Study On Structure And Functional Properties Of An Acidic Polysaccharide Isolated From Boat-fruited Sterculia Seeds

Boat-fruited sterculia seed (Semen Sterculiae Lychnophorae) is a traditional Chinese herb, specified as the ripe seeds of Sterculia lychnophora Hance in the Chinese pharmacopoeia, and is one of the third batch as both edible and medicinal resources published by Ministry of Health, China. Due to the limitation of region and climate, boat-fruited sterculia seed is mainly produced in the tropical zone of Southeast Asia. China is not the original production country of boat-fruited stercilia seed; it was not until the 1970s that Chinese scientists introduced and cultivated long grain seeds from Cambodia and round ones from Thailand. As a result, only in Hainan and Xishuangbanna the trees grew well and blossomed but the seeds were harvested only in Xishuangbanna. Therefore the seed resource in China depends on the import from Southeast Asia. So far boat-fruited sterculia seeds are primarily used for making tea, desserts and healthy buccal tablets etc, and only a little information has been published. Therefore, there is lack of systematic study of its bioactive components. Since polysaccharide is one of the main bioactive components in boat-fruited sterculia seeds, the objectives of the current project were to extract, isolate and purify the polysaccharides with anti-inflammatory bioactivity from boat-fruited sterculia seeds, and to study the structure and functional properties of the bioactive polysaccharide.Chemical composition analysis revealed that boat-fruited sterculia seeds imported from Vietnam contained 12.36% crude protein, 5.89% crude fat, 53.23% carbohydrate and 29.45% reducing sugar. The yield (13.8%), purity (69.7%) and intrinsic viscosity (1.71 dL/g) of crude polysaccharides (W-SP) from water extraction were higher than the polysaccharides (A-SP) extracted from water-insoluble residues with 0.05 mol/L NaOH at 40 oC, indicating that polysaccharides might be partially degraded in NaOH solution possibly due to beta-elimination.Chemical and monosaccharide compositions of W-SP, A-SP and alkali-insoluble mucilage polysaccharides (IMP) were analyzed and the results were as following: the total carbohydrate were 69.74%,44.14% and 80.18%, uronic acid were 14.78%, 7.35% and 5.35%, and protein were 9.12%,13.54% and 3.45%, respectively. They were complex heteropolymers containing a range of neutral sugars, including rhamnose, arabinose, galactose, glucose and small amounts of xylose.Acute anti-inflammatory activity tested in a model of ear edema formation in mice induced by dimethylbenzene was used to monitor the bioactivity of the three fractions. It was found that W-SP exhibited a significant inhibitory effect (inhibition rate 17.84%) compared to that of A-SP (4.15%) and IMP (0.67%). Hence, water extraction was used to obtain the crude polysaccharides and the extraction procedure was optimized using Surface Response Methodology (RSM). The obtained optimal parameters of the extraction procedure of W-SP by RSM were: temperature 60-65°C, time 2.3-3.1 h, and water to solid ratio 75:1. A neutral and an acidic fraction (W-SPN and W-SPA) were obtained from the crude polysaccharides (W-SP) following an ion exchange chromatography on a DEAE-Cellulose column. The neutral polysaccharide W-SPN was composed mainly of glucose (90.18%) and small amounts of protein and ash. The acidic fraction (W-SPA) consisted of galacturonic acids (39.30%), glucuronic acids (1.26%), rhamnose (11.36%), arabinose (17.46%), galactose (15.70%), xylose (0.63%) and glucose (0.35%), with a total carbohydrate of 85.86% and small amounts of protein and ash.Both polysaccharides were identified by gel filtration chromatography on Sepharose CL-6B column and HPSEC as homogeneous in molecular weight: their molecular weights were 586,800 Da and 1,125,000 Da, respectively. W-SPA was identified as a pectic polysaccharide confirmed by monosaccharide composition and FT-IR analysis. The degree of esterification (DE) value of W-SPA was 68% based on a FT-IR method.Acute anti-inflammatory activity of W-SPN and W-SPA was tested in a model of ear edema formation in mice. W-SPA exhibited a much higher anti-inflammatory activity (inhibition rate 26.29%) compared with W-SPN (3.21%). Further chronic anti-inflammatory activity of W-SPA was estimated by a well-accepted model of hyperplasia of granuloma tissue in rats: the inhibition rate of W-SPA was 28.38% which is 82% of the effect of aspirin (inhibition rate 34.38%). The current study revealed that W-SPA presented a significant dose-dependent inhibitory effect at low concentration range on both acute and chronic anti-inflammatory experiments. Therefore, W-SPA was chosen for further studies aimed at elucidation of its detailed structure, which was essential for the understanding of its mechanism of action.Structure characterization of W-SPA was carried out using partial acid hydrolysis and methylation analysis, combined with HPAEC-PAD, GC-MS and 1D & 2D NMR spectroscopy techniques. The backbone of W-SPA was found to contain two characteristic features: the first is homogalacturonan (HG) zones referred to as the'smooth'regions characterized by a linear linkage of 1,4-α-D-GalpA; the second is rhamnogalacturonan-I (RG-I) zones, also called'hairy'regions consisting of a←4)-α-D-GalpA-(1→2)-α-L-Rhap-α-(1→backbone partially substituted at O-4 of the rhamnose units. 20.8% of rhamnosyl residues were only 1,2- linked while 62.2% of residues were 1,2,4- linked (with the O-4 position substitution). On the HG chains carboxyl groups were partially methyl esterified and partially O-acetylated on C-2 and/or C-3. Side chains of W-SPA consisted of araban, galactan and arabinogalactan by the linkages of T-, 1,3-, 1,5-L-Araf and T-, 1,4-, 1,6-, 1,3,6-D-Galp, attached to the backbone via the O-4 position of the rhamnose residues. 55.8% of L-Araf were situated as the nonreducing terminals, which were linked to the O-3 position of 1,3-L-Araf , O-5 position of 1,5-L-Araf and O-3 position of the 1,6-D-Galp units except the O-4 position of the backbone rhamnosyl residues. Araban were composed mainly of 38.5% of 1,3-L-Araf and small amounts of 1,5-L-Araf. 57.2% D-Galp were situated as the nonreducing terminals, and in addition 23.0% of D-Galp units were the linkage of 1,3,6-D-Galp and 3.7% units were 1,6-D-Galp linkage. A small amount of proteins were possibly bonded with the W-SPA molecular chain by O-glycopeptide bonds, however, this speculation needed further confirmation.Solution properties of polysaccharide molecules were investigated by dynamic and static light scattering techniques. W-SPA molecules formed aggregates in pure water, 5 mol/L urea and 100 mmol/L NaNO3 solutions. These aggregates could be only partially eliminated by filtration of the solutions with 0.1μm membrane filter. However, when W-SPA was dissolved in 25 mmol/L NaOH solution at room temperature overnight, there was no observable aggregations: there was only one population of particles with a mean diameter of 51.8 nm in the dynamic light scattering measurement. The Zimm plot from static light scattering measurement for W-SPA in 25 mmol/L NaOH solution was obtained, and weight average molecular weight (Mw), radius of gyration (Rg) and the second virial coefficient (A2) were derived as 9.132×105 Da, 51.85 nm, 2.70×10-4 cm3 mol/g2, respectively. The hydrodynamic radius (Rh) was 39.3 nm by dynamic light scattering measurement. W-SPA in NaOH solution was found to be a branching and exhibited a relatively compact random coiled conformation as evidenced by the characteristic ratioρ=Rg/Rh=1.32. In Mark-Houwink-Sakurada equation,αvalue obtained by HPSEC was 0.586, indicating W-SPA molecule was a little compact random coiled chain, likely a consequence of molecular branching. This observation confirmed the compact random coiled conformation obtained from the characteristic ratioρ.Steady-shear rheological measurements of W-SPA showed a non-Newtonian shear-thinning flow behavior at high concentrations and a Newtonian flow behavior at low concentrations. Similar to other HM pectins, W-SPA could not form a gel on by itself; however, gelation occurred when a co-solute was added under acidic conditions. The mechanical spectra within the linear viscoelastic region were a function of W-SPA concentration and temperature. Both fluid-like (G") and solid-like (G′) behaviors were observed depending on concentration and frequency.