chemical structure and anticoagulant activity of highly pyruvylatedsulfated galactans from tropical...

Please citefrom trop

ARTICLE IN PRESSG ModelCARP-9383; No. of Pages 11Carbohydrate Polymers xxx (2014) xxxxxx

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Carbohydrate Polymers

j ourna l ho me page: www.elsev ier .com/ locate /carbpol

Chemical structure and anticoagulant activity of sulfated galactans from tropical green seaweeds Bryops

Paula X. iz EReinaldoa Ctedra de Qu gronomAv. San Martnb Laboratorio d xactasCiudad Universc Escuela de Bio nueva1051 Caracas, Venezuelad Escuela de Qumica, Facultad de Ciencias, Universidad Central de Venezuela, Av. Paseo de los Ilustres, Ciudad Universitaria, Los Chaguaramos,1450 Caracas, Venezuelae Laboratorio de Ecologa y Taxonoma de Macrtas Marinas, Centro de Botnica Tropical, Instituto de Biologa Experimental,Universidad Central de Venezuela, Apdo.47114, Caracas, Venezuelaf CIHIDECAR-CONICET, Departamento de Qumica Orgnica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina

a r t i c l

Article history:Received 18 JuReceived in re24 SeptemberAccepted 8 OcAvailable onlin

Keywords:Sulfated galacPyruvic acid kGreen seaweeAnticoagulantFibrin formati

1. Introdu

There ispolysaccharthe Bryopsiof them gaUsov, 2007;et al., 2012)

Corresponde Biologa ApAires, Av. San Tel.: +54 11 45

E-mail add1 These auth2 Research M

http://dx.doi.o0144-8617/ this article in press as: Arata, P. X., et al. Chemical structure and anticoagulant activity of highly pyruvylated sulfated galactansical green seaweeds of the order Bryopsidales. Carbohydrate Polymers (2014), http://dx.doi.org/10.1016/j.carbpol.2014.10.030

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2014tober 2014e xxx

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Sulfated and pyruvylated galactans were isolated from three tropical species of the Bryopsidales, Penicilluscapitatus, Udotea abellum, and Halimeda opuntia. They represent the only important sulfated polysac-charides present in the cell walls of these highly calcied seaweeds of the suborder Halimedineae. Theirstructural features were studied by chemical analyses and NMR spectroscopy. Their backbone comprises3-, 6-, and 3,6-linkages, constituted by major amounts of 3-linked 4,6-O-(1-carboxy)ethylidene-d-galactopyranose units in part sulfated on C-2. Sulfation on C-2 was not found in galactans from otherseaweeds of this order. In addition, a complex sulfation pattern, comprising also 4-, 6-, and 4,6-disulfatedgalactose units was found. A fraction from P. capitatus, F1, showed a moderate anticoagulant activity, eval-uated by general coagulation tests and also kinetics of brin formation was assayed. Besides, preliminaryresults suggest that one of the possible mechanisms involved is direct thrombin inhibition.

2014 Elsevier Ltd. All rights reserved.

ction

still scarce information about structures of sulfatedides biosynthesized by green seaweeds belonging todales (Chlorophyta), however, it is known that in mostlactans predominate (Bilan, Vinogradova, Shashkov, &

Chattopadhyay, Adhikari, Lerouge, & Ray, 2007; Ciancia.

ding author at: Ctedra de Qumica de Biomolculas, Departamentolicada y Alimentos, Facultad de Agronoma, Universidad de BuenosMartn 4453, C1417DSE Buenos Aires, Argentina.24 8088/4042.ress: [email protected] (M. Ciancia).ors are equally responsible for this work.ember of the National Research Council of Argentina (CONICET).

Only the sulfated polysaccharides from some species ofthe genus Codium (Bilan et al., 2007; Ciancia et al., 2007;Estevez, Fernndez, Kasulin, Dupree, & Ciancia, 2009; Fariaset al., 2008; Fernndez, Ciancia, Miravalles, & Estevez, 2010;Fernndez, Estevez, Cerezo, & Ciancia, 2012; Fernndez et al.,2013; Love & Percival, 1964; Ohta, Lee, Hayashi, & Hayashi, 2009;Fernndez, Arata & Ciancia, 2014) have been studied in detail.They biosynthesize sulfated galactans constituted by 3-linked -d-galactopyranose residues partially sulfated on C-4 and/or C-6, withramications on C-6 and important amounts of pyruvate formingmainly ve-membered cyclic ketals (S conguration) with O-3 andO-4 of non-reducing terminal -d-galactose residues. A minor partof pyruvate forms six-membered cyclic acetals with O-4 and O-6(R conguration). On the other hand, the major sulfated polysac-charides from Bryopsis plumosa are linear 3-linked -d-galactanshighly pyruvylated and also partially sulfated mainly on C-6 of some

rg/10.1016/j.carbpol.2014.10.0302014 Elsevier Ltd. All rights reserved.idales

Arataa, Irene Quintanab,1, Dilsia J. Canelnc, Beatr S. Compagnonee, Marina Cianciaa,f,,1,2

mica de Biomolculas, Departamento de Biologa Aplicada y Alimentos, Facultad de A 4453, C1417DSE Buenos Aires, Argentinae Hemostasia y Trombosis, Departamento de Qumica Biolgica, Facultad de Ciencias Eitaria Pabelln 2, C1428EHA Buenos Aires, Argentinaanlisis, Facultad de Medicina, Universidad Central de Venezuela, Av. Carlos Ral Villahighly pyruvylatedof the order

. Verad,

a, Universidad de Buenos Aires,

y Naturales, Universidad de Buenos Aires,

, Ciudad Universitaria, Los Chaguaramos,

Please cite icoagfrom trop rs (2

ARTICLE IN PRESSG ModelCARP-9383; No. of Pages 112 P.X. Arata et al. / Carbohydrate Polymers xxx (2014) xxxxxx

of the galactose units. In this galactan, pyruvic acid was forminga ketal linked to O-4 and O-6 (R isomer) of some 3-linked units(Ciancia et al., 2012).

The order Bryopsidales has been divided into two suborders(Lam & Zecorder BryopPenicillus cato the subospecies, preorder, only Caulerpa sp2007; Mackdescribed aterminal- aresidues. Sulinked arabacid was infrom specieactivities, hzation of thGurgel RodRodrigues, 2008; Santo

Antithrotemic theraheparin is side effectsembolism (Anand, Yu1980), anddiseases in ulant treatmof anticoagu

Many distudied pro& Cerezo, 2of action cto the fact protease. Tedge of spetheir interacascade, coagents. Somicant levelssystem; whshow surprPomin, 200sulfation, ancarbohydraanticoagula

In this wgalactan sucapitatus, Utural studyfrom P. capi

2. Experim

2.1. Algal sa

Specimelected in thGmez froCentral de Vtuto Botni

according to Taylor (1960), P. capitatus, in Chichiriviche, stateof Falcn (102424 N, 68151 O) in June 2007; H. opuntia inTucacas, state of Falcn (105126 N, 681842 O) in May 2006;U. abellum in La Cinaga, Ocumare de la Costa, state of Aragua

21

ered wi

coner spezue5, reyzedep wn of n to

poltized

tract

rile ps rsiduetivelt-wation wtratextra

siduetrac

extrae lateated. abmpere wrried

& Ce

n exc

wasg) wpliedhe easin

Finace olsulth, 1d byere dried

emic

totaethod foulfat

whiing term. To eriv this article in press as: Arata, P. X., et al. Chemical structure and antical green seaweeds of the order Bryopsidales. Carbohydrate Polyme

hman, 2006). Codium and Bryopsis, belong to the sub-sidineae, whereas, the species studied in this paper,pitatus, Udotea abellum, and Halimeda opuntia, belongrder Halimedineae which comprises partially calciedsent in tropical and subtropical habitats. From this sub-some structural features of heteroglycan sulfates fromecies were previously reported (Chattopadhyay et al.,ie & Percival, 1961). That from Caulerpa racemosa wass a branched polymer containing, 3-linked galactose,nd 4-linked xylose, and 4- and 3,4-linked arabinoselfate groups, when present, were linked to C-3 of 4-inose and C-6 of 3-linked galactose units. No pyruvicformed (Chattopadhyay et al., 2007). Polysaccharidess of this genus were found to have different biologicalowever, in most of the reports, only minor characteri-e active compounds was carried out (Ghosh et al., 2004;rigues, de Sousa Oliveira Vanderlei, et al., 2011; Gurgelde Queiroz, et al., 2011; Ji, Shao, Zhang, Hong, & Xiong,s Pereira Costa et al., 2012).mbotic agents have been extensively used as a sys-py in cardiovascular or tromboembolic diseases andthe initial choice, nevertheless it can induce several, such as development of thrombocytopenia, arterial(Kelton & Warkentin, 2008), bleeding complicationssuf, Pogue, Ginsberg, & Hirsh, 2003; Kelton & Hirsh,

so on. Furthermore, the incidence of prion-relatedmammals and the increasing requirements of anticoag-ents indicate the need to look for alternative sourceslant and antithrombotic compounds.

fferent sulfated polysaccharides have been thoroughlyving to have anticoagulant effects (Ciancia, Quintana,010). The important differences in their mechanismsould be attributed to the diversity of structures andthat one compound may have more than one targethese differences denote the importance of the knowl-cic structural characteristics of these products and

ction with the different proteins involved in coagulationntributing for the development of new antithrombotice sulfated polysaccharides even though bearing signif-

of sulfation, have scarce effects toward the coagulationile others, even carrying lower sulfation content, canising levels of anticoagulant activity (Mouro, 2004;9). This observation has clearly proved the concept thatd therefore electronegative-charge densities, in marinetes are not the solely structural determinants for thent activities of these molecules.ork we isolated and characterized highly pyruvylated

lfates from three tropical species of the Halimedineae, P.. abellum, and H. opuntia. In particular, a detailed struc-

was carried out on the water soluble polysaccharidestatus and their anticoagulant activity was investigated.

ental

mple

ns of the green macroalgae studied here were col-e coast of Venezuela and identied by Dr. Santiagom Instituto de Biologia Experimental, Universidadenezuela and Dr. Mayra Garcia from Fundacin Insti-co de Venezuela, Universidad Central de Venezuela:

(1028work wwasheepizoicVouchof Ven40585hydrolrst stdilutiodilutiosolublederiva

2.2. Ex

Sterial wathe reexhausand hoextracconcenwater eThe rewas exwater ysis (sewas trfrom Ueach teperatuwas caStortz,

2.3. Io

PA1(100 mwas apH2O. Tof incrlected.presenpheno& Smireplacetions wfreeze

2.4. Ch

Theacid madapteused. S1962),accordwas de(1951)were d1991).ulant activity of highly pyruvylated sulfated galactans014), http://dx.doi.org/10.1016/j.carbpol.2014.10.030

N, 674839 O) in July 2008. The samples used in this in the vegetative state. Thalli of the seaweeds wereth ltered seawater and analyzed for epiphytic andtaminants in a Nikon AFX-II macroscope (Nikon, Japan).ecimens were deposited in the National Herbariumla (Collection Code 200706003, 200606024 and VENspectively). Each algal sample, previously milled, was

in conditions suitable for brillar polysaccharides, aas carried out in 100% TFA for 1 h at 37 C, followed bythe acid to 80%, heating at 100 C for 1 h, and further

2 M to achieve the regular hydrolysis conditions forysaccharides (Morrison, 1988); the sugar mixture was

to the corresponding alditol acetates (see below).

ion of the polysaccharides

lants of P. capitatus were freeze dried. The dry mate-st extracted with methanol at room temperature and

from the alcohol extraction was sequentially andy extracted with water (20 g/L) at room temperatureter. Briey, the residue of the rst room temperatureas removed by centrifugation and the supernatant wasd, dialyzed and freeze-dried. The residue from the rstction was extracted one more time in similar conditions.

from the second room temperature water extractionted twice for 3 h with water at 90 C, giving two hotcts, which gave similar characteristic by chemical anal-er) so they were studied together as one sample, which

with -amylase (Knutsen & Grasdalen, 1987). Thalliellum were extracted in a similar way, but only once atrature. On the other hand, the yield from the room tem-ater extract from H. opuntia was very low, so extraction

out in controlled acid conditions as described by Cases,rezo, 1992, for red seaweed Corallina ofcinalis.

hange chromatography (IEC)

chromatographed on DEAE-Sephadex A-25. The sampleas dissolved in water, centrifuged and the supernatant

to a column (90 1.5 cm id), previously stabilized inrst elution solvent was water and then NaCl solutionsg concentration up to 4 M. Fractions of 4 mL were col-lly, the phase was boiled in 4 M NaCl solution. Thef carbohydrates in the samples was detected by thefuric acid method (Dubois, Gilles, Hamilton, Rebers,956); after obtaining blank readings, the eluant was

another with higher concentration of NaCl. Seven frac-obtained, dialyzed (molecular weight cut off 3500) and

(F1F7).

al analyses

l sugars content was analyzed by the phenolsulfuricd (Dubois et al., 1956), in some cases, modicationr insoluble material (Ahmed & Labavitch, 1977) wase was determined turbidimetrically (Dodgson & Price,le the percentage of pyruvic acid was determinedto Koepsell and Sharpe (1952). The protein contentined by the method of Lowry, Rosenbrough, and Farr,determine the monosaccharide composition, samplesatized to the alditol acetates (Stevenson & Furneaux,


ARTICLE IN PRESSG ModelCARP-9383; No. of Pages 11P.X. Arata et al. / Carbohydrate Polymers xxx (2014) xxxxxx 3

2.5. Desulfation of F1 and F6

The reaction was carried out by the microwave-assisted methoddescribed by Navarro, Flores, and Stortz (2007). The sample (40 mg)was converDMSO contintervals antap water alyolized. Aprevious iso

2.6. Remov

The reacThe sample100 C, the lyophilizeddesulfated F

2.7. Methyl

The polysponding trand methylple was dissused as basand of CH3I were carrieof partially polysaccharmitted to racetates in t& Furneauxhydrolyzedylated sugaacetates (St

2.8. Gas chr

GC of thgasliquid ame ioniz(0.25 mm i.d(Supelco, Bfrom 200 Calditol acetinitial tempto 210 C anrier gas at aThe injector

2.9. GCMS

GCMS a Shimadzu2330 interfJapan) worinjector tema mass rang

2.10. NMR

500 MHzand two-direcorded onreference o

D2O (0.5 mL) four times. Chemical shifts were referenced to inter-nal acetone (H 2.175, CH3 31.1). Parameters for 13C NMR spectrawere as follows: pulse angle 51.4, acquisition time 0.56 s, relax-ation delay 0.6 s, spectral width 29.4 kHz, and scans 25,000. For 1H

pectrctraltand

reatm

10 m and

P 51nd thfreez

ener

testsAsnie (P

bin ti & Bgo, elizerl reaing toed cicchacuband dr th

solutrformmL) i

in qg timtrol

ibrin

rder by /mL)ation

min Inc.,

wernt co

or saere

s therresptwo

numrup

tatis

a wetical n s

ere cered this article in press as: Arata, P. X., et al. Chemical structure and antical green seaweeds of the order Bryopsidales. Carbohydrate Polyme

ted to the pyridinium salt and dissolved in 10 mL ofaining 2% of pyridine. The mixture was heated for 10 sd cooled to 50 C (6). It was dialyzed 3 days againstnd then 24 h against distilled water (MWCO 3500) andn aliquot was methylated as described below withoutlation of the product.

al of pyruvic acid residues from F1

tion was carried out according to Bilan et al. (2007). (50 mg) was heated in 1% CH3COOH (10 mL) for 4 h atsolution was neutralized with NaHCO3, dialyzed, and

to give depyruvylated product (21.5 mg). An aliquot of1 was treated in the same way to give F1desulfdepyr.

ation analysis

saccharide (1020 mg) was converted into the corre-iethylammonium salt (Stevenson & Furneaux, 1991)ated according to Ciucanu and Kerek (1984). The sam-olved in dimethylsulfoxide; nely powdered NaOH wase. For F1, different times between addition of the basewere assayed and also two sequential methylation stepsd out. However, no substantial changes in the patternmethylated derivatives obtained after hydrolysis of theide were observed. The methylated samples were sub-eductive hydrolysis and acetylation to give the alditolhe same way as the parent polysaccharides (Stevenson, 1991). In some cases, methylated samples were also

with 2 M TFA for 2 h at 120 C and the partially meth-rs were converted into the corresponding aldononitrileortz, Matulewicz, & Cerezo, 1982).

omatography

e alditol acetates were carried out on a Agilent 7890Achromatograph (Avondale, PA, USA) equipped with aation detector and tted with a fused silica column. 30 m) WCOT-coated with a 0.20 m lm of SP-2330

ellefonte, PA, USA). Chromatography was performed: to 230 C at 1 C min1, followed by a 30-min hold forates. For the partially methylated alditol acetates, theerature was 160 C, which was increased at 1 C min1

d then at 2 C min1 to 230 C. N2 was used as the car- ow rate of 1 mL min1 and the split ratio was 80:1.

and detector temperature was 240 C.

of the methylated alditol acetates was performed on GC-17A gasliquid chromatograph equipped the SP-aced to a GCMSQP 5050A mass spectrometer (Kyoto,king at 70 eV. He total ow rate was 7 mL min1, theperature was 240 C. Mass spectra were recorded overe of 30500 amu.

spectroscopy

1H NMR, proton decoupled 125 MHz 13C NMR spectra,mensional NMR experiments (HMQC and COSY) were

a Bruker AM500 at room temperature, with externalf TMS. The samples (20 mg) were exchanged in 99.9%

NMR s3 s, speusing s

2.11. T

F3 ((1 mL)(Sigma37 C aL) and

2.12. G

TheStago, bin timthrom(Laffantica Staneutramerciaaccorddepletpolysaand inUSA) aused foSaline also pe(3 mg/formedclottinthe con

2.13. F

In oerated(0.5 IUcoagulwith 1mentsAssaysdifferesulfatetime) wreectthat conal neby thein quad

2.14. S

Dat(Analyas meaples wconsidulant activity of highly pyruvylated sulfated galactans014), http://dx.doi.org/10.1016/j.carbpol.2014.10.030

a: pulse angle 76, acquisition time 3 s, relaxation delay width 6250 Hz and scans 32. 2D spectra were obtainedard Bruker software.

ent with pronase E

g) was dissolved in a phosphate buffer 0.2 M at pH 7.23 mg of protease Type XIV from Streptomyces griseus47) were added. The mixture was agitated for 24 h aten dialyzed (MWCO 68 kDa) against distilled H2O (1e dried.

al coagulation assays

were performed with a coagulometer ST4 (Diagnosticaeres sur Seine, France). Determinations of prothrom-T), activated partial thromboplastin time (APTT), andme (TT) were assayed according to established methodsradshaw, 1995). Reagents were supplied by Diagnos-xcept tromboplastin reagent. Since polybrene, a known

of heparin, is added to the majority of the PT com-gents tromboplastin was extracted from rabbit brain

the method described by Quick (1935). Normal platelettrated plasma (900 L) was mixed with 100 L of eachride samples, in different concentrations (test solution),ted for 1 min at 37 C. Heparin (Sigma, St. Louis, MO,ermatan sulfate (Syntex, Buenos Aires, Argentina) weree comparison of anticoagulant activity of the fractions.ion (0.9% NaCl) was used as control. TT-like assays wereed with puried brinogen (Sigma, St. Louis, MO, USA)

nstead of human plasma. All clotting assays were per-uadruplicate. Results were expressed as ratios betweene of a solution of the anticoagulant and clotting time of.

formation

to perform brin formation studies, clots were gen-addition of thrombin (Wiener, Rosario, Argentina)

to the preincubated plasma (test solution). During the process, optical density (OD) was recorded at 405 nmintervals up to constant values (ELx808, BioTeck Instru-Winooski, VT, USA) (Weisel, Veklich, & Gorkun, 1993).e carried out in polystyrene strips, in the presence ofncentrations (550 g/mL) of F1, heparin, dermatanline solution as control. The curves obtained (OD versus

characterized by three parameters: the lag phase, that time required for initial protobril formation; the slope,onds to the maximum velocity achieved (VMax) and therk OD (ODMax) at the plateau phase, which is inuencedber of protobrils per ber. All assays were performedlicate.

tical analysis

re analyzed using the statistical software Statistix 8Software, Tallahassee, FL, USA). Results were expressedtandard deviation (SD). F1-treated and control sam-

ompared using Students t test, and p values < 0.05 were statistically signicant.



3. Results and discussion

3.1. Extraction and characterization of the sulfatedpolysaccharides

Monosaeach specieponents (Min agreeme-d-xylans& McDoweglucose and

Yields in(Table 1). LoBryopsidaleeven lowerof calcium imum degr1973). Conscase was cain these coincreased frtatus and U38%, respec(Bhm, 197

P. capitaroom tempgalactose wsmall amoucose which-glucans. other handThe ratio gaextract. Theuct (PA2) wextraction wstituted motogether asa low mole 100.3/5.3and terminaregion, and61.5/3.88,34-linked unMoreover, tonly 36% ofurther.

The roommostly conspyruvic acidacid of 1.00found in thwater, obtaalso glucosethe extractperature atas major mtose:sulfatethe residue not analyze

3.2. Structu

PA1 frommatographyand then w

were isolated (Table 2). The major fraction, F1, eluted with water,in spite of the fact that it has a signicant percentage of sul-fate and pyruvic acid, giving a ratio galactose:sulfate:pyruvic acidof 1.00:0.84:0.49. Only a minimum quantity of protein was still

t (1.1respeein des ofwed lfate.ctural melectubmvylavylaed (T

thathievesulthe , ther

4). M wasd aning o co

imped bpercetly, n F1.yruvi

to O, 10

subs, 200t in nkedgalacce o, -danal

of specmerdisplce o

and remted gl as nits. r by

ed todingpectted t

assigCane

et a, 199e ofut in this article in press as: Arata, P. X., et al. Chemical structure and antical green seaweeds of the order Bryopsidales. Carbohydrate Polyme

ccharide composition of milled seaweed material fors, in condition suitable to hydrolyze the brillar com-orrison, 1988), showed xylose as major sugar (5055%),nt with previous ndings that indicated that 3-linked

replace cellulose in seaweeds of these genera (Percivalll, 1981). The other important monosaccharides were

galactose. water soluble polysaccharides were extremely loww yields were also obtained for water extracts of others (Ciancia et al., 2007, 2012), but in these cases, the

yields are due at least in part to the important amountcarbonate deposited (aragonite). For H. opuntia a max-ee of 90% calcication was previously reported (Bhm,equently, extraction of soluble polysaccharides in thisrried out at controlled acid pH (Cases et al., 1992), and,nditions yield of the room temperature water extractom 0.04% to 0.22%, still very low. Besides, for P. capi-. abellum a maximum degree of calcication (56% andtively), not so high, but still important, was informed3).tus was extracted with water sequentially twice aterature and at 90 C (Table 1). In the rst extract (PA1),as the major monosaccharide component, althoughnts of other sugars were also present, mainly glu-

could arise from contaminant polymers, as reserveThe percentage of uronic acids was negligible. On the, proteins were present in important amounts (20.7%).lactose:sulfate:pyruvic acid was 1.00:0.62:0.56 for this

second extraction at room temperature gave a prod-ith only 50% galactose, but 31% of glucose. Moreover,ith hot water gave two extracts (PC1 and PC2) con-

stly by glucose (Table 1), which were further analyzed one fraction. NMR spectra of this fraction, conrmedcular weight 4-linked -glucan structure, as signals at4, 96.8/4.59, and 93.0/5.16, corresponding to 4-linkedl - and -reducing units were present in the anomeric

peaks at 72.4/3.54, 73.63.68, 77.6/3.59, 70.9/3.90, and.76, which were assigned to C-2/H-2C-6/H-6,6 of theits (McIntyre & Vogel, 1993; Synytsya & Novak, 2013).reatment of this sample with -amylase gave PCa withf glucose. Thus, these extracts were not investigated

temperature water extract from U. abellum (UA) istituted by galactose, important amounts of sulfate and

were also present in a ratio galactose:sulfate:pyruvic:0.51:0.56, just a small amount of protein (4.5%) wasis extract. The residue was then extracted with hotining UC, which has important amounts of galactose, but, so only UA was selected for further analyses. Besides,

obtained from H. opuntia by extraction at room tem- low and controlled pH (HA) contained also galactoseonosaccharide component (77.2%) and a ratio galac-:pyruvic acid of 1.00:0.78:0.26. Hot water extraction ofgave only trace amounts of polysaccharides, which wered.

ral studies of sulfated galactans

P. capitatus was fractionated by anion exchange chro- on Sephadex A-25. The sample was eluted with waterith increasing NaCl concentrations and seven fractions

presen46.6%, of protcentagF6 shoand sutively)

Struchemicwas swas sdepyrudepyruanalyztion towas acslight dof F1, dation(Tablecedureshoweindicatsible tunits.

An detectsmall sequenunits ithat plinked 177.0of this& Usovpresento 3-liminal presenmainlylation spectrahand, of anoshow sequenFigs. 1

Thedisulfaas weltose uF1depyassignresponCOSY sattribuscopic2007; CianciaCerezo

Somof F6 bulant activity of highly pyruvylated sulfated galactans014), http://dx.doi.org/10.1016/j.carbpol.2014.10.030

%). Most of the protein appeared in F3 and F4 (32.0 andctively). By treatment of F3 with pronase E, the amountecreased to 22.3%. Taking into account the small per-

these fractions, they were not studied further. F5 andimportant amounts of carbohydrates, mainly galactose,, but small amounts of protein (4.3 and 1.4%, respec-

al analysis was carried out on F1 and F6 byethods and NMR spectroscopy; the latter fraction

ed due to the high yield and sulfate content. F1itted to methylation, desulfationmethylation, andtionmethylation procedures. The desulfated andted derivatives obtained, F1desulf and F1depyr, wereable 3), showing a similar monosaccharide composi-

of the parent sample, and that the expected reactioned. In the case of the depyruvylation procedure, onlyfation occurred. However, for the modied derivativesmethylation reaction gave a certain degree of degra-efore, these results should be taken only qualitativelyoreover, a sequential desulfationdepyruvylation pro-

carried out on F1, and methylation analysis (Table 4) important amount of terminal units for this sample,partial depolymerization. Nevertheless, it was pos-nrm that this galactan has 3-, 6-, and 3,6-linked

ortant amount of non-methylated galactose wasy methylation of F1 in different conditions, but onlyntages were found in the modied derivatives, con-it was attributed to completely substituted galactose

The rst structural evidence from the NMR spectra isc acid is forming a 6-membered ring R-conguration-4 and O-6 of some of the galactose units (signals at1.7, and 26.0/1.41 correspond to C-1 C-2 and C-3/H-3tituent, Table 5, Fig. 1) (Bilan, Vinogradova, Shashkov,6). Methylation analysis showed that this substituent,half of the whole structural units, could be linked

galactose (2-sulfate), and, in minor amounts, to ter-tose residues. These results were conrmed by the

f important quantities of 3-linked -d-galactose and,-galactose 2-sulfate in F1depyr, detected by methy-

ysis, which were conrmed by analysis of the NMRthis derivative (Tables 4 and 5, Fig. 2). On the othertra of the desulfated sample showed the absenceic signals at 103.6/4.79 and 103.5/4.85 ppm, whichacement to higher elds of the carbon signals, con-f the presence of a sulfate group on C-2 (Table 5,2).aining units comprise 3-linked 6-sulfated and 4,6-alactose, the latter, more important in F6 (see later),6- and 3,6-linked, possibly in part 4-sulfated galac-6-Sulfation was very clear in the spectra of F1 and

the presence of a signal at 68.0/4.30 which was C-6 of 3-linked galactose 6-sulfate units. The signal cor-

to C-5/H-5 was deduced by proton correlation in therum. On the other hand, the signal at 75.9/4.99 waso C-4/H-4 of 6-linked galactose 4-sulfate units. Spectro-nment is in agreement with previous data (Bilan et al.,ln, Ciancia, Surez, Compagnone, & Matulewicz, 2014;l., 2012; Ferreira et al., 2012; Stortz, Bacon, Cherniak, &4).

the structural units of F1 are also part of the structure different quantities, with higher degree of sulfation



Table 1Yields and analyses of extracts obtained from Penicillus capitatus (PA, PC), Udotea abellum (UA, UC), and Halimeda opuntia (HA) by extraction with water.

Extracta Yieldb % Carbohydrates %, anhc Sulfate as SO3Na % Monosaccharide composition (moles %)

Rha Fuc Ara Xyl Man Gal Glc

PA1e 0.3 34.2 10.0 2.0 4.6 3.2 5.6 77.0 7.5PA2 0.05 28.2 10.4 1.5 4.9 1.4 5.3 5.9 49.9 30.6PC1 1.3 73.4 2.4 Tr.d Tr. 5.9 93.1PC2 0.5 72.2 2.9 Tr. Tr. Tr. Tr. 6.9 93.8PCa 33.7 n.d. n.d. 1.9 2.1 Tr. 3.5 3.8 52.6 36.0UAe 1.4 55.1 16.8 1.9 1.8 1.6 94.7 Tr.4UC 0.6 44.6 21.2 2.4 Tr. 2.9 4.8 2.7 56.4 30.7HAe 0.22 44.3 21.7 1.0 1.3 2.3 2.1 9.8 77.2 6.1

a Analysis of PC1 and PC2 were similar, so they were worked out together. After treatment with -amylase, PCa was obtained.b For 100 g of the residue from the methanolic extraction. For PCa, yield of the enzymatic treatment of PC.c Values obtained by method for insoluble material.d Tr = traces.e 8.0, 16.3, and 6.2% of pyruvic acid for PA1, UA and HA, respectively.

Table 2Yields and analyses of the fractions obtained by anion exchange chromatography of the room temperature water extract from Penicillus capitatus (PA1).

Fraction Prote

F1b 1.1 F3c 32.0 F4 46.6 F5 4.3 F6b 1.4

a 70.0% of P hich w1% of PA1, so t

b 6.4 and 6.5c After treat

Table 3Yields and ana

Fraction

F1a

F1depyr F1desulf

a Included f

and lower apyruvylatedspectroscopthese units)disulfate unnot detecteof these unF1 and F1de

Results room temp

Table 4Methylation a

Monosaccha

2,3,4,6-Gal 2,4,6-Gal 2,3,4-Gal 4,6-Gal 6-Gal 2,3-Gal 2,4-Gal 2-Gal 3/4-Gal Gal

a Small percpolysaccharideEluant NaCl, M Yielda % Carbohydrates %, anh Sulfate as SO3Na %

60.9 29.3 12.9 0.5 5.3 25.1 9.7 0.75 11.9 24.0 8.7 1.0 5.3 54.8 23.1 1.5 15.6 45.9 35.3

A1 was recovered. Fractions F2, which eluted with 0.25 M NaCl solution, and F7, whey were not included in the table.% of pyruvic acid for F1 and F6, respectively. this article in press as: Arata, P. X., et al. Chemical structure and anticoagical green seaweeds of the order Bryopsidales. Carbohydrate Polymers (2

ment with protease, the carbohydrate content was 34.9% and the percentage of protein,

lyses of the products obtained by depyruvylation and desulfation of F1.

Gal:Sulf:Pyr molar ratio Monosaccharide composition (moles %)

Rha Fuc Ara

1:0.8:0.5 2.4 3.5 0.4 1:0.6:0.0 4.4 2.1 1:0.1:0.5 5.4 3.0 3.8

or comparison.

mounts of pyruvylated units. Accordingly, non-sulfated 3-linked galactose units were not detected by NMRy (absence of the signal at 79.5/4.16 ppm, due to C-3 of, while signals corresponding to 3-linked galactose 4,6-its were clear. Besides, 6-linked galactose 4-sulfate wasd in F6 (peak at 76.0/4.99, corresponding to C-4/H-4its was not found), while it is present in the spectra ofpyr.from methylation analysis and NMR spectra of theerature aqueous extracts from U. abellum and

H. opuntia, ilar also topyruvylatedthe latter sanalysis of monosacchgalactose 6units. The pspond to C-at 70.4/4.04

nalysis of fractions F1, F6 and of their modied derivatives, and of UA and HA.a

ride F1 F1depyr F1desulf F1desulf, depyr

Tr. 15 5 27 7 18 11 20 3 3 5 4 12 12 4 1 Tr. 9 4 15 4 26 33 47 36 21 10 4 4 2 2 Tr. 32 2 1 Tr.

entages of derivatives of glucose, mannose, rhamnose, and xylose were detected in somes and they were not included in the table.in % Monosaccharide composition (moles %)

Rha Fuc Ara Xyl Man Gal Glc

2.4 3.5 0.4 1.6 4.0 82.0 6.23.0 4.0 2.2 5.1 7.9 67.7 10.01.3 1.5 0.5 2.6 4.3 86.5 3.31.6 1.9 0.9 2.6 3.6 87.3 2.20.3 0.7 0.1 1.2 0.7 95.7 1.2

as obtained after boiling the phase in 4 M NaCl, comprised less thanulant activity of highly pyruvylated sulfated galactans014), http://dx.doi.org/10.1016/j.carbpol.2014.10.030

22.3%.

Xyl Man Gal Glc

1.6 4.0 82.0 6.23.8 3.9 80.3 5.52.4 2.1 79.3 4.1

UA and HA (Table 4, Fig. 3), show a similar pattern, sim- those of F1 and F6. Both have important amounts of

3-linked -d-galactose units partially sulfated on C-2,ubstitution being more important for HA. MethylationUA and HA showed 2,4-di-O-methylgalactose as majoraride derivative, which could correspond to 3-linked-sulfate or 3,6-linked galactose, or to a mixture of theseresence of a signal at 68.0/4.30, which could corre-6/H-6 would indicate 6-sulfation. However, the signal,3.83, which was previously assigned to C-6/H6,6 of

F6 F6desulf UA HA

12 3 10 7 9 9 7 3 6 4 5 2 1

24 12 3 730 42 38 3120 7 25 21 3 6 2

13 4 10 21

of the samples, but they were considered to arise from contaminant



Table 5NMR signal assignments (ppm) of substituted galactose units found in galactans from Penicullus capitatus, Udotea abellum and Halimeda opuntia and their modiedderivatives.a

Structural unitb Chemical shifts, ppm Detected clearly in the spectrum ofb

C-1/H-1 C-2/H-2 C-3/H-3 C-4/H-4 C-5/H-5 C-6/H6,6

3G2S 103.6/4.79 79.0/4.40 81.0/4.27 70.1/3.70c 76.0/3.62 62.0/3.70 F1depyr3G 104.9/4.66 70.3/3.67c 83.0/3.81 69.3/4.13 76.0/3.62 62.0/3.70 F1depyr3G6S 104.9/4.66 70.3/3.67c 84.8/3.73 69.3/4.13 74.2/3.84 68.0/4.30c F1depyr3,6G 104.9/4.66 71.1/3.71 83.0/3.81 69.3/4.22 74.2/3.98 70.4/4.04,3.83 F1desulf3G4,6S 105.4/4.53 71.8/3.54 77.2/4.01 78.2/4.79 74.2/3.95 67.8/4.23c F63GP,2S 103.5/4.85 76.2/4.48 77.2/4.39 71.8/4.11 66.9/3.54 65.9/3.85,3.97 F13GP 104.2/4.46 71.9/3.54 79.5/4.16 71.8/4.11 66.9/3.54 66.0/3.85,3.97 F1desulftG 105.4/4.53 71.8/3.53 73.7/3.57 69.5/3.92 76.0/3.62 62.0/3.70 F1depyrtGP 104.2/4.46 72.4/3.64c 72.1/3.62c 71.9/4.05 66.9/3.54 66.0/3.85,3.97 F1desulf

a Signals at 177.0, 101.7, and 26.0/1.41 were assigned to C-1, C-2, and C-3/H-3 of pyruvic acid forming a 6-membered ring R-conguration. They were present in spectraof all the samples, with the exception of F1depyr.

b Fraction where the unit was present in important amounts, bearing full assignment, diagnostic peaks were present in several spectra for each unit.c Assignments could be interchanged.

3,6-linked galactose units was not detected, in the HMQC of UA orin that of HA, indicating that these units are not important. This factwould indicate a low degree of ramication. Also, 3-linked non-sulfated -d-galactose units are present in signicant amounts.In the 13C NMR spectrum of HA there are minor signals in theanomeric region at 102.7 and 99.1, which were not assigned.The fact that small amounts of mannose and glucose are presentin this extract suggests that they could derive from contaminatingstructures.

3.3. Sulfated galactans from the Halimedineae

As far as of water soferent geneinformationrides from tMackie & Pe

These reBryopsidalepolysaccharrides are als

For the seaweeds studied in this paper, galactans representthe only important sulfated polysaccharides, as the only otherpolymers found in the water extracts in signicant amounts are4-linked -glucans, which are neutral reserve polysaccharides,not part of the cell wall. These galactans are obtained in extremelylow yields calculated considering the milled seaweed dry weight.However, taking into account the amount of calcium carbonatereported previously for these calcied seaweeds (Bhm, 1973),yields would be still low, but of the same order as those reportedfor other Bryopsidales (Ciancia et al., 2007, 2012). If any of thesepolymers were suitable for application as biologically active

undsilitythe

on skedt suboxyamouy 4-,ow wole

Fwe know, this is the rst report about structural featuresluble sulfated polysaccharides from these three dif-ra from the Bryopsidales, particularly only very scarce

was previously published about sulfated polysaccha-he suborder Halimedineae (Chattopadhyay et al., 2007;rcival, 1961).sults allow to make some generalizations, namely, thes biosynthesize galactans as major soluble sulfatedides with 3-, 6-, and 3,6-linkages; these polysaccha-o substituted by important quantities of pyruvic acid.

compoavailab

All commof 3-linin par(1-carlower tionallnot knsame m this article in press as: Arata, P. X., et al. Chemical structure and anticoagical green seaweeds of the order Bryopsidales. Carbohydrate Polymers (2

ig. 1. HMQC spectrum of F1, showing signals corresponding to the major units. A detail w, yield would not be a major hindrance due to the of row material.sulfated pyruvylated galactans studied here have

tructural characteristics, namely, (i) major amounts 4,6-O-(1-carboxy)ethylidene-d-galactopyranose unitslfated on C-2, and also possibly terminal 4,6-O-)ethylidene-d-galactopyranose residues, but in muchnts, (ii) a complex sulfation pattern, comprising addi-

6-, and 4,6-disulfated galactose units. Until now, it isether both kind of substitution patterns coexist in the

cule or if they are present in different molecules thatulant activity of highly pyruvylated sulfated galactans014), http://dx.doi.org/10.1016/j.carbpol.2014.10.030

ith the peak of C3/H3 of pyruvic acid ketal is included.



are obtainethem.

There arthe structufrom Caule(Chattopadnose were to single sof the gala(Ciancia et acid ketals obtained bmass specpartial hydlost.

3.4. Anticoa

3.4.1. GlobaAnticoag

weeds was this article in press as: Arata, P. X., et al. Chemical structure and anticoagical green seaweeds of the order Bryopsidales. Carbohydrate Polymers (2

Fig. 2. 13C NMR spectra of F1desulf (a) and F

d together in spite of the efforts carried out to separate

e important differences between these galactans andre previously reported for sulfated polysaccharidesrpa racemosa, which belongs to the same suborderhyay et al., 2007). In the latter, xylose and arabi-detected in important quantities and were attributedtubs or short side chains, sulfate was found at C-6ctose units, as found for galactans from B. plumosaal., 2012) and no reference to the presence of pyruvicwas made. The authors studied the oligosaccharidesy acid hydrolysis of this polymer by MALDI-TOF-trometry, but in conditions necessary to achieverolysis of glycosidic linkages, pyruvic acid would be

gulant activity

l coagulation testsulant activity of polysaccharides from all the sea-

assessed by measuring the prothrombin time (PT),

the activatetime (TT). effect (Supavailable inthis fractionanticoagula

Supplemfound, in th2014.10.03

No clotttions assaypolybrene, made thromform compthe sulfated(Carroll, 19was observ

On the cconcentratiFig. 4). TT For TT, inculant activity of highly pyruvylated sulfated galactans014), http://dx.doi.org/10.1016/j.carbpol.2014.10.030

1depyr (b).

d partial thromboplastin time (APTT), and thrombinAll the samples proved to have similar anticoagulantplementary Table S1). Since F1 from P. capitatus, was

higher quantities, all the studies were focused only on. The activity of the sample was compared with modelnts, heparin and dermatan sulfate (Fig. 4).entary Table S1 related to this article can bee online version, at http://dx.doi.org/10.1016/j.carbpol.0.ing inhibition was observed in PT test at the concentra-ed. In order to avoid possible neutralization effects ofadded in the majority of commercial reagents a home-boplastin was used. Polybrene is a polycation that can

lexes with polyanions, such as heparin and possibly polysaccharide assessed here, blocking their action

99). Even with the homemade thromboplastin, no effected.ontrary, APTT and TT were statistically prolonged, in aon dependent manner, regards to the control (Table 6,increases were higher than those observed in APTT.reases were up to 705 9% and for APTT 197 10%,



were achieinhibition oprocess, meinhibition (polymerizathe lack of kinetics of tences. More

Table 6APTT and TT ra

Samplea

APTTF1 DermatanHeparin

TTF1 DermatanHeparin

a Results web Concentra* Statisticall this article in press as: Arata, P. X., et al. Chemical structure and anticoagical green seaweeds of the order Bryopsidales. Carbohydrate Polymers (2

Fig. 3. 13C NMR spectra of UA (a) and H

ved at 100 g/mL. Prolongation of the APTT suggestsf the intrinsic and/or common pathway of coagulationanwhile the increase of TT indicates either thrombin

direct or mediated by AT and/or HCII) or impaired brintion. Since these results showed anticoagulant activity,prolongation in the PT may be explained by the fasthe assay, which could not allow detecting slight differ-over, prolongation of the TT-like assay, using brinogen

instead of psignal of a ping into accmore assay

Our resbut, when cdermatan sclot format

tios for F1, dermatan sulfate and heparin.

Concentration (g/mL)b

5 10 25

1.0 0.0 1.0 0.0 sulfate 1.0 0.0 1.5 0.0*

7.9 0.1* >10* >1

1.1 0.0 1.3 0.0* sulfate 4.2 0.0* 6,7 0.1* >1

>10* >10* >1

re expressed as ratios between clotting time of a solution of the anticoagulant and clottintion corresponds to the samples in the test solution.y signicant differences regards to the control (p < 0.05). Students t test was used to comulant activity of highly pyruvylated sulfated galactans014), http://dx.doi.org/10.1016/j.carbpol.2014.10.030

A (b).

lasma, was also observed (ratio 4.8 0.1). This is a rstossible direct inhibition of F1 on thrombin activity, tak-ount that AT and HCII were absent in the test. However,s need to be done to conrm this hypothesis.ults demonstrate that F1 exerts anticoagulant effect,ompared to model anticoagulants such as heparin andulfate, F1 proved to be less potent in preventing in vitroion.

50 100

1.3 0.0* 1.9 0.1* 3 0.1*3.6 0.0* 4.0 0.1* 6.3 0.0*0* >10* >10*

2.4 0.1* 4.9 0.1* 8.1 0.1*0* >10* >10*

0* >10* >10*

g time of the control; they are the mean standard deviation (n4).

pare anticoagulant and control samples.



a)

b)

0

50

100

150

200

250

300

350

400

0

TT (s

)

0

50

100

150

200

250

300

350

0

APTT

(s)

Fig. 4. Activatof the fraction

Only a have been view and tsystem. FroanticoagulaBryopsis msuch as, CodCodium latu(Ciancia et active comp2013).

3.4.2. FibrinClot play

structure, astudied. In tcapitatus onshow the kiheparin andproduced sta concentraF1 (10 g/manticoagulasulfate in thwere used.

Moreoveand ODMaxthe time reand the slodiminishedbrin polymstep non-co

a)

5

0

5

0

5

0

0 3 6 9 12 15 18 21 24 27Time (min)

25 g/mL

50 g/mL

5

0

5

0

Control

5 g/mL

10 g/mL

25 g/mL

5

0

5

0

0,35

0,40

0 20 40 60 80 100Concentraon (g/mL)

F1

Dermatan Su lfate

Heparin

20 40 60 80 100Concentraon (g/mL)

F1

Dermatan Su lfate

Heparin

ed partial thromboplastin time (APTT) (a) and thrombin time (TT) (b) F1 from P. capitatus, heparin and dermatan sulfate.

few sulfated polysaccharides from green seaweedsthoroughly studied from both the structural point of

b)

c)

0,1

0,2

0,2

0,3

0,3

0,4

OD

(405

nm)

0,2

0,3

0,3

0,4

OD

(405

nm)

0,1

0,2

0,2

0,3

OD

(405

nm

) this article in press as: Arata, P. X., et al. Chemical structure and anticoagical green seaweeds of the order Bryopsidales. Carbohydrate Polymers (2

heir biological behavior, particularly in haemostaticm Bryopsidales, there are few reports about theirnt activities, only the species Caulerpa okamurai andaxima and several species from the genus Codium,ium fragile, Codium istmocladum, Codium divaricatum,m, Codium vermilara among others were describedal., 2010). However, for species of Codium, the mostounds would be sulfated arabinans (Fernndez et al.,

formation kinetics assayss a crucial role in the hemostatic system, and formation,nd lysis of brin networks are important features to behe present work the effects of sulfated galactans from P.

brin formation process were studied. Table 7 and Fig. 5netics of plasma brin formation in the presence of F1,

dermatan sulfate. All the assayed concentrations of F1atistically signicant changes in kinetics parameters, intion-dependent way. No coagulation was detected withL), which was considered as a positive control of thent effect. Similar effects were detected when dermatane same concentration and a 5 g/mL solution of heparin

r, F1 caused increased lag phase and decreased sloperegards to control. Since the lag phase (which showsquired for initial protobril formation) is increased,pe (that corresponds to the brin formation rate) is, an impaired assembly of brin monomers into the

er would be involved. In the rst polymerizationvalent interactions, in particular, electrostatic bindings

0,15

0,20

0

Fig. 5. Effectsthe kinetics oftime (n = 4) an

are involvecharged mocoagulant awith thosecoagulant c2013).

On the othat the bnetworks. Asure of theto study thGabriel, & H

Kineticsstudy of thunderstandknowledgeegy was evseaweeds.Control

5 g/mL

10 g/mL

3 6 9 12 15 18 21 24 27Time (min)

Control

5 g/mL

10 g/mL

25 g/mL

50 g/mLulant activity of highly pyruvylated sulfated galactans014), http://dx.doi.org/10.1016/j.carbpol.2014.10.030

3 6 9 12 15 18 21 24 27Time (min)

50 g/mL

of F1 from P. capitatus (a), dermatan sulfate (b) and heparin (c) on brin formation. Curves represent the average of OD (405 nm) versusd bars indicate the SD.

d. Therefore, the presence of F1, a highly negativelylecule, could affect brin assembly showing an anti-ctivity. In general, all the results are in accordance

reported for dermatan sulfate, a well known anti-ompound (Lauricella, Castanon, Kordich, & Quintana,

ther hand, the diminished nal optical density suggestsrin bers resulted thinner than those from controllthough mass/length ratio () is a quantitative mea-

ber structure, the nal turbidity (OD) can be usede qualitative features of the brin network (Wolberg,offman, 2002).

of brin formation gives a different insight into thee coagulation process, contributing in this case to theing of anticoagulant activity of F1. To the best of our, this is the rst study where this experimental strat-aluated using sulfated polysaccharides extracted from



Table 7Parameters of the kinetics of brin formation in the presence of F1, heparin anddermatan sulfate.a

Lag phase (min) Slope (min1) ODmax b

Control F1 (g/mL)

5 10 25 50

Dermatan su5 10 25 50

Heparin (g5 10 25 50

a Results web Maximumc n.c.: no clo* Statisticall

the curves par

4. Conclus

The strutans from showing 3-substitutionin detail.

Taking ibrin formaexert anticonisms invol

Recentlymilara wasmechanismthis effect isthe -l-arabrelated galafated on C-2to speculatecould be relsimilar conit would be tion patternin the activplanning mincrease thlant agents,specic strutheir relatioto the undeand to the d

Acknowled

This woCouncil of Agency for 2008-0500)

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ions

ctural characteristics of sulfated and pyruvylated galac-some tropical Bryopsidales have been determined,, 6-, and 3,6-linked units with a very complex patterns of. Particularly, galactans from P. capitatus were studied

nto account results from global coagulation assays andtion assays, it was demonstrated that these galactansagulant effects. Moreover, one of the possible mecha-ved would be direct thrombin inhibition.

a highly sulfated pyranosic arabinan from Codium ver- found to have important anticoagulant activity by a

involving direct thrombin inhibition. It was shown that mostly due to the presence of a sulfate group on C-2 ofinopyranose units (Fernndez et al., 2013). The fact thatctan structures from Codium species, that are not sul-, do not have important anticoagulant action, induces us

that the active structure in the galactans studied hereated to sulfation on C-2 of the galactan chain, which hasguration to that of the latter arabinan. If it were so, then

interesting to obtain galactan fractions where this sulfa- is yet more predominant, giving an important increaseity. In addition, data presented here could be useful in

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Bilan, Mhigh(Bry

Bhm, Eof M

Carroll, Clini

Cases, Mgala3897

CanelnStruand 101,

ChattopCaul68, 4

Ciancia, et almilaMac

Ciancia, sulfaon t

Ciancia, (201seawcoas

Ciucanucarb

Dodgsoncont

Dubois, met28, 3

Estevez,and from

Farias, EA. S.Codi

Fernndmapphyt

Fernndman916

Fernndet a-l-Chem

FernndCodnent this article in press as: Arata, P. X., et al. Chemical structure and anticoagical green seaweeds of the order Bryopsidales. Carbohydrate Polymers (2

odication of other polymers with related structures toe activity, or even for the synthesis of new anticoagu-

more research is still needed. In general, knowledge ofctural characteristics of seaweed polysaccharides andnship with the anticoagulant activity could contributerstanding of the regulation of haemostatic processesevelopment of new antithrombotic therapeutic agents.

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Chemical structure and anticoagulant activity of highly pyruvylated sulfated galactans from tropical green seaweeds of the...1 Introduction2 Experimental2.1 Algal sample2.2 Extraction of the polysaccharides2.3 Ion exchange chromatography (IEC)2.4 Chemical analyses2.5 Desulfation of F1 and F62.6 Removal of pyruvic acid residues from F12.7 Methylation analysis2.8 Gas chromatography2.9 GCMS2.10 NMR spectroscopy2.11 Treatment with pronase E2.12 General coagulation assays2.13 Fibrin formation2.14 Statistical analysis

3 Results and discussion3.1 Extraction and characterization of the sulfated polysaccharides3.2 Structural studies of sulfated galactans3.3 Sulfated galactans from the Halimedineae3.4 Anticoagulant activity3.4.1 Global coagulation tests3.4.2 Fibrin formation kinetics assays

4 ConclusionsAcknowledgementsReferences

chemical structure and anticoagulant activity of highly pyruvylatedsulfated galactans from tropical...

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universidad central

calcied seaweeds

pyruvylated galactans

c tsulfated

botnica tropical

qumica orgnica

tropical species

order bryopsidales