 2b. The presence of
signals at 4.132 and 4.681 corresponds to α-L-fucose and 3-linked α-L-fucose which was the main
constituent of fucoidan [10]. The signal at 3.955 represents the 3-linked ß-D-galactose. The two
signals at 3.662 and 2.103 indicated the existence of α-L-rhamnopyranosyl residues and α-carbon
proton [40].
3
.3. Isolated fucoidan and inflammatory edematous symptoms
As shown Fig.3a, the anti-inflammatory effects upon oral administration of fucoidan from
T. Decurrens on formalin induced inflammatory conditions. The results revealed that fucoidan
administration significantly reduced the licking time (72 to 31 seconds) within 5 min of initial
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observation period (57% reduction) than formalin induced mice (Group II). Extended period of 30
min further reduced the licking time to 13 seconds (76% reduction). Reduction in the licking time
after fucoidan administration was significantly (p ≤ 0.01) different from formalin alone injected
mice. Positive control dexamethasone also significantly reduced the licking time. Formalin
induced nociception has two phases such as first neurologic phase involving nociceptors and later
inflammatory phase involving the action of prostaglandins [41]. Further confirmation on anti-
inflammatory effect of fucoidan is shown in Fig.3b. The visible reduction in swelling in the hind
paw of fucoidan treated mice indicated anti-inflammatory effect of test compound (Fig.4).Coelho-
de-souza [42] noticed that administration of essential oil of Pterodon polygalae on mice
significantly reduced the licking of its paw. Similarly, Zhao [43] reported that exposure of
flavonoids isolated from leaves of Juniperus sabina significantly reduced only the late
inflammatory phase. However, in this study fucoidan isolated from T. decurrens effectively
reduced the impact of formalin induced noxious stimuli in both neurologic and inflammatory
phases of inflammation.
Suppression of inflammatory symptoms during acute and chronic disease conditions is a
therapeutic option for treatment. Many botanicals and marine based biomolecules possess anti-
inflammatory activity[44,45]. Fucoidan isolated from various seaweeds such as Cystoseria [46],
Sargassum wightii, Halophila ovalis [47] and Undaria pinnatifida [48] exhibited in vivo anti-
inflammatory activity against various tested conditions. In the present study, fucoidan isolated
from seaweed T. decurrens at 50 mg dose for 5 h observation period significantly reduced the paw
edema volume (52% reduction) over formalin alone injected mice (Fig.3b). Further, the fucoidan
anti-inflammatory effect increased over time and was almost similar to dexamethasone. The
results clearly revealed that fucoidan isolated from T. decurrens had both anti-nociceptive and
anti-inflammatory activities.
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3
.4Tissue antioxidant enzymes analysis
Maintenance of cellular redox balance is crucial for normal physiology and numerous
studies have highlighted the perturbations that lead to inflammation and disease. It can be useful to
visualize the generation of ROS through different mechanisms that occurs concurrently [49]. Each
oxidant species which results from either normal metabolism or due to oxidative insult is usually
deals with a different combination of potent antioxidant mediators like SOD, CAT, GST, GPx and
GSH. The antioxidant result was displayed in Fig. 5. The major ROS generated include
superoxide anion, hydroxyl radical, nitric oxide as well as peroxides, which in turn damage
cellular components and are a target of cellular antioxidants. We therefore, attempted to assay
cellular levels of different antioxidant enzymes in addition to lipid peroxidation product that is
reliable as an oxidative stress marker. Malondialdehyde (MDA) level was observed in the tissue
homogenate of Swiss albino mice. After formalin administration (Group II; p<0.001) at both 24
and 48 h, animals manifest a significant increase in MDA level 88 and 99%, respectively (Fig.
5
A). However, the MDA level was significantly decreased in group III (p<0.001) animals that
were administered fucoidan and were subjected to formalin injection (Fig. 5A).The results clearly
revealed that fucoidan effectively suppressed the lipid peroxidation.
Superoxide dismutase (SOD) is one of the important endogenous antioxidant enzymes that
catalyze formation of H O from superoxide radical. In this study, formalin administration
2
2
significantly reduced SOD level to 17% (p<0.001) when compared to the control group of animals
Group I (Fig. 5B).). However, the fucoidan treatment significantly increased the SOD level
(
almost similar to Group I (p<0.001) during 48 h period and a similar pattern was also noticed in
catalase level. Catalase enzyme further converts the toxic H O into water and molecular oxygen.
2
2
Thereby catalase level in the body is of great significance during amelioration of oxidative stress.
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After formalin administration animals showed a strong reduction (56%) in CAT level when
compared to control animals (Fig. 5C; Group I). However, the CAT level was significantly
increased in group IV (24 h, p< 0.05; 48 h p< 0.001) animals that were previously administered
with fucoidan and were subjected to formalin injection. In the case of animals belonging to group
III the CAT level was similar to that of control.
Glutathione peroxidase (GPx) enzyme catalyses reduction of hydroperoxides by using thiol
reducing power of glutathione thereby preventing the oxidation of lipids and proteins.
Glutathione-S-transferase (GST) is an important detoxification function to protect cellular
macromolecules by catalyzing the conjugation of reduced glutathione to a wide variety of
endogenous substrates. It also plays an important role as a transport protein and in metabolism of
leukotrienes and prostaglandins. In order to assess the inflammatory status during drug treatment,
the GPx and GST levels were observed in the paw tissue homogenate of Swiss albino mice. After
formalin administration (Group II; p<0.001) animals showed a significant reduction in GPx and
GST level to 62 and 60%, respectively, when compared to control animals (Group I (Fig. 5 D and
E).). However, the animal pre-treatment with fucoidan significantly increases the GPx and GST to
nearly normal level (Group III; p<0.001) as only 1 and 2% reduction were seen respectively over
control animals (Group I). Similarly, the reduced glutathione level significantly decreased (43%;
p<0.001) from the control group. However, the drug treatment elevated GSH levels to almost
normal during the 48-h time period. These results clearly revealed that the restoration of
antioxidants capacity and resistant to formalin induced oxidative stress was observed in fucoidan
treated mice (Fig 5 F). It is known that formalin induces its pro-inflammatory activity through
promoting oxidative stress; oxidant mediated cell damage and as a consequence this leads to
depleted levels of cellular antioxidant enzymes[50].Treatment with fucoidan of T. decurrens
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attenuated the formalin mediated oxidative insult by successfully rescuing the antioxidant
enzymes.
3
.5Effect of isolated fucoidan on p65/NF-κB protein expression
In order to study the molecular mechanism behind anti-inflammatory activity mediated by
fucoidan, we tested its ability to modulate the transcription factor and pro-inflammatory genes that
contribute towards inflammation in formalin induced paw edema condition in mice. Nuclear
factor-κB is an important transcription factor for the induction of pro-inflammatory genes
including that of cytokines and chemokines [51]. The fucoidan isolated from seaweed has both
anti-inflammatory and immunomodulating activities and it has a differential effect on NF-κB
activation. Park [52]noticed that fucoidan from Fucus vesiculosus down regulated the expression
of NF-κB gene in LPS induced inflammation in BV2 microglial cells. Similarly, sulfated
polysaccharides extracted from Sargassum horneri effectively inhibited the NF-κB pathway in
LPS induced RAW 264.7 macrophages [53]. In contrast, fucoidan isolated from New Zealand
Undaria pinatifida possessed immunomodulatory activity by stimulating the release of NO which
increased the expression of iNOS, TNFα, IL-6 and NF-κB [54]. Subsequently, Puet al
[
55]reported that polysaccharides extracted from Poriacocus activates NF-κB thereby functioning
as an immunomodulatory agent in macrophages. Therefore, the present study was designed to
check the effect of fucoidan isolated from T. decurrens on NF-κB expression. The role of NF-κB
as a major regulator in inflammation and was characterized via assaying for p65/NF-κB protein in
all the experimental groups. NF-κB has been known to regulate inflammatory signals by its
subunits that are sequestered in cytosol as an inactive complex. Upon stimulation with pro-
inflammatory signals, IκB-α is phosphorylated and degraded resulting in free NF-κB which is
translocated to nucleus and acts as a transcription factor. It is a common understanding that
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translocation of NF-κB to the nucleus results in transcription of pro-inflammatory genes. We
observed that the level of inactive p65 sub-unit of NF-κB sequestered in cytosol to be lowest in
group II at 48 h. This indicates that translocation of p65 subunit to nucleus activated NF-κB
mediated transcription. On the other hand, group III mice exhibited significant retention of p65
subunit comparable to that control group of animals (Fig. 6).Our results unambiguously
demonstrated the anti-inflammatory effect of fucoidan from T. decurrens by inhibiting the
expression of NF-κB. Conversely, at 24 h there was no significant modulation observed.
3
.6 Effect of isolated fucoidan on inflammatory genes
The pro-inflammatory cytokine IL-1β is a major player known to induce inflammation and
cause tissue destruction by production of MMP-9, while NF-κB is known to be a key transcription
factor that regulates expression of pro-inflammatory genes like IL-1β, MMP-9 and COX-2 [56].
Further, pro-inflammatory genes are pleiotropic that gives a warning signal for tissue damage
during earliest phase of inflammation. It also elevated the acute phase proteins such as C-reactive
protein, serum amyloid and fibrinogen [57].As shown in Fig. 7, mice treated with formalin alone
showed increased expression levels of COX-2,IL-1β and MMP-9 when compared with control
mice. However, fucoidan pre-treatment reduced the expression levels of COX-2, IL-1β and MMP-
9
genes. Subash et al.[16] reported that fucoidan like sulfated polysaccharides isolated from T.
ornata down regulated the COX-2 enzyme. Similarly, fucosterol isolated from edible brown algae
Eisenia bicyclis reduced the expression of iNOS and COX-2 [58]. Recently, fucoidan isolated
from seaweed Undaria pinnatifida exhibited anti-inflammatory activity in carrageenan induced
paw edema in rat model by inhibiting the expression of COX-2 gene [48]. Since COX-2 remains a
potential target for inhibition in inflammation as it induces the production of prostaglandin E2 that
leads to increased level of reactive oxygen species and nitric oxide [59]. Our findings showed that
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administration of fucoidan inhibited the expression of COX-2 gene on formalin induced acute
inflammatory condition.
Matrix metalloproteinase-9 (MMP-9) is a key endopeptidase produced by inflammatory
cells involved in tissue remodeling. They play a key role in cancer, heart disease, neuro
inflammation and various other inflammatory diseases [60,61]. Up regulation of pro-inflammatory
MMP-9 degrades the extra cellular matrix, helps in the migration of leukocyte and release of TNF-
α [62]. Therefore, inhibition of MMP-9 by the fucoidan provides therapeutic opportunity to
control and minimize acute as well as chronic inflammatory conditions. Several studies have
reported that fucoidan administration ameliorates the MMP-9 expression in anticancer [63],
rheumatoid arthritis and atherosclerosis [64]. Bakar et al. [65] reported that edible seaweed extract
of Eucheuma cottonii decreased the expression of MMP-9 gene expression in ovalbumin induced
allergic asthma condition. In the present study, we have for the first time reported that fucoidan
from T. decurrens decreased the expression of MMP-9 gene in formalin induced paw edema in
mice.
Limited research has been carried out to examine the molecular mechanism behind the
anti-inflammatory effect of fucoidan and even no such scientific information is available on T.
decurrens. Based on our results of this study, we noticed that isolated fucoidan from T. decurrens
successfully down regulated the expression of master transcriptor gene NF-κB. Inhibition of NF-
κB transcription by fucoidan molecule at nuclear translocation level (retainment of p65/NFĸB in
the cytosol) indicated effectiveness of the drug molecule to curtail inflammation. The down
regulation of pro-inflammatory genes such as IL-1β, COX-2 and MMP-9 are the positive step for
its anti-inflammatory effect. The transcription factor NF-κB also regulated the expression of IL-
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1
β, COX-2 and MMP-9 as well as boost anti-oxidative defence system. Therefore, blockage of
NF-κB by the seaweed fucoidan gives added advantages for mitigation of inflammatory condition.
3
.7 Effect of isolated fucoidan on IC-21 macrophages
The cytotoxic effect of fucoidan was evaluated in IC-21 macrophages using alamar blue
assay. The different concentrations of fucoidan ranging from 32-500 µg/ml showed cell viability
in a dose dependant manner. The highest activity (70%) was observed at the cells treated with 500
µg/ml concentration of fucoidan for 24 h (Fig. 8).Simultaneously, cells treated with different
concentration of LPS(0.25-5 µg/ml) showed the cell viability at 75-62% (Fig. 9). An optimal
concentration 0.5 µg/ml of LPS was chosen and the cells were pre-treated with 0.5 µg/ml for 5 h
and when post-treated with fucoidan, it reduced LPS induced toxicity in cells (Fig. 10), and it has
been reported that fucoidan has no significant cytotoxicity and also mitigated LPS induced toxic
effect in raw macrophages 264.7 cells [11,66], microglial cells [67] and PC 12 cells [68] and it
showed cell viability up to 400 µg/ml which suggest that fucoidan has minimum toxicity in cells.
Conclusion
For the first time we have reported the anti-nociceptive and anti-inflammatory activities of
fucoidan isolated from T. decurrens. The seaweed fucoidan possesses good antioxidant capacity as
it maintained the equilibrium of LPO, SOD, CAT, GPx, GST and reduced glutathione level to
normal during the acute inflammatory condition created by formalin injection in mice. The anti-
nociceptive activity of fucoidan was established as it significantly lowers the licking time both in
neurologic and inflammatory phases. Inhibition of nociceptive signal by isolated fucoidan useful
in management of pain in acute and chronic conditions. Further, reduction of paw edema in treated
mice confirm the anti-inflammatory activity. The fucoidan exhibited its anti-inflammatory activity
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by upregulation of antioxidative system and suppressed the pro-inflammatory genes via NF-κB
pathway. It leads to reduction in the expression of IL-1β, COX-2and MMP-9 as well as anti-
inflammatory activity against LPS induced IC-21macrophages. Therefore, the isolated fucoidan
could be used as dietary supplements to regulate the symptoms especially pain and inflammation
during various diseases conditions.
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