Designed optimization of a single-step extractionof fucose-containing sulfated polysaccharidesfrom Sargassum sp.

Marcel Tutor Ale & Jørn Dalgaard Mikkelsen &

Anne S. Meyer

Received: 20 March 2011 /Revised and accepted: 18 May 2011 /Published online: 2 June 2011# Springer Science+Business Media B.V. 2011

Abstract Fucose-containing sulfated polysaccharides canbe extracted from the brown seaweed, Sargassum sp. It hasbeen reported that fucose-rich sulfated polysaccharidesfrom brown seaweeds exert different beneficial biologicalactivities including anti-inflammatory, anticoagulant, andanti-viral effects. Classical extraction of fucose-containingsulfated polysaccharides from brown seaweed species typi-cally involves extended, multiple-step, hot acid, or CaCl2treatments, each step lasting several hours. In this work, wesystematically examined the influence of acid concentration(HCl), time, and temperature on the yield of fucose-containing sulfated polysaccharides (FCSPs) in statisticallydesigned two-step and single-step multifactorial extractionexperiments. All extraction factors had significant effects onthe fucose-containing sulfated polysaccharides yield, withthe temperature and time exerting positive effects, and theacid concentration having a negative effect. The modeldefined an optimized single-step FCSPs extraction procedurefor Sargassum sp. (a brown seaweed). A maximal fucose-containing sulfated polysaccharides yield of ∼7% of theSargassum sp. dry matter was achieved by the optimalextraction procedure of: 0.03 M HCl, 90°C, 4 h. HPAEC-PAD analysis confirmed that fucose, galactose, and glucur-onic acid were the major constituents of the polysaccharidesobtained by the optimized method. Lower polysaccharideyield, but relatively higher fucose content was obtained withshorter extraction time. The data also revealed that classicalmulti-step extraction with acid ≥0.2 M HCl at elevated

temperature and extended time had a detrimental effect onthe FCSPs yield as this treatment apparently disrupted thestructural integrity of the polymer and evidently causeddegradation of the carbohydrate chains built up of fucoseresidues.

Keywords Fucoidan . Sargassum . Brown seaweed .

Fucose . Bioactive compound . Extraction method

Introduction

Fucose-containing sulfated polysaccharides, or “fucoidan”,from brown algae may contain differing glycosidic linkagesand are variously substituted with acetate and side branchescontaining fucose or other glycosyl units. These fucose-containing sulfated polysaccharides (FCSPs) can be extractedfrom brown seaweed species such as Fucus, Laminaria, andSargassum (Li et al. 2008; Mori and Nisizawa 1982). A rangeof biological activities have been attributed to FCSPs includinganti-tumoral (Zhuang et al. 1995), anti-viral (Adhikari et al.2006; Trinchero et al. 2009), anti-inflammatory (Blondin et al.1994); and notably anticoagulant effects (Nardella et al. 1996).The potential pharmaceutical and medical applications ofFCSPs have recently directed special interest into utilizationof brown seaweeds as a source of FCSPs (Li et al. 2008;Cumashi et al. 2007).

Some brown seaweed FCSPs have a backbone of 3-linked α-L-fucopyranose, while others have a backbone ofalternating 3- and 4-linked α-L-fucopyranose residues andsulfated galactofucans (Bilan and Usov 2008). The sulfatedgalactofucans are prominently found in various Sargassumspecies (Duarte et al. 2001; Zhu et al. 2003). These sulfatedgalactofucans are mainly built of (1→6)-β-D-galactoseand/or (1→2)-β-D-mannose units with branching points

M. T. Ale : J. D. Mikkelsen :A. S. Meyer (*)Center for Bioprocess Engineering,Department of Chemical and Biochemical Engineering,Technical University of Denmark (DTU),Søltofts Plads—Building 229,DK-2800 Kgs. Lyngby, Denmarke-mail: am@kt.dtu.dk

J Appl Phycol (2012) 24:715–723DOI 10.1007/s10811-011-9690-3

formed by (1→3) and/or (1→4)-α-L-fucose, (1→4)-α-D-glucuronic acid, terminal β-D-xylose and sometimes (1→4)-α-D-glucose (Duarte et al. 2001). Early studies alsoreported the existence of fucoglucuronans having a back-bone of glucuronic acid, mannose, and galactose residueswith side chains of neutral and partially sulfated residuesof galactose, xylose, and fucose; these supposed fuco-glucuronans have been reported to be present inSargassum linifolium (Abdel-Fattah et al. 1974).

Extraction of fucose-containing sulfated polysaccharidesfrom brown seaweeds generally involves multiple, extend-ed aqueous extractions, usually with hot acid (hydrochloricacid), and may include calcium addition to promote alginateprecipitation (Chizhov et al. 1999; Marais and Joseleau2001). It has long been known that extraction time,temperature, and acid concentration/pH may influence bothyields and composition of the resulting fucoidan or FCSPs(Li et al. 2008; Black et al. 1952). Already in 1952, Blacket al. (1952) reported how the use of different extractionmethods influenced the quantity of fucose with a disparityof 20% to 80% of total fucose of Fucus vesiculosusfucoidan and 20% to 55% of total fucose of Pelvetiacanaliculata fucoidan (Black et al. 1952). The influence ofextraction method on fucose-containing sulfated poly-saccharides yield is further exemplified by data for theyield from Laminaria japonica: the yield was only 1.5%of the dry weight (DW) of the seaweed when extractedwith alkaline solution at 95°C for 2 h (Sakai et al. 2002),but 2.3% DW when the extraction was done with water inan autoclave at 120°C for 3 h (Wang et al. 2008). On theother hand, the FCSPs notably fucoidan yield of acombined extract of F. evanescens was 12.9% DW whenextracted four times with 2% CaCl2 solution at 85°C for5 h (Bilan et al. 2002), while cold extraction with 0.4%HCl at 25°C for 5 h yielded 12.0% DW (Zvyagintseva etal. 1999). Typically, the maximum FCSPs yields from(dried) brown seaweeds range from 5–7% DW. Fucoidanyields extracted from F. vesiculosus have thus beenreported to be 7.0% DW; while the fucoidan yieldsobtained from Sargassum horneri and Undaria pinnatifidawere found to be 5.2% DW and 6.8% DW, respectively(Kuda et al. 2002). Despite the existence of early seminalstudies about FCSPs extraction, notably Black et al.(1952), that recommended the use of a three-step hot acidextraction procedure, there is only limited systematicinformation about the influences and apparently complexinteractions of extraction parameters acid, temperature andtime on fucose-containing sulfated polysaccharides yield.

Sargassum is an unexploited brown seaweed genus inthe Phaeophyceae which grows wildly in enormousquantities almost all over the world, but it is particularlyabundant along the coastal regions in south East Asia,where members of this genus are considered as nuisance

seaweeds. In order to initially assess the possible use ofSargassum sp. as a source of FCSPs, we wanted to evaluatesystematically the influence of the extraction parameters,i.e., acid concentration, time, temperature, and maximizethe FCSPs yields, while at the same time attempt arelatively mild extraction procedure. In this present work,we therefore systematically examined the effects ofdifferent combinations of acid, reaction time and temperatureon the fucose-containing sulfated polysaccharides yields froma Sargassum sp. obtained from Vietnam. We also comparedthe composition of the extracted polysaccharides obtained bythe final yield-optimized one-step extraction procedure aswell as an analogous two-step extraction procedure tothose obtained with the classical, state-of-the art multi-step fucoidan extraction methods of Black et al. (1952)and Bilan et al. (2002).

Materials and methods

Chemicals Hydrochloric acid 37%, D-glucose andD-xylose were purchased from Merck. Ethanol 99.8%,trifluoracetic acid 99% (TFA), trichloroacetic acid 99%,diethyl ether, CaCl2, Na2SO4, BaCl2, L-arabinose,L-rhamnose, D-galactose, L-fucose, D-mannose andD-glucuronic acid were from Sigma-Aldrich (Germany).Agarose D-2 was obtained from Hispanagar (Spain). Allchemicals used were analytical grade. Dried Sargassumsp. was obtained from the company Viet Delta Co. Ltd.(Ho Chi Minh City, Vietnam).

Design of experiment Two-step and one-step extractionexperiments were evaluated according to an experimentaldesign objective of response surface modeling (RSM). Acentral composite face centered design was used withprocess modeling and optimization using multiple linearregression modeling. The number of different parametercombinations in each design was 14 with 3 replications ofthe center point. The varying factors were as follows: acidconcentrations, 0, 0.1, and 0.2 M of HCl; extractiontemperatures, 30, 60, and 90°C; and reaction times were1, 3 and 5 h. All extraction experiments were performed induplicate.

General extraction process Sargassum sp. was ground bymilling using an OBH Nordica Coffee Mill 100 watts (OBHNordica Denmark A/S, Denmark) to pass through a 500-μmsieve and pretreated with a MeOH-CHCl3-H2O (4:2:1) mixtureat room temperature to remove colored matter and phenolcompounds prior to extraction. All extractions (Fig. 1a and b)were done in a water bath (Julabo SW22—Germany) withshaking at 200 rpm. 20 mL (HCl) of different concentration(according to the experimental design) were added to a

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centrifuge flask that contained 1 g of ground dried, pretreatedSargassum sp. In the single-step extraction (Fig. 1a), theseaweed was extracted according to the factorial designdescribed in the design of experiment. After extraction, thesuspended seaweed was centrifuged at 10,600×g for 5 min, thesupernatant was removed. Then the residue was washed with5 mL MilliQ water and centrifuged again at 10,600×g for10 min, thereafter the first supernatant and the supernatantfrom the washing were combined (Extract A). A liquid fraction(16 mL) from Extract A was precipitated with 60% (v/v)aqueous ethanol to obtain crude FCSPs. The precipitate waswashed once with water and was coagulated immediately witheither 0.5 M NaCl or 1 M CaCl2 to release and precipitatealginate. The sticky precipitate was discarded and thesupernatant was centrifuged again at 10,600×g for 10 min.The pellet was collected and transferred to an Eppendorf tube.Each Eppendorf tube was weighed prior to pellet transfer, andthen centrifuged at 10,600×g for 5 min; the pellet was washedwith alcohol and diethyl ether and then finally dried overnightat 50°C. The amount of dried crude FCSPs yield wastranslated to dry weight (in mg) of the total extract volume(in mL) from the original dry seaweed solution (in grams dryweight per milliliter) and the FCSPs yield (in mg g-1 dryweight) was thus based on the original seaweed dry weight.For the two-step extraction (Fig. 1b), the seaweed residue ofthe single-step extraction (Residue A, Fig. 1b) was extractedonce more by addition of 10 mL HCl (of a concentrationaccording to the experimental design) and the suspendedseaweed was treated the same way as described above to

obtain Extract B. Extract A and B were then combined, andcrude FCSPs was isolated by the same procedure as describedabove (Fig. 1b).

Benchmark experiment A comparative study between theoptimized conditions of the two-step extraction and theone-step extraction respectively, obtained from the multi-variate models, and two other extraction conditions fromknown methods (Black et al. 1952; Bilan et al. 2002) wascarried out: The Sargassum sp. was pretreated as mentionedabove prior to extraction. The RSM optimized two-stepextraction condition, 0.07 M HCl at 90°C for 3 h (twice),was designated as Method 1. The RSM optimized one-stepextraction condition, 0.03 M HCl at 90°C for 4 h (once), or1 h (once), was designated as Method 1a. Method 2 was thebenchmark extraction of Bilan et al. (2002) which, in short,involved extraction of the ground, dried, pretreated Sargas-sum sp. four times with a 2% CaCl2 solution at 85°C for 5 h(Bilan et al. 2002). Method 3 was the benchmarkextraction of Black et al. (1952): for this extraction, theground, dried, pretreated Sargassum sp. was extractedthree times with 0.17 M HCl at 70°C for 1 h with washing,centrifugation and pH reduction from pH 2.5 to 1.9 aftereach extraction step (Black et al. 1952). After extraction,all extracts were precipitated with ethanol, washed withH2O and treated with 1 M CaCl2 to precipitate alginate. Ineach case, the supernatant was then centrifuged, precipi-tated with ethanol, centrifuged again, and the pellet wasdried overnight at 50°C.

Dry Milled AlgaDilute acid extraction: 0.1 M & 0.2 M HClReaction time: 1 h, 3 h, 5 hTemperature: 30, 60, 90 °C

Dilute acid extraction: 0.1 M & 0.2 M HClReaction time: 1 h, 3 h, 5 hTemperature: 30, 60, 90 °C

Extract A Residue A

Extract B

Residue B

Extract A+B

EtOHSupernatant

Precipitate

Dry Milled AlgaDilute acid extraction: 0.1 M & 0.2 M HClReaction time: 1 h, 3 h, 5 hTemperature: 30, 60, 90 °C

Extract A Residue A

EtOHprecipitation

EtOHSupernatant

Precipitate

CoagulationWashing and drying

Fucose-containingsulfated polysaccharides

ba

EtOHprecipitation

CoagulationWashing and drying

Fucose-containingsulfated polysaccharides

Fig. 1 Schematic diagram of a single-step (a) and two-step extraction (b) of fucose-containing sulfated polysaccharides from Sargassum sp.

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Acid hydrolysis Dried Sargassum sp. powder and extractedpolysaccharide samples (50 mg) were subjected to acidhydrolysis using 2 M TFA at 121°C for 2 h (Arnous andMeyer 2008). After hydrolysis, the mixture was freezedried, and the dried powder was resolubilized and centri-fuged at 10,000×g for 10 min to collect the supernatantthen filtered using a 0.2 μm syringe tip filter (Sun Sr. USA)prior to HPAEC-PAD analysis (see below). The monosac-charide recoveries were determined for arabinose, rham-nose, fucose, galactose, glucose, xylose, and glucuronicacid and used as correction factors for the quantitativemonosaccharide assessment principally as describedpreviously (Arnous and Meyer 2008).

Compositional analysis The separation and quantificationof monosaccharides of the acid hydrolyzed polysacchar-ides were done by HPAEC-PAD using a BioLC systemconsisting of GS50 gradients pumps/ED50 electrochem-ical detector/AS50 chromatography compartment coupledto an AS50 autosampler (Dionex Corp., USA). Separa-tions were performed using a CarboPacTM PA20(3 mm×150 mm) analytical column (Dionex Corp.)according to Thomassen and Meyer (2010). The quanti-fication was carried out using the external monosaccharidestandards: L-arabinose, L-rhamnose, L-fucose, D-galactose,D-glucose, D-mannose, D-xylose and D-glucuronic acid.Data were collected and analyzed with Chromeleon 6.80SP4 Build 2361 software (Dionex Corp.). The sulfateanalysis was performed by a turbidometric method usingagarose-barium reagent as described by Jackson andMcCandless (1978).

Statistical and data analysis The analyses of varianceswere performed using Minitab 15 (Minitab, Inc., UK) witha significance value of P≤0.05. The program Moddeversion 7.0.0.1 (Umetrics AB, Sweden) was used as anaid for the design of experimental templates and for theevaluation of the effects and the interactions by multiplelinear regression analysis.

Results

Polysaccharide compositional profile

The HPAEC analysis of the TFA hydrolysate of Sargassumsp. showed that the polysaccharide profile was dominatedby fucose (29±3.7), galactose (14±2.1), and glucuronicacid (39±3.5)mg·g−1 seaweed dry weight, with a sulfatecontent of (155±5)mg·g−1 seaweed dry weight, while otherminor components such as mannose, rhamnose, glucose,arabinose, and xylose were also detected (data not shown).

This composition is in accordance with early studies ofsulfated polysaccharides from S. linifolium (Abdel-Fattah etal. 1974) and typical for the Sargassaceae.

Evaluation of extraction—two steps

Optimizing two-step extraction To determine the optimalextraction of fucose-containing sulfated polysaccharides,the seaweed was extracted twice and the effects of differenttreatment factors were tested systematically, i.e., acid, 0 to0.2 M HCl; temperature, 30 to 90°C; and time, 1 to 5 h(Fig. 1b). According to the model, the predicted maximumyield of 7.1% DW, was achieved using 0.07 M HCl/90°C/3 hextracted twice. The predicted extraction yield obtained whenthe two-step extraction was done without acid, at 30°C for 1 hproduced 2.9±0.8% DW only, while the harshest extractiontreatment of 0.2 M/90°C/5 h resulted in a yield of 3.1±0.6%DW from the combined Extract A and B, respectively.

Benchmarking Assessment of the optimal parametersobtained from the model was carried out by performinganother extraction experiment using the optimum of theresponse surface model. Subsequently, a comparativeextraction study was also done to verify the feasibility ofthe model. Two extraction methods were compared to thepredicted optimal extraction condition: Method 1 was thepredicted maximum optimal condition (i.e., 0.07 M HCl,90°C, and 3 h-two-step extraction); Method 2 was asdescribed by Bilan et al. (2002); and Method 3 was asdescribed by Black et al. (1952). The data showed that thetotal yields obtained by Method 1 were higher thanpredicted by the model, namely ∼8.5% DW, and that thetotal polysaccharide yields with the two-step extractionsMethod 1 and 2 were similar, but much higher than whatwas obtained with Method 3 (∼5% DW; Fig. 2). The dataalso showed that the major part of the yield was obtainedduring the first extraction. The results of these experiments

Fig. 2 Polysaccharide yield (% DW) of Sargassum sp. extractedusing different methods. Method 1 was extracted using 0.07 M HCl/90°C/3 h (two-step); Method 2 as described by Bilan et al. (2002) andMethod 3 as described by Black et al. (1952)

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thus showed that the yield of the first extraction (Extract A)of Method 1 (7.6±0.31% DW) was higher, p<0.05, thanthe corresponding yields of the benchmark methodsMethod 2 (6.2±0.08% DW) and Method 3 (4.2±0.09%DW), respectively (Fig. 2). Based on this result, it wasdecided to evaluate whether a higher polysaccharide yieldcould be obtained using only a single-step extraction ofMethod 1 type. A second statistical experiment wastherefore performed to define the optimum acid concentra-tion, temperature and incubation time for this single-stepextraction (Fig. 1a, Extract A only).

Optimization of polysaccharide yield—single-stepextraction

Multiple linear regression analysis of the data obtained in thestatistically designed single-step extraction study showed thatan increase in extraction time and temperature significantlyincreased the polysaccharide yield, whereas a decrease of acidconcentration also significantly increased the yield (Table 1).Moreover, acid and acid interaction, acid and time interac-tion and acid and temperature interaction each had asignificant effect on the polysaccharide yield (Table 1).According to the model, the maximum polysaccharide yieldof ∼8% DW from Sargassum sp. would be obtained at0.03 M HCl/90°C/4 h, whereas the lowest polysaccharideyield was at 30°C or 60°C with 0.2 M HCl (Fig. 3).

The quality of the model was confirmed by the averagevalue of the center points (5.76±1.01%DW) being close tothe coefficient of the constant (6.28±0.29%DW). Theregression model was given as y=6.28−1.17 x1+0.23 x2+0.63 x3−1.46 x1 x1−0.35 x2 x2+0.31 x3 x3−0.36 x1 x2−0.51 x1 x3−0.06 x2 x3 (x1 is acid concentration; x2 is time;

x3 is temperature; Table 1). The summary of the fit and thepredictability of the model for FCSPs yield were satisfac-tory with R2=0.928; Q2=0.853, model validity=0.891;and model reproducibility=0.889 (Fig. 3).

Plots of the polysaccharide yields and fucose contentsobtained in response to extraction time and temperaturefurther illustrated the effects of the different treatmentfactors during single-step extraction (Fig. 4). With no acidtreatment, the yield of polysaccharide rose when extractiontime and temperature increased, while high acid treatment(0.2 M HCl) gave low yield with no significant differenceswith elevated time and temperature (Fig 4a). After each ofthe different extraction treatments of the Sargassum sp.seaweed, the recovered polysaccharide was sequestered andsubjected to TFA hydrolysis and thereafter HPAEC toanalyze the monomer content. The results showed thatthe fucose content was higher in the polysaccharideextracted with 0.2 M HCl than without acid when thetreatment time was 1 or 3 h, but at 90°C/5 h, the fucoselevel was higher with no acid than with 0.2 M HCl(Fig. 4b). The influence of acid treatment (0.2 M HCl)during extraction was thus observed to have a negativeeffect on the fucose content when time and temperaturewere elevated (Fig. 4b).

Comparative extraction analysis

The optimal condition of the single-step extraction pre-dicted by the model (i.e., 0.03 M HCl/90°C/4 h) was usedin an actual extraction experiment designated as Method 1afor comparative analysis of the monosaccharide composi-tion with the benchmark extraction methods of Bilan et al.(2002): Method 2 and Black et al. (1952): Method 3. Themonosaccharide composition of the polysaccharide showedthat fucose, galactose, and glucuronic acid were thedominant monomers (Table 2). Clearly, the fucose contentof the statistically optimized one-step extraction methodwas significantly higher than the fucose levels in thefucoidan (or FCSPs) obtained by both Method 2 and 3(Table 2). Method 2 had the lowest glucuronic acid contentamong the three methods (Table 2); this was probably dueto the interaction of CaCl2 after each round of extraction.The sulfate content of the fucoidan polysaccharide washowever lower with the optimized one-step 4-h extractionthan that obtained in Method 2 and 3.

Investigation of extended extraction time

Based on the predicted optimized extraction model (Fig. 3)and the raw data (Fig. 4), it was decided to evaluate theinfluence of extended extraction time on the extractedpolysaccharide composition. Extended extraction was car-ried out for up to 46 h using the predicted one-step optimal

Table 1 Multiple linear regression results of the parameters andinteractions on the polysaccharide yield using the single-stepextraction

Parameters and interactions % DW

Coefficient P values

Acid (x1) −1.17 3.46×10−10

Time (x2) 0.23 0.045

Temperature (x3) 0.63 7.11×10−6

Acid×acid −1.46 1.96×10−7

Time×time −0.35 No effect

Temperature×temperature 0.31 No effect

Acid×time −0.36 0.006

Acid×temperature −0.51 0.0004

Time×temperature −0.06 No effect

Constant 6.28 2.56×10−22

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treatment, i.e., 0.03 M HCl/90°C. The duration ofextraction time influenced the polysaccharide yield(Fig. 5a). Hence, the total polysaccharide yield increaseduntil 8 h of extraction (using 0.03 M HCl/90°C) and theyield then reached a plateau of approximately 9% DW(Fig. 5a). The amount of fucose dropped steadily as theduration of the extraction time increased whereas glucur-onic acid increased (Fig. 5a). Apparently, the duration ofextraction time also resulted in an almost linear increase inthe mannose and galactose content of the extractedpolysaccharide, but did not affect the xylose, glucose,and rhamnose contents (Fig. 5b). In addition, the extendedextraction time significantly decreased the sulfate contentuntil 8 h of extraction (Fig. 5b). These results indicatedthat obtaining a high yield of a fucose-rich fucans fromSargassum sp. having a limited glucuronic acid (andgalactose and mannose) content was a compromise, andthe data confirmed that an extraction time of 3 h was thebest compromise to achieve high yields and a high fucoselevel (Fig. 5a). It is tempting to speculate that some “true”fucoidan is released in the early minutes of the extraction,that the acid catalyzed loss of α-fucosyl linkages relativeto the other glycosidic bonds during extended treatmentmay confound the picture. One could interpret theevidence as indicating that the product isolated containeda sulfated fucose-containing heteroglycan, possibly havinga glucuronan primary structure with extensive fucosyl sidebranches which are cleaved and lost as extraction time isextended. A sulfated fucan (a fucoidan) may occur inSargassum sp., but further elucidation of fucose-containing sulfated polysaccharides from Sargassum sp.is required. With a lower total polysaccharide yield

requirement, a relatively higher fucose content could beobtained with a shorter extraction of 1 h as compared to a4 h extraction (Table 2 and Fig. 5).

Discussion

Statistically designed optimization of FCSPs extraction wasconducted to produce a model that provided an understand-ing of the complex influences and interactions of theextraction factors temperature, time, and acid concentration,and in turn allowed prediction of the optimal extractiontreatment to obtain high FCSPs yield. The model predictedthat 0.07 M HCl/90°C/3 h produced high FCSPs yieldusing two-step extractions. However, careful assessment ofthe data obtained after the first and second steps at theseoptimal extraction conditions showed that an additionalextraction step to produce Extract B (Fig. 1b) could beomitted since its yield was very low compared to Extract A(Fig. 2). Hence, a new optimized extraction design wasperformed for a single-step extraction using the samefactors settings, i.e., acid, 0 to 0.2 M HCl; temperature,30 to 90°C; and time, 1 to 5 h. The maximum yieldproduced by this new optimal condition (0.03 M HCl/90°C/4 h) was ∼7.0% DW. Previously reported multi-stepextraction results of fucose-containing polysaccharidesfrom S. horneri and U. pinnatifida were 5.17% DW and6.77–15.10% DW (Kuda et al. 2002); from S. ring-goldianum it was 200 mg from fresh 150 g algal fronds,approximately equivalent to 0.67% DW (Mori and Nisizawa1982). Hence, the result of multiple linear regressions of theparameters and interaction on the fucose-containing poly-

Fig. 3 The 3D response surface plots at three different temperatureseach at the optimal extraction condition and maximum fucose-containing polysaccharide yield (% DW) of the single-step extraction

procedure as a function of time (h) and HCl concentration (M). R2=0.928; Q2=0.853; model validity=0.891; model reproducibility=0.889 respectively

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saccharide yield for the one-step extraction was in agreementwith the available data.

The composition obtained from HPAEC-PAD analysis ofthe TFA hydrolysate of Sargassum sp. agrees with theprevalent polysaccharide structure among Sargassumspecies; hence, in the case of S. patens and S. stenophyl-lum (Duarte et al. 2001; Zhu et al. 2003) the structure hasbeen found to be a linear backbone of (1→6)-β-D-galactose and/or (1→2)-β-D-mannose units with branchedchains formed by (1→3) and/or (1→4)-α-L-fucose, (1→4)-α-D-glucuronic acid, terminal β-D-xylose and sometimes(1→4)-α-D-glucose. However, as pointed out by Percivaland McDowell (1967): "Fucoidin, first isolated and namedby Kylin (1913) was more systematically named fucoidan".The algal source in this case was F. vesiculosus, and theKylin product has been established as a fucan sulfate(currently termed fucoidan as it was isolated from brown

seaweed). Percival and McDowell understood and empha-sized that fucoidan referred to polysaccharides consistingalmost entirely of fucose and ester sulfate. Fucose-containingheteropolysaccharides (e.g., glucuronoxylofucans) were trea-ted as quite different entities from fucoidan. Painter (1983)provided the following definition: "Natural polysaccharidesbuilt up essentially of sulfated α-L-fucose residues areknown as fucoidans". This working definition of fucoidanhas been retained by polysaccharide chemists to the present(Mabeau et al. 1990; Chevolot et al. 1999; Berteau andMulloy 2003).

The optimization of the one-step method illustrated thata single-step extraction with the combination of low acidconcentration, 0.03 M HCl or below, and temperature near90°C was sufficient to produce a satisfactory FCSPs(fucoidan) yield. The integrity of the polysaccharide wasbest conserved at low acid treatment (Fig. 4b), since the useof 0.2 M HCl apparently broke the integrity of thepolysaccharide molecules resulting in a decline of fucoseat elevated time and temperature (Fig. 4b). This indicatedthat the higher acid levels might have caused a loosening ofthe cell wall matrix allowing local penetration of the acidinto the fucoidan in the intercellular spaces resulting inpartial degradation of FCSPs or notably fucoidan (Kloareg1984; Mabeau et al. 1990). The effect of acid might havebeen enhanced as dried seaweed material was used in theexperiment, where it can absorb and expand abruptlyduring hydration (Phillips et al. 2002). The degradation ofcarbohydrate chains built up of fucose was recognizedwhen the duration of extraction time increased (Fig. 5). Ourresult is in agreement with Ponce et al. (2003) who reportedthat longer extraction time led to poorer fucose content. In

Table 2 Analysis of monosaccharide after 2 M TFA hydrolysis of thesulfated polysaccharide fractions of Sargassum sp. isolated bydifferent extraction methods

Monomers(mg/g DW)

Method 1a(1 step—1 h)

Method 1a(1 step—4 h)

Method 2 Method 3

Fucose 113±0.9 97±2 49.2±1.7 54.8±1.7

Rhamnose 6.2±0.6 2.9±0.3 6.29±0.07 nd

Galactose 39±6 39 ±1.9 25.8±0.30 36±1.34

Glucose 11.1±0.2 3.7±0.4 10.9±0.13 13.7±1.25

Mannose 11.3±1.6 11.1±1.7 2.41±0.03 5.76±0.21

Xylose 15.7±1.1 11.4±1.4 5.12±2.36 6.8±1.7

Gluc Acid 85±11.7 97±2.7 10.0±0.12 45±5.8

Sulfate 362±16.5 308±11 341±5.2 327±8.5

Data given as average values±standard deviation, n=3. Method 1aextraction by using the optimized model of the one-step extraction(0.03 M HCl/90°C) with an extraction time of either 1 h or 4 h asindicated. Method 2 as described by Bilan et al. (2002), and Method 3as described by Black et al. (1952). General linear model significantlydifferent (P<0.05)

Temperature [°C]

Po

lysa

cch

arid

e yi

eld

[%

DW

]

2

4

6

8

10 0 M HCl0.2 M HCl

Time [h]

30 60 90

1 3 5

Fu

cose

[m

g/g

DW

Po

lysa

cch

arid

e]

0

2

4

6

8

10

12

14

16

18

0 M HCl0.2 M HCl

Fig. 4 Polysaccharide yield (a) and fucose content (b) of differentcombination of extraction treatment (i.e., acid, time and temperature)of fucose-containing polysaccharide seaweed (Sargassum sp.). Datagiven as mean±SD, n=3

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addition, longer extraction time at higher temperatures ledto higher polysaccharide yield with lower amount of sulfateand higher proportion of glucuronic acid (Ponce et al. 2003;Duarte et al. 2001). The same trend was also noticed in ourpresent work. The glucuronic acid increased while sulfatedecreased with time, i.e., the sulfate content was highestwhen glucuronic acid was lowest (Fig. 5a, b). This was alsoobserved for fucoidan extraction from S. ringgoldianum(Mori and Nisizawa 1982).

In conclusion, a simple and practical method forrecovering a suite of complex fucose-containing sulfatedpolysaccharides from Sargassum sp., has been established.Clearly, yield and chemical composition of the product arestrongly affected by the method of extraction as was to beexpected. The yield data are gravimetric only and so pertainto a crude mixture of biopolymers extracted. The evidencepresented shows that the extracted polysaccharide productis heterogeneous at any time it is analyzed, although thecomposition varies with the duration of extraction. Themonomeric composition shows that fucose and sulfate wereimportant components of the polysaccharide mixture as

isolated. An optimized one-step extraction treatment toobtain high yields of a fucose-containing sulfated polysac-charide from Sargassum sp. was developed, and the effectof different treatment parameters on the integrity of thepolysaccharide was established. The results confirmed thatSargassum sp. may be a good source of fucose-containingsulfated polysaccharides. The data also demonstrated thevulnerability of fucose-containing sulfated polysaccharidesto harsh extraction conditions and confirmed that theextraction method significantly influences the yields andnot least the polysaccharide composition of the extractedpolysaccharide. Furthermore, the main conclusions confirmthe long known facts that cell wall polymers of brown algaeare complex, and that the yields and chemical nature ofpolysaccharides recovered from such seaweeds are mark-edly influenced by the conditions used to extract them. It isimportant to emphasize this point as it has a major bearingon any study in which such products are being evaluatedfor biological activity. It is our belief that the modelobtained may be applied to other FCSPs or fucoidan-containing types of brown seaweed.

Po

lysa

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arid

e Y

ield

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cose

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nic

Aci

d [

mg

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W]

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300Polysaccharide YieldFucoseGlucuronic Acid

Extraction Time [h]0 10 20 30 40 50

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, Man

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lfat

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GalactoseMannoseXylose GlucoseRhamnoseSulfate

a

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Fig. 5 Polysaccharide yield andmonosaccharide content as aresult of prolonged reaction timeusing the predicted optimizedone-step extraction condition0.03 M HCl/90°C. a Polysac-charide yield, fucose andglucuronic acid, b galactose,mannose, xylose, glucose,rhamnose and sulfate

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