【作者】 李勤凡；

【导师】 王建华；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2005， 博士

【摘要】 疯草是豆科黄芪属和棘豆属有毒植物的总称,也是世界范围内危害草原畜牧业最严重的一类毒草。在我国,疯草广泛分布于西北、华北和西南的广大草原,危害面积达1100万hm~2。近年来,国内外在疯草的危害、有毒成分、毒理机制、防除和利用等方面的研究取得了重要进展,但迄今还没有控制疯草蔓延的特效方法,致使疯草中毒问题日趋严重,成为草原畜牧业的大患。冰川棘豆(Oxytropis.glacialis)是疯草的一种,也是青藏高原特有草种之一,主要分布在西藏阿里地区,本文就冰川棘豆毒素的毒性,苦马豆素(SW)的提取,2,2,6,6-四甲基哌啶酮(TMPD)和SW 的细菌降解等方面研究进行总结。1.按体重0.38 g/(kg﹒d)给健康山羊灌服TMPD,试验的21 d 山羊出现中毒症状,42 d 后逐渐出现走路时容易跌倒,倒地后不易起立。血液RBC、LC 显著降低(P <0.05或P <0.01),PCV、Hb 稍有降低,淋巴细胞空泡变性;血清UBN 含量、LDH 和ALT 活性升高(P <0.05 或P <0.01),AMA 活性显著降低(P <0.05 或P <0.01),表明TMPD 对心、肝、肾、脑等组织造成损害。病理组织学检查发现,小脑、大脑、肝等组织细胞出现空泡变性。山羊TMPD 中毒的症状、血常规指标、血液生化指标及病理变化与冰川棘豆中毒相似,证明TMPD 是冰川棘豆中的主要有毒成分之一。2.家兔按10 g/(kg﹒d)饲喂冰川棘豆草粉,从第40 天起,试验组家兔开始出现以中枢神经系统机能紊乱为特征的中毒症状。从试验第4 天开始试验组家兔陆续出现淋巴细胞空泡变性,而在40 d 后才出现中毒症状,其他指标与山羊冰川棘豆中毒一致,说明家兔的耐受性较山羊高,也属敏感动物,其胃肠道内不可能存在降解冰川棘豆毒素的细菌菌株。试验性冰川棘豆中毒家兔血清AMA 活性明显下降,说明冰川棘豆的有毒成分中可能含有吲哚兹啶生物碱,动物冰川棘豆中毒是TMPD 和其他有毒成分共同造成的。3.首次从冰川棘豆中提取纯化出SW。用甲醇提取冰川棘豆生物碱,提取率为5.5%。用氯仿、乙酸乙酯、正丁醇分段萃取,称重表明:冰川棘豆生物碱主要集中在正丁醇部分。薄层层析,Ehrlich’s 试剂显色,可以看出:氯仿部分含有10 种生物碱,乙酸乙酯部分含有7 种生物碱,正丁醇部分含有5 种生物碱。氯仿部分用于GC-MS 分析,共检测出17 种生物碱,其中已鉴定结构的有9 种,未鉴定结构的有8 种。取正丁醇部分进行柱层层析,梯度洗脱,分段收集,合并同类组分后,薄层层析监测,Ehrlich’s 试剂显色,显色明显的组分用氨性氯仿处理,减压油浴升华,得到白色针状结晶。白色针状结晶,经TLC、气相色谱、熔点检测,红外光谱、质谱、核磁共振谱等光谱鉴定,确定更多还原

【Abstract】 Oxytropis and Astragalus species known as Locoweeds are responsible for Locoism and other problems. Locoweeds are mainly distributed on the vast rangeland, exceeding 11 million hectares, in northwest, north and southwest China. Resent years, Locoweeds have been deeply researched all over the world .However, there is no effective method to control Locoism. Locoism has become an important disease on the vast rangeland. O.glacialis is one kind of Locoweeds that only distributed in the area of Ali, Tibet. In this paper, researching the toxicity of O.glacialis’toxin, isolation of swainsonine(SW) from O.glacialis and degradation toxin with bacteria. The results are as follows. 1. TMPD was feed to four goats constantly at 0.38g/(kg﹒d). 21 days later, some goats appeared the poisoning symptoms. 42 days later, some goats were stumbled, felled down easily, could not stand up. The blood routine items results showed that the number of RBC and LC declined sharply (P <0.05 or P <0.01), PCV, Hb decreased slightly, Lymphocytes occurred vacuolation in the blood. Biochemistry items showed that the BUN, LDH and ALT raised obviously(P <0.05 or P <0.01),while serumα-mannosides activity reduced obviously (P <0.01 or P<0.05). These results proved that heart, livers, kidney, brain and other viscera were damaged by toxin of O.glacialis. Histopathological changes showed that heart, liver and other viscera were vacuolation. The TMPD was a main toxicant in the O.glacialis . 2. Seventeen rabbits were divided into control group (n=6) and experimental group (n=11). In the experimental group, rabbits were fed with O.glacialis of 10g/(kg ﹒day).Blood biochemical indexes were measured respectively during feeding. The results indicated that the activities of serum AKP,GOT and BUN value of poisoned rabbits were significantly higher (P <0.05 or P <0.01), while serum α-mannosides activity significantly lower (P <0.01).Lymphocytes occurred vacuolation in the blood from 4th day. Histopathological examination showed that heart, liver, spleen, lungs and kidney were all vacuolized. These results proved that brain, livers, kidney, heart and other viscera were damaged by toxin of O.glacialis. Rabbit have better ability to bear the toxin of O.glacialis than other susceptible animals and may not exist degrade bacteria in their gastrointestine. The toxin may be contain indolizidine alkaloids. Animal O.glacialis poisoning was caused by TMPD and other toxin. 3. It was the first times to separated and purified the SW from O.Glacialis. The total alkaloid was extracted by hot methanol from O.glacialis powder with an ratio of 5.5%. The extraction was further extracted respectively with chloroform,ethyl acetate and butanol. The most alkaloids were maximum polarity in the butanol phase. With TLC checking on alkaloids,there were 10 alkaloids in chloroform phase, 7 alkaloids in ethyl acetate and 5 alkaloids in butanol phase. The alkaloids in chloroform phase analyzed by GC-MS. There were 17 alkaloids among those 7 kinds were defined the structure and other are not. Applied the crude alkaloid extracted with butanol to a silica gel column, eluted with different proportion ethyl acetate and methanol, monitored by TLC, incorporated the same fraction. Sublimated the Ehrlich’s colorable fraction and obtained white needles crystal in the end.The chemical structures were identified as SW by means of MP and IR, MS, 1HNMR, 13CNMR. 4. It was the first times to establish a method of determine the TMPD by ultraviolet-spectrophoto-metric method. The method is not need high condition, simple and convenient, can fast quantitative analysis the TMPD. 5. Separated the bacteria from the soil in which locoweed grows, and selected the bacteria by culture medium using TMPD as sole carbon source.12 colonies of bacteria that could degrade TMPD, then resorted to repeat sifting and separation, at last chose four colonies of bacteria as the experimental targets.The Grame staining and watching through light microscope for the four colonies chosen, Three of them are G-bacillus and the other is G+ coccus. 6. The degradation ratio of 12 strains bacteria to TMPD was measured. After measurement, there were 4 individuals bacterium had relatively high degradation rate to TMPD. The No 2# had highest degradation ratio that was 82.54% after cultured 30 days later, and the other 3 strains bacteria’s were more then 60%. Tested the degradation ratio under different culturing conditions. The results showed that the initial stage concentration as 200 mg/L and the fostering temperature as 30℃was helpful to microbe developed degradation. The liquid culture media was extracted by butanol, and analyzed by GC-MS. There were 8 kindsof alkaloids and 5 degradations most were esters. The mechanism of degradation was complex and further studies are needed. 7. Used 4 colonies of bacteria as the experimental targets to degrade SW. Continuously culturing for 9 days, centrifugal 10 minutes at 7500 r/min. extracted by butanol. The content of SW in the extracted matter was detected. The results proved that 3 strains of No 2#,6# and 11# bacteria were not ability to degrade SW. No 9# strain bacterium could degrade SW, the degradation ratio was 14.2%. The results pave a way for eliminating the toxin contained in locoweed.更多还原