【作者】 王建辉；

【导师】 贺建华；

【作者基本信息】 湖南农业大学 ， 饲料作物生产与应用， 2007， 博士

【摘要】 为了人类和动物健康，开发和使用无残留、无污染、无毒副作用的抗生素替代品和抗氧化剂显得日益重要。我国是植物资源大国，植物种类繁多，针对性地对某些植物进行抗氧化性及其抗生素替代的可行性研究具有很强的实用价值。杜仲作为我国重要中草药，因其茎叶富含绿原酸、桃叶珊瑚甙等药用成分；桐粕过95％乙醇回流提取，初步鉴定其富含多酚类物质，两者均是开发天然抗氧化剂的重要来源。本文通过动物饲养、屠宰试验和抗氧化动力学特征研究对杜仲提取物（EUOE）的抗氧化性及其抗生素替代作用进行了探讨；通过分离纯化、药物高通量筛选、体外抗氧化试验及小鼠高血脂造模试验，以体内外试验从生物整体或细胞水平对桐粕活性物质抗氧化性与降血脂作用进行了评价，主要试验结果如下：试验一杜仲提取物对猪生产性能影响研究旨以探索杜仲提取物（EUOE）对猪生长性能、胴体品质及肌肉氨基酸含量的影响，试验选用胎次相同、体重相近的42日龄杜×长×大三元杂交猪300头，平均初始重为9.96±0.05kg，随机分成5个处理，每个处理6个重复，每个重复10头猪。试验分成3个阶段，阶段Ⅰ：9.96-20 kg；阶段Ⅱ：20-60 kg；阶段Ⅲ：60-90 kg。试验日粮分别在基础日粮中添加杜仲提取物0.1500，2000，2500 mg／kg或黄霉素12mg／kg（阶段Ⅰ）或8mg/kg（阶段Ⅱ和Ⅲ）。试验结果表明在猪日粮中添加EUOE能一定程度地提高其生长性能，改善猪只免疫水平和抗腹泻能力，影响猪只血液生理生化指标，改善胴体质量和猪肉品质，具体结果如下：1500mg／kgEUOE显著降低阶段Ⅰ和Ⅱ日均腹泻评分（P＜0.05），显著提高了阶段Ⅱ猪群日均增重（ADG）（P＜0.05），降低了阶段Ⅱ和Ⅲ料重比（F／G）（P＜0.05）。EUOE（除2000mg/kg）和黄霉素组均能显著提高试验全期ADG（P＜0.05），动物日均采食量存在增加趋势（0.05＜P＜0.10）。EUOE能显著降低血清尿素氮（SUN）水平（P＜0.05），显著提高阶段Ⅱ和Ⅲ血清球蛋白水平（P＜0.05）。EUOE1500，2000mg／kg的添加显著提高了猪肌内脂肪含量（P＜0.05），其不同添加水平间无显著差异（P＞0.05）；而黄霉素的添加显著降低了肌内脂肪含量（P＜0.05）。黄霉素和EUOE组均能不同程度地降低胴体皮厚（0.05＜P＜0.10），同时各日粮处理有降低滴水损失，增加眼肌面积、肉色评分和肌肉大理石纹评分的趋势（0.05＜P＜0.10），且EUOE处理组能显著提高猪肉肌肉总氨基酸和鲜味氨基酸含量（P＜0.05）。这表明EUOE能不同程度地改善猪胴体品质和肌肉质量，其中以1500mg／kg添加效果最佳。试验二EUOE对浓缩饲料氧化动力学影响研究通过EUOE对猪浓缩饲粮抗油脂过氧化试验研究发现，添加抗氧化剂可以显著降低饲料过氧化值（POV）。饲粮油脂水平越高，POV越高；饲粮油脂水平越高，需添加的抗氧化剂水平也越高；各种抗氧化剂对添加油脂的饲粮均能表现出良好的抗氧化活性，但对猪用浓缩饲料中油脂抗氧化活性仅在前21天表现良好效果，28天后基本无效；根据POV比较各抗氧化剂的抗氧化活性，乙氧基喹啉（I）最好，EUOE的抗氧化活性较低；不同抗氧化剂及其添加水平氧化动力学方程各有不同，其中不同EUOE添加水平及油脂添加水平各组氧化反应均以一级反应为主。试验三桐粕活性成分的提取、分离与结构鉴定综合利用NKA大孔树脂和聚酰胺层析分离，对桐粕95％乙醇提取物过NKA大孔树脂的50％乙醇洗脱部分继续追踪分离活性成分，经高效液相色谱确定化合物纯度后，结合其理化性质，并通过UV、¹H-NMR、¹³C-NMR、DEPT等光谱解析，证明晶体为双四氢呋喃类木脂素，化学名称为（±）3，3｀-bisdemethyl pinoresinol，即（±）3，3｀-双去甲基松脂醇，该物质在桐粕中第一次发现。试验四桐粕提取物抗肿瘤与降糖降脂活性筛选对桐粕提取物各组分进行高通量筛选发现，70％乙醇洗脱部分在浓度为50μl／ml和10μl／ml时，对28SC肿瘤细胞生长有抑制作用；水洗脱部分在浓度为10、5μl／ml，70％乙醇洗脱部分在浓度为5μl／ml时，对HL-60肿瘤细胞生长有抑制作用；晶体1和桐粕95％乙醇提取物经50％乙醇洗脱部分在浓度为50μg/ml时对SMMC-7721细胞有很强的抑制作用；桐粕95％乙醇提取物50％乙醇洗脱部分具有明显的抗糖尿病活性，在浓度为5μg／ml时对PPARa、PPARd、PPARγ，RXR受体的激活倍数分别为1.29，3.88，7.06和1.95。试验五桐粕95％乙醇提取物不同乙醇洗脱部分体外抗氧化试验研究本试验对桐粕95％乙醇提取物不同乙醇浓度梯度洗脱部分进行体外抗氧化试验，研究发现各洗脱部分均具有较强的还原能力和抗脂质过氧化功能。不同洗脱部分中总酚含量存在显著差异（P＜0.05），从50％乙醇洗脱部分的47.68 mg/g到水洗脱部分的34.51mg/g不等；对DPPH·的清除率各异，50％乙醇梯度洗脱部分高达90.37％；当浓度=0.8 mg/ml时，三种不同乙醇梯度洗脱部分的还原能力显著强于维生素C（0.2mg／ml）和BHT（0.2mg/ml）；50％乙醇洗脱部分对邻苯三酚自氧化的最大抑制率为96.67％，且在同浓度条件下与维生素C（0.20mg/ml）的抑制作用相当（P＞0.05）；相同浓度（0.50 mg／ml）条件下，50％乙醇洗脱部分对亚油酸过氧化的抑制效果与维生素C相当（P＞0.05）；当浓度为0.80 mg/ml时，50％乙醇洗脱部分对羟自由基的清除率最高达96.01％；随着洗脱部分浓度的增加，对H₂O₂诱导的红细胞氧化溶血的抑制作用增强，不同剂量水平下均以50％乙醇梯度洗脱部分最佳。这说明按照该试验工艺制备的桐粕提取物是一种优良的天然抗氧化剂和自由基清除剂。试验六桐粕提取物降脂保肝作用研究选取50只昆明SD小鼠按体重随机分为5个处理即空白对照组，高脂模型组和50％乙醇洗脱部分3个组（1000 mg/kg BW，500 mg／kg BW，250 mg／kg BW），每个处理10只小鼠，进行高血脂模型构建，3周造模成功后，除空白对照组给予普通饲料外，其他4组均给予高脂饲料，同时50％乙醇洗脱部分各组以1000 mg／kg BW，500 mg/kg BW，250 mg/kg BW剂量灌喂桐粕提取物95％乙醇提取物50％乙醇洗脱部分，空白和高脂对照组灌服等容量的生理盐水。试验研究发现，桐粕95％乙醇提取物50％乙醇洗脱部分能通过提高血中抗氧化防御功能、调节血脂水平、改变血液流变学参数等，发挥抗动脉硬化（AS）的活性。饲喂桐粕95％乙醇提取物经50％乙醇洗脱物后，高、中、低3个剂量组血清TC、TG、LDL-C含量、LDL-C／HDL-C和Apo-AI／Apo-B比值、冠心指数和动脉硬化指数均有显著降低（P＜0.05）；高、中、低3个剂量组血清和肝脏SOD、GSH-Px水平、TAC和羟自由基清除能力均显著升高（P＜0.05），MDA水平显著降低（P＜0.05）；高、中、低3个剂量组显著降低高脂血症小鼠的全血粘度、血浆粘度、红细胞压积、血沉、纤维蛋白原、红细胞刚性指数和聚集指数显著升高（P＜0.05），红细胞变形指数显著升高（P＜0.05）。更多还原

【Abstract】 In the last 20 years, active oxygen and free radicals have become the hot topicsof modern life science, and the appraisal and selection of natural resources with strongantioxidative activities have become the new trends of biology, medical and foodscience and technology. And with the passing epoch of antibiotics, the search ofeffective and harmless antibiotic substitute has become the basic point for thesustainable development of husbandry. Therefore, for the sake of the health of humanbeing and animals, the exploitation of antibiotic alternatives without remnant,pollution, toxic and side effects becomes increasingly important. Botanicals are thecritical resource of natural antioxidants and feed additives. China holds the title ofbotanic kingdom with various species, so it is obviously meaningful to research on thecertain uninvestigated or being little investigated herbals.Eucommia ulmoides Oliv, as an important Chinese herbal medicine, abound withiridoids such as aucubin, eucommiol, geniposide, geniposidic acid, phenols such aschlorogenic acid, lignans, coniferin, syringin, koaburaside, flavonoids and terpenes.Tong-seed meal extract extracted with 95% ethanol was reported abundant withpolyphenols, so both are important sources for the development of natural feedadditives and antioxidants. Though animal feeding and slaughter tests and oxidationkinetic characteristictest of pig concentrate, the substitution role of EUOE asantioxidants and antibiotics was investigated and the antioxidation and hypolipidemiceffects of the Tong-seed meal extract were appraised at whole organism level and celllevel in vitro and in vivo according to the high throughput screening. The activesubstance was extracted from the Tongmeal extract, and its main active componentswere further isolated and purified and their basic structures were identified. The mainexperimental results were as follows:（1） To investigate the effect of Eucommia ulmoicles Oliv Extract （EUOE） ongrowth performance, carcass characteristics, meat qualities and muscular amino acidcontents, a feeding trial was conducted with three hundred 42-d Durocx （LandracexYorkshire） crossbred piglets with average weight 9.96±0.05 kg. They were randomlyassigned to five treatment groups （six replicates per group, ten heads per replicate）.The trial was spit into three phases: PhaseⅠ（9.96-20kg BW）; PhaseⅡ（20-60kgBW） and PhaseⅢ（60-90kg BW）. The piglets were fed on the basal dietsupplemented with graded EUOE （0, 1500, 2000, 2500mg/kg） or flavomycin（12mg/kg for PhaseⅠand 8mg/kg for PhaseⅡandⅢ） respectively, the diet supplemented with 1500 mg/kg EUOE showed significantly better results of ADDS（PhaseⅠ） （P＜0.05）, ADG, F/G and ADDS （PhaseⅡ） （P＜0.05）, F/G （PhaseⅢ） （P＜0.05）than that of the control, and there was a trend of increase on ADFI with the gradedlevel of EUOE supplementation （P=0.074） （PhaseⅢ）. As for the overall phase, ADGof 1500, 2500mg/kg EUOE and Flavomycin treatments were significantly differentfrom the control （P＜0.05）, no significant difference existed on other indices but atrend is detected on ADFI （P=0.086）. Compared with the control, TSC and SUNcontents of all the EUOE treated groups were significantly decreased during all thethree periods （P＜0.05）. SG contents were increased during PhaseⅡ,Ⅲ（P＜0.05）.There was a trend that EUOE supplementation improved the carcass characteristicsand meat quaiities by decreasing the drip loss, skin thickness and increasing lion eyearea （0.05＜P＜0.10）. TMAA contents of EUOE and Flavomycin treatments weresignificantly increased, while DMAA contents of Flavomycin was significantlylowered but that of 1500 mg/kg EUOE was significantly increased in pork muscle（P＜0.05）. Furthermore, the examined DMAA and TMAA contents were decreasedwith the graded EUOE level （P＞0.05）. These results indicated that EUOEsupplementation and Flavomycin had no significant difference on the growthperformance （P＞0.05）, but better results of EUOE on carcass qualities and DMAAcontents were observed. The comparatively optimal dietary EUOE level was1500mg/kg. It suggested that EUOE could be an attractive antibiotic alternative foranimal nutrition, although further experiments are necessary to verify the results.（2） Antioxidant capacity comparison of four kinds of antioxidants includingEUOE showed that significant difference was found with the addition of antioxidants.The higher the addition level of feed fat, the higher the feed POV, and the higherantioxidant level was needed. All the antioxidants activities was significantlyincreased by the antioxidants whether or not the level of the fat level of the pigconcentrate but the activities of the botanical feed mainly exhibited the first 21st day,and no significant effect were detected after the 28th day. Based on the POV curves,EMQ（I） showed the best antioxidant capacity, while the EUOE activity was ratherlower. Different antioxidants and their addition levels differed the oxidation kineticsequations of the concentrate, as to EUOE, different EUOE levels and the fat levels ofpig concentrate mainly caused the Class One reaction of oxidation.（3） One monomer was isolated from 50% ethanol fraction of 95% ethanol extractof Tongmeal through NKA-9 resin. With application of HPLC and concerned physical and chemical characteristics, the structure of the monomer was identified by means ofUV, IR, MS, ¹HNMR and ¹³CNMR. It was named as （±）3,3｀-bisdemethylpinoresinol,which was first purified from Tong-seed meal.（4） High throughput screening was used to detect the anti-diabetes, anti-hyper-lipidenmia and anti-tumor capacities of the extracts. The results showed: 70%ethanol fraction at the levels of 50μl/ml and 100μl/ml had the abilities of inhibitoryrole on 28SC tumor cell and water fraction at the levels of 10μl/ml and 50μl/ml, and70% ethanol fraction at the level of 50μl/ml on HL-60 tumor cell. The monomer and50% ethanol fraction showed strong inhibitory effect on SMMC-7721 at the level of50μg/ml. 50% ethanol fraction had the anti-diabetes activity at the level of 50μg/mlwith the activation multiple to PPARa、PPARd、PPARγ, RXR receptor at 1.29, 3.88,7.06 and1.95 respectively.（5） After the experiments of the different ethanol fraction on antioxidantcapacities under different system in vitro, the total phenol contents differedsignificantly （P＜0.05） from 47.68 mg/g of 50% ethanol fraction to 34.51mg/g ofwater fraction. The scavenging capacity of DPPH·differed from one another at thehighest ability of 90.37% of 50% ethanol fraction. The deoxidizing capacity of thethree fractions of 30%, 50% and 70% ethanol fraction were significantly higher thanthat of the vitamin C （0.2 mg/ml） and BHT （0.2 mg/ml） at the level of more than 0.8mg/ml. And the inhibitory abilities of pyrogallol auto-oxidation of the three fractionsamounted to 96.67% with the equivalent ability of vitamin C at the same level of 0.20mg/ml （P＞0.05）. As to the inhibitory capacity of linoleic acid peroxidation, at thelevel of 0.50 mg/ml, 50% ethanol fraction and vitamin C had the similar effects（P＞0.05）. At the level of 0.80 mg/ml, the clear ability of 50% ethanol fraction tohydroxyl radicals amounted to 96.01%. As the increase of the concentration of thethree fractions, the hemolysis inhibitory percent of mice RBC induced by H₂O₂increased, and at the different concentrations, 50% ethanol showed the best effect.（6） Fifty Kunming SD mice were randomly arranged into 5 treatments （10 miceper treatment） to conduct the anti-hyperlipidenmia test based on the body weight. Onetreatment was fed on the common diet, while the other treatments were fed on high-fat feed. After the success of hyperlipidenmia model, three treatments fed on high-fatfeed were lavaged with 50% ethanol fraction at the levels of 1000, 500, 250 mg/kgBW respectively. The experimental resuies showed that 50% ethanol fraction couldeffectively decrease blood lipids level of SD mice with hyperlipemia, improve blood lipid metabolism, reduce the formation of atherosclerosis, effectively increaseantioxidative level of mice body, and improve antioxidase activities. The three levelsof 50% ethanol fraction significantly decreased TG, TC, LDL-C, ApoB, R-CHR,TC-HDL-C/HDL-C and LDL-C/HDL-C （P＜0.05）, and significantly increased HDL-Cand Apo-AI level （P＜0.05）, and significantly increased mice serum and liver TAC,SOD、GSH-Px activities、hydroxyl radicals scavenging capacities, decreased MDAcontents （P＜0.05）. The three levels of 50% ethanol fraction could significantlydecrease the levels of blood viscosity, plasma viscosity, fibrinogen, hematocrit,indices of erythrocyte rigidity, aggregation （P＜0.05）, and increase indexes oferythrocyte deformability （P＜0.05）.更多还原