论文标题:疯草（甘肃棘豆）生物碱系统分析及其毒性的比较病理学研究
The Systematic Analysis and Toxic Comparative Pathology Research of Locowed (Oxytropis Kansuensis) Alkaloid Omposition
论文作者 赵宝玉
论文导师 曹光荣；程东亮,论文学位 博士,论文专业 临床兽医学
论文单位 西北农林科技大学,点击次数 242,论文页数 191页File Size12840k
2001-11-01论文网 http://www.lw23.com/lunwen_3477392/ 疯草,甘肃棘豆,披针叶黄华,生物碱,分析,毒性, 比较病理学,GC－MS,HI－MS,山羊
Locoweed,Oxytropis Kansuensis,Thermopsis lanceolate, Alkaloid,Analysis,Toxicity,Comparative pathology,GC-MS,HI-MS,Goat
本试验通过对疯草(甘肃棘豆)生物碱成分的系统分析及其毒性的比较病理学研究，取得了以下结果： 1．疯草(甘肃棘豆)生物碱成分的提取：本试验对疯草(甘肃棘豆)生物碱进行了系统提取，结果表明，疯草(甘肃棘豆)含有小极性、中等极性和大极性生物碱，从各极性段所得的量可以看出，主要以大极性生物碱为主；各极性段生物碱经TLC检查，不含臭豆碱、黄华碱等喹诺里西啶生物碱，而含有苦马豆素等吲哚里西啶生物碱，在大极性生物碱中苦马豆素是其主要成分。 2．疯草(甘肃棘豆)生物碱成分柱层析分离：采用硅胶柱层析对疯草(甘肃棘豆)生物碱进行了分离，得到2个单体化合物(结晶A、结晶D)和B、C、E、F段生物碱混合物。结晶A为淡黄色针状结晶，有紫色荧光，改良碘化铋钾显色弱；结晶D为黄色片状结晶，有紫黑色荧光，硫酸乙醇显黄色，改良碘化铋钾显褐色；B段改良碘化铋钾显色呈现5个桔红色斑点；C、E、F段Ehrllich’s试剂显色呈现5～8紫红色斑点。 3．疯草(甘肃棘豆)生物碱结构鉴定：采用GC—MS联用技术对疯草(甘肃棘豆)小极性及中等极性生物碱进行了结构鉴定，从中鉴定出5，6，7，8-四氢吲哚里西啶、1，2，3，4-四氢喹啉、N-苯基-2-吡啶丙酰胺、1-乙酰基-9氢-吡啶[3，4-B]吲哚、3-甲氧基-4，7-甲基-1-氢-异吲哚、吡略烷类、二苯胺、3-丁基吲哚里西啶等41种生物碱成分；采用HI—MS分析技术对疯草(甘肃棘豆)大极性生物碱经乙酰化、锌还原处理前后比较分析，从中鉴定出5种生物碱成分即：苦马豆素(Swainsonine)、氧化氮苦马豆素(Swainsonine N-oxide)、斑荚素(Lentiginosine)、倒千里光裂碱(Retroecine)和2-羟基吲哚里西啶(2-Hydroxyindozidine)，其中倒千里光裂碱和2-羟基吲哚里西啶为首次在甘肃棘豆中发现：结晶A经IR、MS、1HNMR初步鉴定为N-苯甲酰基甘氨酸甲酯，结晶D经UV、IR、1HNMR、13CNMR鉴定为5，7—二羟基—4′—甲氧基黄酮，为首次在甘肃棘豆中分离到。三 疯草（甘肃棘豆）生物碱系统分析及其毒性的比较病理学研究 4．变异黄蔑、宽苞棘豆与甘肃棘豆生物碱的比较分析：采用TLC技术对变异黄尾 宽苞棘豆及甘肃棘豆生物碱进行了比较分析，结果表明，这三种疯草生物碱种类基本相似，都属于叼跺里西睫生物碱：从各极性段量的多少可以看出，三种疯草生物碱主要集中在正丁醇部分，且苦马豆素为其主要成分。 5．疯草（甘肃棘豆）生物碱的毒性研究：每天按S00mg八g剂量给小鼠、雏鸡灌服疯草（甘肃棘豆）总生物碱，在灌服的第15－20d可复制出疯草中毒的典型病变，主要表现为肝、肾等实质器官的空泡变性，表明疯草（甘肃棘豆）主要有毒成分是苦马豆素。 6．披针叶黄华生物碱提取、分离与结构鉴定：采用醇类溶剂提取法对披针叶黄华生物碱进行了提取、分离与鉴定，结果表明，披针叶黄华干草中生物碱含量为1．90％；总生物碱经GC－－MS联用分析技术，从中鉴定出臭豆碱、黄华碱、鹰瓜豆碱、金雀花碱、N－甲基金雀花碱、白羽扇豆碱等12种睦诺里西陡生物碱，其中Dodecah叩ro－7，14Methanodiprpdoll，2－a：1’，2’－E］［1．＊diazocine、1－Ace…－l，2，3，4tetrahydro－5－（－plperdinyl）prpdine、7，14－Methano－ZH，6H－iprpdo［1，2－a：1’，2’－ e］ ［1a山一 儿1－m一hy卜3－（－d汀。phmp口7，1个＊＊m。＊－4H，6H刁lp加加［1，2－a：1 ’，2e，51diazs种化合物为首次从该植物中发现。 7．披针叶黄华的毒性研究：从细胞水平和亚细胞水平对披针叶黄华中毒山羊病 理学进行了研究，结果表明，病理组织学变化以实质器官细胞颗粒变性为主；特别是 肝脏结缔组织和胆管增生；超微结构变化以肝脏胶原纤维增生为特征，这是肝硬化 的典型病变。这些病变与疯草中毒的典型病变完全不同。
The experiment has achieved the follwing results through the systematic analysis and toxic comparative pathology research of locoweed (0. kansuensis) alkaloid composition.1.The extraction of alkaloid from Oxytropis kansuensisThe systematic extraction result of alkaloid of 0. kansuensis indicates that a Kansuensis contains alkaloids of minimum, neutral and maximum polarity,of which maximum polarity alkaloid hold the firstplace.With TLC checking on alkaloid from different polarity phase, it doesn" t contain quinolizidine alkaloids-anagyrine, thermopsine etc, but indolizidine alkaloids-swainsonine and so on. Swainsonine is the main composition in maximum polarity alkaloids.2.The fractionation of alkaloid from O.kansuensis by column chromatographyWe applied silica gel column chromatography to get the fractionation of alkaloid from a kansuensis and get 2 monomer compound (crystal A and crystal D) and alkaloid mixture of B,C,E,F phase. Crystal A is a light yellow needle with purple fluorescence and test with ameliorated dragendorff reagent shows faint colour. Crystal D is a yellow plate with purple and dark fluorescence, tests with sulpharic alcohol and ameliorated dragendorff reagent show yellow and brown respectively.The test withameliorated dragendorff reagent in phase B shows 5 orange spots. Test with Ehrlich"s reagent shows 5 to 8 red purple spots in C,E and F phase.3.The identification of alkaloid from O.kansuensis41 kinds of alkaloid in the maximum polarity from 0. kansuensis were identified by GC-MS. The maximum polarity of alkaloid were treated by acetylation and zinc reduction and 5 kinds of alkaloid composition were identified by HI-MS. they are swainsonine, swainsonine N-oxide, lentiginosine,retroecine and 2-hydroxyindozidine,the last two are discovered for the first time from the plant.Crystal A is initially identified as N-benzoylglycine methyl ester through IR, MS, 1HNMR crystal D is identified as 5, 7-dihydroxy-4" -methoxyflavone through UV, IR, 11-INMR, "3CNMR, the compound was discovered for the first time from the plant.4.Related analysis of alkaloids from A. variabilis,Q. Iatiracteata and0. Kansuensis by thin layer chromatographyA related analysis of alkaloids from A. variabilis, 0. latiracteata and 0. Kansuensis by means of TLC indicated that the types of alkaloids of these three locoweeds are alike,as they all belong to indolizidine alkaloid. From the quantity in different phase of polarity, it can find that the alkalods of these three locoweeds mainly localized in n-butanol segment with swainsonine as its main composition.5.The study on toxicity of alkaloid from O.kansuensisPour mouse and sixteen chickes were dreched with 800mg/kg of alkaloid from 0. kansuensis per day. In 15th to 20th day, the typical pathological change from locoweed poisoning can be duplicated. The liver, kidney and other solid organs cytoplasmic vacuolation indicated that the main toxicity composition is swainsonine.6.The extraction, isolation and identification of alkaloids formThe rmops is Ianceo lateThe extraction, isolation and identification of alkaloids from Thei-mopsis lanceolate indicate that dried Thermopsis lanceolate contains 1. 90% of alkaloid. 12 types of quinolizidine alkaloids were identified through the GC-MS from total alkaloid, of which 5 kinds of compound were discovered for the first time in this plant.7.The study on toxicity of Thermopsis IanceolateThe research on the pathology of poisoning goat indicated that the pathologic histology changes are mainly granular degenration in viscera cell in particular and hyperplasia of connective tissue and bile duct of liver.The ultrastructural change of liver shows collagenous fiber hyperplasia as its feature which is the typical change of cirrhosis of the lives. These pathological changes are totally different from locoweed poisoning.
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