The Isolation, Purification And Functional Properties Of Polysaccharides In Semen Plantaginis

Semen plantaginis has the dual usages as medicine and as food. It is an approved raw material for healthy food by the Minister of Public Health of China. The polysaccharides which are located in the outer seed-coat (husk) are one of the main active components of Semen plantaginis. The polysaccharides in Semen Plantaginis have many biological activities including laxation effect, hypoglycemic effect, hypolipidemic effect, anti-inflammatory and immunological activity. The polysaccharides in Semen Plantaginis as a hydrophilic colloid were widely used in food and pharmaceutical industries. Many previous researches were focused on the structure, bioactivity and application of the polysaccharides in Semen Plantaginis in foreign countries especially Europe and America, However, few studies were found in China. Therefore, in our paper, the polysaccharides extracted from the seeds of Ji'an Plantago asiatica L were studied. On the basis of systematic analysis on the extraction, isolation, purification and primary structure of this polysaccharides, its antioxidant activity and functional properties were studied preliminarily. The main results in the paper were concluded as follows:1. A suitable method for the determination of the contents of polysaccharides in Semen Plantaginis by the phenol-H₂SO₄ colorimetry was established, and xylose was used as a standard. The two colorimetry methods and seven standards were compared and analyzed, respectively. The results showed that the colored compounds of xylose and Semen Plantaginis polysaccharides by phenol-H₂SO₄ colorimetry were more stable and had the similar maximum absorption wavelength. It means that the determination value of the content of polysaccharides in Semen Plantaginis by the phenol-H₂SO₄ colorimetry method with xylose as a standard was more near to the true content. The optimum conditions and operating steps are as follows. Firstly, 3 % phenol solution 0.5 mL is added into the sample solution 2 mL. Then sulfuric acid 4 mL is added in. After shaking up rapidly and sufficiently, the mixture is stayed for 35 min at room temperature. Finally the absorbency at 480 nm is determined. The content of polysaccharides in Semen Plantaginis was evaluated as 9.02 % by the optimization method. The average recovery rate was 98.9 % with 1.79 % of RSD (n=6). This method is suitable for the determination of polysaccharides in Semen Plantaginis. It can also be regarded as the reference for the determination of polysaccharides with high pentoses content in other plants.2. The preparation process of polysaccharides in Semen Plantaginis was studied. The extraction conditions that influenced the extraction yield of polysaccharides were optimized by orthogonal design, the optimum extraction conditions were: 6 h at 100℃, the ration of material to solvent is l:35(w/v), giving the yield of polysaccharides at 8.5%. The optimum precipitation conditions were determined by single factor test, the best precipitation conditions were as follows: the concentration ratio 2, 60% alcohol precipitating for 6 h at low temperature. The optimum conditions are economic, simplicity and feasible, providing a theoretical reference for the industrialization of the preparation of polysaccharides in Semen Plantaginis.3. The two methods of Sevage method and Enzyme combined with Sevage method were used to remove proteins from the crude polysaccharides in Semen Plantaginis. By comparing the effects, it was found that by Enzyme combined with Sevage method, both the protein removal rate and polysaccharide yield were higher, and the purity of Semen Plantaginis polysaccharides after deproteinated was higher, too. The Sephacryl-400 gel column chromatography in series with the AKTA purifier 100 system was used to purify the polysaccharides in Semen Plantaginis. A main component, named SPG {Semen Plantaginis Glycoprotein), was obtained which was found to be a homogeneous glycoprotein component by HPLC. The molecular weight (Mw) of SPG was 1898584 Da determined by HPGPC.4. The physicochemical properties of SPG were studied. The results showed that SPG was brown powder and dissolvable in water. SPG contained protein of 2.92% and uronic acid of 14.58%. Arabinose, xylose, mannose, ribose, glucose and galactose were found to be the compositional monosaccharides in SPG by GC. Xylose and arabinose are the two main compositional monosaccharides. By UV spectrum and IR spectrum, SPG was identified as an acid glycoprotein, containing theβ- pyranoid ring.5. The antioxidant effects of different Semen Plantaginis Polysaccharides (Semen Plantaginis Water Extract, SPWE; Crude Semen Plantaginis Polysaccharides, CSPP; Semen Plantaginis Glycoprotein, SPG) were studied in vitro by different testing systems , which included DPPH radical, superoxide anion radical, hydroxyl radical and the lipid peroxidation of yolk liporeotein system. The results showed that SPWE, CSPP, SPG all had certain antioxidant activities. The correlativity of their effects and the doses are positive, in a certain concentration range. The SPG showed high antioxidant activities in those four antioxidant systems, especially in the lipid peroxidation of yolk liporeotein system. SPG showed comparable antioxidant effects with Vc at the same concentration of 2 mg / mL.6. The rheological property, gelation property and thermally stability of polysaccharides in Semen Plantaginis were studied by rheometer, TA-XT2i instrument, DSC and TGA/SDTA analysis respectively. It was found that the polysaccharides solution had pseudoplasticity. Apparent viscosity increased with the increase of concentration; the increasing of temperature can reduce the viscosity of the polysaccharides; the viscosity is not affected by the pH 5 toll; It has better compatibility with NaCl, Ca²⁺ obviously improve its viscosity, and the ability is correlated to the concentration of Ca²⁺ ions. The polysaccharides gel was the thermal reversible gel. Dissolution temperature, concentration, pH, salt all have obvious effects on the gel strength. The results of DSC showed no obvious enthalpic transitions for the gel, which indicated no obvious melting point (T_melt) and gel point (T_gel) for the gel. The results of TGA/SDTA analysis showed the polysaccharides had good thermal stability. All the above results showed that the polysaccharides in Semen Plantaginis had wide application potential in the food, pharmaceutical industries and so on.