【作者】 徐超；

【导师】 樊明涛； 胡新中；

【作者基本信息】 西北农林科技大学 ， 食品科学， 2013， 博士

【摘要】 燕麦β-葡聚糖具有降低心血管疾病风险的功能特性，但对燕麦β-葡聚糖的药代动力学特性和抗疲劳功能特性尚不清楚。本试验从定莜四号裸燕麦麸皮中制备燕麦β-葡聚糖，对其纯度、分子量大小、结构进行了鉴定；利用异硫氰酸荧光素标记β-葡聚糖，探索其在大鼠体内代谢、组织分布和排泄状况；利用Trolox当量的方法评价燕麦β-葡聚糖（OG）和荧光标记燕麦β-葡聚糖（OG-FITC）在体外的抗氧化能力；利用大鼠的耐力训练动物模型，评价燕麦β-葡聚糖的抗疲劳作用及对运动疲劳的防护机制。本研究取得以下主要结果：（1）燕麦麸皮利用碱提酸沉、酶法去杂和去离子水透析相结合，经冷冻干燥制备的燕麦β-葡聚糖样品的得率为8.22%，提取率为77.40%，纯度为92.58%，重均分子量为1.92×105Da。红外吸收光谱（Fourier Translation Infrared spectroscopy，FTIR）分析表明所得样品具有燕麦β-葡聚糖的特有结构。（2）利用异硫氰酸荧光素（FITC）与燕麦β-葡聚糖（OG）分子之间的共价偶联，成功地得到了含有荧光因子FITC的荧光标记燕麦β-葡聚糖（OG-FITC）。OG-FITC为橙红色粉末状固体，与OG具有相同的溶解性； OG-FITC中FITC的取代度为0.68%；OG和OG-FITC的重均分子量（Mw）分别为192.2kDa和192.4kDa，表明荧光标记物FITC对OG的分子量影响很小。（3）燕麦β-葡聚糖的药代动力学分析表明：大鼠单次灌胃给予300mg/kg OG-FITC，血药浓度—时间曲线数据采用非房室模型计算药代动力学参数，揭示燕麦β-葡聚糖在大鼠体内呈线性动力学特征。燕麦β-葡聚糖在大鼠体内吸收代谢较慢，半衰期较长，生物利用度相对较高。排泄试验表明，燕麦β-葡聚糖在体内代谢吸收时间主要集中在4-10h，且在10h左右排泄速率达到最高，燕麦β-葡聚糖主要以粪便的形式代谢排出体外。（4）燕麦β-葡聚糖组织分布试验表明：大鼠单次灌胃给予300mg/kg OG-FITC，灌胃后2h，OG-FITC能分布到所有主要组织中，在胃和肠道中分布最高，在心脏和肝脏组织中也有一定分布；给药后6h，胃和肠道组织中的浓度下降最明显，而在大脑、脾和肌肉中浓度明显上升，表明燕麦葡聚糖在这个阶段主要参与了脾脏和骨骼肌组织的吸收代谢；灌胃后24h，大部分组织中燕麦β-葡聚糖的浓度均明显降低，附睾脂肪中的β-葡聚糖降低不大，可能与脂肪在正常的机体生理活动中较少的参与新陈代谢有关。脑组织中能够检测到大量燕麦β-葡聚糖的存在，表明燕麦β-葡聚糖能够穿过血脑屏障，参与大脑的神经调控。（5）燕麦β-葡聚糖的耐力运动评价结果表明：与对照组（NC）相比，燕麦β-葡聚糖组（OG）可以极显著增加大鼠的力竭跑步时间，降低血清尿素氮水平、血清肌酸激酶活力（P＜0.01）；显著增加肝脏糖原含量、血清游离脂肪酸含量和乳酸脱氢酶活力，降低血清尿素氮水平（P＜0.05）。提示燕麦β-葡聚糖具有显著的抗疲劳特性，与中等强度训练相结合能够更好的增加机体对运动强度的适应性，提高机体耐力，延缓疲劳的发生。（6）燕麦β-葡聚糖对运动大鼠体内氧化应激的研究表明：与NC组大鼠相比，燕麦β-葡聚糖显著提高OG组大鼠血清过氧化氢酶活力的作用（P＜0.05），极显著提高OG组大鼠骨骼肌组织匀浆的SOD酶活力（P＜0.01）；显著降低OG组大鼠血清羟自由基的含量（P＜0.05），极显著降低OG组大鼠骨骼肌组织匀浆的MDA含量（P＜0.01）。燕麦β-葡聚糖具有降低大鼠运动引起的氧化应激程度，减少肝脏和肌肉组织的细胞损伤的作用；与运动训练相结合能更好的增强大鼠机体的抗氧化体系，维持机体的氧化—抗氧化体系的平衡。（7）燕麦β-葡聚糖及其荧光标记物的体外抗氧化结果得出：DPPH自由基清除能力分别为33.07μmol TE/g和35.55μmol TE/g，清除羟自由基的能力分别为52.95μmolTE/g和46.45μmol TE/g，抗氧化能力指数分别为35.53μmol TE/g和34.07μmol TE/g。表明燕麦β-葡聚糖及其荧光标记物具有较强的自由基清除能力，两者在自由基清除能力方面不存在显著差异（P>0.05），表明荧光标记对其体外抗氧化特性影响较小。（8）综上研究表明，燕麦β-葡聚糖的荧光标记物具有稳定、方便快捷、准确检验生物样本的特性，药代动力学结果揭示其在肝脏、肌肉和脂肪中均有存在；抗疲劳研究提示其可通过直接增加血清游离脂肪酸、肝糖原和肌糖原，参与体内的能量代谢，间接调节乳酸脱氢酶的提高和肌酸激酶的降低，减少乳酸和尿素氮等代谢产物的累积或加速消除；其抗氧化作用能够增加机体抗氧化酶活力的提高和减少脂质过氧化物的产生，提高了机体对氧化应激的适应程度。表明燕麦β-葡聚糖可以直接参与能量代谢和提高机体的抗氧化能力，从而具有提高机体运动耐力，延缓疲劳发生的作用。更多还原

【Abstract】 Oat β-glucan has the feature of reducing the risk of cardiovascular disease, however, it isnot clear about its metabolic and anti-fatigue effects. In this study, oat β-glucan was isolatedfrom oat bran, which is made from “Ding you si” naked oat. Its purity, molecular weight andstructure were identified. By using fluorescein isothiocyanate labeled β-glucan, itsmetabolism, tissue distribution and excretion in rats were investigated. Also, in vitro anti-oxidant abilities of oat β-glucan（OG）and fluorescent labeling of oat β-glucan（OG-FITC）were evaluated using Trolox equivalent method. Moreover, endurance training model in ratwas used to estimate anti-fatigue effect and the protective mechanism of oat β-glucan. Themain results of this study were obtained as follows:（1）Oat β–glucan was isolated from inactivated enzyme oat bran, the sample was madeby the method of alkali dissolution and aci deposition, purified by adding different enzymeand dialysis by deionized water, then freeze dried. The yield of oat β-glucan was8.22%.Extraction rate was77.40%, and the purity was92.58%. The average molecular weight was1.92×105Da. IR analysis showed that the samples had the characteristic structure of oatβ-glucan.（2）Oat β-glucan （OG） has been successfully labeled with fluorescein isothiocyanateby the covalent coupling between fluorescein isothiocyanate （FITC） and oat β-glucan.OG-FITC was orange red powder, and had the same solubility as OG； substitution degree ofFITC in OG-FITC was0.68%. The average molecular weight （Mw） weight of OG andOG-FITC were192.2kDa and192.4kDa, respectively, suggesting that fluorescence markerFITC had little effect on the molecular weight of OG.（3）Study on pharmacokinetics of oat β-glucan showed that: After rats were orallyadministered with300mg/mg OG-FITC, plasma concentration-time data was used to do thecalculation of pharmacokinetic parameters by using a compartmental model. Results showedthat oat β-glucan in rats were metabolized slowly, and had longer absorption half-life andhigher bioavailability. Oat β-glucan was mainly metabolized in the4-10h, and the excretionrate reached the highest around10h. Oat β-glucan was mainly excreted from feces after oraladministration． （4）Results on tissue distribution indicated that: OG-FITC was widely distributed totissues of most organs in rats2h after intravenous administration of300mg/kgOG-FITC．The concentration in stomach and intestine plasma was the most highest in alltissues, and also was abundant in liver and heart．The concentration in stomach and intestinedegraded rapidly after6h，and increased rapidly in brain, spleen, and skeletal muscle,suggesting that oat β-glucan was involved mainly in the spleen and skeletal musclemetabolism. For most tissues，oat β-glucan concentrations had significantly decreased in24hafter oral administration, while the oat β-glucan concentration in fat was not decreasedsignificantly. This may be associated with the fact that fat was less participated in metabolism.β-Glucan was also be detected in brain，which means β-glucan could cross the blood brainbarrier and participate in the brain’s neural regulation.（5）Evaluation on anti-fatigue effect of oat β-glucan suggested that: Compared withcontrol group （NC）, oat β-glucan group （OG） could increase the exhaustive running time,and reduce serum urea nitrogen and serum creatine kinase of rats significantly （P <0.01）.Also, it could increase the liver glycogen contents, serum free fatty acid content and lactatedehydrogenase enzyme activity, and reduce serum urea nitrogen levels significantly （P <0.05）. Results showed that oat β-glucan has significant anti-fatigue properties, by combinedwith oat β-glucan and moderate intensity training, it could improve the body’s endurance,delay fatigue.（6）Research on antioxidantive of oat β-glucan in vitro and in vivo showed that: Oatβ-glucan has strong antioxidant properties in vitro, which can effectively scaveng DPPH,Hydroxyl free radical, and owned higher oxygen radical absorbing capacities compared withTrolox. The results also showed that the antioxidant properties of β-glucan after fluorescentlabeling treatment did not change significantly. Results in vivo indicated that Oat β-glucancould reduce exercise-induced oxidative stress levels, and decrease cell injury of liver andmuscle tissue in rat model. Compared with the NC group, oat β-glucan treatment （OG）could enhance serum catalase activity significantly （P <0.05）, increase SOD activity ofskeletal muscle homogenates significantly （P <0.01）； and reduce serum hydroxyl radicalcontent （P <0.05） and MDA content in skeletal muscle homogenates significantly （P <0.01）. Oat β-glucan combined with exercise training could reduce exercise-induced oxidativestress levels and decrease cell injury of liver and muscle tissue much better than β-glucanalone in rats.（7）All in all, OG-FITC has excellent properties on stability, effective and exactlyinspection in biology sample test. First, study on pharmacokinetics showed that oat β-glucancan distribute in liver, skeletal muscle and epididymis fat. Second, research on anti-fatigue indicated that oat β-glucan can directly supply energy by improving the content of glycogenin liver and skeletal muscle and serum free fatty acid, and indirecty reduce/remove harmfulmetabolites such as decrease the lactid acid in serum by improve the activity of lacticdehydrogenase and reduce the content of urea nitrogen in serum. Third, the antioxidant of oatβ-glucan can improve adaptment of body on exercise induced oxidative stress, by increase theactivity of antioxidant enzyme and decrease the production of lipid peroxide. In conclusion,oat β-glucan could significantly enhance exercise capacity and prolong occurance ofexercise-induced-fatigue by directly engage in energetic metabolism and improve antioxidantcapacity of rats.更多还原