| Sulfated oligosaccharides and polysaccharides widely exist in nature, and they exhibit diverse biological activities, such as anticoagulation, antioxidant, antitumor, antiviral, anti-inflammatory, anti-bacteria and immunostimulatory properties. However, due to the presence of sulfate anion groups, sulfated oligosaccharides and polysaccharides have high polarity. It is still difficult to analyze and prepare sulfated oligosaccharides and polysaccharides. In the thesis, the sulfated oligosaccharides and polysaccharides with a series of derivatization were analyzed by mass spectrometry (MS), high performance liquid chromatography coupling with electrospray ionization mass spectrometry (HPLC-ESI-MS) and gas chromatography coupling with mass spectrometry (GC-MS). The detailed structures of sulfated oligosaccharides obtained by enzymatic hydrolysis were well unraveled. Furthermore, the structural characteristics and their antioxidant activities of κ-carrageenan oligosaccharides degraded by different methods were comparatively investigated. In addition, a new method was established for sulfation pattern determination of sulfated polysaccharides. This study makes a fundament for the deep understanding the structure-function relationships of sulfated polysaccharides and oligosaccharides. The major results were summarized as followings:1. The structural information of κ-carrageenan oligosaccharides and the catalytic dynamics in enzymatic hydrolysis by a novel κ-carrageenase were well analyzed and unraveled. The novel κ-carrageenase was purified from Pedobacter hainanensis. The κ-carrageenase had a molecular weight of -55 kDa. Under the optimum condition of 40 ℃ and pH 7.0, the κ-carrageenase yielded the maximum enzymatic activity of 700.53 units per mg protein. κ-Carrageenan was then degraded with the κ-carrageenase. The degradation products were first analyzed by TLC and HPLC, and the results indicated that the κ-carrageenase degraded κ-carrageenan to even-numbered polymerization degree of oligosaccharides; and among the degradation products, tetrasaccharide was the major. The structures of all the components degraded from different time courses were then determined and assigned by ESI-MS and CID MS/MS, and the results revealed that each of the carrageenan oligosaccharides was consisted of An-G4S-type neocarrabiose units, which composed of a 3,6-anhydro-a-D-galactose (An) residue in the non-reducing terminus and a P-D-galactose-4-sulfate (G4S) residue in the reducing terminus. These results demonstrated that the κ-carrageenase cleaved κ-carrageenan at the internal β-1,4 linkage of κ-carrageenan. This novel enzyme offers an alternative approach to produce κ-carrageenan oligosaccharides.2. Four kinds of κ-carrageenan oligosaccharides were obtained by the degradation of parent κ-carrageenan using free radical depolymerization, mild acid hydrolysis, κ-carrageenase digestion and partial reductive hydrolysis, respectively. Their structural types were accurately elucidated by MS and their antioxidant activities were comparatively assayed by in vitro and cellular antioxidant assay systems. ESI-MS and CID MS/MS results demonstrated that the oligosaccharides prepared by different methods have various and different chemical structures. The κ-carrageenan oligosaccharides obtained from H2O2 depolymerization contained a series of even-and odd-numbered oligosaccharides, and generated four series of different oligosaccharides, such as even-numbered oligosaccharides-(G4S-An)n-, with An at the reducing terminus and G4S at the non-reducing terminus; odd-numbered oligosaccharides-An-(G4S-An)n-, with An at the reducing terminus and the non-reducing terminus; even-numbered oligosaccharide acids-(G4S-AnOOH)n-, with AnOOH at the reducing terminus and G4S at the non-reducing terminus; and odd-numbered oligosaccharides-An-(G4S-AnOOH)n-, with AnOOH at the reducing terminus and An at the non-reducing terminus. The κ-carrageenan oligosaccharides prepared by HC1 hydrolysis mainly contained odd-numbered oligosaccharides-G4S-(An-G4S)n-, with G4S at the reducing end and the non-reducing terminus; and minor even-numbered oligosaccharides-(G4S-An)n-, with An at the reducing terminus and G4S at the non-reducing terminus were also found.The κ-carrageenan oligosaccharides obtained from enzymatic degradation only contained a series of even-numbered oligosaccharides-(An-G4S)n-, with G4S at the reducing terminus and An at the non-reducing terminus. Additionally, The partial reductive hydrolysis produced even-numbered oligosaccharide alditols-(G4S-Anol)n-, with Anol at the reducing terminus and G4S at the non-reducing terminus, which made a great distinguish from the sequence of enzymatic preparations. The antioxidant activities of different degradation oligosaccharides were investigated by several antioxidant assays, including in vitro systems, e.g. superoxide radical scavenging activity (’O2-), hydroxyl radical scavenging activity (OH), reducing power, DPPH radical scavenging activity, and the cellular antioxidant activity. These results indicated that the methods of depolymerization had an influence on the antioxidant activities of κ-carrageenan oligosaccharides. The antioxidant activities of κ-carrageenan oligosaccharides were related to the polymerization degree (Dp), reducing sugar and sulfate groups contents, and structure of the reducing end. Among all of the hydrolysates, H2O2 hydrolysates exhibited much higher antioxidant abilities in all assay systems. These results provide a scientific basis for the deep study on the relationships between structure and antioxidant activities of κ-carrageenan oligosaccharides.3. A new method was established for sulfation pattern determination of sulfated polysaccharides. This method was applied to qualitative analysis and quantitative measurements of sulfate groups on polysaccharides. Two sulfated Lycium barbarum polysaccharides with different degrees of sulfation (LbGp1-OL-SL with 13.7% sulfate content, and LbGp1-OL-SH with 27.4% sulfate content) were employed to establish the analysis method. The sulfation patterns were analyzed only using a GC-MS strategy. With a series of sequential chemical derivatizations, the sulfated polysaccharides were converted to their partially methylated alditol acetates (PMAAs). All the sulfate groups on the sulfated polysaccharide molecules were replaced by acetyl groups, maintaing the positional information of the original sulfation pattern. Through comparing the relative molar ratio from methylation analysis between Lycium barbarum polysaccharide and sulfated derivatives, the pattern of sulfate groups were well deduced. As a result, in LbGp1-OL-SL,12.65% of sulfation occurred on 5-C of Ara,0.69% and 0.34% of sulfate groups were found at C-4 and C-6 Gal, respectively; however, in LbGp1-OL-SH,24.96% of sulfation located on 5-C of Ara, and only 0.40% and 2.02% of sulfate groups were substituted at C-4 and C-6 Gal, respectively. This method provides a great methodological reference frame for accurately structural determation of sulfated polysaccharides, which could be useful for identifying the influence of sulfation modification on the sulfated polysaccharides. In addition, this method would be helpful for the further study on the relationships between sulfation pattern and function of κ-carrageenanpolysaccharides. |
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