单一聚合度和特定N-乙酰化壳寡糖的分离及抗氧化活性研究
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摘要
壳寡糖是壳聚糖的降解产物，具有多种生物活性，如抗肿瘤、抗氧化、抑菌、免疫调节等。然而，以往的文献报道中，均是采用含有不同分子量和乙酰度的壳寡糖混合物进行的生物活性研究，这使得很难明确具体哪个或哪些寡糖分子在生物活性中发挥作用，也限制了对机理的深入研究。因此，为了进一步研究壳寡糖的生物活性，制备特定聚合度和乙酰度的壳寡糖是非常必要的。但由于壳寡糖单体间电荷密度和分子量差别较小，使得其分离纯化非常困难，尤其对聚合度大于4的壳寡糖的分离更是困难，另外，单一聚合度不同乙酰度壳寡糖的制备方法和相关生物活性也未见报道。
本文对单一聚合度、不同乙酰度的壳寡糖的制备、分离和抗氧化活性进行了研究，并通过X射线衍射（XRD）、红外光谱（FT-IR）、紫外可见光谱（UV-vis）、元素分析、核磁共振波谱（NMR）、质谱（MS）、高效液相色谱（HPLC）等分析手段对降解和分离纯化产物的理化性质进行了全面的解析；确定了单一聚合度、不同乙酰度壳寡糖的色谱分离条件，建立了高分辨分离全脱乙酰化壳寡糖的离子交换色谱模型，并研究了特定聚合度和乙酰度壳寡糖的抗氧化活性，获得主要研究结果如下：
1．微波辅助壳聚糖氧化降解工艺：微波辐射能够有效的加快壳聚糖的氧化降解速率，并使其在更低的过氧化氢浓度和温度下进行。降解产物的XRD分析表明壳聚糖降解过程中其晶形结构被改变，当壳聚糖分子量降至5.5kDa以下时变为无定形态，此时降解产物有较好的水溶性。紫外可见光谱分析表明降解过程中由于断链和开环会有羰基的产生且褐变加剧，但氧化副反应的羧基没有从FT-IR谱图中观察到。另外，元素分析结果表明降解过程中脱胺副反应被有效的抑制，产物保持了壳聚糖的基本化学结构。
2．确定了特定聚合度壳寡糖的制备色谱分离条件：离子交换色谱能够依据氨基数量的差异对不同聚合度的壳寡糖进行有效的分离。采用CM Sephadex C-25对低聚合度全脱乙酰化壳寡糖进行分离，可以得到六个高纯度的单一聚合度壳寡糖，通过ESI/MS和HPLC分析，结果表明六个壳寡糖组分分别为壳二糖，壳三糖，壳四糖，壳五糖，壳六糖和壳七糖，纯度分别为的纯度99.3%，98.9%，98.3%，99.1%，99.0%和92.9%。获得的所有单一聚合度壳寡糖的纯度均大于以往文献的报道，各个分离组分产率分别为75，60，60，55，35和20mg/天，基本可以满足壳寡糖生物活性实验的要求。另外，我们建立了一个高分辨的壳寡糖分离色谱模型，壳寡糖的容量因子k和聚合度DP之间符合方程㏑k=0.686+0.926㏑DP,(R2=0.997)，该公式对于更高聚合度壳寡糖单体的分离以及单一聚合度壳寡糖单体的放大制备具有指导意义。采用CM Sepharose Fast Flow首次完成了聚合度大于6的全脱乙酰壳寡糖的分离，得到五个窄聚合度的全脱乙酰化壳寡糖组分，其寡糖聚合度分布为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%)和DP>12。
3．单一聚合度、特定乙酰度壳寡糖的制备工艺：采用离子交换色谱可依据氨基数量对合成的N-乙酰化壳寡糖混合物进行有效的分离，本文共分离得到8种单分子量N-乙酰化壳寡糖，包括N-乙酰壳三糖、N,N‘-二乙酰壳三糖、N-乙酰壳六糖、N,N‘-二乙酰壳六糖、N,N‘,N‘‘-三乙酰化壳六糖、N,N‘,N‘‘,N‘‘‘-四乙酰壳六糖、N,N‘,N‘‘,N‘‘‘,N‘‘‘‘-五乙酰壳六糖、N,N‘,N‘‘,N‘‘‘,N‘‘‘‘,N‘‘‘‘‘-六乙酰壳六糖。一级质谱分析表明分离得到的N-乙酰化单分子量壳寡糖是相对纯的。另外，采用ESI-MS/MS对分离得到的两种乙酰化壳三糖进行糖链序列分析，结果表明N-乙酰壳三糖和N,N‘-二乙酰壳三糖的异构体主要组分分别为DDA和ADA。而六种不同乙酰度的壳六糖除N,N‘,N‘‘,N‘‘‘,N‘‘‘‘,N‘‘‘‘‘-六乙酰壳六糖之外，其它几种壳六糖成分是非常复杂的，可能含有多种糖链序列不同的异构体，具体成分分析还有待进一步研究。
4．抗氧化活性研究：对分离得到的每一种特定聚合度和乙酰度的壳寡糖进行了抗氧化活性实验，包括羟自由基清除活性，超氧阴离子清除活性和还原能力测定。结果表明，低聚合度的壳寡糖具有更强的羟自由基清除活性和还原能力，而超氧阴离子清除活性最强的壳寡糖聚合度为10-12，结合其成分分析进一步推测壳十一糖有可能是最佳的清除超氧阴离子的壳寡糖分子。对制备的两种部分乙酰化壳三糖和全脱乙酰化壳三糖的抗氧化活性进行比较，结果显示高乙酰度的壳三糖具有更强的抗氧化活性，这说明N-乙酰基对壳寡糖的抗氧化活性起重要作用。
本文提出的壳寡糖单体制备方法还适用于其它聚合度和乙酰度壳寡糖的制备，获得的研究结果包括壳寡糖抗氧化活性与其聚合度和乙酰度之间的关系等，为进一步筛选壳寡糖的生物活性、研究其相关的活性机制以及寡糖在食品、医药等行业的广泛应用奠定了基础。
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.
本文对单一聚合度、不同乙酰度的壳寡糖的制备、分离和抗氧化活性进行了研究，并通过X射线衍射（XRD）、红外光谱（FT-IR）、紫外可见光谱（UV-vis）、元素分析、核磁共振波谱（NMR）、质谱（MS）、高效液相色谱（HPLC）等分析手段对降解和分离纯化产物的理化性质进行了全面的解析；确定了单一聚合度、不同乙酰度壳寡糖的色谱分离条件，建立了高分辨分离全脱乙酰化壳寡糖的离子交换色谱模型，并研究了特定聚合度和乙酰度壳寡糖的抗氧化活性，获得主要研究结果如下：
1．微波辅助壳聚糖氧化降解工艺：微波辐射能够有效的加快壳聚糖的氧化降解速率，并使其在更低的过氧化氢浓度和温度下进行。降解产物的XRD分析表明壳聚糖降解过程中其晶形结构被改变，当壳聚糖分子量降至5.5kDa以下时变为无定形态，此时降解产物有较好的水溶性。紫外可见光谱分析表明降解过程中由于断链和开环会有羰基的产生且褐变加剧，但氧化副反应的羧基没有从FT-IR谱图中观察到。另外，元素分析结果表明降解过程中脱胺副反应被有效的抑制，产物保持了壳聚糖的基本化学结构。
2．确定了特定聚合度壳寡糖的制备色谱分离条件：离子交换色谱能够依据氨基数量的差异对不同聚合度的壳寡糖进行有效的分离。采用CM Sephadex C-25对低聚合度全脱乙酰化壳寡糖进行分离，可以得到六个高纯度的单一聚合度壳寡糖，通过ESI/MS和HPLC分析，结果表明六个壳寡糖组分分别为壳二糖，壳三糖，壳四糖，壳五糖，壳六糖和壳七糖，纯度分别为的纯度99.3%，98.9%，98.3%，99.1%，99.0%和92.9%。获得的所有单一聚合度壳寡糖的纯度均大于以往文献的报道，各个分离组分产率分别为75，60，60，55，35和20mg/天，基本可以满足壳寡糖生物活性实验的要求。另外，我们建立了一个高分辨的壳寡糖分离色谱模型，壳寡糖的容量因子k和聚合度DP之间符合方程㏑k=0.686+0.926㏑DP,(R2=0.997)，该公式对于更高聚合度壳寡糖单体的分离以及单一聚合度壳寡糖单体的放大制备具有指导意义。采用CM Sepharose Fast Flow首次完成了聚合度大于6的全脱乙酰壳寡糖的分离，得到五个窄聚合度的全脱乙酰化壳寡糖组分，其寡糖聚合度分布为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%)和DP>12。
3．单一聚合度、特定乙酰度壳寡糖的制备工艺：采用离子交换色谱可依据氨基数量对合成的N-乙酰化壳寡糖混合物进行有效的分离，本文共分离得到8种单分子量N-乙酰化壳寡糖，包括N-乙酰壳三糖、N,N‘-二乙酰壳三糖、N-乙酰壳六糖、N,N‘-二乙酰壳六糖、N,N‘,N‘‘-三乙酰化壳六糖、N,N‘,N‘‘,N‘‘‘-四乙酰壳六糖、N,N‘,N‘‘,N‘‘‘,N‘‘‘‘-五乙酰壳六糖、N,N‘,N‘‘,N‘‘‘,N‘‘‘‘,N‘‘‘‘‘-六乙酰壳六糖。一级质谱分析表明分离得到的N-乙酰化单分子量壳寡糖是相对纯的。另外，采用ESI-MS/MS对分离得到的两种乙酰化壳三糖进行糖链序列分析，结果表明N-乙酰壳三糖和N,N‘-二乙酰壳三糖的异构体主要组分分别为DDA和ADA。而六种不同乙酰度的壳六糖除N,N‘,N‘‘,N‘‘‘,N‘‘‘‘,N‘‘‘‘‘-六乙酰壳六糖之外，其它几种壳六糖成分是非常复杂的，可能含有多种糖链序列不同的异构体，具体成分分析还有待进一步研究。
4．抗氧化活性研究：对分离得到的每一种特定聚合度和乙酰度的壳寡糖进行了抗氧化活性实验，包括羟自由基清除活性，超氧阴离子清除活性和还原能力测定。结果表明，低聚合度的壳寡糖具有更强的羟自由基清除活性和还原能力，而超氧阴离子清除活性最强的壳寡糖聚合度为10-12，结合其成分分析进一步推测壳十一糖有可能是最佳的清除超氧阴离子的壳寡糖分子。对制备的两种部分乙酰化壳三糖和全脱乙酰化壳三糖的抗氧化活性进行比较，结果显示高乙酰度的壳三糖具有更强的抗氧化活性，这说明N-乙酰基对壳寡糖的抗氧化活性起重要作用。
本文提出的壳寡糖单体制备方法还适用于其它聚合度和乙酰度壳寡糖的制备，获得的研究结果包括壳寡糖抗氧化活性与其聚合度和乙酰度之间的关系等，为进一步筛选壳寡糖的生物活性、研究其相关的活性机制以及寡糖在食品、医药等行业的广泛应用奠定了基础。
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.
引文
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