The system of low-molecular-weight carrageenans and agaroids from the room-temperature-extracted fraction of Kappaphycus alvarezii

The room-temperature-extracted fraction from the red seaweed Kappaphycus alvarezii consists mainly of low-molecular-weight carrageenans, with structural dispersion around a basic kappa-pattern. This dispersion results from: (a) low percentages of 3,6-anhydrogalactose and the presence of precursor units; (b) important quantities of 6-O-methyl beta-D-galactose (4-sulfate) residues; (c) significant amounts of iota-repeating structure, and (d) small amounts of non-sulfated and disulfated beta-D-galactose residues. Significant quantities of alpha-L-galactose units suggest the presence of agaroids, as it has been reported in several other carrageenophytes.     

A novel high-performance anion-exchange chromatographic method for the analysis of carrageenans and agars containing 3,6-anhydrogalactose

A novel method has been developed to determine the sugar composition of 3,6-anhydrogalactose-containing polysaccharides, such as carrageenan and agar. The method is based on reductive hydrolysis with a methylmorpholine-borane complex in the presence of acid and subsequent high-performance anion-exchange chromatography analysis of the alditols without any derivatization. The method was validated by 13C NMR analysis of six carrageenans and three agars and by a previously used method based on derivatization to alditol acetates and gas-liquid chromatography analysis. The new method was found to be superior to the gas-liquid chromatography method as the analysis time was less than half. Also it was found to be more accurate and reproducible and no derivatization was required. The analysis of the six different carrageenan samples revealed that homogeneous mu- and nu-carrageenan, theoretically without 3,6-anhydrogalactose residues, cannot be isolated from red seaweeds. Consequently, the question arose if mu- and nu-carrageenans at all are present in seaweeds and if the current hypotheses regarding biosynthesis of carrageenans in the seaweeds are correct. The data demonstrated that carrageenans are highly complex natural polysaccharides, which are more irregular than assumed hitherto. The new analytical technique will permit elucidation of the detailed structure of seaweed polysaccharides and determination of their structure-property relationships.     

Microwave-assisted extraction of the Carrageenan from Hypnea musciformis (Cystocloniaceae,Rhodophyta)

Hypnea musciformis (Wulfen in Jacqu.) J. V. Lamour. (Rhodophyta) was investigated for its carrageenan production. Traditionally, the desulfation process for carrageenans has been promoted by an alkaline treatment of up to 3 h by conventional heating during carrageenan extraction. New extraction techniques based on microwave irradiation may accelerate this reaction with the advantages of reduced consumption of solvents, energy, and extraction time, suggesting the feasibility of this method as a “Green” technology. In this study, aqueous- and alkali-treated carrageenans from H. musciformis collected along Quintana Roo coast of Yucatan Peninsula (Mexico) were extracted by conventional method and by microwave-assisted extraction (MAE). Microwave irradiation in closed vessels was used to carry out the alkaline modification. The influence of temperature (85, 95, and 105 °C) and extraction time (10 and 20 min) in MAE was investigated in terms of yield, sulfate, and 3,6-anhydrogalactose contents, and Fourier transformed infrared spectra. Although lower carrageenan yields were obtained during MAE extraction, the κappa/iota hybrid carrageenan obtained by this novel method is comparable to that extracted by conventional technique. At the maximum temperature used for MAE (105 °C), an increase of 3,6-anhydrogalactose as well as an increase of the κappa-proportion was observed indicating that MAE could be an adequate procedure for carrageenan extraction of H. musciformis; however, further extraction parameters should be tested to optimize extraction.     

Comparative study of carrageenans from reproductive and sterile forms of <Emphasis Type="Italic">Tichocarpus crinitus</Emphasis> (Gmel.) Rupr (Rhodophyta,Tichocarpaceae)

A comparative study of the structure and properties of the sulfated polysaccharides (carrageenans) isolated from the vegetative and reproductive forms of the red alga Tichocarpus crinitus was performed. The polysaccharides were separated into the gelling (KCl-insoluble) and non-gelling (KCl-soluble) fractions by precipitation with 4% KCl. The total content of polysaccharides extracted from the reproductive form of the alga was 1.8-fold more than that extracted from the vegetative form, and in the first case, the gelling polysaccharides mostly accumulated. The gelling polysaccharides from the vegetative form have the highest molecular weight (354 kD). According to the results of FT-IR and 13C-NMR spectroscopy, the gelling polysaccharide fractions from both forms are kappa/beta carrageenans. The differences concern the content of the kappa- and beta-disaccharide units and the presence of a small content of the sulfated disaccharide segments (precursors of the kappa-carrageenans) in the polysaccharide from the reproductive form of the alga. The non-gelling polysaccharide fractions from both forms of the plant are mixtures of sulfated galactans with a low content of 3,6-anhydrogalactose.     

The chemistry and immunochemistry of carrageenans from Eucheuma and related algal species

Carrageenans from several species of Eucheuma have been fractionated into KCI-soluble and KCI-insoluble fractions and analyzed by the usual chemical procedures. An anti-K-carrageenan, the reactivity of which is directed to K-structures (i.e., 3-linked galactose 4-sulphate, and 4-linked 3,6-anhydrogalactose) was used to analyze these carrageenans immunochemically. The antibody preparation shows only a small amount of cross-reactivity with i-type carrageenans and thus could be used to distinguish K- and i-type carrageenans, the latter having an index of homology of less than 0.2. A comparison of chemical and immunochemical data yielded further information as to the nature of the carrageenan-anti-carrageenan interaction, as well as elucidating the finer structure of carrageenans.     

Cell wall composition of the carrageenophyte Kappaphycus alvarezii (Gigartinales, Rhodophyta) partitioned by wet sieving

The cortical and medullary cells of Kappaphycus alvarezii fractions were screened by wet sieving after aqueous extraction of carrageenans. The cell populations obtained showed a clear partition between these two cell types. The main monosaccharide in hydro-insoluble cell walls was cellulosic glucose (70% dry weight), the crystallinity of which was shown torange from 20% in the cortical cells to 45% in the large medullary cells (over 250 μm diameter). Minor monosaccharides in the insoluble fraction were galactose, 3,6-anhydrogalactose (indicating presence of residual carrageenans), mannose and xylose. However, the major part of the remaining galactose probably originated from another galactoglycan strongly linked to insoluble polymers in the large medullarycell walls. The mannose concentration was maximum in the cortical cells and decreased with increasing size of the medullary cells. Thus, besides cellulose, two other types of polysaccharides were detected in insolublecell walls, mannoglycans and galactoglycans in cortical and medullary cellwalls, respectively. This revised version was published online in August 2006 with corrections to the Cover Date.     

Influence of tissue source and growth rates on dry weight and carrageenan composition of Chondrus crispus (Gigartinales,Rhodophyta)

Carrageenan analyses were conducted on vegetative female clones of Chondrus crispus that were cultured to provide tissues with differing growth rates. Tissue dry weights increased from apex to base of fronds. Total carrageenan contents were lower in apical 1 to 2 cm segments than elsewhere in the frond, except when the alga was grown at high photon irradiances. Clone 373A contained more carrageenan than clone G8. The proportion of 0.3 M KCl-soluble polymers in the total native carrageenans varied from 44 to 92%, being highest in older tissues of fronds cultured at high photon irradiances. The apical 1 cm segments contained less KCl-soluble carrageenans than other tissues from the corresponding fronds. The KCl-soluble carrageenans, when alkali-modified and refractionated, afforded the expected kappa-iota carrageenan in > 79% yields. The remainder consisted of a polymer containing 23.1% SO₃Na and 8.4% 3,6-anhydrogalactose. Lambda carrageenan was not detected. Variations in carrageenan distribution between the apical region and other parts of the frond may reflect the increasing influence of medullary tissue developed as the immature cells differentiate.     

Carrageenan from Sarconema scinaioides (Gigartinales, Rhodophyta) of Indian waters

Carrageenan was extracted from the red seaweed Sarconema scinaioides of Indian waters and was characterized. The crude carrageenan as well as its alkali modified derivative was composed of 3,6-anhydro galactose, 6-O-methyl galactose as well as galactose moieties in various proportions. Linkage analysis exhibited that these two carrageenan samples consisted of 4-linked 3,6-anhydrogalactose residue sulphated at position 2, and 3-linked galactose residue sulphated at position 4. The physicochemical and rheological data along with molecular weight data, FT-IR, 1D and 2D NMR (¹H, ¹³C, COSY and HSQC) spectrometry suggested that the polysaccharide was composed predominantly of iota- along with a small amount of its precursor nu (ν)-carrageenan, unlike the hybrid carrageenans (iota-, pyruvated- and kappa-carrageenans) from this seaweed reported in the literature. This Indian seaweed species would be a potentially important source of iota-carrageenan.     

Selective Hydrolysis of the 3,6-Anhydrogalacotosidic Linkage in Red Algal Galactan: A Combination of Reductive Acid Hydrolysis and Anhydrous Mercaptolysis

Here we report a simple method for the structural analysis of red algal galactan containing 3,6-anhydrogalactose. Structural heterogeneity in the galactan was demonstrated by this method. For selective hydrolysis of 3,6-anhydrogalactosidic linkages in the galactan, conditions for reductive mild acid hydrolysis were examined by characterizing the resulting oligosaccharide alditols by anhydrous mercaptolysis. Residues other than alditols at the reducing ends, including labile 3,6-anhydrogalactose, were liberated quantitatively as diethyl dithioacetal derivatives, whereas alditols at the reducing ends were not derivatized and were liberated as alditols intact. The liberated sugars were then separated and measured quantitatively by gas-liquid chromatography. Heating of agarose in reductive hydrolysis with 0.3 M trifluoroacetic acid in the presence of an acid-stable reducing agent, 4-methyl morpholine borane, at 80 °C for 90 min and for 90 °C for 45 min was found to be optimum for the selective hydrolysis of 3,6-anhydrogalactosidic bonds, without detectable cleavage of other glycosidic bonds.     

Potential energy surfaces of carrageenan models: carrabiose,beta-(1 --> 4)-linked D-galactobiose,and their sulfated derivatives

The adiabatic conformational surfaces of several beta-linked disaccharides, which correspond to the repeating structures of carrageenans, were calculated using the MM3 force-field. The studies were carried out on the disaccharide beta-D-Galp-(1 --> 4)-alpha-D-Galp and eight sulfated derivatives, as well as on carrabiose (beta-D-Galp-(1 --> 4)-3,6-An-alpha-D-Galp) and five sulfated derivatives. The presence of 3,6-anhydrogalactose does not change the main features of the maps, although it increases the flexibility of the glycosidic linkage. Sulfation neither produces a striking effect on the map shape, nor a shift on the global minimum, which always remains with psi (theta(C-1'-O-4-C-4C-5)) in trans orientation, and phi (theta(O-5'-C-1'-O-4-C-4)) with a value close to -80 degrees. This effect differs from that occurring on the alpha linkage of equivalent disaccharides, for which the sulfation pattern on the beta-galactose unit shifts the global minima to different positions. A reduction in the flexibility (originated in a deepening of the global minimum well) is observed by sulfation on position 2 of the beta-D-galactose unit, and by sulfation of position 6 of the alpha-D-galactose unit (when the beta-D-galactose unit is 4-sulfated). Within the compounds containing 3,6-anhydrogalactose, the effect of sulfation is even less noticeable. The calculated low-energy regions on carrabiose derivatives agree with X-ray diffraction data on carrageenan fibers and on peracetylated carrabiose dimethyl acetal, and with NOE calculations carried out on kappa-carrabiose.     

CO-OCCURRENCE OF CARRAGEENANS AND AGAROIDS IN GYMNOGONGRUS TORULOSUS (GIGARTINALES, PHYLLOPHORACEAE) FROM ARGENTINA

The red seaweeds, Gigartina skottbetgii and Sarcothalia crispata , have commercial value as raw material for the industrial production of phycocolloids (carrageenans) in Argentina. The third alga with potencial possibilities for the carrageenan production is Gymnogongrus torulosus. Herein, we report the study of the polysaccharide system present in Gymnogongrus torulosus , which contribute to the estimation of the importance of this algae in the seaweed industry. Analysis of the hot water-soluble extract (C1), by FT-IR, methylation and ¹³C NMR, showed mainly the presence of iota/kappa carrageenan hybrid (molar ratio ∼ 2:1). The soluble fractions obtained after KCl fractionation (F3, 16.1 % of C1) and soluble after alkaline treatment and KCl fractionation (F3T3, 34.6% of F3T) gave negative optical rotations (−15.5 and −55.4, respectively), considerably lower than those reported for kappa/iota carrageenans (from 56.1 to 66.5). These fractions (F3 and F3T3) have significant quantities of L-galactose (11.1% and 29.8%) and L-3,6-anhydrogalactose (19.2% and 4.3%). The direct relationship between the optical rotation and the percentage of L-galactose indicated that its structural influence is similar in all the fractions. The results suggest that Gymnogongrus torulosus biosynthesizes a polysaccharide system with co-occurrence of carrageenans and agaroids in the same thallus.     

Immunochemistry of kappa-type carrageenans from certain red algae

Carrageenans from female and male gametophytic plants of the alga Rhodo-glossum californicum, female plants of Chondrus crispus and Gigartina pistillata, and male plants of Iridaea cordata and a Gigartina species from San Francisco Bay were fractionated into potassium chloride-soluble and -insoluble components and were analysed chemically. An anti-K-carrageenan, the reactivity of which is directed to K-type structures (i.e., 3-linked d-galactose 4-sulphate and 4-linked 3,6-anhydro-D-galactose residues) was used to analyse these carrageenans immunochemically. The potassium chloride-insoluble carrageenans from these species were found to be highly reactive K-type carrageenans. The potassium chloride-soluble carrageenans were less reactive to anti-K-carrageenan and, in addition, showed reactivity to an anti-λ-carrageenan preparation. The chemical and immunochemical data suggest that the potassium chloride-soluble carrageenans contain either λ- or μ-carrageenan, as a high proportion of the precursors to the 3,6-anhydro-D-galactose are 4-linked D-galactose 2,6-disulphate residues, and no increase in immunological reactivity to anti K-carrageenan was observed upon alkali treatment.     

Structural analysis and antiviral activity of a sulfated galactan from the red seaweed Schizymenia binderi (Gigartinales, Rhodophyta)

Aqueous extraction of gametophytic Schizymenia binderi afforded a polysaccharide composed of galactose and sulfate groups in a molar ratio of 1.0:0.89 together with uronic acids (6.8 wt%) and minor amounts of other neutral sugars. Alkali-treatment of the polysaccharide afforded a polysaccharide devoid of 3,6-anhydrogalactose. 13C NMR spectroscopy of the desulfated alkali-treated polysaccharide showed a backbone structure of alternating 3-linked beta-D-galactopyranosyl and 4-linked alpha-galactopyranosyl units that are predominantly of the D-configuration and partly of the L-configuration. Methylation, ethylation and NMR spectroscopic studies of the alkali-treated polysaccharide indicated that the sulfate groups are located mainly at positions O-2 of 3-linked beta-D-galactopyranosyl residue and at position O-3 of 4-linked-alpha-galactopyranosyl residues, the latter is partially glycosylated at position O-2. The sulfated galactan from S. binderi exhibited highly selective antiviral activity against Herpes simplex virus types 1 and 2, with selectivity indices (ratio cytotoxicity/antiviral activity) >1000 for all assayed virus strains. This compound was shown to interfere with the initial adsorption of viruses to cells.     

Chemical screening of Brazilian marine algae producing carrageenans

The yield, IR spectra, 3,6-anhydro-d-galactose and sulfate content are given for several species of putative carrageenophytes from different sites on the Brazilian coast. Kappa carrageenan was found in Hypnea musciformis and Gigartina spp. ; all the other species tested yield an iota type. The lowest carrageenan yield was found in Cryptonemia crenulata (5%), and the highest in Gigartina sp. (72%); 3,6-anhydrogalactose content was highest in Hypnea musciformis and Gigartina spp., whereas sulfate was lowest in H. musciformis. Slight variations in the yield of carrageenans were found during the period of observation (March 1988 to May 1989), but no clear pattern could be recognized. No significant variation was observed in plants from different collection sites.     

Methylation analysis of carrageenans from the seaweed Iridaea undulosa

Two carrageenans from Iridaea undulosa, isolated by precipitation of the crude polysaccharide at O.70–1.05 M and 1.55–1.65 M KCl concentrations, were studied by methylation analysis. Acid hydrolysis of the methylated derivative of the less soluble carrageenan (molar ratio galactose: 3,6-anhydrogalactose: sulphate 1.00: 0.50: 1.20) yielded major amounts of 2,6-di-O-methylgalactose (51.3 mol %), 4,6-di-O-methylgalactose (25.6%) and 4-O-methylgalactose (51.3mol%), 4,6-di-O-methylgalactose (25.6%) and 4-O-methylgalactose (13.4%). Minor quantities of 3-O-methylgalactose (4.6%) and 6-O-methylgalactose (3.2%) were found together with traces of 2,3,6- and/or 2,4,6-tri-O-methylgalactose, 2-O-methylgalactose and galactose. Oxidative acid hydrolysis produced 3,6-anhydro-2-O-methylgalactonic acid and 3,6-anhydrogalactonic acid in a molar ratio 3.5-4.0:1.0. The methylated derivative of the more soluble carrageenan (molar ratio galactose:3,6-anhydrogalactose:sulphate 1.00:0.04:1.43) gave on acid hydrolysis, 2,3,4,6-tetra-O-methylgalactose (4.6%), 2,3,6-tri-O-methylgalactose (4.2%), 2,4,6-tri-O-methylgalactose (10.7%), 4,6-di-O-methylgalactose (24.1%), 3,6-di-0-methylgalactose (8.0%), 2,3-di-O- methylgalactose (3.4%), 2,4-di-O-methylgalactose (4.6%), 2,6-di-O-methylgalactose (4.2%), 3-O-methylgalactose (19.5%),4-O-methylgalactose (9.6%),6-O-methylgalactose(3.1%),galactose (3.4%)and traces of 2-O-methylgalactose.     

Polysaccharides from the red seaweed Bostrychia montagnei: chemical characterization

Bostrychia montagnei was submitted to aqueous extraction at 25 and 85 °C. The purified polysaccharide extracts represent ∼ 17% of the dried alga. Galactose is the principal monosaccharide component of these extracts (60.8–70.4 mol%). 3,6-Anhydrogalactose and its 2- O-methyl derivative are also present in smaller amounts (16.2–22.0 mol%), as well as other methylated sugars, such as 6- O- (6.5–7.8 mol%) and 2-O-methylgalactose (0.2–2.1 mol%). Xylose (4.1–8.1 mol%) and glucose (0.7–2.6 mol%) were also detected. The aqueous extracted polysaccharides (25 °C) were separated by anion-exchange chromatography into six sulphated galactan fractions with negative specific rotations and another two with high xylose contents and positive specific rotations. The sulphated galactans all have an agar type backbone modified by partial O-methyl substitution on O-6 or O-2 of the galactosyl units. The latter substitution is also present in varying degrees of 3,6-anhydrogalactose. This revised version was published online in June 2006 with corrections to the Cover Date.     

Chemical structure analysis of water-soluble sulfated polysaccharide fromSchizymenia dubyi (Rhodophyta,Gigartinales)

Chemical and spectroscopic methods showed that the water-soluble polysaccharide extracted fromSchizymenia dubyi from Sicily was composed of 1/0.75/1.3 galactose, glucuronic acids and sulfate groups; 45% of total galactose was present as the L-form and no 3,6-anhydrogalactose was detected. The structural characteristics of this galactan of molecular weight 290 000 were close to sulfated polysaccharide with 1,3-, 1,4- and terminal-linked galactose units and secondary ramifications in 1,3,6; 1,4,6; 1,3,4 and 1,6. Permethylation analysis suggested the presence of sulfate groups on positions O-2 and/or O-3 of 1,4-linked galactose and on O-2 and/or O-4 of 1,3-linked residues.Author for correspondence     

Determination of the configuration of 3,6-anhydrogalactose and cyclizable alpha-galactose 6-sulfate units in red seaweed galactans

A combination of two reported procedures was used in order to determine the configuration of the 3,6-anhydrogalactose present in red seaweed polysaccharides. A mild hydrolysis (to cleave only 3,6-anhydrogalactosyl linkages) was followed by a reductive amination with a chiral amine. Then, the total hydrolysis proceeded, followed by a new step of reductive amination. In this way, using (S)-alpha-methylbenzylamine as the chiral amine, it was possible to separate and quantitate both enantiomers of 3,6-AnGal and its 2-O-methyl ether as their diastereomeric acetylated aminoalditols. On the other hand, using (S)-1-amino-2-propanol, even though the derivatives of both enantiomers of 3,6-AnGal are not separated, the mixture can be safely quantitated with respect to galactose. Furthermore, a one-pot technique was developed to carry out an alkaline treatment of the polysaccharides, followed by the double hydrolysis-reductive amination procedure, which is useful to determine the proportions of both enantiomers of 6-sulfated 4-linked galactose units in the native polysaccharides. The unexpected presence of small amounts of units of this type belonging to the D-series in a porphyran sample is revealed by this novel procedure.     

Polysaccharide structure of tetrasporic red seaweed Tichocarpus crinitus

Sulfated polysaccharide isolated from tetrasporic plants of Tichocarpus crinitus was investigated. The polysaccharide was isolated by two methods: with water extraction at 80 °C (HT) and with a mild alkaline extraction (AE). The extracted polysaccharides were presented by non-gelling ones only, while galactose and 3,6-AG were the main monosaccharides, at the same time amount of 3,6-AG in AE polysaccharides was the similar to that of HT. According to methods of spectroscopy and mass spectrometry, the polysaccharide from tetrasporic T. crinitus contains main blocks of 1,3-linked β-d-galactopyranosyl-2,4-disulfates and 1,4-linked 3,6-anhydro-α-d-galactopyranosyl while 6-sulfated 4-linked galactopyranosyl resudies are randomly distributed along the polysaccharide chain. The alkaline treatment of HT polysaccharide results in obtaining polysaccharide with regular structure that composed of alternating 1,3-linked β-d-galactopyranosyl-2,4-disulfates and 1,4-linked 3,6-anhydro-α-d-galactopyranosyl residues. Native polysaccharide (HT) possessed both high anticoagulant and antiplatelet activity measured by fibrin clotting and platelet aggregation induced by collagen. This activity could be connected with peculiar chemical structure of HT polysaccharide which has high sulfation degree and contains also 3,6-anhydrogalactose in the polymer chain.     

Alkaline modification of carrageenans. Part III. Use of mild alkaline media and high ionic strengths

The cyclization reaction (formation of 3,6-anhydro--d-galactose and 3,6-anhydro--d-galactose 2-sulfate units from -d-galactose 6-sulfate and 2,6-disulfate residues, respectively) of carrageenans follows a pseudo-first-order kinetics. This reaction can be carried out, at reasonable rates, in sodium hydroxide solutions less concentrated than those usually employed or by using a milder base such as sodium carbonate. Rate constants were also determined at a fixed sodium hydi oxide concentration but using different ionic strengths. No specific salt effects were observed at a given sodium hydroxide concentration and ionic strength.