J. Chil. Chem. Soc, 52, N° 1 (2007)

BLOCK STRUCTURE IN ALGINIC ACID FROM LESSONIA VADOSA (LAMINARIALES, PHAEOPHYTA)

 

BETTY MATSUHIRO^1*, SIMONET E. TORRES¹ AND JUAN GUERRERO²

¹Departamento de Ciencias del Ambiente, ²Departamento de Ciencias de los Materiales, Facultad de Química y Biología, Universidad de Santiago de Chile, Casilla 40, Correo 33, Santiago, Chile., e-mail: bmatsuhi@lauca.usach.cl

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ABSTRACT

Alkaline extraction of Lessonia vadosa blades collected in Fuerte Bulnes (southern Chile) afforded sodium alginate in 14.5% yield. Partial acid hydrolysis of the sodium alginate gave three fractions which were characterised by one- and two-dimensional nuclear magnetic resonance techniques. Fraction 1 (17.8 % yield) showed to be mainly composed of alternate heteropolymeric block (MG) and in minor proportions of polymannuronic sequences. Analysis of ¹³C NMR and 2D HSQC spectra indicated that Fraction 2 (64.5% yield) is a polymannuronic enriched fraction. The minor amount of guluronic acid residues in this fraction showed by 2D ¹H/¹H NOESY NMR spectrum to be present in a 1→4 homopolyguluronic sequence; moreover, the interresidue correlation data indicated that MG blocks were not present. Results are in agreement with data obtained by FT-IR spectroscopy in the solid state. Full assignments of ¹H and ¹³C signals in the NMR spectra indicated that Fraction 3 (17.7%) presented a regular homopolyguluronic block structure.

Keywords: brown seaweeds, alginic acid, block composition, Lessonia vadosa, NMR.

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INTRODUCTION

Alginic acid constitutes the major structural polysaccharide of brown seaweeds (Phaeophyta). It is a linear copolymer of l-»4 linked |3-D-mannopyranuronic acid (M) and l-»4 linked a-L-gulopyranuronic acid (G) residues, the two uronic acids can be arranged in heteropolymeric and homopolymeric blocks¹"³. The chemical composition of alginic acids from members of the genus Lessonia has been reported⁴"⁷. Differences in the mannuronic acid to guluronic ratio (M/G) and block composition in the alginic acids of Lessonia vadosa collected in different localities and different seasons were previously reported⁸. No relation between M/G valúes and block composition were found. One dimensión 'H and ¹³C NMR spectroscopies have been used to characterize alginic acids and their fractions⁹"¹³. We now report a study of the fine chemical structures of the fractions obtained by partial hydrolysis of sodium alginate from Lessonia vadosa by two-dimensional nuclear magnetic resonance techniques.

Materials and methods

Lessonia vadosa Searles was collected in south Fuerte Bulnes (53° 37’55.6” S, 70°55’17.9” W) in spring, 2003. A sample was deposited in Sala de Colecciones, Departamento de Ciencias y Recursos Naturales, Universidad de Magallanes, Punta Arenas, Chile. D-galacturonic acid and D-glucuronolactone used as standards, were from Sigma Chemical Co., St. Louis, Mo, USA. FT-IR spectra in KBr pellets were registered in the 4000-400 cm"¹ región using a Bruker IFS 66v instrument¹⁴. One and two-dimensional NMR spectra were recorded at 70 °C on a Bruker Avance 400 spectrometer equipped with a z-gradient inverse broad band probé of 5 mm diameter, operating at 400.13 MHz ('H) and 100.62 MHz (¹³C) after isotopic exchange with D₂0 (3 x 0.75 mL) using D₂0 as solvent with MeOH as internal reference (6¹³C: 49.50 ppm, 6'H 3.340 ppm). All the two-dimensional experiments were acquired using pulse field gradient incorporated into NMR pulse sequences. The two dimensional homonuclear 'H/'H correlation spectroscopy (COSY) and nuclear Overhauser effect (NOESY ) spectra were acquired with 128 x 2040 data point with a spectral width of 1200 Hz and processed in a 1024 x 1024 matrix to give a final resolution cióse to 2.3 Hz/point in the two dimensions. The two dimensional heteronuclear-single quantum coherence correlation (2D 'H/¹³C HSQC) spectra with 128 x 1024 data point and processed in a 1024 x 1024 matrix to give a final resolution cióse to 2.3 Hz/point in 'H and cióse to 2.4 Hz/point in ¹³C. The number of scans (ns) in each experiment was dependent on the sample concentrations.

Extraction and purification

Ground blades (~ 2 mm size) of Lessonia vadosa (120 g) were stirred for 10 min. with 1 L of petroleum ether (b.p. 40-60°C). The supernatant was concentrated in vacuo and the extraction process was repeated until no more solid residue was obtained (five times) in the concéntrate. The residual petroleum ether was evaporated at room temperature for 72 h and the algae was treated with 1.6 L of 98% ethanol and 0.4 L of 37% aqueous formaldehyde. After 72 h, the solid was decanted and dried at room temperature. Extraction was performed with 3% sodium carbonate solution (2 L) at 50 ºC during 4 h. The mixture was centrifuged (4000 x g) and the supernatant solution was dialysed against distilled water using Spectra/Por membrane (MWCO 3500). The resulting solution was concentrated in vacuo, poured over 0.5 L of 98% ethanol and centrifuged. The solid obtained was dried in an oven at 60 ºC for 48 h. The extraction process was repeated two more times. The extracts were pooled and purified as previously described⁶.

Total hydrolysis

The alginate sample (5 mg) and 4.5 mL of 90% formic acid in a sealed tube were heated for 6 h at 100 ºC in an oven. The resulting solution was diluted with 20 mL of distilled water, refluxed for 2 h and concentrated in vacuo⁷. The residue was dissolved in distilled water and the M/G ratio was determined by HPLC with a Whatman Partisil 10-Sax column (250 x 4.6 mm) on a Merck-Hitachi L-6000 equipment with a L-4000A UV detector^5,15. The hydrolysis was conducted in duplicate.

Fractionation of sodium alginate

Purified sodium alginate (0.250 g) in 25 mL of water was refluxed with 0.75 mL of 3.0 M HCl under N₂ for 0.3 h. After cooling, the mixture was centrifuged (3000 x g) and the supernatant solution was neutralized with 1.0 M NaOH and poured over 100 mL of ethanol. The precipitate was dissolved in distilled water and freeze-dried (Fraction 1). The insoluble material obtained by centrifugation was refluxed with 0.3 M HCl for 2 h under N₂. The precipitate was separated by centrifugation and neutralized with 1.0 M NaOH and, then the pH was decreased to 2.85 by addition of 1.0 M HCl. The soluble fraction was neutralized, dialysed against distilled water and freeze-dried (fraction 2). The precipitate was dissolved by neutralization, dialysed and freeze-dried (fraction 3). The fractionation was conducted in duplicate.

RESULTS

Alkaline extraction of blades of Lessonia vadosa afforded after three purifications, sodium alginate in 14.5 dry weight percent. Its FT-IR spectrum showed four bands in the finger print region, at 948.0 cm^-1 assigned to the C-O stretching vibration of α-guluronic acid residues, at 898.9 cm^-1 assigned to the anomeric vibration of β-mannuronic acid residues, and at 814.5 and 781.5 cm^-1 due to deformation bands of COH, CCH and OCH groups of guluronic acid residues in homopolymeric blocks^7,8.16. Total hydrolysis and HPLC analysis showed an M/G ratio of 0.64. By partial acid hydrolysis of the sodium alginate three fractions were obtained with 75% of mass recovery. Fraction 1 (F-1), (17.8% yield, M/G 1.25) presented in the 1000-700 cm^-1 region of the FT-IR spectrum, a band at 961.3 cm^-1assigned to C-O stretching vibration of the uronic acids residues in heteropolymeric blocks and its second-derivative spectrum presented a band at 818.9 cm^-1 assigned to deformation vibrations of mannopyranuronic acid residues in homopolymeric blocks. Fraction 2 (F-2), (64.5% yield), soluble at pH 2.85, presented in the FT-IR spectrum the deformation vibration of anomeric C-H group at 892.8 cm^-1 and the band at 819.6 cm^-1 attributed to β-homopolymannuronic blocks. Furthermore, the FT-IR spectrum of F-2 showed a small band around 775 cm^-1 which was observed at 780.6 cm^-1 in the second-derivative spectrum, assigned to α-L-guluronic acid in a homopolymeric sequence. Fraction 3 (F-3), (17.7% yield), insoluble at pH 2.85 showed by total hydrolysis and HPLC analysis the presence of a single component, with a retention time that corresponded to guluronic acid. The FT-IR spectrum of F-3 presented a band at 903.7 cm^-1 assigned to anomeric C-H deformation vibration of α-guluronic acids residues, and the bands at 947.9, 811.2 and 781.6.cm^-1 previously found in the whole alginate.

The fractions were analysed by ¹H and ¹³C spectroscopy. Two dimensional NMR spectra were collected to confirm the fine structure of the hetero-and homopolymeric fractions. Figure 1 shows the ¹³C/¹H HSQC NMR spectrum of F-1. The major ¹³C signals were assigned to mannuronic acid and guluronic acid in the sequence GMG and MGM⁹. In the anomeric region connectivities between two ¹³C/¹H systems were observed. The 102.08/4.60 ppm correlation was assigned to C-1/H-1 of mannuronic acid residues in the triad GMG and the 100.44/4.97 ppm correlation was assigned to C-1/H-1 of guluronic acid residue in the MGM triad.

 

Correlations for the rest of ¹³C/¹H systems were obtained as shown in Table 1. The third signal in the anomeric zone of the ¹³C spectrum at 100.95 ppm, and the signal at 70.96 ppm were assigned to C-1 and C-2 of mannuronic acid residue, respectively in the centre of GMM triad. The mole fraction of guluronic acid (F_G) of 0.44 was calculated from the integration ratio of anomeric protons in the ¹H NMR spectrum (figure not shown) and the value is very close to that deduced from the M/G ratio.

Two dimensional ¹³C/¹H HSQC spectrum of F-2 (Figure 2) allowed the full identification of the ¹³C/¹H systems of a homopolymannuronic acid fraction as indicated in Table 1. ¹H/¹H 2D NOESY experiment showed interresidue cross-peaks of the anomeric proton of L-guluronic acid with H-4 of the near by L-guluronic acid residue as well as with H-4 of D-mannuronic acid residue (Figure 3). Assignments of the rest of cross-peaks are indicated in Figure 3.

Through ¹³C/¹H HSQC spectrum (Figure 4) all proton and carbon signals of F-3 can be assigned. Results are shown in Table 1.

DISCUSSION

Alkaline extraction of Lessonia vadosa blades, followed by exhaustive purification afforded sodium alginate in good yield, similar to the yields reported previously for sodium alginates from the same algae collected in different seasons at different localities⁸. Removal of lipids with petroleum ether before extraction, allowed the preparation of pure sodium alginate which was characterized by FT-IR spectroscopy. The spectrum presented in the finger print or anomeric region, the characteristic bands of alginic acids similar to those of alginic acids samples extracted from L trabeculata⁷.

IR spectroscopy has been used to characterize alginic acids. According to Mackie¹⁷ alginates presented in the IR spectra two characteristic bands, at 808 cm^-1 assigned to mannuronic acid residues, and at 787 cm^-1 assigned to guluronic acid residues. The FT-IR spectra and the second-derivative spectra of polysaccharides, gave more information than classical IR^14,18,19. In the analysis by FT-IR spectroscopy of alginates from Lessonia trabeculata, both bands were assigned to guluronic acid residues and a new band which appeared at 822 cm^-1 was assigned to mannuronic acid residues⁷. The guluronic acid content in the sodium alginate was lower than in the sodium alginate (M/G 0.33) from L. vadosa collected at the same locality and in same season two years before, but similar to the value reported for sodium alginate extracted from holdfast (M/G 0.66)⁸. Partial acid hydrolysis of the sodium alginate allowed the preparation of three fractions. According to Haug et al.²⁰, the first fraction obtained by partial hydrolysis with 0.3 M HCL of sodium alginate was mainly composed of heteropolymeric blocks (MG), the fraction soluble at pH 2.85 was an enriched fraction in polymannuronic acid (MM) and the fraction insoluble at pH 2.85 was enriched in polyguluronic acid (GG).It is important to establish the content of guluronic acid in homopolymeric blocks since the gel forming capacity of sodium alginate depends on the presence of homopolyguluronic acid chains²¹. Block composition is quite different from the sodium alginate with M/G 0.33 extracted from blades of L. vadosa previously studied^8. Although the sodium alginate from holdfast of L. vadosa collected in south Fuerte Bulnes in spring showed a very similar M/G value (0.66) to that found in this work, the guluronic acid residues were almost in the heteropolymeric fraction, and the sodium alginate (M/G 0.63) extracted from stipes of L. vadosa collected in spring at the same locality is composed of almost the same proportions of the three blocks⁸. Venegas et al.⁶ studied alginates from L. trabeculata collected in different localities. They found that the sample obtained from blades with M/G value of 0.90 was mainly composed by polymannuronic blocks while the sample from blades with M/G value of 0.79 contained similar proportions of the three blocks. In the sample obtained from blades with low value of M/G (0.33), the guluronic acid residues were in the homopolymeric fraction (55 %) whereas the alginate from stipe of L. trabeculata with M/G value of 0.38, the guluronic acid residues were mainly present in the heteropolymeric fraction (78.6%). Larsen et al.¹³ studied block distribution of alginates from Cystoseria trinide and Sargassum latifolium by ¹H NMR spectroscopy, they found that both alginates contained the same proportions of homoguluronic fractions even that the M/G values were 0.48 and 0.80, respectively. These findings support the assumption that no relation between M/G ratio and block composition could be established.

In this work, the fractions obtained by partial hydrolysis were analysed by FT-IR spectroscopy. F-1 showed the characteristic bands of heteropolymeric fraction and a band in the second-derivative spectrum which may indicate the presence of mannuronic acid in a homopolymeric sequence. The FT-IR spectrum of F-2 suggested the presence of homopolymannuronic acid blocks and in minor proportions of homopolyguluronic acids blocks as deduced by the signal at 780.6 cm^-1 in the second-derivative spectrum. Results of total hydrolysis and FT-IR analysis indicated that F-3 is composed of homopolyguluronic blocks. In contrast, fraction 3 obtained by partial hydrolysis of sodium alginate from L. trabeculata showed in the FT-IR spectrum one extra signal at 822.2 cm^-1 in the second-derivative FT-IR spectrum which indicated the presence of mannuronic acid residues in the homopolyguluronic fraction⁷.

Fine structures of the fractions were studied by ¹H and ¹³C NMR and by 2D ¹H/¹H COSY, ¹³C/¹H HSQC and NOESY NMR techniques. ¹³C chemical shift values differ generally in ~ 1.7 ppm with those published by Grasdalen et al.⁹. Two-dimensional ¹H/¹H COSY spectrum showed only partial assignments of H-1-H-2 correlations (figures not shown). However, ¹³C/¹H HSQC spectrum allowed to trace connectivities between ¹³C and ¹H atoms and the full assignments of the signáis was achieved. (Figure 1 and Table 1). Data presented on Table 1 indicated that F-1 is mainly composed of altérnate heteropolymeric block structure. The altérnate MG sequence is interrupted by MM diads, results are in accordance with the slight excess of mole fraction (F_M=0.56) of mannuronic acid. The ¹³C NMR and 2D HSQC spectra of F-2 showed ten major signáis which can be ascribed to a polymannuronic sequence carrying guluronic acid residues. Two dimensional 'H/'H NOESY experiments provide valuable information about through-space interactions of protons²². In the'H/'H NOESY NMR spectrum (Figure 3) of F-2, interresidue correlation of anomeric protón with H-4 ascribed to guluronic acid residues is indicative of the presence of l-»4 homopolyguluronic sequence. It can be also seen interresidue correlation of the anomeric protón of guluronic acid with H-4 of mannuronic acid residues which indícate that both uronic acids were present in homopolymeric sequences, moreover, the interresidue correlation of H-l of guluronic acid with H-4 of mannuronic acid is indicative that the homopolyguluronic sequence was linked l-»4 to mannuronic acid residues. These results indicated that both homopolymeric block structures are present in the polymer and do not constitute a mixture of polymers. According to Panikkar and Brasch¹² the remaining G residues in homopolymannuronic blocks are associated with heteropolymeric blocks still attached to the former. However, in the FT-IR spectrum of F-2 the absorbance due to altérnate heteropolymeric MG fraction was not present and its second-derivative spectrum clearly showed the characteristic band, attributed to a-L-gulopyranuronic acid residues in homopolymeric blocks. It can be pointed out that 2D NMR analysis, corroborates the FT-IR results and strongly suggest that the signáis in the second-derivatives spectra give valuable information about the block structures. For comparative purposes, fraction 2 obtained by partial hydrolysis of sodium alginate with a high content of mannuronic acid, from blades of Lessonia vadosa collected in Puerto del Hambre in winter⁸ was also analysed by 2 D NMR. 'H NMR chemical shiñ are almost coincident for both fractions soluble at pH 2.85, and are in good agreement with those valúes reported by Steginsky et al.²³ for homopolymeric fractions of alginic acid from the bacteria Azotobacter vinelandii. ¹³C chemical shifts were also in good agreement and allow to conclude that even in alginic acids from L. vadosa with high content of mannuronic acid, guluronic acid can be found forming homopolymeric sequence in the fraction soluble at pH 2.85.

The ¹³C NMR spectrum of F-3 (figure not shown) presented six intense signáis which indicated the presence of a highly regular structure, valúes differ in -0.3-0.4 with those reported by Heyraud et al.²⁴ for oligoguluronates obtained by acid and enzymatic hydrolysis of polyguluronic acid fraction from commercial sodium alginate. Differences may be due to the different temperature applied in the NMR experiments and/or to the conformation adopted by the polymer. HSQC spectrum (figure 4) allowed the full assignments of 'H chemical shifts (Table 1), valúes are similar to those reported by the same authors for the trisaccharide obtained by acid and enzymatic hydrolysis of polyguluronic blocks from commercial sodium alginate. Fraction F-3 would correspond to a homopolyguluronic block and agrees with the structure in solid phase proposed taking into considerations the two characteristic bands (—811 and 780 cm"¹) in the FT-IR spectrum. On the other hand, the second-derivative FT-IR spectrum of the fractions insoluble at pH 2.85 of three sodium alginate samples from L. trabeculata with different M/G valúes (1.73, 0.64, and 0.43) showed the characteristic band of mannuronic acid residues⁷. These results may indícate that composition of fractions is not the same in alginic acid samples from different Lessonia species.

In conclusión, 2D NMR spectroscopy allowed to confirm the structures proposed by FT-IR in the solid state of the fractions prepared by partial hydrolysis of alginic acids. Characterization of fucans and alginic acids from different species of the genus Lessonia²⁵ by 2D NMR techniques will help to find polymeric fractions with specific properties and new industrial applications.

ACKNOWLEDGEMENTS

The financial support of FONDECYT (Grant 1010594) and of Dirección de Investigaciones Científicas y Tecnológicas of Universidad de Santiago de Chile is gratefully acknowledged. The authors thank Grant MECESUP USA-0007 for the NMR equipment facilities, and Dr. Andrés Mansilla (Universidad de Magallanes, Chile) for the collection and identification of the algal material.

REFERENCES

1.    Haug, A., B. Larsen and O. Smidsrod. Acta Chem. Scand. 21: 691-704 (1967).

2.    Painter, T. Algal polysaccharides. In: (G.O. Aspinall, ed) The polysaccharides, Vol. II, Academic Press, Orlando, pp.196-285, 1983.

3.    Craigie, J.S., E.R. Morris, D.A. Rees and D. Thom. Carbohydr. Polym. 4, 237-252 (1984).

4.    Percival E.E., M.F. Venegas Jara and H. Weigel. Phytochemistry 22, 1429-1432 (1983).

5.    Matsuhiro, B. and D.E. Zambrano. Bol. Soc. Chil. Quím. 34, 21-25 (1989).

6.    Venegas, M., B. Matsuhiro and M. Edding. Bot. Mar. 36, 47-51(1993).

7.    Chandía, N.P., B. Matsuhiro and A.E. Vásquez. Carbohy. Polym. 46, 81-87 (2001).

8.    Chandía, N.P., B. Matsuhiro, E. Mejías and A. Moenne. J. Appl. Phycol. 16, 127-133 (2004).

9.    Grasdalen, H. Carbohydr. Res. 118, 255-260 (1983).

10.  Grasdalen, H., B. Larsen and O. Smidsrod. Carbohydr. Res. 89, 179-191 (1981).

11.  Panikkar, R. and D.J. Brasch. Carbohydr. Res. 293, 119-132 (1996).

12.  Panikkar, R. and D.J. Brasch. Carbohydr. Res. 300, 229-238 (1997).

13.  Larsen, B., D.M.S.A. Salem, M.A.E. Sallam, M.M. Misheikey and A.I. Beltagy. Carbohydr. Res. 338, 2325-2336 (2003).

14.  Cáceres, P.J., C.A. Faúndez, B. Matsuhiro and J.A. Vásquez. J. Appl. Phycol. 8, 523-527 (1997).

15.  Gacesa, P., A. Squirre and P. Winterburn. Carbohydr. Res. 118. 1-8 (1983).

16.  Mathlouthi, M. and J.L. Koenig. Adv. Carbohydr. Chem. 44, 7-66 (1986).

17.  Mackie, W. Carbohydr. Res. 20, 413-415 (1971).

18.  Matsuhiro, B. Hydrobiologia 326/327, 481-489 (1996).

19.  Matsuhiro, B., P.Rivas. J. Appl. Phycol. 5, 45-51 (1993).

20.  Haug, A., B. Larsen and O. Smidsrod. Carbohydr. Res. 32, 217-225 (1974).

21.  Grant, G., E.A. Morris, D.A. Rees, P.J.C. Smith and D. Thom. F.E.B.S. Lett. 32, 195-198 (1973).

22.  Agrawal, P.K. Phytochemsitry 31, 3307-330 (1992).

23.  Steginsky, C.A., J.M. Beale, H.G. Floss and R.M. Mayer. Carbohydr. Res. 225, 11-26 (1992).

24.  Heyraud, A., C. Gey, C. Leonard, C. Rochas, S. Girond and B. Kloareg. Carbohydr. Res. 289, 11-23 (1996).

25.  Chandía, N.P., B. Matsuhiro, J.S. Ortiz and A. Mansilla. J. Chil. Chem. Soc. 50, 501-504.(2005).

(Received: October 26, 2006 - Accepted: December: 18, 2006)