【作者】 赵春艳；

【导师】 敖长金；

【作者基本信息】 内蒙古农业大学 ， 动物营养与饲料科学， 2008， 博士

【摘要】 本文通过分离纯化沙葱中的黄酮类化合物,以昆明种小白鼠为动物模型,研究饲喂沙葱黄酮对机体免疫及抗氧化能力的影响。试验共分三个部分:第一部分试验确定沙葱黄酮最佳提取纯化条件,应用75%乙醇提取沙葱中黄酮类化合物,提取温度为70℃、提取时间为2h/2次。应用聚酰胺层析柱分离纯化,用30%、50%、70%和95%的乙醇溶液洗脱,用液相色谱-电喷雾离子化-质谱串联技术(LC-ESI-MS-MS)鉴定沙葱黄酮的结构,结果如下:30%乙醇洗脱出8种沙葱黄酮类化合物,分别是①山奈素-4′-O-3,7-O-O-二葡萄糖苷(m/z609)、②槲皮素-7-O-3-O-芸香糖苷(m/z609)、③山奈素-4′-O-7-O-葡萄糖-3-O-二葡萄糖苷(m/z771)、④7-O-5, 4′-二甲氧基-3′-羟基黄酮醇(m/z329)、⑤3′,4′-环氧基-7-O-5-甲氧基黄酮醇(m/z327)、⑥槲皮素-7-O-3-O-二葡萄糖苷(m/z625)、⑦槲皮素-7-O-3-O-葡萄糖苷(m/z463)、⑧山奈素-7-O-3-O-葡萄糖苷(m/z447);50%乙醇洗脱出7种沙葱黄酮类化合物,分别是①木犀草素-5′-O-葡萄糖-4-羟基苯丙酸(m/z593)、②槲皮素-7-O-3-O-芸香糖苷(m/z609)、③山奈素-4′-O-3,7-O-O-二葡萄糖苷(m/z609)、④7-O-5, 4′-二甲氧基-3′-羟基黄酮醇(m/z329)、⑤3′,4′-环氧基-7-O-5-甲氧基黄酮醇(m/z327)、⑥槲皮素-7-O-3-O-葡萄糖苷(m/z463);⑦山奈素-7-O-3-O-葡萄糖苷(m/z447);70%乙醇洗脱出3种沙葱黄酮类化合物,分别是①7-O-5, 4′-二甲氧基-3′-羟基黄酮醇(m/z329)、②3′,4′,3,4-四羟基-6-O-类黄酮(m/z 273)、③3′,4′-环氧基-7-O-5-甲氧基黄酮醇(m/z327);95%乙醇洗脱出2种沙葱黄酮类化合物,分别是①3′,4′-环氧基-7-O-5-甲氧基黄酮醇(m/z327)、②7-O-5, 4′-二甲氧基-3′-羟基黄酮醇(m/z329)。第二部分试验应用纯昆明种小白鼠480只(体重:20±2g),随机分为4组,每组120只(雌雄各半),分笼饲养。第1组为对照组,第2组为添加75.36mg/kg·BW·d沙葱组,第3组为添加75.36mg/kg·BW·d75%乙醇提取的沙葱总黄酮粗提物组,根据预试验的结果第4组为添加75.36mg/kg·BW·d30%乙醇洗脱纯化后沙葱黄酮添加组,预饲期5d,正式试验期14d,正式试验期内2次宰杀采血并分离血清,剖离肝脏、胸腺和脾脏称重。试验结束时,沙葱组和沙葱黄酮纯化组的血清T-SOD、GSH-Px含量显著高于对照组和沙葱黄酮粗提物组(P<0.05),沙葱黄酮纯化组和沙葱黄酮粗提物组的血清CAT含量显著高于对照组(P<0.05),试验各组的血清MDA含量低于对照组,但差异不显著(P>0.05),试验组血清T-AOC含量显著高于对照组(P<0.05)。试验组肝脏T-SOD含量显著高于对照组(P<0.05),试验组之间差异不显著(P>0.05),沙葱黄酮纯化组的肝脏GSH-Px含量显著高于其它各组(P<0.05),对照组、沙葱组和沙葱黄酮纯化组的肝脏CAT含量显著高于沙葱黄酮粗提物组(P<0.05),沙葱组和沙葱黄酮纯化组肝脏MDA含量显著低于对照组和沙葱黄酮粗提物组(P<0.05),沙葱黄酮纯化组肝脏T-AOC含量高于其它各组,但差异不显著(P>0.05)。试验结果显示:沙葱黄酮可提高血清和肝脏中抗氧化酶类的含量和活性,降低体内脂质过氧化产物的含量,从而提高机体的抗氧化能力。第三部分试验设计同第二部分,试验结束时,试验各组的脾脏指数高于对照组,但差异不显著(P>0.05),沙葱黄酮纯化组胸腺指数显著高于其它各组(P<0.05);沙葱黄酮纯化组的血清ACP含量显著高于其它各组(P<0.05),试验各组肝脏ACP含量显著高于对照组(P<0.05);试验各组碳廓清指数高于对照组,但差异不显著(P>0.05),沙葱黄酮纯化组的吞噬指数、血清溶菌酶含量显著高于其它各组(P<0.05);沙葱黄酮纯化组血清lgG含量显著高于对照组(P<0.05);沙葱黄酮纯化组CD19~+细胞数量高于其它各组,但差异不显著(P>0.05);沙葱黄酮纯化组CD4~+细胞数量显著高于对照组和沙葱组(P<0.05),对照组CD8~+细胞数量高于其它各组(P>0.05);试验组血清NO含量均高于对照组,其中沙葱黄酮粗提物组显著高于对照组和沙葱黄酮纯化组(P<0.05),高于沙葱组但差异不显著(P>0.05)。试验结果显示:沙葱黄酮纯化组能提高免疫器官指数、血液和肝脏的ACP和溶菌酶的含量,从而增强吞噬细胞消化和降解异物的能力,并且沙葱黄酮纯化组能显著提高小鼠的碳廓清指数(κ)和吞噬系数(α),因此沙葱黄酮在增强机体非特异性免疫机能方面发挥着重要作用;沙葱黄酮纯化组还可提高小鼠外周血中IgG的含量和CD19~+细胞(B细胞)百分率,表明沙葱黄酮通过增加机体抗体形成细胞的数量或增强抗体形成细胞的功能,而提高机体体液免疫功能;通过提高外周血中CD4~+细胞和CD8~+细胞数量而增强机体的细胞免疫机能。沙葱黄酮还可通过提高血液中生物信息传递分子――NO的含量,活化巨噬细胞发挥非特异性杀菌和杀肿瘤作用,在T淋巴细胞和B淋巴细胞的参与下进行复杂的免疫活动。更多还原

【Abstract】 Is this paper, Kunming mice were used to study the effect of flavonoids separated and purified from Allium mongolium Regel on immity and antioxidant function in body. The experiment was divided into three parts:The first part of the experiment determined the best extracting and purifying conditions of the flavonoids from Allium mongolium Regel,75% ethanol c used to extract flavonoids in Allium mongolium Regel,extraction temperature was 70℃,extraction time was 2 hours/2 times. Polyamide column chromatography was used to separate and purify flavonoids,using 30%,50%,70% and 95%ethanol washed and then the structure of flavonoids from Allium Mongolium Regel was identified by liquid chromatography -ESI-tandem mass spectrometry technology (LC-ESI-MS-MS).The results showd that:eight kinds of flavonoids from Allium mongolium Regel were separated by 30% ethanol They are,kaempferol-4′-O-3,7-O-O-2glucosidase(m/z609), quercetin-7- O- 3-O -rutaceaeglucoside(m/z609),kaempferol-4’-O-7-O-glucose-3-O-2glucosidase(m/z771),7-O-5,4′-dimethoxy-3’-hydroxyflavonol(m/z329),3′,4′-epoxy-7-O-5-methoxy-flavonol(m/z327),quercetin-7-O-3-O-2glucosidase(m/z625),quercetin-7-O-3-O-glucoside(m/z463) and kaempferol-7-O-3-O-glucoside(m/z447) respectively; seven kinds of flavonoids from Allium mongolium Regel were separated by 50% ethanol they are, Luteolin -5’-O-glucose-4-hydroxy phenylpropionate(m/z593),quercetin-7-O-3-O-rutaceae glucosi -de(m/z609),kaempferol-4’-O-3,7-O-O-2glucosidase(m/z609),7-O-5,4′-dimethoxy-3’-hydroxyflavonol(m/z329),3′,4′-epoxy-7-O-5-methoxy-flavonol(m/z327),quercetin-7-O-3-O-glucoside(m/z463) and kaempferol-7-O-3-O-glucoside(m/z447) respectively; three kinds of flavonoids from Allium mongolium Regel were separated by 70% ethanol they are,7-O-5,4′-dimethoxy-3’-hydroxy flavonol(m/z329),3’,4’,3,4- 4hydroxy-6-O–flavonoid (m/z273) and 3′,4′- epoxy -7- O- 5- methoxy-flavonol(m/z327) respectively; two kinds of flavonoids from Allium mongolium Regel were separated by 95% ethanol they are,3′,4′-epoxy-7-O-5- methoxy-flavonol(m/z327) and 7-O-5, 4′-dimethoxy -3’- hydroxyl flavonol(m/z329) respectively.In the second part of the experiment,480 (weight 20±2g) pure-blooded Kongming mice, which were divided randomly into four groups and each group 120 (half female and half male), were raised in different cages which were named control group, 75.36mg/kg·BW·d Allium mongolium Regel supplementation group,75.36mg/kg·BW·d flavonoid extract supplementation group,75.36mg/kg·BW·d flavonoids purification supplementation group. Preparatory experimental period lasted for 5 days and the formal experimental period lasted for 14 days. After two slaughters being done during the formal experimental period, two slaughter experiment were conducted,the blood was collected and serum was isolated ,and live thymus and spleen was dissected and weighed.The result show that, the content of T-SOD and GSH-Px in serum to Allium mongolium Regel group and flavonoids purification group was higher than that of control group and flavonoid extract group(P<0.05). The content of CAT in serum of flavonoids purification group and flavonoid extract group was higher than that of control group(P<0.05).The content of MDA in serum of experiment groups was lower than that of control group, but the difference was not significant(P>0.05).The content of T-AOC in serum of experiment groups was higher than that of control group(P<0.05).The content of T-SOD in liver of experiment groups was higher than that of control group(P<0.05), but the difference was not significant in experiment groups(P>0.05).The content of GSH-Px in liver of flavonoids purification group was higher than that of other groups(P<0.05).As compared to the control group, The CAT content in liver of flavonoid extract supplementation group was significant lower(P<0.05).The content of MDA in liver of Allium mongolium Regel group and flavonoids purification group were lower than that of control group and flavonoid extract group(P<0.05).The content of T-AOC in liver of flavonoids purification group was higher than that of other groups, but the difference didn′t reach statistical significant(P>0.05).From the results we conduded that flavonoid of Allium mongolium Regel can enhance the content and activity of antioxidant enzymes in serum and liver and reduce the content of lipid peroxidation in vivo, thereby enhance the body’s antioxidant capacity.The design of the third part of experimental was the same as the second part. The results showed, the spleen index of experiment groups were higher than that of control group, but the difference was not significant (P>0.05).The thymus index of flavonoids purification group was higher than that of other groups(P<0.05).The content of ACP in serum of flavonoids purification group was higher than that of other groups(P<0.05); The carbon clearance index of experiment groups was higher than that of control group, but the difference was not significant (P>0.05).The phagocytic index and lysozyme content in serum of flavonoids purification group were higher than other groups(P< 0.05),the content of lgG in serum to flavonoids purification group was higher than that of control group(P<0.05).The number of CD19~+ of flavonoids purification group was higher than that of other groups, but the difference was not significant (P>0.05).The number of CD4~+ of flavonoids purification group was higher than that of control group and Allium mongolium Regel group(P<0.05).The number of CD8~+ of control group was higher than that of other groups(P>0.05).The content of NO in serum of experiment groups was higher than that of control group, the content of NO in serum of flavonoid extract group was higher than that of control group(P<0.05), flavonoids purification group(P<0.05) and Allium mongolium Regel group(P>0.05).The results showed that flavonoids purification supplementation group can enhance immune organs index, the content of ACP and lysozyme in blood and liver, thereby enhance the capability of phagocytes to digest and degradate of foreign body. Meanwhile, carbon dissection index(κ) and engulfed coefficient(α) of mice in flavonoids purification group was significantly increased,so flavonoid of Allium mongolium Regel played an important role in enhancing the body non-specific immune function.Flavonoid of Allium mongolium Regel can improve the content of IgG and the percentage of CD19~+ (B cell) in the peripheral blood of mice. The results indicated that flavonoid of Allium mongolium Regel stronghten the immune function of body fluid; Through increasing the number of antibody in the body formatting cell and enhancing the function antibody formation of cell.Through increasing the cell numbers of CD4~+ and CD8~+ in blood, flavonoid of Allium mongolium Regel enhanced humoral immune function. Flavonoid of Allium mongolium Regel activated macrophages by creasing the content of NO,which was biological information transmission and play the non-specific sterilization and killing tumor function of macrophages and carry through complex immunization activities with participation with T lymphocytes and B lymphocyte.更多还原