Carbohydrate Constituents of the Marine Algae of Sri Lanka Bart 11 . Composition and Sequence of Uronate Residues in Alginates from some Brown Seaweeds

The ratio of mannuronic acid residues to guluronic acid residues (M/Q ratio) of sodium alginate extracted from Cystoseira trinodis. Turbinaria conoidcs and Sargassum sp. was determined using high resolution IH-NMR spectroscopy. The intensities of the signals due to H-5 of guluronate residues and H-1 of both guluro1 nate and mannuronate residues were used. The alginate samples were found to "& be rich in guluronate residues, and the polymer chains are likely to be composed of long blocks -of guluronate residues, short blocks of mannuronate residues and G a small proportion of blocks containing both uronide residues.


Introduction
Alginic acid is a mucilaginous polysaccharide which has been found in all species of brown seaweeds examined, but is not present in any other plant tissue.9 The polysaccharide is a linear glycuronan which consists of (1+4)-linked residues of D-mannuronic acid and L-guluronic acid arranged in a block fashion in the polymer chain.Blocks containing one type of residue (MM blocks and GG blocks) are separated by segments in which the two residues alternate (Figure 1).5,6,7Physical properties of alginates depend on its uronic acid composition, i,e. the ratio of mannuronic acid residues to guluronic acid residues (M/G ratio), and also upon tne relative proportion of the tmee types of blocks (MNI, GG and MG).Both the M/G ratio and the monomer sequence distribution changes from one species of brown alga to another.
Penman and Sandersons found that 1H-NMR spectroscopy could be used to distinguish between signals due to H-1 and H-5 in the guluronic acid residues and H-1 from mannuronic acid residues in homopolymeric blocks obtained by partial hydrolysis of alginates.Grasdalen et ale using high resolution 1H-NMR spectroscopy were able to distinguish between H-5 of guluronic acid residues with a mannuronic acid neighbour (GM sequence) from those with a guluronic acid neighbour (GG sequence).In this paper we describe the results obtained using this method, in analysing alginate samples isolated from four species of brown .seaweedscollected from the coastal regions of Sri Lanka.

Results and D i i i o n
The 1H-NMR spectra of the partially depolymerised alginate samples were interpreted using the method described by Grasdalen et al. 2 The intensities of (i) the doublet centred at 5.1 ppm due to the H-1 of the G-residues (IA) (see Figure 2), (ii) the singlet at 4.7 ppm due to H-1 of the M-residues and H-5 of GM residues.(IB) and (iii) the singlet at 4.5 ppm due to H-5 of GG residues (Ic) were measured.The M/G ratios as well as the doublet frequencies were calculated and are given in Table 1 for the alginates from the four species of brown algae examined by us.
The 1H-NMR spectra of the samples from Turbinaria conoides and Sargassum sp.(oval) were re-run and amplified.In these two samples the intensities of the signals A, B and C were also obtained by planimetry.These values were found to be different and are considered to be more accurate than those obtained by integration.They were also found to agree with preliminary results obtained from 13C-NMR spectroscopy3,4 where intensities of the signals were calculated by planimetry (see Table 1).The results indicate that all four alginate samples are rich in guluronic acid residues, and the doublet frequencies give an idea of the block character of each alginate.Therefore these four samples of sodium alginate probably contain long blocks of G, shorter blocks of M and very little alternate MG and GM blocks.
Sodium alginate was isolated from four species of brown seaweeds Cystoseira trinodis, Turbinaria conoides and two unidentified species of Surgassum referred to as Sargassum sp.(linear) and Sargassum sp.(oval) with respect to the shape of their fronds.These seaweeds were washed, sundried and milled.Samples of seaweed (50 g) were extracted successively as follows (i) twice with 2% CaClz solution (300 ml) at room temperature for 4h; (ii) twice with 2% CaC12 solution (300 ml) at 70" C for 4 h; (iii) four times with dil.HCl(300 ml, pH 2) at 70°C for 4h; (iv) five times with 3 % Na2C03 solution (300 ml) at 50" C for 4h.The combined Na,C03 extract was poured with stirring into ethanol (6 1).The precipitate was filtered, dried, dissolved in water and stirred with 2% CaCI, solution until precipitation was complete.The calcium alginate was suspended in 0.5 M HCI, stirred occasionally for 3h and filtered.The filtrate was tested for Ca++ ions.
The residue was washed with 0.5 M HCI until the filtrate was free of Ca++ ions.
The alginic acid was suspended in water and tiirated with 0.1M NaOH until the pH reached 7, when all the alginic acid was dissolved.The solution was dialysed for two days and then freeze dried to give a white powder.The M/G ratio and monomer sequence distribution of each sample were determined by PMR spectroscopy (t --6 secs).The spectra were recorded at 90" C in order to increase the spectral resolution and to shift the solvent peak upfield away from the low field spectral region.1H-Chemical shifts were expressed in ppm downfield from the internal standard sodium 3-(trimethylsilyl) propane sulphonate.The area under each peak in the low field region was found by integration, and in two cases by planimetry .

Calculation
The M/G ratios and the doublet frequencies were calculated as follows.Quantitatively the mole fraction of G(FG) and the doublet frequency (FGG) are related to the intensities (I) of the respective lines by the following relationships.
The mole fraction of M is derived from the normalization condition t