止血用壳聚糖的质量和安全控制研究
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摘要
甲壳素和壳聚糖不但生物相容性好，止血效果优异，而且可生物降解，因此在止血材料方面有大量的研究并已有一定的应用。然而现有标准难以保证组织脏器止血用壳聚糖的安全和性能；我国也已有壳聚糖等可吸收材料的临床不良反应报道。因此研究止血用壳聚糖的质量和安全控制方法，开展止血用壳聚糖的质量标准研究，对于保障止血用壳聚糖的质量有重要的学术意义，并为促进壳聚糖在止血材料等领域等研究和应用提供基础。
残留蛋白质可能引起过敏反应影响壳聚糖作为止血材料的安全性，而碱提等脱蛋白方法为非均相反应，且蛋白质与壳聚糖结合强，因此脱蛋白效率较低。此外碱提等方法可能导致壳聚糖分子量降低。为此采用固定化酶技术研究了壳聚糖在溶液中脱除蛋白质残留的方法：通过研究不同硅原比例对固定化酶的固定化率、酶活力和蛋白选择性的影响，确定了以四甲氧基硅烷：氨丙基三乙氧基硅烷（TMOS:ATPES）90:10对胃蛋白酶的固定条件。固定化酶的蛋白酶活力为游离酶的125.3%±14.3%，蛋白质底物选择性也提高为游离酶的2.7倍。同时研究了温度和pH对固定化酶蛋白酶活力和酶选择性的影响，并建立了以pH4.5，45℃条件下固定化酶对壳聚糖溶液温孵160min的脱除蛋白质方法。该方法可有效脱除壳聚糖中53.8-80.4%残留蛋白质，使蛋白质残留量降低至0.12%～0.34%，脱蛋白效果显著优于碱提法。
受到壳聚糖的干扰，壳聚糖中微量蛋白质残留难以灵敏、准确的定量测定。为减少壳聚糖的干扰，研究了水解后芴甲氧羰酰氯（FMOC-Cl）柱前衍生HPLC法测定壳聚糖中微量蛋白质残留。研究了大量壳聚糖水解产物干扰时氨基酸的FMOC-Cl衍生条件，及16种氨基酸和氨基葡萄糖的FMOC-Cl衍生产物在不同pH条件的色谱行为，建立了壳聚糖中微量蛋白含量的方法。该方法对各氨基酸的线性关系良好（r>0.99），精密度（RSD）1.6%～8.5%，定量限1.9～10.9ng·mL~(-1)，回收率84.4%～98.6%。与考马斯亮蓝染色法、元素分析法和FMOC-Cl柱前衍生LC-MS/MS法相比方法灵敏、准确。
病毒污染会严重威胁壳聚糖作为止血材料的安全性，而相关的研究报道很少。对壳聚糖中病毒的表征和灭活方法进行了研究：以猪细小病毒（PPV）和牛病毒性腹泻病毒（BVDV）为指示病毒，研究了γ射线辐照和NaOH-乙醇溶液等处理方法对壳聚糖病毒滴的灭活以及壳聚糖分子量、脱乙酰度的影响。发现辐照剂量增强，壳聚糖分子量显著降低，但30kGy时仍无法完全灭活病毒（ΔlgTCID_(50)<4.0）；NaOH-乙醇溶液可降低病毒滴度，且NaOH浓度是最主要影响因素。建立了8mol·L-1NaOH的10%乙醇溶液35℃处理1h后γ射线辐照5kGy的病毒灭活方法，该方法可有效灭活病毒（ΔlgTCID_(50)>4.0），并仅引起壳聚糖分子量降低8.4%。
受水解中的褐变降解影响，壳聚糖含量测定准确度较低，回收率约90%。为提高含量测定的准确度，研究不同比例盐酸和磷酸条件下氨基葡萄糖和壳聚糖褐变，并确定了5-羟甲基糠醛和脱氧果糖嗪等主要褐变产物，建立了盐酸-磷酸（4.5:1.5mol·L~(-1)）水解结合柱前衍生HPLC法测定壳聚糖含量的方法。采用盐酸-磷酸（4.5:1.5mol·L~(-1)）可抑制壳聚糖水解中的褐变降解，使回收率由盐酸水解的5.1%提高至97.7%，同时方法在20～300μg·mL~(-1)范围内线性良好（r>0.99），精密度高（RSD2.9%-3.9%）。
因缺乏止血用壳聚糖的质量标准，对壳聚糖的各主要质量指标及其测定方法进行了比较和评价，并参考国内外壳聚糖标准及外科植入物的质量要求，提出了止血用壳聚糖质量标准，为我国止血用壳聚糖的质量控制提供基础。
论文建立了固定化胃蛋白酶脱除蛋白质残留纯化壳聚糖的方法，其效果显著优于非均相的碱提法；建立了灵敏、准确的柱前衍生HPLC法测定壳聚糖中微量蛋白质残留；建立了盐酸-磷酸水解柱前衍生HPLC法测定壳聚糖含量，抑制了水解中的褐变，显著提高了方法的回收率，对于壳聚糖的全面质量控制有重要的意义。
Chitin and chitosan are not only biocompatible and outstanding in hemostasis effect, but alsobiodegradable. Therefor they have been widely studied as hemostatic materials. However, present qualitycontrol methods are inadequate to ensure the safety and efficiency of chitosan. The adverse effect ofchitosan and other absorbable material were also reported recently. Researching on the safety andeffectiveness related quality factors of chitosan, and establishing the standard of chitosan for hemostasisusage is of great importance to ensure the quality of chitosan for hemostasis usage, and will provide a solidtheoretical basis for the research and application of chitosan as a hemostatic material.
Protein residue could possibly cause allergy reaction and therefore affect the safety of chitosan forhemostasis usage. Meanwhile alkai extraction and other deproteinization methods were inefficient becausethe deproteinization was hetergenous reaction. The deproteinization could also induce the degradation ofchitsoan. Deproteinization of chitosan with immobilized enzyme was studied. Enzyme was immobilizedusing sol-gel method with tetramethoxysilane (TMOS) and3-aminopropyltriethoxysilane (APTES) assilanes for immobilization. The immobilization condition was studied by estimating the immobilizationefficiency, activity and protein selectivity. The optimum condition was immobilizing pepsin withTMOS:APTES (90:10), with enzyme activity125.3%±14.3%to free enzyme and substrate selectivityincreased2.7times. Chitosan deproteinization with chitosan solution incubated with immobilized pepsin atpH4.5,45oC for160min. After the deproteinization with immobilized pepsin,53.8%-80.4%protein inchitosan was removed and the residue protein content lowered to0.12%~0.34%, which was significantlysuperior to traditional NaOH deproteinization.
As chitosan influenced the determination of protein residue in chitosan, in present workfluorenylmethyloxycarbonyl chloride (FMOC-Cl) derivation and HPLC method was studied to minimizethe influence of chitosan. The derivation of amino acids in large amount of glucosamine, chitosanhydrolysis product, and the chromatographic behavior of the derivation products were optimized and thedetermination of protein residue in chitosan was studied and established. The established method was goodin calibration (r>0.99), precision (RSD1.6%-8.5%), sensentive (LOQ1.9-10.9ng·mL~(-1)) and accurate(recovery84.4-98.6). The method was also proved superior to Bradford method, LC-MS/MS method andelemental analysis.
Virus in chitosan was another safety related factor of chitosan but few related study reported. Theinactivation of virus in chitosan with γ irradition or NaOH-ethanol incubation was evaluated and studied,with Porcine parvovirus (PPV) and Bovine viral diarrhea virus (BVDV) as indicators. Irradation ofchitosan wiht γ ray for5kGy, after incubation with8mol·L~(-1)NaOH consisting10%ethanol at35oC for1hour was proved effective in inactivation of virus in chitosan（ΔlgTCID_(50)>4.0） and only inducedmarginal degradation of chitosan for8.4%.
Because the browning degradation of chitosan in acid hydrolysis, the recovery of chitoan inquantification was about90%in most reports, which was inadequate for quantification of chitosan inhemostasis application. The quantification of chitosan was studied by optimizing and evaluating thebrowning degradation of glucosamine and chitosan in hydrolysis with hydrochloric acid and phosphoricacid. Minimum brwoning degradation was proved with hydrochloric acid and phosphoric acid (4.5:1.5inmolar ratio) hydrolysis. Chitosan was quantified with hydrochloric acid and phosphoric acid (4.5:1.5inmolar ratio) hydrolysis and FMOC-Cl derivation HPLC determination. The method was linear at20-300μg·mL-（1r>0.99）and with good precision. The accuracy (recovery97.7%) was significantly superiorto the traditional HCl hydrolysis (recovery85.1%) and can be used for the quality control of chitosan forhemostatic usage.
As lack proper standard of chitosan for hemostasis usage, the main quality related factors of chitosanwere evaluated, and the standard of chitosan was then proposed to ensure the quality control of chitosan forhemostasis usage, with the consult of current chitosan standard and the general requirements of implantmaterials.
The present work studied on the deproteinization and purification of chitosan, which was significantlysuperior to NaOH deproteinization. Studied and established a sensitive and accurate pre-column derivationHPLC method for the determination of trace protein residue in chitosan. Studied on the HCl-H3PO4hydrolysis coupled pre-column derivation HPLC method for the quantification of chitosan, this improvedthe recovery of the method. These were meaninful to the total quality control of chitosan.
残留蛋白质可能引起过敏反应影响壳聚糖作为止血材料的安全性，而碱提等脱蛋白方法为非均相反应，且蛋白质与壳聚糖结合强，因此脱蛋白效率较低。此外碱提等方法可能导致壳聚糖分子量降低。为此采用固定化酶技术研究了壳聚糖在溶液中脱除蛋白质残留的方法：通过研究不同硅原比例对固定化酶的固定化率、酶活力和蛋白选择性的影响，确定了以四甲氧基硅烷：氨丙基三乙氧基硅烷（TMOS:ATPES）90:10对胃蛋白酶的固定条件。固定化酶的蛋白酶活力为游离酶的125.3%±14.3%，蛋白质底物选择性也提高为游离酶的2.7倍。同时研究了温度和pH对固定化酶蛋白酶活力和酶选择性的影响，并建立了以pH4.5，45℃条件下固定化酶对壳聚糖溶液温孵160min的脱除蛋白质方法。该方法可有效脱除壳聚糖中53.8-80.4%残留蛋白质，使蛋白质残留量降低至0.12%～0.34%，脱蛋白效果显著优于碱提法。
受到壳聚糖的干扰，壳聚糖中微量蛋白质残留难以灵敏、准确的定量测定。为减少壳聚糖的干扰，研究了水解后芴甲氧羰酰氯（FMOC-Cl）柱前衍生HPLC法测定壳聚糖中微量蛋白质残留。研究了大量壳聚糖水解产物干扰时氨基酸的FMOC-Cl衍生条件，及16种氨基酸和氨基葡萄糖的FMOC-Cl衍生产物在不同pH条件的色谱行为，建立了壳聚糖中微量蛋白含量的方法。该方法对各氨基酸的线性关系良好（r>0.99），精密度（RSD）1.6%～8.5%，定量限1.9～10.9ng·mL~(-1)，回收率84.4%～98.6%。与考马斯亮蓝染色法、元素分析法和FMOC-Cl柱前衍生LC-MS/MS法相比方法灵敏、准确。
病毒污染会严重威胁壳聚糖作为止血材料的安全性，而相关的研究报道很少。对壳聚糖中病毒的表征和灭活方法进行了研究：以猪细小病毒（PPV）和牛病毒性腹泻病毒（BVDV）为指示病毒，研究了γ射线辐照和NaOH-乙醇溶液等处理方法对壳聚糖病毒滴的灭活以及壳聚糖分子量、脱乙酰度的影响。发现辐照剂量增强，壳聚糖分子量显著降低，但30kGy时仍无法完全灭活病毒（ΔlgTCID_(50)<4.0）；NaOH-乙醇溶液可降低病毒滴度，且NaOH浓度是最主要影响因素。建立了8mol·L-1NaOH的10%乙醇溶液35℃处理1h后γ射线辐照5kGy的病毒灭活方法，该方法可有效灭活病毒（ΔlgTCID_(50)>4.0），并仅引起壳聚糖分子量降低8.4%。
受水解中的褐变降解影响，壳聚糖含量测定准确度较低，回收率约90%。为提高含量测定的准确度，研究不同比例盐酸和磷酸条件下氨基葡萄糖和壳聚糖褐变，并确定了5-羟甲基糠醛和脱氧果糖嗪等主要褐变产物，建立了盐酸-磷酸（4.5:1.5mol·L~(-1)）水解结合柱前衍生HPLC法测定壳聚糖含量的方法。采用盐酸-磷酸（4.5:1.5mol·L~(-1)）可抑制壳聚糖水解中的褐变降解，使回收率由盐酸水解的5.1%提高至97.7%，同时方法在20～300μg·mL~(-1)范围内线性良好（r>0.99），精密度高（RSD2.9%-3.9%）。
因缺乏止血用壳聚糖的质量标准，对壳聚糖的各主要质量指标及其测定方法进行了比较和评价，并参考国内外壳聚糖标准及外科植入物的质量要求，提出了止血用壳聚糖质量标准，为我国止血用壳聚糖的质量控制提供基础。
论文建立了固定化胃蛋白酶脱除蛋白质残留纯化壳聚糖的方法，其效果显著优于非均相的碱提法；建立了灵敏、准确的柱前衍生HPLC法测定壳聚糖中微量蛋白质残留；建立了盐酸-磷酸水解柱前衍生HPLC法测定壳聚糖含量，抑制了水解中的褐变，显著提高了方法的回收率，对于壳聚糖的全面质量控制有重要的意义。
Chitin and chitosan are not only biocompatible and outstanding in hemostasis effect, but alsobiodegradable. Therefor they have been widely studied as hemostatic materials. However, present qualitycontrol methods are inadequate to ensure the safety and efficiency of chitosan. The adverse effect ofchitosan and other absorbable material were also reported recently. Researching on the safety andeffectiveness related quality factors of chitosan, and establishing the standard of chitosan for hemostasisusage is of great importance to ensure the quality of chitosan for hemostasis usage, and will provide a solidtheoretical basis for the research and application of chitosan as a hemostatic material.
Protein residue could possibly cause allergy reaction and therefore affect the safety of chitosan forhemostasis usage. Meanwhile alkai extraction and other deproteinization methods were inefficient becausethe deproteinization was hetergenous reaction. The deproteinization could also induce the degradation ofchitsoan. Deproteinization of chitosan with immobilized enzyme was studied. Enzyme was immobilizedusing sol-gel method with tetramethoxysilane (TMOS) and3-aminopropyltriethoxysilane (APTES) assilanes for immobilization. The immobilization condition was studied by estimating the immobilizationefficiency, activity and protein selectivity. The optimum condition was immobilizing pepsin withTMOS:APTES (90:10), with enzyme activity125.3%±14.3%to free enzyme and substrate selectivityincreased2.7times. Chitosan deproteinization with chitosan solution incubated with immobilized pepsin atpH4.5,45oC for160min. After the deproteinization with immobilized pepsin,53.8%-80.4%protein inchitosan was removed and the residue protein content lowered to0.12%~0.34%, which was significantlysuperior to traditional NaOH deproteinization.
As chitosan influenced the determination of protein residue in chitosan, in present workfluorenylmethyloxycarbonyl chloride (FMOC-Cl) derivation and HPLC method was studied to minimizethe influence of chitosan. The derivation of amino acids in large amount of glucosamine, chitosanhydrolysis product, and the chromatographic behavior of the derivation products were optimized and thedetermination of protein residue in chitosan was studied and established. The established method was goodin calibration (r>0.99), precision (RSD1.6%-8.5%), sensentive (LOQ1.9-10.9ng·mL~(-1)) and accurate(recovery84.4-98.6). The method was also proved superior to Bradford method, LC-MS/MS method andelemental analysis.
Virus in chitosan was another safety related factor of chitosan but few related study reported. Theinactivation of virus in chitosan with γ irradition or NaOH-ethanol incubation was evaluated and studied,with Porcine parvovirus (PPV) and Bovine viral diarrhea virus (BVDV) as indicators. Irradation ofchitosan wiht γ ray for5kGy, after incubation with8mol·L~(-1)NaOH consisting10%ethanol at35oC for1hour was proved effective in inactivation of virus in chitosan（ΔlgTCID_(50)>4.0） and only inducedmarginal degradation of chitosan for8.4%.
Because the browning degradation of chitosan in acid hydrolysis, the recovery of chitoan inquantification was about90%in most reports, which was inadequate for quantification of chitosan inhemostasis application. The quantification of chitosan was studied by optimizing and evaluating thebrowning degradation of glucosamine and chitosan in hydrolysis with hydrochloric acid and phosphoricacid. Minimum brwoning degradation was proved with hydrochloric acid and phosphoric acid (4.5:1.5inmolar ratio) hydrolysis. Chitosan was quantified with hydrochloric acid and phosphoric acid (4.5:1.5inmolar ratio) hydrolysis and FMOC-Cl derivation HPLC determination. The method was linear at20-300μg·mL-（1r>0.99）and with good precision. The accuracy (recovery97.7%) was significantly superiorto the traditional HCl hydrolysis (recovery85.1%) and can be used for the quality control of chitosan forhemostatic usage.
As lack proper standard of chitosan for hemostasis usage, the main quality related factors of chitosanwere evaluated, and the standard of chitosan was then proposed to ensure the quality control of chitosan forhemostasis usage, with the consult of current chitosan standard and the general requirements of implantmaterials.
The present work studied on the deproteinization and purification of chitosan, which was significantlysuperior to NaOH deproteinization. Studied and established a sensitive and accurate pre-column derivationHPLC method for the determination of trace protein residue in chitosan. Studied on the HCl-H3PO4hydrolysis coupled pre-column derivation HPLC method for the quantification of chitosan, this improvedthe recovery of the method. These were meaninful to the total quality control of chitosan.
引文
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