微波固态合成低聚糖及其对小鼠抗氧化和免疫活性研究
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摘要
微波固态合成低聚糖以单糖或双糖为反应物,其反应速度快,合成产率高且清洁无污染。本研究以甘露糖和葡萄糖为反应物,在微波加热条件下固态合成低聚糖混合物,探索了合适的反应条件,对合成产物进行了初步的分离,并对合成初产物进行成分分析,研究其抗氧化和免疫活性。
优化实验表明利用微波固态合成葡低聚糖的最适反应条件为:微波功率1000瓦,微波处理时间9 min,引发剂的添加量30%,催化剂的添加量为4%。合成产率为76.56%,高效液相色谱(HPLC)分析表明:初产物葡寡糖中二糖占20.81%,三糖占15.50%,四糖占10.84%,五糖占7.79%,六糖及其以上占21.63%。利用微波固态合成甘露低聚糖的最适反应条件为:微波功率1000瓦,微波处理时间4min,引发剂的添加量15%,催化剂的添加量为3%,合成产率为86.50%。高效液相色谱(HPLC)分析表明:初产物甘露低聚糖各成分组成如下:单糖占13.50%,二糖占3.82%,三糖占7.56%,四糖占6.84%,五糖占4.77%,六糖占5.54%,七糖及其以上占57.97%。
微波固态合成的甘露低聚糖体外可有效清除羟自由基(?OH)且IC50 =4mg/mL。动物实验表明,高脂模型与衰老模型小鼠体内氧化应激增加,经甘露低聚糖饲喂之后,结果证实,与模型对照组比,甘露低聚糖日粮组小鼠的血浆、肝脏、胰腺、小肠、十二指肠、大肠可较对照组(高脂日粮组)显著提高羟自由基(NO-)的清除率,显著增强超氧化物岐化酶(SOD)、过氧化氢酶(CAT)、Na+-K+-ATP、谷胱甘肽过氧化物酶(GSH-Px)的活性和谷胱甘肽(GSH)的含量(P<0.05),提高小鼠总抗氧化能力(T-AOC)及减少脂质过氧化产物丙二醛(MDA)的含量(P<0.05)。故合成的甘露低聚糖可有效清除小鼠体内自由基、增强抗氧化酶活性,从而增强小鼠的抗氧化水平。
另外,动物衰老造模后,组织各项免疫指标显著下降(P<0.05)。与衰老对照组相比,甘露低聚糖日粮饲喂小鼠,显著提高衰老小鼠的肝脏、肾脏、胸腺和脾脏指数及血清中IgG、IgA、IgM水平(P<0.05);同时,饲喂甘露低聚糖日粮的衰老模型小鼠在ConA刺激下胸腺T细胞的增殖指数和脾脏淋巴细胞转化率也较衰老对照组显著提高(P<0.05)。故合成甘露低聚糖日粮可提高衰老模型小鼠的体液免疫和细胞免疫水平。
Microwave-assisted solid-state synthesis of oligosaccharides offers us an ideal resolution with the advantages of rapidity, high yield and less environmental pollution. In the present work, we optimized the reaction conditions of microwave-assisted solid-phase synthesis of oligosaccharides (glucooligosaccharides (GOS) and mannooligosaccharides (MOS)). And the effects of MOS on the scavenging rate of free radicals, the antioxidant enzymes and immunity activities in mice were investigated.
Optimal experiment show that the optimal condition of microwave-assisted solid-phase synthesis of GOS as follows: 1000 w of microwave power, 9 min of microwave irradiation time, 30% water as initiator, 4% phosphoric acid as the catalyst. The yield of product was 76.56%. The product was characterized by HPLC. The components of synthetic product was determined as follows: 20.81% disaccharides, 15.50% trisaccharides, 10.84% tetrsaccharides, 7.79% pentsaccharides, 21.63% oligosaccharides with higher molecule weight beyond hexsaccharides. The optimal condition of microwave-assisted solid-phase synthesis of MOS as follows: 1000 w of microwave power, 4 min of microwave irradiation time, 15% water as initiator, 3% phosphoric acid as the catalyst. The yield of product was 86.50%. The product was characterized by HPLC. The components of synthetic product was determined as follows: 13.50% mannosaccharides, 3.82% disaccharides, 7.56% trisaccharides, 6.84% tetrsaccharides, 4.77% pentsaccharides, 5.54% hexsaccharides, 86.50% with higher molecule weight beyond heptasaccharide.
The results indicate that MOS could efficiently scavenge the hydroxyl radical in vitro with the IC50 of 4mg/ml. The animal experiment show that high-fat diet or d-gal administration can significantly increased oxidative stress (P<0.05) in untreated model mice compared to the normal control mice. The MOS diet administration can significantly enhance the level of glutathione (GSH), the activities of superoxide dismutase (SOD), catalase (CAT), Na+-K+-ATP and glutathione peroxidase (GSH-Px) and reduce NO- and MDA levels (P<0.05) in plasma, liver, small intestine, large intestine, stomach and pancreas of high-fat mice compared to those of the untrated model control mice (P<0.05).
The experiment was also designed to investigate the effect of MOS on immune activities of mice. The results show that some immune indexs in aged mice were significantly decreased (P>0.05) compared to the young control. The MOS diet administration can significantly increased the weight of thymus, spleen, liver and kidney and the levels of serum lgG, LgA and lgM (P<0.05). Moreover, all mice feeded with MOS diet show a significant increase in the stimulation index (SI) (with/without ConA) of thymus and spleen (P<0.05). It is therefore believed that the MOS diet can enhance the activity of antioxidant enzymes and improve immunity activity in mice.
优化实验表明利用微波固态合成葡低聚糖的最适反应条件为:微波功率1000瓦,微波处理时间9 min,引发剂的添加量30%,催化剂的添加量为4%。合成产率为76.56%,高效液相色谱(HPLC)分析表明:初产物葡寡糖中二糖占20.81%,三糖占15.50%,四糖占10.84%,五糖占7.79%,六糖及其以上占21.63%。利用微波固态合成甘露低聚糖的最适反应条件为:微波功率1000瓦,微波处理时间4min,引发剂的添加量15%,催化剂的添加量为3%,合成产率为86.50%。高效液相色谱(HPLC)分析表明:初产物甘露低聚糖各成分组成如下:单糖占13.50%,二糖占3.82%,三糖占7.56%,四糖占6.84%,五糖占4.77%,六糖占5.54%,七糖及其以上占57.97%。
微波固态合成的甘露低聚糖体外可有效清除羟自由基(?OH)且IC50 =4mg/mL。动物实验表明,高脂模型与衰老模型小鼠体内氧化应激增加,经甘露低聚糖饲喂之后,结果证实,与模型对照组比,甘露低聚糖日粮组小鼠的血浆、肝脏、胰腺、小肠、十二指肠、大肠可较对照组(高脂日粮组)显著提高羟自由基(NO-)的清除率,显著增强超氧化物岐化酶(SOD)、过氧化氢酶(CAT)、Na+-K+-ATP、谷胱甘肽过氧化物酶(GSH-Px)的活性和谷胱甘肽(GSH)的含量(P<0.05),提高小鼠总抗氧化能力(T-AOC)及减少脂质过氧化产物丙二醛(MDA)的含量(P<0.05)。故合成的甘露低聚糖可有效清除小鼠体内自由基、增强抗氧化酶活性,从而增强小鼠的抗氧化水平。
另外,动物衰老造模后,组织各项免疫指标显著下降(P<0.05)。与衰老对照组相比,甘露低聚糖日粮饲喂小鼠,显著提高衰老小鼠的肝脏、肾脏、胸腺和脾脏指数及血清中IgG、IgA、IgM水平(P<0.05);同时,饲喂甘露低聚糖日粮的衰老模型小鼠在ConA刺激下胸腺T细胞的增殖指数和脾脏淋巴细胞转化率也较衰老对照组显著提高(P<0.05)。故合成甘露低聚糖日粮可提高衰老模型小鼠的体液免疫和细胞免疫水平。
Microwave-assisted solid-state synthesis of oligosaccharides offers us an ideal resolution with the advantages of rapidity, high yield and less environmental pollution. In the present work, we optimized the reaction conditions of microwave-assisted solid-phase synthesis of oligosaccharides (glucooligosaccharides (GOS) and mannooligosaccharides (MOS)). And the effects of MOS on the scavenging rate of free radicals, the antioxidant enzymes and immunity activities in mice were investigated.
Optimal experiment show that the optimal condition of microwave-assisted solid-phase synthesis of GOS as follows: 1000 w of microwave power, 9 min of microwave irradiation time, 30% water as initiator, 4% phosphoric acid as the catalyst. The yield of product was 76.56%. The product was characterized by HPLC. The components of synthetic product was determined as follows: 20.81% disaccharides, 15.50% trisaccharides, 10.84% tetrsaccharides, 7.79% pentsaccharides, 21.63% oligosaccharides with higher molecule weight beyond hexsaccharides. The optimal condition of microwave-assisted solid-phase synthesis of MOS as follows: 1000 w of microwave power, 4 min of microwave irradiation time, 15% water as initiator, 3% phosphoric acid as the catalyst. The yield of product was 86.50%. The product was characterized by HPLC. The components of synthetic product was determined as follows: 13.50% mannosaccharides, 3.82% disaccharides, 7.56% trisaccharides, 6.84% tetrsaccharides, 4.77% pentsaccharides, 5.54% hexsaccharides, 86.50% with higher molecule weight beyond heptasaccharide.
The results indicate that MOS could efficiently scavenge the hydroxyl radical in vitro with the IC50 of 4mg/ml. The animal experiment show that high-fat diet or d-gal administration can significantly increased oxidative stress (P<0.05) in untreated model mice compared to the normal control mice. The MOS diet administration can significantly enhance the level of glutathione (GSH), the activities of superoxide dismutase (SOD), catalase (CAT), Na+-K+-ATP and glutathione peroxidase (GSH-Px) and reduce NO- and MDA levels (P<0.05) in plasma, liver, small intestine, large intestine, stomach and pancreas of high-fat mice compared to those of the untrated model control mice (P<0.05).
The experiment was also designed to investigate the effect of MOS on immune activities of mice. The results show that some immune indexs in aged mice were significantly decreased (P>0.05) compared to the young control. The MOS diet administration can significantly increased the weight of thymus, spleen, liver and kidney and the levels of serum lgG, LgA and lgM (P<0.05). Moreover, all mice feeded with MOS diet show a significant increase in the stimulation index (SI) (with/without ConA) of thymus and spleen (P<0.05). It is therefore believed that the MOS diet can enhance the activity of antioxidant enzymes and improve immunity activity in mice.
引文
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