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Sulfated Galactans of Champia indicaand Champia parvula (Rhodymeniales,Rhodophyta) of Indian WatersSanjay Kumar a , Devaki Nandan a , Ramavatar Meena a , KamaleshPrasad a & Arup K. Siddhanta aa Marine Biotechnology & Ecology Discipline, Central Salt & MarineChemical Research Institute, Council of Scientific & IndustrialResearch (CSIR), G.B.Marg, Bhavnagar, 364002, Gujarat, India

Available online: 27 Jun 2011

To cite this article: Sanjay Kumar, Devaki Nandan, Ramavatar Meena, Kamalesh Prasad & Arup K.Siddhanta (2011): Sulfated Galactans of Champia indica and Champia parvula (Rhodymeniales,Rhodophyta) of Indian Waters, Journal of Carbohydrate Chemistry, 30:1, 47-60

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Journal of Carbohydrate Chemistry, 30:47–60, 2011Copyright C© Taylor & Francis Group, LLCISSN: 0732-8303 print / 1532-2327 onlineDOI: 10.1080/07328303.2011.587924

Sulfated Galactans ofChampia indica andChampia parvula(Rhodymeniales, Rhodophyta)of Indian Waters

Sanjay Kumar, Devaki Nandan, Ramavatar Meena,Kamalesh Prasad, and Arup K. SiddhantaMarine Biotechnology & Ecology Discipline, Central Salt & Marine Chemical ResearchInstitute, Council of Scientific & Industrial Research (CSIR), G.B.Marg,Bhavnagar-364002, Gujarat, India

Sulfated galactans of the red seaweed species Champia indica and Champia parvula ofIndian waters were extracted and purified by ion exchange chromatography. These werecharacterized by infrared and 13C NMR spectroscopy as well as by GC-MS analysis ofalditol acetate derivatives produced by reductive hydrolysis/acetylation of sulfated anddesulfated and their methylated samples. The sulfated galactans of these Champiaspecies contained alternating β-(1→3)-linked galactopyranosyl units with sulfation atthe 2-position and α-(1→4)-linked galactopyranosyl units having sulfation at both the2- and 3-positions. Other minor substitutions included 6-O-methyl ether of the β-(1→3)-linked galactose residues only in Champia parvula.

Keywords Champia indica; Champia parvula; Rhodophyta; Sulfated galactans; GC-MS; 13C NMR

INTRODUCTION

Sulfated galactans are the major matrix-phase linear polymers of the red sea-weeds, composed of alternating dimeric unit β-(1→3)-linked galactopyranose(Galp) and α-(1→4)-linked 3,6-anhydro Galp or α-(1→4)-linked Galp alongwith sulfated ester residues at different carbon atoms.[1] These galactans may

Received September 16, 2010; accepted May 11, 2011.Address correspondence to Arup K. Siddhanta, Marine Biotechnology & Ecology Dis-cipline, Central Salt & Marine Chemical Research Institute, Council of Scientific &Industrial Research (CSIR), G.B.Marg, Bhavnagar-364002, Gujarat, India. E-mail:aks@csmcri.org

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also bear glycosyl substitutions, most commonly known as single xylopyranosyl(Xylp) or mono-O-methyl galactopyranosyl (Me-Galp) residues branching fromthe main galactan chain.[2] Pyruvate groups too are found in carrageenans,which form a cyclic acetal between the 4- and 6-positions of (1→3)-linked-Galp units.[2c] Carrageenans are the major texturizing ingredients used infood industry, pharmaceutical, cosmetics, printing, and textile formulations.[3]

Kappa (κ) and iota (ι) carrageenans are used as gel-forming substances, whilelambda (λ) carrageenan is used as a thickening agent.[4] The λ-carrageenandid not show gelling behavior due to 4C1 conformations of the β-D-Galp unit.[5]

Besides the industrial applications, derivatives of carrageenans as well aslow-molecular-weight carrageenans and their conjugates have been reportedto show anti-HIV activity.[6] Carrageenan has also been reported as a po-tent inhibitor of papilloma virus infection.[7] Many biological activities suchas anticoagulant,[8] antiherpetic,[9] antithrombotic,[10] antitumor,[11] and antivi-ral[12] properties have been reported from the red algal sulfated galactans. Invivo antitumor and immunomodulation activities of different molecular weightλ-carrageenans from Chondrus ocellatus have been reported by Zhou et al.[13]

Recently sulfated polysaccharides from Champia feldmannii have been re-ported to exhibit antitumor properties.[14] Galactans from Champia novae-zealandiae, Champia laingii, and Champia chatamensis have been isolatedand characterized using 13C NMR spectroscopy and linkage analysis.[15]

Champia parvula has been found to be particularly sensitive to heavy metaltoxicity to arsenite and arsenate[16] as well as to hexavalent chromium.[17]

We report herein the isolation and characterization of sulfated galactans ofChampia indica and Champia parvula of Indian waters. To our knowledge,this is the first such report on chemical investigation of Indian Champiaspecies. The results of this study would be useful in bioprospecting of galactan-producing red seaweed species.

RESULTS AND DISCUSSION

The yields, total sugar, protein and sulfate contents as well as weight-averagemolecular weights of the crude, sulfated, and desulfated polysaccharides ofChampia indica (CICrude, CISul, and CIDes) and Champia parvula (CPCrude,CPSul, and CPDes) are given in Table 1. The sulfate contents in CISul (33.4%w/w) and CPSul (35.2% w/w) were higher than those of CICrude (29.7% w/w) andCPCrude (31.4% w/w), respectively, as expected.

Monosaccharides and Linkage AnalysisThe GC-MS profile of alditol acetates revealed that CICrude, CISul, and

CIDes were composed mainly of galactose and small amounts of 6-O-methylated

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Table 1: Physicochemical characteristics of crude, sulfated, and desulfatedpolysaccharides of Champia indica and Champia parvula

Champia indica Champia parvula

CICrude CISul CIDes CPCrude CPSul CPDes

Yielda

(% ±SD)14.1 ( ± 1.0) 12.7 ( ± 0.8) 5.8 ( ± 0.6) 13 ( ± 1.0) 11.4 ( ± 0.9) 5.1 ( ± 0.4)

Total sugarb(% ±SD)

42.2 ( ± 0.8) 44.1 ( ± 1.0) 54.7 ( ± 0.7) 43.5 ( ± 1.0) 45.9 ( ± 0.7) 56.1 ( ± 0.8)

Proteinb

(% ±SD)3.8 ( ± 0.1) 2.9 ( ± 0.1) ND 4.0 ( ± 0.2) 3.1 ( ± 0.3) ND

Sulfateb

(% ±SD)29.7 ( ± 0.9) 33.4 ( ± 1.0) ND 31.4 ( ± 1.0) 35.2 ( ± 1.0) ND

Mw (×105) 15.1 21.25 1.9 10.8 22.6 2.1

ND = Not done; SD = standard deviation of three replicates.a Yield w.r.t. as received dry seaweed.b Yield w.r.t. polysaccharide.

galactose (<3 mol%), while CPCrude, CPSul, and CPDes were composed of galac-tose and 6-O-methylated galactose in the following ratios viz. 87.2: 8.8, 86.2:12.6, and 94.4: 5.6 (mol%), respectively, along with small amounts of glucose(<3 mol%, cf. Table 2). 3,6-Anhydro galactose (3,6-AnGal) residues were alsoencountered in CICrude and CPCrude in lesser amounts (<2 mol%). The totalsugar, sulfate, and 6-O-methylated galactose contents in the polysaccharidesof Champia parvula (CPCrude and CPSul) were higher than those of polysaccha-rides of Champia indica (CICrude and CISul).

Linkage analysis of the CISul and CPSul (Table 3) showed the presence of(1) 1,2,3,5-tetra-O-acetyl-4,6-di-O-methyl galactitol, which indicated linkagesat the C-2 and C-3 positions and was assigned to (1→3)-linked 2-sulfatedgalactopyranosyl residues as reported in λ-carrageenan,[18] and (2) 1,2,3,4,5-penta-O-acetyl-6-O-methyl galactitol, which showed linkages at the C-2,C-3, and C-4 positions (Fig. 1) and would be consistent with (1→4)-linked

Table 2: Monosaccharide compositions (mol%) of crude, sulfated, and desulfatedpolysaccharides of Champia indica and Champia parvula

Champia indica Champia parvulaConstituentmonosaccharides CICrude CISul CIDes CPCrude CPSul CPDes

6-O-Me-Galactose

1.9 2.8 — 8.8 12.6 5.6

Galactose 96.3 97.2 100 87.2 86.2 94.43,6-Anhydro-

galactose1.8 — — 1.1 — —

Glucose — — — 2.9 1.2 —

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Table 3: Linkage analysis of (mol%) of sulfated and desulfated polysaccharides ofChampia indica and Champia parvula

Champia indica Champia parvula

Deduced linkagesa CISul CIDes CPSul CPDes

→3)-Galp (1→ 4.3 33.2 8.5 42.2→4)-Galp (1→ 2.1 29.9 1.8 32.9→6)-Galp (1→ — 4.7 — —→2,3)-Galp (1→ 22.1 3.2 30.7 6.9→3,4)-Galp (1→ 8.9 7.3 7.2 6.4→4,6)-Galp (1→ 0.8 4.2 2.7 1.5→2,3,4)-Galp (1→ 42.6 6.8 47.3 8.2→2,4,6)-Galp (1→ 11.2 3.1 1.8 1.9→3,4,6)-Galp (1→ 7.2 7.6 — —Terminal Galp (1→ 0.8 — — —

a →3)-Galp (1→ means a (1→3)-linked galactose unit determined as 2,4,6-tri-O-methyl-1,3,5-tri-O-acetyl-galactitol and so on.

2,3-disulfated galactopyranosyl units as reported from the Champia speciesof New Zealand.[15]

To determine the positions of sulfate esters in CISul and CPSul, linkageanalyses of their desulfated polysaccharides (CIDes and CPDes) were also done.The linkage analysis of CIDes showed the presence of (1→3)-linked galactose(Gal); (1→4)-linked Gal; 3,4-linked Gal; 2,3,4-linked Gal; and 3,4,6-linked Galin 33.2, 29.9, 7.3, 6.8, and 7.6 mole%, respectively, while that of CPDes showedthe presence of (1→3)-linked galactose (Gal); (1→4)-linked Gal; 2,3-linkedGal; 3,4-linked Gal; and 2,3,4-linked Gal in 42.2, 32.9, 6.9, 6.4, and 8.2 mol%

Figure 1: Structures of CISul, CPSul, and λ-carrageenan.

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Sulfated Galactans of Champia indica and Champia parvula 51

(Table 3), respectively. Low levels of (1→6)-linked Gal; 2,3-linked Gal; 4,6-linked Gal; and 2,4,6-linked Gal in CIDes and CPDes were also detected.

In CISul and CIDes the 2,3-linked Gal was estimated to be 22.1 and3.2 mol%, and (1→3)-linked Gal to be 4.3 and 33.2 mol%, respectively(Table 3). This suggests that the sulfate group was located on C-2 of (1→3)-linked galactose in CISul. A similar observation was made for 2,3,4-linked Galunits being present in 42.6 and 6.8 mol% in CISul and CIDes, respectively. The(1→4)-linked Gal was estimated to be 2.1 and 29.9 mol%, respectively, con-firming that the C-2 and C-3 positions of (1→4)-linked Gal units were sulfatedin CISul. The proportion of 2,4,6-linked galactose units got reduced to 3.1 (inCIDes) from 11.2 mol% (in CISul) after desulfation, suggesting that a fraction of(1→4)-linked Gal units were sulfated at the C-2 and C-6 atoms as reported inλ carrageenan.[18b] Similar sets of observations were registered in the case ofCPSul and CPDes in regard to the decreases in 2,3-linked galactose and 2,3,4-linked galactose (in CPSul), with concomitant increases in (1→3)- and (1→4)-linked galactose (in CPDes), respectively. Besides this, a few other variouslylinked galactose units (<3 mol%) (e.g., 4,6-linked and 2,4,6-linked galactose,etc.) were also encountered in linkage analyses of CISul, CIDes, CPSul, and CPDes

(Table 3).[15] The electron ionization mass spectra (EIMS) of various partiallymethylated alditol acetates (PMAA) of galactose units obtained from linkageanalyses of CISul, CIDes, CPSul, and CPDes are shown in Figure 2.

FTIR and 13C NMRThe FTIR spectra of CICrude, CISul, CPCrude, and CPSul polysaccharides are

given in Figure 3. The appearance of IR bands at 934 cm−1 (in CICrude) and at935 cm−1 (CPCrude) indicated the presence of 3,6-AnGal units, which were sup-plemented by the GC-MS data (Table 2). The presence of 2-sulfated-galactosyland sulfate esters in CICrude, CISul, CPCrude, and CPSul was confirmed by thefollowing bands at 835 and 839 (νas C O C) and at 1251 and 1257 cm−1

(νantias S O), respectively.[19] Other respective bands at 1031 and 1032, 1138and 1145, and 1627 and 1647 cm−1 were due to the presence of primary alcoholgroups of the pyranose ring, vibration of the sulfate group, and bound water ofthe polysaccharide, respectively.[19]

The 13C NMR resonances of CISul and CPSul were assigned to the repeat-ing units, which are depicted in Figures 4 and 5, respectively. Difficulties wereencountered in obtaining well-resolved 13C NMR spectra due to the high viscos-ity of aqueous carrageenan solutions, even at high temperatures (ca. 70◦C).[20]

The chemical shifts (δ in ppm) were similar to those of carrageenans reportedearlier[21] and were in good agreement with those reported for the polysaccha-rides of Champia species of New Zealand.[15] In each spectrum two anomericcarbon resonances were identified at δ 102.9 and 97.2 and 103.0 and 97.2 ppm,which were assigned to C-1’s of β-(1→3)-linked 2-sulfated galactosyl (G2S) and

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Figure 2: EIMS patterns at m/z 60 to 280 of various partially methylated alditol acetates(PMAA) of galactose (Gal) units obtained after linkage analysis of CISul, CIDes, CPSul, andCPDes; 2,4,6-Me3 Gal (A1); 2,3,6-Me3 Gal (A2); 2,3,4-Me3 Gal (A3); 2,3,4,6-Me4 Gal (A4); 4,6-Me2Gal (A5); 2,6-Me2 Gal (A6); 2,3-Me2 Gal (A7); 6-Me Gal (A8); 3-Me Gal (A9); 2-Me Gal (A10).

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Sulfated Galactans of Champia indica and Champia parvula 53

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600

cm-1

d

c

b

a

Figure 3: FT-IR spectra of (a) CICrude, (b) CPCrude, (c) CISul, and (d) CPSul.

Figure 4: 13C NMR spectrum of CISul.

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Figure 5: 13C NMR spectrum of CPSul.

α-(1→4)-linked 2,3-disulfated galactosyl (L2S,3S) residues of each, respec-tively. The 13C NMR spectra of the CISul and CPSul exhibited 14 and 13 majorcarbon resonances, respectively. The signal for the 6-O-methyl galactose ap-peared at δ 59.8 ppm in CPSul, which was not observed in CISul. Assignmentsof C-2, C-3, C-4, C-5, and C-6 carbon resonances of G2S and L2S,3S residues inCISul and CPSul were done, which were in good agreement with those reportedearlier.[15,18,22] Downfield shifts by δ 7 to 8 ppm due to the presence of a sulfategroup were observed with the signals of C-2 of G2S as well as with those of C-2and C-3 of L2S,3S in the spectra of CISul and CPSul, respectively, with respectto their nonsulfated forms.[18b,22,23] These results complemented those obtainedin the linkage analyses of the CISul and CPSul (Table 3).

The signal at δ 68.9 ppm was assigned to C-6 of α-(1→4)-linked galactosylunits in CISul, which resembled those of λ-carrageenan. The carbon resonanceat δ 69.1 ppm was assigned to nonsulfated C-3 of the α-(1→4)-linked 2-sulfatedgalactosyl unit of λ-carrageenan.[18,22,23]

In summary, CISul polysaccharide consisted of alternating β-(1→3)-linked 2-sulfated galactosyl residues, α-(1→4)-linked 2,3-disulfated galactosylresidues, and significant amounts of α-(1→4)-linked 2,6-disulfated galactosylresidues. The CPSul polysaccharide, on the other hand, consisted of alternating

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β-(1→3)-linked 2-sulfated galactosyl residues, α-(1→4)-linked 2,3-disulfatedgalactosyl residues, and β-(1→3)-linked-6-O-methyl-galactosyl units.

Similar sulfation patterns in galactans characterize the chemotaxonomicfeatures of this genus of Rhodophycean seaweeds as it is found in this in-vestigation as well as reported by other groups elsewhere. However, thepresence of (1→4)-linked 2,6-disulfated galactose units (11.2 mol%) inChampia indica, a structural component of λ-carrageenan, differentiates itfrom Champia parvula of Indian waters and other species of this genus re-ported in the literature.[15]

MATERIALS AND METHODS

Champia indica and Champia parvula (Rhodymeniales, Rhodophyta) were col-lected from Okha (22.28◦N, 69.04◦E) Gujarat and Diu (20.42◦N, 70.58◦E) at thewest coast of India, respectively. The seaweed thalli were washed with sea wa-ter to remove impurities and air dried. The voucher specimens (AL-II-112-12and AL-II-112-01) were deposited with the CSMCRI Herbarium at Bhavnagar.Borane 4-methyl morpholine complex (MMB) was purchased from Aldrich. Allchemicals used in this study were purchased from S. D. Fine Chemicals Mum-bai and were AR grade except isopropyl alcohol (LR).

Extraction and Purification of Sulfated PolysaccharidesDried seaweed samples (50 g) were soaked for 12 h at rt in demineral-

ized (DM) water (1:30 w/v) containing 2% methanol for preventing bacterialcontaminations, to obtain cold water-soluble components. The residue was re-suspended in DM water (1500 mL) and autoclaved at 110◦C for 2 h. Theextracts were then homogenized and centrifuged and crude polysaccharideswere isolated by precipitation of supernatants with isopropyl alcohol (1:2 v/v)to get crude hot water polysaccharides, designated as CICrude and CPCrude

from Champia indica and Champia parvula, respectively. The crude hot waterpolysaccharides (CICrude and CPCrude) were further purified using DEAE col-umn chromatography to obtain sulfated polysaccharides designated as CISul

and CPSul. To a 1% aqueous solution of crude hot water polysaccharides, DEAE(10 g suspended in 20 mL water) was added under stirring at rt. After stirringfor 10 h at rt, the contents were centrifuged at 8000 rpm (20 min) to get DEAEpellets, which were soaked in 4 M NaCl (400 mL) for 24 h under stirring atrt. Then the contents were centrifuged at 8000 rpm for 20 min (× 2) to get su-pernatants, dialyzed against distilled water till the dialysate showed negativesilver nitrate test for halide ions. The retentates were freeze dried to affordCISul and CPSul, which were subjected to various analyses.

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Desulfation of CISul and CPsul

Solvolytic desulfation of CISul and CPSul was carried out following themethods reported by Nagasawa et al.[24] An aqueous solution of CISul and CPSul

(1%, 20 mL) was passed through the IR 120-H+ resin column, and the eluentswere collected over 20% pyridine (20 mL), dialyzed against distilled water, andlyophilized to get the pyridinium salt. The latter were treated with 89:10:1dimethyl sulfoxide–methanol–pyridine at 80◦C for 4 h. The partial desulfatedpolysaccharides were isolated by dialysis against distilled water and the re-tentates were freeze dried to afford the partially desulfated polysaccharides,named CIDes and CPDes.

Chemical AnalysisTotal carbohydrate contents of CICrude, CISul, CIDes, CPCrude, CPSul, and

CPDes were determined by the method described by Dubois et al.[25] Pro-tein content was calculated from the percentage nitrogen (% N) estimated byKjeldahl’s method on a KEL PLUS-KES 201 Digestion unit attached to a KELPLUS-CLASSIC DX Distillation unit (M/s PELICAN equipments, Chennai, In-dia) using the factor 6.25.[26] Sulfate contents were determined by inductivelycoupled plasma spectrophotometry on a Perkin-Elmer (ICP)-OES Optima 2000DV machine following the method as described in the literature.[27] All analy-ses were done in triplicate and the mean value was considered.

Monosaccharide Composition AnalysisReductive hydrolysis of the polysaccharides was done following the meth-

ods reported by Stevenson and Furneaux, followed by acetylation for monosac-charide composition analysis of CICrude, CISul, CIDes, CPCrude, CPSul, andCPDes.[28] The resulting alditol acetates were analyzed by GC-MS on a capil-lary column (length 25 m, diameter 0.22 mm, thickness 0.25 µm, BP 225 ona Shimadzu GCMS-QP2010 machine) using a temperature program of 160◦C(3 min hold) to 230◦C (10◦C/min), ion source temperature 240◦C, split ratio1:30, and flame ionization detector (FID).[29] Quantification was carried outfrom the peak area calculation. Helium was used as carrier gas (1 mL/min).The detector condition was 70 eV, 60–400 amu.

Methylation analysisThe samples CISul, CIDes, CPSul, and CPDes were permethylated using

methyl iodide and powdered sodium hydroxide in dimethyl sulfoxide as re-ported by Ciucanu and Kerek.[30] Permethylated alditol acetates (PMAAs) wereobtained by reductive hydrolysis and acetylation.[28] The resulting partiallymethylated alditol acetates (PMAA) were analyzed by GC-MS on a capillary

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Sulfated Galactans of Champia indica and Champia parvula 57

column as mentioned above using the same temperature program. The identi-fication of PMAAs was done on the basis of retention time and mass fragmenta-tion patterns.[31] Quantification was carried out from the peak area calculation.Helium was used as carrier gas (1 mL/min), with the detector working at70 eV (60–400 amu).

Molecular Weight DeterminationThe weight-average molecular weights (Mw) of CICrude, CISul, CIDes,

CPCrude, CPSul, and CPDes were determined by gel permeation chromatog-raphy (GPC) on a Waters Alliance 2695 machine with RI 2414 detector. A20-µL aliquot of 0.5% solutions were injected on to the column and 0.1% aque-ous NaNO3 was used as the mobile phase with a flow rate 0.6 mL/min.[32]

Oven and flow cell temperatures were maintained at 45◦C for all measure-ments. Dextrans of different molecular weights were used as standards forcalibration. Area measurements and calculations of molecular weights weredetermined using Empower 2 Software (USA).

FT-IR and 13C NMRFTIR spectra of CICrude, CISul, CPCrude, and CPSul were recorded on a

Perkin-Elmer Spectrum GX (FT-IR System, USA) using the KBr disc method.13C NMR spectra of CISul and CPSul in D2O (50 mg/mL) were recorded at 70◦Con a Bruker Avance-II 500 spectrometer operating at 125 MHz. Chemical shifts(δ in ppm) were measured relative to internal DMSO (at δ 39.43 ppm).

ACKNOWLEDGMENTS

Authors are grateful to Ministry of Earth Sciences, New Delhi, for financialsupport (MoES/9-DS/6/2007-PC IV; dated March 5, 2008) as well as for award-ing fellowships to SK and DN. Sincere thanks are accorded to Dr. V. A. Mantriof CSMCRI for identification of the seaweed species.

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