-
Please citefrom trop
ARTICLE IN PRESSG ModelCARP-9383; No. of Pages 11Carbohydrate
Polymers xxx (2014) xxxxxx
Contents lists available at ScienceDirect
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
e i n f o
ly 2014vised form
2014tober 2014e xxx
tanetald
activityon
a b s t r a c t
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,
-
Please cite icoagfrom trop rs (2
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.
-
Please cite icoagfrom trop rs (2
ARTICLE IN PRESSG ModelCARP-9383; No. of Pages 114 P.X. Arata et
al. / Carbohydrate Polymers xxx (2014) xxxxxx
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
-
Please citefrom trop
ARTICLE IN PRESSG ModelCARP-9383; No. of Pages 11P.X. Arata et
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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
-
Please citefrom trop
ARTICLE IN PRESSG ModelCARP-9383; No. of Pages 116 P.X. Arata et
al. / Carbohydrate Polymers xxx (2014) xxxxxx
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.
-
Please citefrom trop
ARTICLE IN PRESSG ModelCARP-9383; No. of Pages 11P.X. Arata et
al. / Carbohydrate Polymers xxx (2014) xxxxxx 7
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%,
-
Please citefrom trop
ARTICLE IN PRESSG ModelCARP-9383; No. of Pages 118 P.X. Arata et
al. / Carbohydrate Polymers xxx (2014) xxxxxx
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.
-
Please citefrom trop
ARTICLE IN PRESSG ModelCARP-9383; No. of Pages 11P.X. Arata et
al. / Carbohydrate Polymers xxx (2014) xxxxxx 9
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
-
Please citefrom trop
ARTICLE IN PRESSG ModelCARP-9383; No. of Pages 1110 P.X. Arata
et al. / Carbohydrate Polymers xxx (2014) xxxxxx
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)
References
Ahmed, A. E. Rnation of c
Anand, S., Yusuf, S., Pogue, J., Ginsberg, J., & Hirsh, J.
(2003). Relationship ofactivated partial thromboplastin time to
coronary events and bleeding inpatients with acute coronary
syndromes who receive heparin. Circulation, 107,28842888.
Bilan, M. I., Vinogradova, E. V., Shashkov, A. S., & Usov,
A. I. (2006). Isolation and pre-nary copsida. I., Vily pyropsida.
L. (19arine W. E. cal Pa. R.,
ctans 390
, D. J., Ccture Laure
7057adhyaerpa r074M., Qu. (2007ra witromolM., Quted p
hose oM., Al2). Cheed Bt. Jour, I., & Kohydr, K. S.,ent
ofM., Gihod of503
J. M., in sit
the g. H. C.
(2008um istez, P. Vping ia). Jouez, P. nan fr919.ez, P. l.
(201arabinistry,
ez, P. ium sps of th0.0 0.0 61.5 2.1 0.382 0.004
4.1 0.9* 7.3 0.1* 0.278 0.006*n.c.c n.c. 0.210 0.007*n.c. n.c.
0.210 0.004*n.c. n.c. 0.200 0.000*
lfate (g/mL)2.4 1.0* 3.0 0.8* 0.244 0.005*n.c. n.c. 0.207
0.001*n.c. n.c. 0.211 0.001*n.c. n.c. 0.213 0.003*
/mL)n.c. n.c. 0.208 0.005*n.c. n.c. 0.207 0.002*n.c. n.c. 0.211
0.001*n.c. n.c. 0.219 0.001*
re expressed as mean standard deviation (n = 4). optical
density.tting detected.y signicant difference (p < 0.05).
Students t test was used to compareameters of the anticoagulant and
control samples.
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
limi(Bry
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.
gements
rk was supported with grants from National ResearchArgentina,
CONICET (PIP 559-2010) and the NationalPromotion of Science and
Technology, ANPCYT (PICT, Argentina, and FONACIT-2012000830,
Venezuela.
., & Labavitch, J. M. (1977). A simplied method for accurate
determi-ell wall uronide content. Journal of Food Biochemistry, 1,
361365.
(Serial VolAcademic
Ferreira, L. G., NE. R. (2012from the rdrate Rese
Ghosh, P., Adh(2004). InCaulerpa r
Gurgel RodrigMagalhesactivity ofcupressoid
Gurgel Rodrigde Souza dependencupressoid634639.
Ji, H., Shao, Hcharides iactivity. Jo
Kelton, J. G., &Seminars H
Kelton, J. G., &torical perulant activity of highly
pyruvylated sulfated galactans014),
http://dx.doi.org/10.1016/j.carbpol.2014.10.030
haracterization of a highly pyruvylated galactan from Codium
yezoenseles, Chlorophyta). Botanica Marina, 49, 259262.nogradova,
E. V., Shashkov, A. S., & Usov, A. I. (2007). Structure of
auvylated galactan sulfate from the pacic green alga Codium
yezoenseles, Chlorophyta). Carbohydrate Research, 342, 586596.73).
Studies on the mineral content of calcareous seaweeds. Bulletin
Science, 23, 177190.(1999). Thromboplastins, heparin, and
polybrene. Americal Journal ofthology, 111(4), 565.Stortz, C. A.,
& Cerezo, A. S. (1992). Methylated, sulfated xylo-from the red
seaweed Corallina ofcinalis. Phytochemistry, 31,0.iancia, M.,
Surez, A. I., Compagnone, R. S., & Matulewicz, M. C.
(2014).
of highly substituted agarans from the red seaweeds Laurencia
obtusancia liformis (Rhodomelaceae, Ceramiales). Carbohydrate
Polymers,13.
y, K., Adhikari, U., Lerouge, P., & Ray, B. (2007).
Polysaccharides fromacemosa: Purication and structural features.
Carbohydrate Polymers,15.intana, I., Vizcargnaga, M. I., Kasulin,
L., de Dios, A., Estevez, J. M.,). Polysaccharides from the green
seaweeds Codium fragile and C. ver-
h controversial effects on hemostasis. International Journal of
Biologicalecules, 41, 641649.intana, I., & Cerezo, A. S.
(2010). Overview of anticoagulant activity ofolysaccharides from
seaweeds in relation to their structures, focusingf green seaweeds.
Current Medicinal Chemistry, 17, 25032529.berghina, J., Arata, P.
X., Benavides, H., Leliaert, F., Verbruggen, H., et
al.aracterization of cell wall polysaccharides of the coencocytic
greenryopsis plumosa (Bryopsidaceae, Chlorophyta) from the
Argentinenal of Phycology, 48, 326335.erek, K. (1984). A simple and
rapid method for the permethylation ofates. Carbohydrate Research,
134, 209217.
& Price, R. G. (1962). A note on the determination of the
ester sulphate sulphated polysaccharides. Biochemistry Journal, 84,
106110.lles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F.
(1956). Colorimetric
determination of sugars and related substances. Analytical
Chemistry,56.Fernndez, P. V., Kasulin, L., Dupree, P., &
Ciancia, M. (2009). Chemicalu characterization of macromolecular
components of the cell wallsreen seaweed Codium fragile.
Glycobiology, 19, 212228., Pomin, V. H., Valente, A. P., Nader, H.
B., Rocha, H. A. O., & Mouro, P.). A preponderantly 4-sulfated,
3-linked galactan from the green algahmocladum. Glycobiology, 18,
250259.., Ciancia, M., Miravalles, A. B., & Estevez, J. E.
(2010). Cell wall polymern the coenocytic macroalga Codium
vermilara (Bryopsidales, Chloro-rnal of Phycology, 46, 556565.V.,
Estevez, J. M., Cerezo, A. S., & Ciancia, M. (2012). Sulfated
-d-om the green seaweed Codium vermilara. Carbohydrate Polymers,
87,
V., Quintana, I., Cerezo, A. S., Caramelo, J. J., Pol-Fachin,
L., Verli, H.,3). Anticoagulant activity of a unique sulphated
pyranosic (13)-an through direct interaction with thrombin. Journal
of Biological
288, 223233.V., Arata, P. X., & Ciancia, M. (2014). Chapter
9: Polysaccharides fromecies: Chemical structure and biological
activity. Their role as compo-e cell wall. In Jacquot, J-P. &
Gadal, P. (Serial Eds.) & Bourgougnon, N.. Ed.), Advances in
Botanical Research, Vol. 71. Sea plants, pp. 253278.
Press, Elsevier Ltd, Oxford, Great Britain.oseda, M. D., Gonc
alvez, A. G., Ducatti, D. R. B., Fujii, M. T., & Duarte, M.
). Chemical structure of the complex pyruvylated and sulfated
agaraned seaweed Palisada agellifera (Ceramiales, Rhodophyta).
Carbohy-arch, 347, 8394.ikari, U., Ghosal, P. K., Pujol, C. A.,
Carlucci, M. J., Damonte, E. B., et al.
vitro anti-herpetic activity of sulfated polysaccharide
fractions fromacemosa. Phytochemistry, 65, 31513157.ues, J. A., de
Sousa Oliveira Vanderlei, A., Fac anha Bessa, E., de Arajo, F.,
Monteiro de Paula, R. P., Lima, V., et al. (2011).
Anticoagulant
a sulfated polysaccharide isolated from the green seaweed
Caulerpaes. Brazilian Archives of Biology and Technology, 54,
691700.ues, J. A., de Queiroz, I. N. L., Gomes Quinder, A. L.,
Cunha Vairo, B.,Mouro, P. A., & Barros Benevides, N. M. (2011).
An antithrombin-t sulfated polysaccharide isolated from the green
alga Caulerpaes has in vivo anti- and prothrombotic effects. Cincia
Rural, 41,
., Zhang, C., Hong, P., & Xiong, H. (2008). Separation of
the polysac-n Caulerpa racemosa and their chemical composition and
antitumorurnal of Applied Polymer Science, 110, 14351444.
Hirsh, J. (1980). Bleeding associated with antithrombotic
therapy.ematology, 17, 259291.
Warkentin, T. E. (2008). Heparin-induced thrombocytopenia: A
his-spective. Blood, 112, 26072616.
-
Please cite icoagfrom trop rs (2
ARTICLE IN PRESSG ModelCARP-9383; No. of Pages 11P.X. Arata et
al. / Carbohydrate Polymers xxx (2014) xxxxxx 11
Knutsen, S. H., & Grasdalen, H. (1987). Characterization of
water extractablepolysaccharides from norwegian Furcellaria
lumbricalis (Huds) Lamour (Gigarti-nales, Rhodophyceae) by IR and
NMR spectroscopy. Botanica Marina, 30,497505.
Koepsell, H. J., & Sharpe, E. S. (1952). Microdetermination
of pyruvicand -ketoglutaric acids. Archives of Biochemistry and
Biophysics, 38,443449.
Laffan, M. A., & Bradshaw, A. E. (1995). Investigation of
haemostasis. In J. V. Dacie,& S. M. Lewis (Eds.), Practical
haematology (pp. 297315). New York: ChurchillLivingstone.
Lauricella, A. M., Castanon, M. M., Kordich, L. C., &
Quintana, I. L. (2013). Alterationsof brin network structure
mediated by dermatan sulfate. Journal of Thrombosisand
Thrombolysis, 35, 257263.
Lam, D. W., & Zechman, F. W. (2006). Phylogenetic analyses
of the Bryopsidales(Ulvophyceae, Chlorophyta) based on rubisco
large subunit gene sequences.Journal of Phycology, 42, 669678.
Love, J., & Percival, E. J. (1964). The polysaccharides of
the green seaweed Codiumfragile: Part III. A -1, 4-linked mannan.
Journal of the Chemical Society,33453349.
Lowry, O. H., Rosenbrough, N. J., & Farr, A. L. (1951).
Protein measurements with theFolin phenol reagent. Journal of
Biological Chemistry, 193, 265275.
Mackie, I. M., & Percival, E. (1961). Polysaccharides from
the green seaweeds ofCaulerpa spp. Part III. Detailed study of the
water-soluble polysaccharides of C.liformis: comparison with the
polysaccharides synthesised by C. racemosa andC. sertularioides.
Journal of the Chemical Society, 30103015.
McIntyre, D. D., & Vogel, H. J. (1993). Structural studies
of pullulan by nuclear mag-netic resonance spectroscopy. Starch,
45, 406410.
Morrison, I. M. (1988). Hydrolysis of plant cell walls with
triuoroacetic acid. Phy-tochemistry, 27, 10971100.
Mouro, P. A. (2004). Use of sulfated fucans as anticoagulant and
antithromboticagents: Future perspectives. Current Pharmaceutical
Design, 10, 967981.
Navarro, D. A., Flores, M. L., & Stortz, C. A. (2007).
Microwave-assisted desulfation ofsulfated polysaccharides.
Carbohydrate Polymers, 69, 742747.
Ohta, Y., Lee, J.-B., Hayashi, K., & Hayashi, T. (2009).
Isolation of sulfated galactanfrom Codium fragile and its antiviral
effect. Biological & Pharmaceutical Bulletin,32, 892898.
Percival, E., & McDowell, R. H. (1981). Algal walls:
composition and biosynthesis. InW. Tanner, & F. A. Loewus
(Eds.), Encyclopedia of plant physiology (vol. 13B) (pp.277316).
Berlin: Springer.
Pomin, V. H. (2009). An overview about the structure-function
relationship of marinesulfated homopolysaccharides with regular
chemical structures. Biopolymers,91, 601609.
Quick, J. (1935). The prothrombin time in haemophilia and in
obstructive jaundice.Journal of Biological Chemistry, 109,
7374.
Santos Pereira Costa, M. S., Silva Costa, L., Lima Cordeiro, S.,
Almeida-Lima, J.,Dantas-Santos, N., Dantas Magalhes, K., et al.
(2012). Evaluating the possibleanticoagulant and antioxidant
effects of sulfated polysaccharides from the trop-ical green alga
Caulerpa cupressoides var. abellata. Journal of Applied
Phycology,24, 11591167.
Synytsya, A., & Novak, M. (2013). Structural diversity of
fungal glucans. CarbohydratePolymers, 92, 792809.
Stevenson, T. T., & Furneaux, R. H. (1991). Chemical methods
for the analysis ofsulphated galactans from red algae. Carbohydrate
Research, 210, 277298.
Stortz, C. A., Bacon, B. E., Cherniak, R., & Cerezo, A. S.
(1994). High-eld NMR spec-troscopy of cystocarpic and tetrasporic
carrageenans from Iridaea undulosa.Carbohydrate Research, 261,
317326.
Stortz, C. A., Matulewicz, M. C., & Cerezo, A. S. (1982).
Separation and identication ofO-acetyl-O-methyl-galactononitriles
by gasliquid chromatography and massspectrometry. Carbohydrate
Research, 111, 3139.
Taylor, W. R. (1960). Marine algae of the eastern tropical and
subtropical coast of theAmericas. Michigan: The University of
Michigan Press.
Weisel, J., Veklich, Y., & Gorkun, O. (1993). The sequence
of cleavage of brinopep-tides from brinogen is important for
protobril formation and enhancementof lateral aggregation in brin
clots. Journal of Molecular Biology, 232, 285297.
Wolberg, A. S., Gabriel, D. A., & Hoffman, M. (2002).
Analyzing brin clot structureusing a microplate reader. Blood
Coagulation and Fibrinolysis, 13, 533539. this article in press as:
Arata, P. X., et al. Chemical structure and antical green seaweeds
of the order Bryopsidales. Carbohydrate Polymeulant activity of
highly pyruvylated sulfated galactans014),
http://dx.doi.org/10.1016/j.carbpol.2014.10.030
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