Preparative Separation And Antioxidant Activity Of Chitooligosaccharides With Single Degree Of Polymerization And Well-defined N-acetylation

Chitooligosaccharides (COS), the hydrolyzed product of chitosan, has been reported topossess diverse bioactivities, such as antitumor activity, antioxidant activity, antimicrobial activity,immunity modulatory effect and so on. However, previous studies on biological activities of COSwere mostly performed using mixtures with different molecular weights and various degrees ofacetyltion. It is difficult to know which COS molecules play a leading role in the biological assayand the study of the related mechanism is limited. Therefore, in order to further understand thebioactivities of COS, the preparation of COS with narrow degree of polymerization (DP) andwell-defined degree of acetylation (DA) is required. However, due to small difference of chargedensity and molecular weight between chitooligosaccharides, the separation of COS with singleDP is very difficult, especially for those oligomers with DP>4. In addition, the method ofpreparation of N-acetylated COS with single DP is still unknown.This study focuses on the preparation, separation and antioxidant activity of COS withdifferent DP and various DA. The prepare COS and their separated fractions were characterizedwith various analytical techniques, including X-ray diffraction (XRD), fourier-transform infraredspectroscopy (FT-IR), ultraviolet–visible spectroscopy (UV-vis), elemental analysis, nuclearmagnetic resonance spectroscopy (NMR), mass spectrometry (MS), high-performance liquidchromatography (HPLC). We developed a chromatographic procedure for the separation of COSwith single DP and well-defined DA and established a high-resolution chromatographic separationmodel of fully deacetylated COS. Additionally, the antioxidant activities of COS with narrow DPand well-defined DA were investigated. The major results were as follows:1．Microwave-assisted oxidative degradation technique of chitosan was developed: oxidative degradation of chitosan was accelerated by microwave irradiation under the condition of lowtemperature and low concentration of H2O2. XRD analysis demonstrated that the crystallinestructure of chitosan was altered with the degradation going on and when the degraded chitosanbecame amorphous (5.5kDa), the product had high water solubility. The result of UV-vis showcarbonyl groups formed during degradation due to chain scission and ring open of chitiosan andbrown compounds increased but no absorbance band corresponding to–COO-was observed inFT-IR spectra. Elemental analysis indicated deamination reaction occurred in the degradationprocess but was inhibited partially. These results suggested that the degraded products remainedthe essential chemical structure of chitosan.2．The chromatographic separation conditions of COS with well-defined DP were specified:ion-exchange chromatography could be applied to prepare COS with different DPs according tothe number of amino group on the sugar chains. The prepared COS with low DP was separatedwith CM Sephadex C-25column and six highly purified glucosamine oligomers with single DPswere isolated. ESI/MS and HPLC analysis revealed that the six isolated fractions contained99.3%dimer,98.9%trimer,98.3%tetramer,99.1%pentamer,99.0%hexamer, and92.9%heptamer,respectively. The purities of separated single COSs were higher than those reported in prior studies.The yields of a single round of separation were75,60,60,55,35, and20mg for glucosaminedimers, trimers, tetramers, pentamers, hexamers, and heptamers, respectively, which are adequateto satisfy the needs of most chitooligomers bioactivity assays. Furthermore, a chromatographicseparation model for GlcN homomers was established. The capacity factor (k) of glucosamineoligomers and their degrees of polymerization (DPs) exhibited a good correlation, represented bythe equation㏑k=0.786+0.846㏑DP,(R2=0.997), which is instructive in the separation ofCOSs with higher DP and scale-up preparation of single COS. Additionally, the prepared fullydeacetylated COS with high DP (>6) were firstly separated by CM Sepharose Fast Flow columnand five COS fractions with narrow DP were obtained, which mainly contained glucosamineoligomers with DP6-7(41.31%,50.22%), DP7-8(22.47%,70.13%), DP9-10(53.06%,27.99%),DP10-12(18.45%,49.36%,22.31%), and DP>12, respectively.3．The preparation and separation technique of COS with single DP and well-defined DA wasestablished: the N-acetylated COS was synthesized and separated by ion-exchangechromatography according to the number of amino group. In this paper, eight N-acetylated COS with single Mwwere prepared, including N-acetylchitotriose, N, N‘-diacetylchitotriose,N-acetylchitohexaose, N, N‘-diacetylchitohexaose, N, N‘, N‘‘-triacetylchitohexaose, N, N‘, N‘‘,N‘‘‘-tetraacetylchitohexaose, N, N‘, N‘‘, N‘‘‘, N‘‘‘‘-pentaacetylchitohexaose, N, N‘, N‘‘, N‘‘‘,N‘‘‘‘, N‘‘‘‘‘-hexaacetylchitohexaose. All separated fractions were judged to be relative pure basedon the ESI-MS spectra. In addition, The sequence analysis of ESI-MS/MS revealed that the mainisobaric components of the N-acetylchitotriose and N, N‘-diacetylchitotriose were DDA and ADA,respectively. However, the separated chitohexaoses were complex except N, N‘, N‘‘, N‘‘‘, N‘‘‘‘,N‘‘‘‘‘-hexaacetylchitohexaose and their isobaric components need to be further studied.4．The antioxidant activities of the separated COSs with well-defined DP and DA werestudied, including hydroxyl and superoxide radical scavenging activity and reducing power. Therelationship between antioxidant activity of COS and their DA and DP was investigated. Theresults indicated that the COS with low DP showed better effect of scavenging hydroxyl radicaland reducing power than that with high DP. There existed optimal chitooligomers with narrow DPranging from10to12and the chitooligomers with DP11was speculated to be optimal.Furthermore, The antioxidant activities of two partially acetylated chitotrioses and originalchitotriose were investigated and the N, N‘-diacetylchitotriose exhibited with high DA the highestantioxidant activity, revealing that the N-acetylation play an important role in the antioxidantactivity of chitooligosaccharides.In this study, a separation technique of COS was developed, which also can be used toprepare of other COS with single DP and well-defined DA for bioactivity assay. Furthermore,we revealed that the relationship between antioxidant activity of COS and their DA and DP.These results are instructive to the further bioactivity research of COS and its application in foodand medicine fields.