Synthesis and Physiochemical Properties of Sulphated Tamarind (Tamarindus indica L.) Seed Polysaccharide

Abstract

Tamarind seed polysaccharide (TSP) is a neutral water-soluble galactoxyloglucan isolated from the seed kernel of Tamarindus indica with average molecular weight (Mw) 600-800 kDa. The high viscosity of TSP slows solubilisation, and the absence of charged substituent hinders the formation of electrostatic interactions with biomolecules. TSP was sulphated in a one-step process using dimethylformamide as a solvent, and sulphur trioxide-pyridine complex as a sulphating reagent. Studies of chemical structure, molecular weight distribution and viscosity were conducted to characterise the synthesised products. The sulphation degree was established by conductimetric titration; the sulphate group distribution was studied by NMR spectroscopy and liquid chromatography-mass spectrometry, and sulphated TSP oligomers were obtained by enzymatic degradation with cellulase and/or xyloglucanase. Sulphated products showed higher solubility than TSP, Mws in the range of 700-1000 kDa, a sulphation degree of two to four per subunit and pseudoplastic behaviour. A preliminary study of mucoadhesion revealed the unexpected interaction of S-TSP with mucin, providing a route by which sulphated TSP interactions with biomolecules may be influenced.

Keywords: LC-MS; NMR; enzymatic hydrolysis; molecular weight; structural characterization; sulphated polysaccharide; tamarind seed polysaccharide; viscosity.

Figure 1

Chemical structure of tamarind seed polysaccharide (TSP) isolated from the seed kernel of Tamarindus indica. TSP is composed of a β-(1,4)-d-glucan backbone, with α-(1,6)-d-xylose branches, partially substituted with β-(1,2)-d-galactose. The coloured residues and abbreviations in bold correspond to the observed monosaccharide, while the linked monosaccharides are shown in parentheses. Glc—glucose; Xyl—xylose; Gal—galactose.

Figure 2

FT-IR spectra of TSP in black, S-TSP_1 in dark blue, S-TSP_2 in blue and S-TSP_3 in light blue. The new bands assigned to S=O and C–O–S stretching vibrations are signed with arrows.

Figure 3

Chromatographic profile (red—refractive index; black—low laser light scattering; green—right angle light scattering; blue—viscometer) of pristine TSP (a) and S-TSP_1 (b).

Figure 4

Viscosity curves of TSP in black, S-TSP_1 in dark blue, S-TSP_2 in blue and S-TSP_3 in light blue at 10 mg/mL at 20 °C.

Figure 5

¹H-¹³C HSQC superimposition of the anomeric region of TSP in black and hydrolysed TSP, with xyloglucanase in red. The superscript corresponds to the carbon number of the observed monosaccharide, which is in bold, while the monosaccharide linked is in the parentheses. NR—non-reducing end; Glc—glucose; Xyl—xylose; Gal—galactose.

Figure 6

¹H-¹³C HSQC superimposition of TSP in black and hydrolysed TSP with xyloglucanase in red. The superscript corresponds to the carbon number of the observed monosaccharide, which is in bold style, while the monosaccharide linked is in the parentheses. * signals assigned to the arabinose residue. Glc—glucose; Xyl—xylose; Gal—galactose.

Figure 7

HILIC/ESI-QTOF-MS chromatograms of TSP hydrolysed by cellulase on the top and TSP hydrolysed by xyloglucanase on the bottom. Hex—hexose (glucose or galactose, 162 Da), P—pentose (xylose, 132 Da); the numbers in subscript indicate the number of hexoses and pentoses within the detected oligosaccharide: ox indicates the oxidized minor components (−2 Da), most likely related to the C4-oxidized oligomers, as previously reported by Sun et al. [29].

Figure 8

IPRP-HPLC/ESI-QTOF-MS chromatogram of S-TSP hydrolysed by cellulase and xyloglucanase. Hex—hexose (glucose or galactose, 162 Da), P—pentose, (xylose, 132 Da), S—sulphate (SO₃-, 80 Da); the numbers in subscript indicate the number of hexoses and pentoses within the detected oligosaccharide. The most abundant oligomers Hex₆P₃S_x and Hex₅P₃S_x are underlined and in bold. $—oligomers with the intensity lower than 500 (the intensity of the highest peaks Hex₆P₃S₁/Hex₅P₃S₁ and Hex₆P₃S₂/Hex₅P₃S₂ are higher than 1 × 10⁴).

Figure 9

LC-MS chromatogram (a) and MS spectra of the most abundant peaks co-eluted in the regions of mono (b), bi- (c), tri- (d), tetra- (e), penta- (f) and hexasulphated (g) oligomers. Because various oligomers with the same sulphation degree, including positional isomers, are co-eluted, the mass spectra were averaged for their retention time range: 13.9–17.6 min ((b), Hex_xP_yS₁); 18.6–22.6 min ((c), Hex_xP_yS₂); 24.6–27.7 min ((d), Hex_xP_yS₃); 31.0–34.3 min ((e), Hex_xP_yS₄); 36.4–38.0 min ((f), Hex_xP_yS₅); 41.7 ± 0.5 min ((g), Hex_xP_yS₆). Hex—hexose (glucose or galactose, 162 Da), P—pentose, (xylose, 132 Da), S—sulphate (SO₃⁻, 80 Da); DBA—dibutylamine (129 Da, adducts are indicated with blue arrow); the numbers in subscript indicate the number of hexoses and pentoses within the detected oligosaccharide. The m/z values and the corresponding ion forms are reported in Table S3.

Figure 10

¹H-¹³C HSQC superimposition of TSP in black hydrolysed with cellulase and S-TSP_1 hydrolysed with xyloglucanase and cellulase in blue. Circled signals are related to sulphation. Galactose anomeric signals, α and β, are reported in the figure. The superscript corresponds to the carbon number of the observed monosaccharide, which is in bold, while the monosaccharide linked is in the parentheses. * signals assigned to the cellulose enzyme; Gal—galactose.

Figure 11

Viscosity curves: (a) TSP (10 mg/mL) in black and TSP with mucin (2.5 w/w) in red; (b) S-TSP_1 (10 mg/mL) in blue and S-TSP_1 with mucin (2.5 w/w) in red.