【作者】 童德文；

【导师】 曹光荣； 程东亮；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2000， 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400万ha。疯草所含主要有毒成分吲跺兹定生物碱—苦马豆素，能导致大批家畜中毒死亡，更为严重的是引起母畜流产、不孕、胎儿畸形和公畜不育，造成了巨大经济损失。尽管近20多年国内外在疯草有毒成分、毒理机制、化学防除和脱毒利用等的研究方面取得了重要进展，但迄今还没有快速检测分析苦马豆素的方法和特效的控制疯草蔓延的方法，致使疯草中毒问题日趋严重，成为草原畜牧业的大患。因此，探索苦马豆素（SW）—BSA合成及其免疫原性研究对用免疫的方法预防动物疯草中毒具有十分重要的科学意义。本试验系统地研究了苦马豆素（半抗原）纯品的制备，在化学模型（倒千里光裂碱—BSA合成）研究基础上，进行了SW—BSA的合成及其免疫原性研究。结果如下：1 甘肃棘豆中苦马豆素的分离、鉴定 本试验是为苦马豆素免疫原合成提供原料。用甲醇浸泡甘肃棘豆粉，回收甲醇浸泡液至侵膏，1MHCl溶解后上732型强酸性阳离子交换柱，1MNH₄OH洗脱并挥发至干，得到总生物碱。总生物碱经氨性氯仿提取后，通过两个途径分离苦马豆素。一个途径是氨性氯仿提取物上硅胶（180μ）柱，氯仿∶甲醇∶氨水∶水（70∶26∶2∶，V/V）混合溶液洗脱，每10mL收集一份，共收集160管，薄层层析和抑制α-甘露糖甙酶活性监测，合并同类部分，挥干后在90℃，-0.094Pa真空条件下升华。另一个途径是用高效液相色谱柱分离，色谱条件是：反相（C18硅胶）柱，用水和甲醇的混和液洗脱，在25min内用5％～100％甲醇线性梯度洗脱，苦马豆素一般出现在80％～100％的范围内，表现为一个宽峰。升华Ⅱ部分得到一种白色细针状结晶70mg，植物中的提取率为0.0014％。高效液相色谱柱分离后回收溶液也得到一种白色细针状结晶1.04mg，植物中的提取率为0.0052％。经TLC、MP、IR、MS鉴定分析，确定所分离到的结晶为苦马豆素。2 豆类丝核菌总生物碱的提取及苦马豆素含量测定 用PAD、三叶草汁糖琼脂培养基和豆芽汁糖琼脂培养基培养7-1、7-3、3-1三株豆类丝核菌，结果只有菌株7-1和菌株7-3在PAD和三叶草汁糖琼脂培养基上生长良好。将复活的菌株接种于改良Czapek’s培养基，置25～27℃培养箱培养14d。结果表明液体 2000届 西北农林科技大学攻读博士学位研究生学位（毕业）论文 培养时菌种7－3菌丝体产量高于菌株7l。收集菌株7·l和7－3菌丝体，自然干 燥后乙醇索氏提取，回收乙醇至糖浆状，H。O：CH。CI。u：l，V／V)革取，水 层调pH10 后，再用H＊O：CH。CI。（3：l，VfV）革取，水层调pH7，浓缩后上732 型强酸性阳离子交换树脂，用 IM NHOH洗脱，收集氨洗液，调 pH7，浓缩 后冻干，得到一种浅黄色粉末。取苦马豆素标准品 100 p g及浅黄色粉末提取 物 10mg，分别用 lmL毗唆溶解，取 0＊mL加 BSTFAO．ltnL，50℃水浴 30min。 取反应液二poe，直接进样作气相色谱（co）。结果表明在相同的气相色谱条件 下，在相同出峰时间＊．2～smin内，标准样品的峰形与总生物碱的峰形完全一 致，可以判定总生物碱中含有苦马豆素。7－l 菌株总生物碱平均峰高为 116．75tnmtheloX7习茵株总生物碱平均峰高为138．56mln加司X标准品平均 峰高为 197．78mm…叫卜根据计算得出总生物碱中苦马豆素含量分别为 5．903mg／g和7刀06mg／g。试验结果证实，豆类丝核菌能够生物合成苦马豆素， 而且含量比已报道的疯草中含量高，这就为大量生产苦马豆素以利于批量主产 苦马豆素一BSA疫苗摸索了条件。 3 倒千里光裂碱－－SA的合成研究 单猪屎豆碱扣 加入带八结晶水的氢氧化 钡39．sg，加水200mL，加热回流3hr，溶液冷却，通二氧化碳气体使碳酸钡沉 淀完全，过滤，用 IM HCI酸化滤液至刚果红试纸变蓝，用乙醚连续荤取，水 溶液通过强碱性阴离子交换树脂柱，用蒸馏水淋洗，直至洗脱液呈中性，减压 抽干，用适量丙酮回流提取3次，过滤，回收丙酮，残留物用丙酮重结晶，得 到柱状结晶7二Zg，产率为72％。经TLC、MP、MS鉴定结晶物就是倒千里光 裂碱。倒千里光裂碱与用滨乙酸制备的活性酯反应，得到一种白色粉末状物 32．06mg，产率为82％，根据MP测定、IR鉴定结果判定，白色粉末状物为所 要制备的倒千里光裂碱一活性酯结合的季铰盐。季铰盐再与BSA反应，产物透 析、冻干后呈白色针状结晶 97．15mg，按 BSA计算，产率为 87％，UV鉴定结 果与正常的BSA相比，其空间构象发生明显改变，280urn处光吸收值显著下降， 表明季铰盐己与BSA结合。这一试验结果为苦马豆素一BSA的合成提供了理 想的化学模?更多还原

【Abstract】 Oxytropis and Astragalu.s species known as Locoweeds are responsible for Locoism and other problems. Locoweeds in china are mainly distributed on the vast rangeland, exceeding 4 million hectares, in northwest, north and southwest China. Locoweeds contain the indolizidine alkaloid, swainsonine (SW). which has most serious toxicity, resulting neurological damage. abortion. fetal abnormality and male infertility. Besides containing toxin, Locoweeds are excellent herbage for animals. However, there is not the effective method to control Locoism. Locoism has become an important disease on the vast rangeland. In this paper. Attempt to protectively immunize animals against Locoism with SW-BSA was fulfilled. The results are as follows. I The isolation and identification of swainsonine from O.kansuensis Bunge The raw material for synthesis of SW-BSA was supplied by this test. O.kansuensis Bunge powder was dipped in methanol. The residue following evaporated of the methanol was dissolved in I N4 HCI and applied to a strong cation-exchange resin and eluted with I M NH4OH. The eluted solution was collected and concentrated to afford the crude alkaloid. Further isolation was conducted by two ways. One was extracting the crude alkaloid with ammoniated chloroform, applied the residue to a silica gel column, Eluted with chloroform: methanol: ammonia: water (70:26:2:2,V/V), each lOmL to collect, monitored by TLC and the activity of a -mannosidase, incorporated and evaporated the same fraction. The other was preparation by HPLC, using reverse phase (C-I 8 silica) and water/methanol, starting at 5% methanol and a linear gradient to 100% in 25 minutes. TLC plates sprayed with Ehrlich’s reagent showed three single fraction, 65 -79 tube (H)and 106 120 (III) tube became visible as pink spots, Rr value were 0.44 and 0.12 respectively, fraction II was the sante as standard sample. 25 3 tube (I) became visible as purple spots, R value was 0.60. fraction I and II retained strong a -mannosidase inhibiting activity, III inhibiting effect was poor. Sublimed II under vacuum (-0.094pa) at 90t to obtain 70mg crystal of white needles, the yield of approximately 0.0014% based on dry plant weight. 1.04mg crystal was obtained by HPLC too, but the yield was 0.0052%. The crystal was swainsonine identified by TLC, MP, IR and MS. 2 Extraction of alkaloid from Rliizoctonia leguminicola and determination of swainsonine concentration Cultured the strains of 7-1, 7-3 and 3-1 R. leguminicola on the medium of PAD, bean sprout juice-sugar-agar, clover juice-sugar-agar. Strains 7-I and 7-3 were reviviscent and cultured on Czapek’s medium at 25-27XD for 14d. The mold mycelium yield of strain 7-3 is more than 7-1. Collected mycelium, air dried, and extracted with ethanol (Soxhlet, 24hr) .The extract was concentrated to a syrup, shaken ( 3X ) with H20 CH2CI, (V/V) ,the water extract was brought to pHIO and again extracted ( 3X ) with H.,O CH.,Cl, ( V/V). The water layer was brought to pH7 and absorbed (30 mm) on a strong cation-exchange resin (H), eluted with I M NH4OH, and the filtrate was dried. The residue was brought pH7 and lyophilized to obtain a light brown powder. After dissolved 100 t g standard sample and 10mg powder in lmL pyridine, 0.ImL of the pyridine was applied to GC. The powder contains swainsonine, for the peak of the powder is consistent with the standard sample at the same GC condition. The peak of strain 7-I was 116.75mm(n10), strain 7-3 was 138.56mm(n10), the standard sample was I97.78mm(nI0) The concentration of swainsonine in powder of 7-1 and 7-3 were 5.9O3mgI更多还原