versión impresa ISSN 0366-1644
Bol. Soc. Chil. Quím. v.47 n.3 Concepción sep. 2002
SOLUBLE POLYSACCHARIDES FROM Rhodymenia:
CHARACTERIZATION BY FT-IR SPECTROSCOPY
Betty Matsuhiro* and Luis G. Miller
Departamento de Ciencias Químicas, Facultad de Química y Biología,
Universidad de Santiago de Chile, casilla 40, correo 33, Santiago, Chile,
(Received: April 8, 2002 - Accepted: June 10, 2002)
The soluble polysaccharides of few species of the family Rhodymenaceae (Rhodophyta) have been studied. According to the literature members of this family are agarophytes. Dried samples of tetrasporic, cystocarpic and gametophytic Rhodymenia howeana and tetrasporic R. corallina were analysed by FT-IR spectroscopy. Results indicate that these seaweeds are not agar producers. Aqueous extraction followed by chemical and FT-IR analysis showed that the extracts are very similar in composition. Carrageenotype structures in the polysaccharides were not found.
KEY WORDS: agarans, carrageenans, Rhodymeniales, sulfated polysaccharides.
Los polisacáridos solubles de muy pocas especies de la familia Rhodymenaceae (Rhodophyta) han sido estudiados. Según la literatura, estos polisacáridos presentarían la estructura de agar. El análisis por espectroscopia de IR-TF de muestras secas y molidas de individuos de las fases tetraesporofítica, cistocárpica y gametofítica de Rhodymenia howeana y de la fase tetraesporofítica de R. corallina indica que no son productoras de agar. La extracción acuosa y posterior caracterización por métodos químicos y espectroscopia de IR-TF muestran que los polisacáridos son muy similares entre sí y constituyen mezclas de polímeros sulfatados donde tampoco están presentes estructuras típicas de carragenanos.
PALABRAS CLAVES: agaranos, carragenanos, Rhodymeniales, polisacáridos sulfatados.
The main soluble polysaccharides of Rhodophyceae are galactans. They are based on linear chains of alternating 3-linked b-D-galactopyranosyl (A unit) and 4-linked a-galactopyranosyl (B unit).The configuration of the B unit can be D or L which leads to the classification in carrageenan and agar types galactans, respectively. The A units are usually substituted by methyl groups, sulfate hemiesters, and pyruvic acid ketals. The B units can be partially or wholly substituted by 3,6-anhydro rings, and may be methylated or sulfated (1).
Members of the orders Gelidiales and Gracilariales are the most important agar resource. According to Duckworth and Yaphe (2), agar is essentially a mixture of the neutral polymer agarose, pyruvated agarose and sulfated galactans (Fig. 1). Agar contains around 3.5% of sulfate groups.
Fig.1. Structures of red seaweeds polysaccharides. A: agarose, B: sulfated galactans of agar, C: k-carrageenan, D: l-carrageenan.
Carrageenans are produced by members of the order Gigartinales, they are typically more sulfated than agars. In k-carrageenan, the B unit is 3, 6-anhydro-D-galactopyranose, and in l-carrageenan, the B unit is D-galactopyranose-2,6-disulfate (3-5) (Fig. 1). According to some authors (6,7) gametophytes of carrageenan producing seaweeds of the families Gigartinaceae and Phyllophoraceae contain predominantly gelling polysaccharides of the k-carrageenan family while diploid tetrasporophytes contain l-carrageenan.
Concurrence of agar and carrageenans structures has been reported in several red algal polysaccharides of the Cryptonemiales and Ceramiales (8). Takano et al. (9) fractionated the funoran from Gloiopeltis furcata into four fractions, being the main fraction 6-sulfated-agarose. In the minor fractions agaroid and carrageenan backbones were found.
Members of Rhodymenia of the order Rhodymeniales have received little attention because they do not produce phycocolloids of industrial interest. According to Fredericq et al. (10) on the basis of plastid-encoded rbcL sequences analysis, Rhodymeniales is a possible agarophyte order. Usov and Klochokova (11) by chemical characterization of the polysaccharides from Rhodymenia pertusa proposed that species of the genus Rhodymenia are agarophytes. Although, Whyte (12) had shown by ion-exchange chromatography that the polysaccharide from Rhodymenia pertussa contained a neutral glucan and sulfated galactans. Semesis y Dawes (13) isolated from Rhodymenia pseudopalmata a sulfated galactan with 22.1% of 3,6-anhydrogalactose.
According to Etcheverry (14), the genus Rhodymenia in the Chilean coasts is represented by the species, R. corallina, R. howeana, R. palmatiformis, R. cuneifolia, and R. peruviana. Although for central Chile, R. corallina, R. flabellifolia, R. peruviana, and R. skottsbergii were described. (15). In this work, the characterization by infraùSd spectroscopy and by chemical methods of the soluble polysaccharides from different stages of the life cycle of Rhodymenia howeana, and tetrasporic Rhodymenia corallina is presented.
Materials and methods
Rhodymenia howeana Dawson and Rhodymenia corallina (Bory) Greville were collected in winter at La Herradura Bay (29º71'S, 72º21'O), and Tongoy Bay (30º15'S, 71º30'O), respectively. Samples were sorted by nuclear phases in Departamento de Biología Marina, Universidad Católica del Norte, Coquimbo. Specimens with different phenologies were deposited in Sala de Sistemática y Colecciones of Universidad Católica del Norte. The FT-IR spectra of algal samples and polysaccharides in KBr pellets were recorded in a Bruker IFS 66v instrument. The second-derivative spectra were obtained with OPUS/IR versión 1.44 software incorporated into the hardware of the instrument (16). Commercial agar was from Merck. Polyacrylamide gel electrophoresis (PAGE) was conducted according to Usov and Arkhipova (17). Gas-liquid chromatography (GLC) of alditol acetates was carried out in a Shimadzu GC-14B gas chromatohraph equipped with a flame ionization detector using a SP-2330 column (15 m x 0.25 mm). Liquid chromatography (HPLC) was conducted in a Hitachi-Perkin Elmer 655-A chromatograph using a Partisil SAX column.
Sulfate content was determined by sulfur microanalysis in Facultad de Química, Pontificia Universidad Católica de Chile. 3,6-anhydrogalactose was determined according to the method of Yaphe and Arsenault (18). The content of uronic acids was determined by the sulfamate/m-hydroxydiphenyl assay using D-glucuronic acid as standard.
The dried ground seaweed (50 g) was stirred with distilled water (1 L) for 3h at 95ºC and filtered through muslim. The residue was extracted twice in the same conditions but with 0.5 L of water. The filtrates were centrifuged, dialysed against distilled water, concentrated in vacuo and freeze-dried.
The polysaccharide was heated with 2M trifluoroacetic acid during 6h a 100ºC. The solvent was removed and the resulting syrup was applied to a column of DEAE Sephadex A-25 (Cl-) and eluted with water, until the eluant monitored by phenol-sulfuric acid reagent (20) became free of carbohydrates, and then with 10% formic acid. The fraction eluted with water was concentrated to dryness, reduced with NaBH4, acetylated and analysed by GLC. The acidic fraction was concentrated in vacuo, the formic acid removed by repeated additions of water and concentrations, and examined by HPLC for uronic acids.
Fractionation with cetrimide
The polysaccharide (0.800g) was disolved in 80 mL of distilled water, treated with 50 mL of a 3% aqueous solution of cetrimide and stirred for 12 h at 40ºC. The mixture was centrifuged and the precipitate was washed with distilled water. The precipitate was dissolved in 4M NaCl and poured into 5 volumes of ethanol. The supernatant solution remainning after removal of the precipitate was treated with 10% KI aqueous solution until no further precipitation occurred. The mixture was centrifuged and the supernatant was dialysed against distilled water, concentrated and poured into ethanol.
A solution of the acidic fraction in distilled water was added to a DEAE-Sephadex A-50 column and eluted with water until the eluant monitored by phenol-sulfuric acid reagent (20) became polysaccharide-free. The column was then eluted stepwise with increasing concentrations of KCl (0.3-4.0 M), 0.5M NaOH, and 6.0M urea.
The polysaccharide was treated with NaBH4-NaOH according to the technique reported by Matulewicz and Cerezo (21).
RESULTS AND DISCUSSION
The FT-IR spectra of dried, ground samples of cystocarpic, tetrasporic and gametophytic Rhodymenia howeana are very similar. All of them present bands assigned to sulfate group at ~1230 cm-1, ~620 cm-1 and ~580 cm-1. In the second-derivative mode they present bands at 990 cm-1 due to C-O, C-C, HC6-C vibrations of the pyranosyl ring, at around 860 cm-1 assigned to the deformation of a anomeric C-H vibration, and at 740 cm-1 assigned to skeletal mode of vibration of anomeric carbons. The characteristic agar signals at 790 and 717 cm-1 were not present. This results suggests that R. howeana is not an agarophyte (16).
For R. corallina, only individuals in the tetrasporic phase were found. The FT-IR spectrum of the seaweed is very similar to those of R. howeana samples and to cystocarpic and tetrasporic R. howeana collected in summer (22).
It is worth to mention that R. howeana is isomorphic with the carrageenophyte Stenogramme interrupta (Phyllophoraceae, Gigartinales). The two species can be differentiated by FT-IR spectroscopy (22).
Hot water extraction of R. howeana and R. corallina afforded polysaccharides in similar yields to those found for unsorted R. howeana (23) but with lower yields than those reported for agar and carrageenan types extracts (24). The sulfate content of the polysaccharides are similar for those reported for carrageenan-types phycocolloids (Table I). By spectrophotometric analysis it was found that the polysaccharides do not contain 3,6-anhydrogalactose.
The normal FT-IR spectra of the polysaccharides show bands at 1260, 836, 615 and 580 cm-1 assigned to sulfate groups. The second-derivative spectra show new bands at 988 cm-1 due to the stretching vibration of the pyranosyl ring, at 864 cm-1 attributed to a-anomeric C-H deformation, and at 840 cm-1 and 823 cm-1 assigned to an axial secondary and equatorial primary sulfate group, respectively. At lower wave numbers, two bands assigned to S-O vibrations, at 623.7 and 581.9 cm-1 are present (16). In figure 2 the normal and the second-derivative FT-IR spectra of the polysaccharide extracted from cystocarpic Rhodymenia howeana are shown. For comparison the second-derivative FT-IR spectrum of a commercial sample of agar with the two characteristic clusters of signals between 1000-600 cm-1 is presented.
Fig. 2. FT-IR spectra of the polysaccharide extracted from cystocarpic Rhodymenia howeana. A: normal spectrum, B: second-derivative spectrum.
Fig. 3. Second-derivative FT-IR spectrum of commercial agar.
Since by FT-IR spectroscopy the polysaccharides from different nuclear phases of R. howeana show very similar properties, the chemical characterization was conducted on the polysaccharide from tetrasporic plants.
Fractionation by cetrimide of the polysaccharide afforded a soluble fraction in 3% yield. Acid hydrolysis followed by GLC analysis of the alditol acetates indicated the presence of xylose, glucose, mannose and galactose in the molar ratio 1.0:1.0:1.5:2.5. The presence of glucose in the neutral fraction may be due to the hydrolysis of floridean starch. On the other hand, the presence of a neutral xylomannogalactan is rather unusual in red seaweed. The insoluble fraction in cetrimide (45% yield) was shown by PAGE to be no-homogeneous. It was separated into six fractions by chromatography on DEAE Sephadex-50. All the fractions contain significant amounts of glucuronic acid (Table II). The presence of uronic acids in red seaweds polysaccharides had been reported in few cases. From Palmaria decipiens of the order Palmariales, a neutral xylan and a xylogalactan with 4.8 % uronic acids were obtained (23). Takano et al. (24) isolated from Lomentaria catenata of the family Champiaceae (Rhodymeniales) two sulfated polysaccharides fractions with agar-carrageenan backbone structures carrying D-glucose and
|1.5M KCl|| |
|2.OM KCl|| |
|4.OM KCl|| |
|6.OM Urea|| |
|5.OM NaOH|| |
D-glucuronic acid as single units branching.
Fraction 1 by hydrolysis and GLC of the alditol acetates showed the presence of glucose, mannose and galactose in the molar ratio 1.0:2.0:2.3.
Fraction 4 is composed of glucose and galactose in the molar ratio 1.0:3.0. Alkaline treatment of this fraction followed by FT-IR analysis and spectrophotometric determinations, showed a small decrease in the content of sulfate groups but no formation of 3,6-anhydro residues. The results obtained indicates that 4¾1-a-galactopyranosyl residues sulfated at C-6 are not present in the polymer. This type of residue is common in agarans and carrageenans.
Fraction 6 is composed by galactose, glucose, mannose and xylose in the molar ratio 1.0:2.9:3.0:5.8.
It is noteworthy the variation in compositoin of the fractions. Only fraction 4 seems to be composed of a sulfated galactan, probably contaminated by floridean starch.
The phycocolloid obtained by aqueous extraction of Rhodymenia howeana is a complex mixture of polysaccharides that differs from those of agar and carrageenans families. No significant variations in the composition and FT-IR properties of the polysaccharides with the life cycle of Rhodymenia howeana were found. The presence of glucuronic acid in the sulfated fractions, may have a taxonomic significance.
Financial support of Dirección de Investigaciones Científicas y Tecnológicas of Universidad de Santiago de Chile is gratefully acknowledged.
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