Structural analysis of a sulfated polysaccharidic fraction obtained from the coenocytic green seaweed Caulerpa cupressoides var . lycopodium

Researches on structural chemistry of sulfated polysaccharides (SPs) have been mainly focused on red and brown algae. Caulerpa cupressoides var. lycopodium (Chlorophyta) contains three SPs fractions (Cc-SP1, CcSP2 and Cc-SP3). Cc-SP1 and Cc-SP2 had anticoagulant (in vitro) and antiand prothrombotic, antinociceptive and/or anti-inflammatory (in vivo) effects. However, their structural features have not yet been investigated. This study analyzed the chemical composition, elemental microanalysis and structural features by infrared (IR) and nuclear magnetic resonance (H NMR) spectroscopy of Cc-SPs. Fractionation of SPs by DEAE-cellulose yielded Cc-SP1, Cc-SP2 and CcSP3 containing differences among the relative proportions of sulfate (14.67-26.72%), total sugars (34.92-49.73%) and uronic acid (7.15-7.22%). Carbon (21.76-29.62%), sulfate (2.16-4.55%), nitrogen (0.85-1.57%) and hydrogen (4.57-5.86%) contents were obtained using a CHN equipment. Data from IR indicated occurrence of sulfate ester, galactose-6-sulfate, uronic acid and glycoside linkages. For H NMR spectrum of the soluble Cc-SP1 fraction, it was mainly found β-galactopyranose residues and CH3 group. The results showed that Cc-SPs fractions have some structural features similar to others studied Caulerpaceae SPs.

The genus Caulerpa Lamouroux (1809) includes species generally occurring in tropical and subtropical marine waters.Approximately one hundred species have been described and are important contributors to the algal biomass of coral reefs and lagoons (TRI, 2009).Polysaccharides from Caulerpa genus consisting of sulfate, galactose, glucose, arabinose and xylose, and small amounts of mannose and rhamnose and traces of fucose residues have been identified with medicinal importance (GHOSH et al., 2004;HAYAKAWA et al., 2000;JI et al., 2008;MAEDA et al., 2012).From the Brazilian species Caulerpa cupressoides var.lycopodium, three SPs fractions (Cc-SP 1 , Cc-SP 2 and Cc-SP 3 ) have been recently isolated.Cc-SP 2 showed anticoagulant (in vitro), anti-and prothrombotic (in vivo) (RODRIGUES et al., 2011a), antinociceptive and anti-inflammatory (in vivo) (RODRIGUES et al., 2012) effects.Cc-SP 1 and Cc-SP 3 had no anticoagulant effect, but an antinociceptive action of Cc-SP 1 was recently investigated (RODRIGUES et al., 2013b).Nevertheless, only one study reported the structure features of its SPs (Cc-SP 2 ) (RODRIGUES et al., 2013a).In the present study, some structural properties of Cc-SP 1 were investigated.

Marine alga and SPs and chemical analyses
The green seaweed C. cupressoides var.lycopodium (Vahl) C. Agardh (Caulerpaceae, Bryopsidales) was collected on the seashore from Flecheiras beach, Ceará State, Brazil.A voucher of this specimen has been deposited in the Prisco Bezerra Herbarium (Department of Biology, Federal University of Ceará, Brazil).The crude SP was extracted from the dehydrated algal tissue (room temperature) by papain digestion (60°C, 6h), and then subjected to fractionation by anion-exchange chromatography on a DEAE-cellulose column using a NaCl gradient (0→1 M, with 0.25 M of intervals).Fractions (Cc-SP 1 , Cc-SP 2 and Cc-SP 3 eluted with 0.5, 0.75, and 1 M of NaCl, respectively) were obtained, as described by Rodrigues et al. (2011a).Quantitative determination of sulfate, total sugars and contaminant proteins of the SPs fractions were carried out.The uronic acid content of the SP fractions was determined by carbazole-sulfuric acid method using spectrophotometric analysis (AMERSHAM BIOSCIENCES ULTROSPEC 1100) at 525 nm, using glucuronic acid as standard.These experimental protocols were performed, as previously described (RODRIGUES et al., 2011a).Nitrogen, carbon, hydrogen and sulfate contents were determined by elemental microanalysis using a CHN equipment Perkin Elmer model 2400 based on Maciel et al. (2008).

Structural analysis Infrared (IR) spectroscopy
To study structural features, Fourier Transform IR (FT-IR) spectra of the SP fractions were determined using a SHIMADZU IR spectrophotometer (model 8300) between 4000 and 500 cm -1 .The samples (5 mg) were pressed on KBR pellets.

Nuclear magnetic resonance (NMR) spectroscopy
One dimension 1 H NMR spectrum of a SP fraction from C. cupressoides var.lycopodium (Cc-SP 1 ) was recorded using a Bruker DRX 800 MHz apparatus with a triple resonance (5 mm).About 3 mg of sample was dissolved in 0.5 mL 99.9% deuterium oxide.The spectrum was recorded at 60°C with HOD suppression by presaturation.Chemical shifts were given relative to external trimethylsilyl-propionic acid standard at 0 ppm (FARIAS et al., 2008).

Chemical analyses
The fractionation of the crude SP obtained from C. cupressoides var.lycopodium performed on a DEAEcellulose column resulted in different chemical proportions of sulfate and total sugars contents among the SPs fractions, while the uronic acid content was almost equal among them (Table 1).
Although proteins have not been detected, analyses revealed the presence of nitrogen in SP fractions (Table 1).Values ranged from 0.85 to 1.57%.These data suggested the presence of amino acids of the proteins in the SPs fractions, indicating the hypothesis that C. cupressoides var.lycopodium may be capable of biosynthesizing proteoglycans as described for Caulerpa species (GHOSH et al., 2004;JI et al., 2008) and in animals (RODRIGUES et al., 2011b).It could indicate a correlation between proteins levels and nitrogen content (MARINHO-SORIANO et al., 2006).The nitrogen content of SPs fractions (C.cupressoides var.lycopodium) was similar to that described for G. birdiae (1.22%) by Maciel et al. (2008), but higher than obtained for the red seaweed G. cornea (0.41-0.47%) (MELO et al., 2002).Fractions Cc-SP 1 , Cc-SP 2 and Cc-SP 3 also showed levels of 29.62, 21.76 and 22.5% for carbon; and 5.86, 4.57 and 4.75% for hydrogen, respectively (Table 1).Maciel et al. (2008) found 40% carbon content in the cold extract for G. birdiae.Further investigations should be conducted to infer the effect of these molecules on the bacterial growth (AMORIM et al., 2012).
Therefore, low values were recorded in Cc-SP 1 when compared to those data found in Cc-SP 2 and Cc-SP 3 (Table 1 and Figure 1), revealing thus the occurrence of distinct SPs and that the employment of different NaCl molarities was important for C. cupressoides var.lycopodium SPs separation (DEAEcellulose) (ARAÚJO et al., 2011).Some SPs possessing highly complex and heterogeneous structures have been isolated from aquatic organisms.Bilan et al. (2004) investigated the presence of a highly regular fucoidan composed of alternating 3-linked α-L-fucopyranose 2,4-disulfate and 4-linked α-L-fucopyranose 2-sulfate residues from F. distichus (Phaeophyta).Marine angiosperms (Ruppia maritima, Halodule wrightii and Halophila decipiens) were described to have sulfated galactans.Those found in R. maritima were constituted by a regular tetrasaccharide repeating unit that appeared to have an intermediate chemical structure compared to SPs obtained from marine invertebrates and red seaweeds (AQUINO et al., 2005).More recently, SPs obtained from the microalgae Arthrospira platensis were demonstrated to have a preponderance of rhamnose present in their chemical structures (MAJDOUB et al., 2009).
The present study, together with literature data, point out galactose as a highly conserved structural sugar in Caulerpaceae (CHATTOPADHYAY et al., 2007a;GHOSH et al., 2004) and could be of taxonomic significance (AMORIM et al., 2012;AQUINO et al., 2005;DANTAS-SANTOS et al., 2012;POMIN;MOURÃO, 2008;USOV, 1998).Murano et al. (1997) investigated SPs (carrageenans) extracted from S. filifomris and Agardhiella subulata (Rhodophyta) from Mar Piccolo, Italy.The authors separately analyzed different crude SPs extracts by IR and NMR spectroscopic analysis showing similar polysaccharide structure backbone among them, but some irregularities notably were attributed to 6-sulfated 4-linked precursor units (galactose-6sulfate). Differences in the precursor content could be derived from variations of season, growth conditions and life cycle of these macroalgae species.Galactose-6-sulfate is a natural biological precursor which can be converted to 3,6-anhydrogalactose (CAMPO et al., 2009), which may be found in SPs from some red seaweeds species with commercial interests (MACIEL et al., 2008;SILVA et al., 2010).According to Campo et al. (2009), the use of alkaline extraction for red seaweeds SPs increases the functional ability of the gel as thickening, gelling and stabilizing agents for biotechnological applications.

NMR
For a more detailed structural investigation of Cc-SP1, which showed to be soluble in D 2 O solution, 1 H NMR spectroscopy was carried out (Figure 2).This method gives valuable structural information of polysaccharides (CAMPO et al., 2009).The experiment was performed at high temperature (60 °C) to increase the solubility of the Cc-SP 1 solution (CAMPO et al., 2009;SILVA et al., 2010).However, its structural analysis was very difficult and not fully examined.Chemical shifts of Cc-SP 1 showed an evident anomeric proton signal at δ H 4.65 ppm assigned to H-1 of the sugar residues (CHATTOPADHYAY et al., 2007b), with value of coupling constant of ̴ 8.34 Hz.H-1 would correspond to the β-configuration of galactopyranoses (FARIAS et al., 2008), and glucose and/or xylose residues could be linked with the same molecule (CHATTOPADHYAY et al., 2007b).In contrast, glucan showing α-configuration was isolated from C. racemosa (Chlorophyta) by Chattopadhyay et al. (2007a).Anomeric signals located at the region ranging from δ H ̴ 3.35 to 4.38 ppm could be attributed to protons of the C-2˗C-5 of the sugar residues (QI et al., 2012); of uronic acid at δ H ̴ 3.78 (COO -) (LI et al., 2012); and discrete peaks assigned from δ H ̴ 4.5 to 4.8 ppm would indicate sulfated sugar residues (ROBIC et al., 2009) and/or of uronic acid in polymeric blocks (SINHA et al., 2010).The absence of low-field signals (δ H > 5) could confirm that the polymer consisted of β-pyranoses based on Bilan et al. (2007) and Chattopadhyay et al. (2007a).The signal at δ H 1.42 ppm suggested the presence of CH 3 group in rhamnopyranose residues of the sample (BILAN et al., 2007;FARIAS et al., 2008;QI et al., 2012).
Although significant progresses in research on the structural chemistry of algae SPs had occurred in recent years, the structural heterogeneity of these compounds is still considered the major limitation to determine their precise chemical features (JIAO et al., 2011).There is also a lack of analytical methods to elucidate fine structures of these polymers (CAMPO et al., 2009).Each algal species could be a potential source of SPs exhibiting novel structures (ARAÚJO et al., 2011;BILAN et al., 2004BILAN et al., , 2007;;CIANCIA et al., 2012;FENORADOSOA et al., 2009;MAZUMDER et al., 2002;POMIN;MOURÃO, 2008).The study of the chemical structures and their molecular targets are essential steps to the design of new biomaterials for food and pharmacological uses (AMORIM et al., 2011;CAMPO et al., 2009;FONSECA et al., 2008;LEITE et al., 1998;LI et al., 2012;QUINDERÉ et al., 2014;SMIT, 2004).In the present study, the employment of high temperature (60°C) to increase the solubility of the solution containing SPs from C. cupressoides var.lycopodium(Cc-SP 1 ) could affect the proton directly linked to carbons involved in glycosidic linkages.Possibly, it justified the absence of low-field signals (δ H > 5) in the 1 H NMR spectrum of Cc-SP 1 (Figure 2), although the signal correspondent to the residual water has been observed (data not shown) (CAMPO et al., 2009).Single structural difference due to sugar type and anomeric configuration could promote great changes on biological action of SPs (POMIN; MOURÃO, 2008).In a recent report, it was demonstrated that a SPs fraction with anticoagulant effect from C. cupressoides var.lycopodium (Cc-SP 2 , Table 1) contained various polysaccharides of different molecular weights (RODRIGUES et al., 2013a).Based on these our previous findings, low molecular weights SPs (oligosaccharides) could be used to a more detailed structural investigation of these molecules, and arousing thus a new importance to gain insight into the complexity of these polysaccharides (CAMPO et al., 2009).

Conclusion
The coenocytic green seaweed Caulerpa cupressoides var.lycopodium contains sulfated polysaccharides fractions that possess some structural features similar to others studied Caulerpa sulfated polysaccharides.However, the structural analysis by 1 H NMR technique from a polysaccharidic fraction indicates new importance to gain insight in the study of Caulerpaceae.

Figure 2 .
Figure 2. 1 H NMR spectrum of the Cc-SP 1 fraction obtained by anion-exchange chromatography (DEAE-cellulose) from the green seaweed Caulerpa cupressoides var.lycopodium.(A) Signal assigned to the β-configuration of galactopyranoses; (B) Signal corresponding to the proton of methyl group of the sugar residues.
a -Dosage by Dodgson and Price' method using NaSO3 as standard; b -Dosage by Dubois et al.' method using D-galactose as standard; c -Dosage by Dische' method using glucuronic acid as standard; d -Dosage by Bradford' method using bovine serum albumin (-not detected); e -sulfate, f -carbon, g -hydrogen and h -nitrogen were determined by elemental microanalysis using a CHN equipment Perkin Elmer model 2400(MACIEL et al., 2008).