Fucoidan  in  the  Preven0on  and  Treatment  of  Chronic  Disease    

Rebecca  Behr  Advanced  Food  Science  Prof.  Eileen  Fitzpatrick  Russell  Sage  College  

Abstract  Fucoidan is a term used to describe fucan-containing sulfated polysaccharides, which can be extracted from brown marine algae. Fucoidan offers several beneficial bioactive properties for humans including antioxidant, anticoagulant, antitumor, and antiviral. Recently, there has been an increase in fucoidan research and the results have been promising for use in the treatment of chronic disease. Fucoidan has yet to be approved for medical treatment, other than in research, due to certain inconsistencies. Extraction methods vary greatly across studies, which is problematic, as it affects the final structure of the molecules and thus the bioactivities. The aim of this paper is to outline current research and findings regarding the various activities of fucoidan, while emphasizing the significant differences between extraction techniques.

Introduc0on  •  Marine algae have been documented as being used in oriental medicine

for more than 1,000 years (Smit, 2004; Wijesekara, 2010). •  The class Phaeophyta, in particular, contains sulfated polysaccharides,

termed fucan or fucoidan (heterofucans), which are becoming increasingly studied in biomedical research (Barros Gomes Camara, et al., 2011).

•  In the last 10 years, there has been a spike in fucoidan research. It can be credited to the polysaccharide’s potential health benefits, such as antioxidant, anti-inflammatory, anticoagulant, antithrombotic, antitumor, and antiviral (Tutor Ale, et al., 2011).

•  Fucoidan’s properties make it a potential treatment for Cardiovascular disease (CVD) and cancer. Current treatments can be painful, with negative side effects.

•  The mechanism behind fucoidan’s beneficial activities has yet to be fully understood.

An0coagulant  and  An0oxidant  Ac0vi0es  

•  Freinbichler, et al. claim that oxidative stress and the resulting inflammation has been linked to diabetes, atherosclerosis and cancinogenesis (as cited in Barros Gomes Camara, et al., 2011).

•  Oxidative stress can be prevented through the use of antioxidants. •  Synthetic compounds, such as BHA, BHT, and TBHQ have possible

cancinogenic and toxic health effects (as cited in Barros Gomes Camara, et al., 2011).

•  Fucoidan has showed promise as an antioxidant in several applications and has no adverse health effects (Barros Gomes Camara, et al., 2011).

•  In atherosclerosis and CVD, anticoagulants are used to prevent thromboembolic disorders.

•  Some of the most commonly used anticoagulants are unfractionated heparins, which have negative side effects (Barros Gomes Camara, et al., 2011).

An0coagulant  and  An0oxidant  Ac0vi0es  

•  In 2011, Barros Gomes Camara, et al. published a study in Brazil, which sought to isolate sulfated polysaccharides from Canistrocarpus cervicornis, and evaluate their anticoagulant and antioxidant activities, in vitro.

Mar. Drugs 2011, 9

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2.3.3. Chelating Effect on Ferrous Ions

Antioxidants inhibit interaction between metals and lipids through the formation of insoluble metal complexes with ferrous ion or generation of steric resistance. Furthermore, transition metal ions can react with H2O2, generating hydroxyl radicals, the most reactive free radicals [33].

The chelating effect on ferrous ions exhibited by sulfated polysaccharides extracted from C . cervicornis is shown in Figure 3. The results revealed that heterofucan CC-0.3 did not display significant ferrous chelating capacity, while CC-1.2 and CC-2.0 showed moderate activity at the maximum concentration tested, chelating 33.3% and 39.7% of ferrous ions, respectively. CC-0.5, CC-0.7, and CC-1.0 showed similar dose-dependent activity, reaching maximum activity (about 47.0%) at a concentration of 2.0 mg/mL. This was only 1.8 times lower than EDTA activity at the same concentration under the same experimental conditions (data not shown). The purification process did not increase the chelating effect of the heterofucans compared to the chelating effect of sulfated polysaccharide-rich extract from C . cervicornis [2].

F igure 3. Ferrous chelating activity of sulfated polysaccharides from the brown seaweed C . cervicornis. Each value is the mean ± standard deviation of three determinations: a,b,c Different letters indicate a significant difference (p < 0.05) between each concentration of the same sulfated polysaccharide.

The results obtained by heterofucans, with the exception of CC-0.3, were higher than those shown previously by Costa and co-workers where a sulfated polysaccharide-rich extract from C . cervicornis had a chelating capacity of about 30.0% at a concentration of 2.0 mg/mL [2]. These heterofucans still had superior activity to that of sulfated polysaccharides extracted from Laminaria japonica [34], low molecular and high sulfated derivatives from polysaccharides extracted from Ulva pertusa [32], and sulfated polysaccharide-rich extracts from the algae Dictyopteris delicatula, Dictyota menstrualis, Sargassum filipendula, Spatoglossum schroederi, Gracilaria caudata, Caulerpa cupressoides and Codium isthmocladum [2].

• The researchers concluded that heterofucans, specifically CC-0.7 and CC-1.2, have strong anticoagulant and antioxidant activities, and therefore are “potential multipotent drugs” (Barros Gomes Camara, et al., 2011).

Ferrous chelating activity of sulfated polysaccharides from the brown seaweed C. cervicornis.

An0tumor  and  An0metasta0c  Ac0vi0es    

•  Metastasis occurs when cancer cells break off the primary tumor and move through the blood and lymphatic vessels to invade other organs. This phenomenon causes up to 90 percent of cancer-related deaths (Lee et al., 2012).

•  A South Korean study conducted by Lee, J. Kim, and E. Kim (2012) sought to assess and understand the mechanism of fucoidan anti-metastatic effects, using a highly metastatic form of human lung cancer, denoted A549.

•  At concentrations of 400-1,000 µg/ml, “fucoidan inhibited cell proliferation and decreased cell viability in a concentration-dependent manner”

An0tumor  and  An0metasta0c  Ac0vi0es    

Figure 1. Effect of fucoidan on MMP-2 activity of A549 cells. (A) Subconfluent A549 cells were incubated for 48 hr in the absence or presenceof fucoidan (0, 12.5, 25, 50, 100, and 200 mg/ml) in serum-free RPMI media. The conditioned media were collected, and their MMP-2 activity wasestimated using gelatin zymography. (B) MMP-2 activity by analysis of zymography was quantified by measuring the band intensities using Image Jsoftware. (C) Total cell lysates were subjected to Western blot analysis probed with anti-MMP-2 antibody. (D) Expression of MMP-2 by analysis ofwestern blot was quantified by measuring the band intensities using Image J software. The data shown are the means 6 SD of six experiments.Significant difference from control group, *p,0.05 and **p,0.01.doi:10.1371/journal.pone.0050624.g001

Fucoidan Inhibits Cancer Cell Metastasis

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Western BlottingThe protein components (50 mg) were separated on 12% SDS-

polyacrylamide gel, transferred to polyvinylidene difluoridemembranes (Bio-Rad, C.A. USA), and subsequently subjected toimmunoblot analysis using specific primary antibodies for over-night at 4uC. After washing, the membranes were incubated withhorseradish peroxidase-conjugated secondary antibody (Cell Sig-naling Technology, Beverly, MA) for 1 hr at room temperature.The blots were then visualized by using the enhanced chemilu-minescence method (ECL, Amersham Biosciences, Buckingham-shire, UK) on blue light-sensitive film (Fujifilm Corporation,Tokyo, Japan). If necessary, the blotted membranes were strippedand reprobed using other primary antibodies. Densitometryanalysis was performed with a Hewlett-Packard scanner andNIH Image software (Image J).

Statistical AnalysisThe results are expressed as the mean 6 standard deviation

(S.D.). One-way analysis of variance (ANOVA) was used toevaluate the significance of difference between the two meanvalues. *P,0.05 and **P,0.01 were considered to be statisticallysignificant.

Results

Fucoidan Inhibits the Activity of MMP-2 in A549 HumanLung Cancer CellsIn order to determine non-cytotoxic concentration of fucoidan,

A549 cells were incubated with various concentrations of fucoidan(0–1000 mg/ml) for 24 hr or 48 hr, and then cell viability wasdetermined using MTT assay (data not shown). Fucoidan did notshow any significant toxic effect on A549 cells within theconcentration range of 0–200 mg/ml. At higher concentrations(400–1000 mg/ml), however, fucoidan inhibited cell proliferationand decreased cell viability in a concentration-dependent manner.Therefore, the present study applied the treatment of fucoidan atconcentrations equal to or lower than 200 mg/ml, at which nosignificant cytotoxic effect of A549 cells was observed.The degradation of extracellular matrix (ECM) is crucial for

cellular invasion, indicating the inevitable involvement of matrix-degrading proteinases for the process. Hence, it was assessed theeffect of fucoidan on MMP-2 activity, as a key molecule of ECMdegradation, by gelatin zymography. As shown in Figure 1A,fucoidan suppressed MMP-2 activity in a concentration-depen-dent manner. On the other hand, the impact of fucoidan on theMMP-9 activity was inconclusive, since there is only an extremelylow level of intrinsic MMP-9 activity detectable in A549 cells (datanot shown). MMP-2 activity was reduced by 36%, 53%, and 72%at 50, 100, and 200 mg/ml, respectively, after treatment with the

Figure 2. Effects of fucoidan on migration of A549 cells. (A)A549 cells were plated on 12-well and grown to 90% confluence inRPMI containing 10% FBS. Cells were scratched with a pipette tip andthen treated with fucoidan (0, 50, 100, and 200 mg/ml). (B) Cellmigration distance was estimated by measuring the width of thewound. The data shown are the means 6 SD of six experiments.Significant difference from control group, *p,0.05 and **p,0.01.doi:10.1371/journal.pone.0050624.g002

Figure 3. Effect of fucoidan on invasion of A549 cells. (A) Effectsof fucoidan on cancer cells invasion were examined using Cell InvasionAssay kit (BD Bioscience, San Jose, CA, USA). For this, A549 cells wereseeded onto the upper chamber of Matrigel-coated filter and incubatedin the absence or the presence of fucoidan (0, 50, 100, and 200 mg/ml)for 48 hr. The invading cells on the lower surface of the membranewere visualized by crystal-violet staining and observed with lightmicroscope. (B) The amount of invading cells was quantified usingImage J software. The data shown are the means 6 SD of threeexperiments. Significant difference from control group, *p,0.05 and**p,0.01.doi:10.1371/journal.pone.0050624.g003

Fucoidan Inhibits Cancer Cell Metastasis

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From these results the researchers concluded that because fucoidan is indeed an anti-metastatic reagent and shows low side effect in normal cells, further in vivo studies and clinical investigations are needed to apply fucoidan as a new cancer therapy.

Effect of fucoidan on MMP-2 activity of A49 cells

An0viral  Ac0vi0es  •  Hepatitis C Virus (HCV) is extremely virulent, devastating to the liver,

and chronic infection can cause liver cirrhosis (LC), which can lead to liver cancer (Chen, et al., 2013). No vaccine exists.

•  In Japan, Mori, et al. conducted a study, which assessed the antiviral activity of fucoidan on HCV, both in vivo and in vitro (2012).

•  Researchers found from the in vitro testing that fucoidan inhibited HCV replication, as indicated by luciferase (LUC) activity, but did not exhibit cytotoxic effects, as cell viability was not significantly affected (Mori, et al., 2012).

•  There was no significant decrease in HCV RNA levels or clinical symptoms in the participants, however, none of the patients showed progression of LC during the 12-month treatment. It is important to note, however, that the patient pool was small and contained non-responders to standard treatments (Mori, et al., 2012).

An0viral  Ac0vi0es  

Eight patients were not eligible for IFN treatment be-cause of LC, complication (depression), or advanced age. Seven patients had received IFN therapy in the past. Six patients were non-responders to IFN and 1 discontinued therapy because of side effect (depression). All patients assessed the tolerability as excellent.

During fucoidan treatment, 11 patients received a -

hide Bio Co., Okinawa, Japan) was given orally as cap-sules containing 166 mg of dry extract from C. okamura-nus Tokida12 mo. Informed consent was obtained from all patients enrolled in the study, after a thorough explanation of the

Test parametersThe outcome parameters included the course of alanine aminotransferase (ALT), aspartate aminotransferase (AST), quantitative HCV RNA levels, subjective symp-toms associated with CHC, LC, and HCC (such as fa-tigue, abdominal discomfort, depression, and dyspepsia), safety, and compliance. Data on all clinical parameters were documented at each visit. HCV RNA levels were determined using the AMPLICOR GT HCV Monitor

a lower limit of quantitation of 0.5 kIU/mL at a linear range up to 850 kIU/mL.

Statistical analysis Data are expressed as mean ± SD. The results of bio-chemical tests and HCV RNA levels were compared by the Student’s t test. A P

!"#$%Fucoidan suppresses HCV replication To assess the effects of fucoidan on intracellular replica-

tion of the HCV genome, HCV subgenomic replicon cells FLR3-1 were cultured in the presence of various concentrations of fucoidan in the medium. The luciferase activities of the FLR3-1 cells showed a concentration-dependent suppression of replication of HCV replicon by fucoidan. The WST-8 assay showed that fucoidan had negligible effect on cell viability (Figure 1). These results suggest that fucoidan inhibits HCV replication but does not have cytotoxic effects.

Effect of fucoidan therapy on HCV RNA and alanine aminotransferase levels Changes in HCV RNA and serum ALT levels in patients treated with fucoidan are shown in Figure 2. The mean HCV RNA for the 15 patients was 736 ± 118 kIU/mL (range, 100-850 kIU/mL) before fucoidan therapy. As shown in Figure 2A, fucoidan tended to reduce the mean HCV RNA level with time relative to the baseline,

However, HCV RNA increased after 11 and 12 mo. Bio-chemical tests showed that the mean serum ALT level, but not AST, correlated with the mean HCV RNA level, although the decrease in ALT level was not significant relative to the baseline (Figure 2B). Whereas the above changes were not associated with improvement in clinical symptoms in every patient, none of the patients showed progression of LC or adverse events.

'(#)$##(*+It is estimated that 170 million people worldwide are infected with HCV[3], and some 2 million (1%) of these reside in Japan[23]. Of the HCC cases in Japan, around 80% are caused by HCV infection. The increase in the number of HCC patients contributes to the increase in total deaths in Japan from HCC. This trend is expected to continue until 2015[23]. The general strategies followed in the treatment of CHC include eradication of HCV and suppression of hepatitis.

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Extrac0on  Methods  and  Molecular  Structure  

•  The relationship between the beneficial activities of fucoidan and the structure of the molecules has not yet been elucidated.

•  According to a review by Tutor Ale, et al., this understanding has been delayed by the lack of extraction and purification standards and protocol in fucoidan research (2011).

•  The authors also state that the methods of extraction affect the final structure of the fucoidan molecules, which has an effect on their biological properties

•  It has been noted across various studies, that the degree of sulfation and size of the fucoidan molecules has a direct impact on their antitumor and anticoagulant properties (Tutor Ale, et al., 2011).

•  Tutor Ale, et al., suggest that standard extraction procedures should be developed, which include “hydrolysis treatment, purification and fractionation methodology, preferably with specific steps adapted to the particular botanical order of the seaweed” (2011).

Conclusions  

Current research has shown that fucoidan, extracted from brown algae, has anticoagulant, antioxidant, antitumor, and antiviral properties. Fucoidan is a potential effective treatment for chronic disease, such as CVD and cancer, and is a more healthful alternative to current treatments. Despite ample research on the topic, standard protocol for fucoidan extraction and purification must be developed, so that fucoidan can be better understood and eventually approved for use in disease prevention and treatment (Tutor Ale, et al., 2011).

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