【作者】 陈绍淑；

【导师】 赵宝玉；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2004， 硕士

【摘要】 小花棘豆为豆科棘豆属草本植物，是疯草的一种。一方面，它可供药用，具麻醉、镇静、止痛之功效，用于关节炎、牙痛、神经衰弱及小鼠肉瘤等疾病的治疗；另一方面，又可导致家畜中毒，诱发疯草病。小花棘豆引起中毒的主要成分是生物碱。为了更好地评价小花棘豆的毒理和药理活性并筛选出小花棘豆的有效成分，本研究对小花棘豆的生物碱成分进行了系统分析及其毒性和抗肿瘤活性的测定，并取得了以下结果。 1．小花棘豆生物碱成分的提取 利用 95％乙醇进行热回流，从 １５　ｋｇ 小花棘豆中提取出浸膏，浸膏用 1％HCl酸化，过滤，得酸水液。酸水液用氯仿萃取除去杂质后，用 NaOH 调 pH 至 ９～１０，得碱水液。碱水液依次用氯仿、乙酸乙酯和正丁醇萃取，回收溶剂分别得氯仿部分、乙酸乙酯部分和正丁醇部分。TLC 初步分析各部分生物碱。气相色谱检测各萃取段苦马豆素的含量。结果表明，小花棘豆热回流出膏率为14.7%，氯仿、乙酸乙酯和正丁醇各段萃取所得总生物碱依次为 3.01g，5.15g 和119.57g ，计算出碱率为 0.85％；ＴＬＣ 检测表明小花棘豆中至少含有 9 个吲哚里西啶生物碱，其中氯仿部分和乙酸乙酯部分的生物碱种类较多；而且与苦马豆素标准品比较，各部分均有与苦马豆素显色相似的点。气相色谱测定各萃取段的苦马豆素含量分别为 5.03%、8.34%和 11.08%。 ２．小花棘豆小极性生物碱的分离和 GC-MS 分析 将小花棘豆的氯仿和乙酸乙酯萃取段合并，应用柱层析分离技术将其分成若干个极性段，TLC 检查生物碱集中的极性段用于 GC-MS分析，结果表明，小花棘豆中至少含有 9个小极性生物碱。 3．小花棘豆中苦马豆素的分离、纯化与鉴定 应用硅胶柱层析法对小花棘豆的正丁醇部分进行分离、纯化，得到一个白色针状结晶的单体化合物，通过熔点和波谱技术（MS，IR 和 NMR） 对分离到的单体化合物进行结构鉴定，确定该白色针状结晶为苦马豆素，计算提取率为 0.002%。 4．小花棘豆正丁醇部分的毒性研究 每天按 1600mg/kg、800mg/kg、400mg/kg剂量分别给小鼠灌服小花棘豆正丁醇提取部分，在灌服 60d 后，病理组织学及超微结构检查，主要表现有肝、肾等实质器官的细胞胞质空泡变性。这与苦马豆素中毒引起的病理变化一致，表明小花棘豆主要有毒成分是苦马豆素。 5．苦马豆素的抗肿瘤活性研究 通过细胞生长曲线的绘制，分析苦马豆素对S180细胞生长的抑制作用；通过 MTT 比色法，测定苦马豆素对 Eca-109、N-28、N-45 和SGC-7901 细胞增殖的抑制率；通过流式细胞术（FCM）检测苦马豆素对 S180 细胞周期的影响。结果为 S180 细胞经不同浓度的 SW 处理后均出现细胞增殖被抑制；MTT 检测<WP=6>ii 　　西北农林科技大学 ２００４ 届硕士学位论文：小花棘豆生物碱成分系统分析及其活性研究　　　结果表明 2.5 mg/L SW对 Eca-109细胞生长的抑制率为 50.38%；10mg/L SW对 N-28、N-45 和 SGC-7901 细胞抑制率分别为 25.90% 19.31%和 21.91%；FCM 检测表明，经苦马豆素作用 24h后 S180细胞的 G0-G1 期细胞增多，S期细胞减少。 通过本研究，得到以下结论： Ⅰ　生物碱的系统分析表明，小花棘豆正丁醇萃取段的生物碱含量较多，而氯仿和乙酸乙酯的萃取段的生物碱种类较多。气相色谱测定表明氯仿、乙酸乙酯和正丁醇萃取段的苦马豆素含量分别为 5.03%，8.34%和 11.08%。 Ⅱ　GC-MS 显示，小花棘豆的氯仿和乙酸乙酯萃取段至少含有 9 个小极性生物碱。 Ⅲ　首次从小花棘豆中分离到一种白色针状结晶的单体化合物，结构鉴定表明其是吲哚里西啶生物碱-苦马豆素，计算其提取率为 0.002%。 Ⅳ　通过给小鼠灌服小花棘豆生物碱的正丁醇萃取部分，复制出以实质器官的细胞空泡变性为特征的疯草中毒的病理学变化。　 Ⅴ 苦马豆素对小鼠肉瘤 S180细胞和 Eca-109 细胞有一定的抑制作用，对 N-28、N-45 和 SGC-7901 细胞的作用较差。对 S180 细胞的作用主要是阻止细胞进入 S 期，从而抑制细胞的增殖。更多还原

【Abstract】 Oxytropis glabra DC. is herbaceous plant that is part of genera Oxytropis, and yet one oflocoweed. On the one hand, Oxytropis glabra DC. may be for medicinal purposes, providingthe efficacy of anaesthesia, calming, and relieving pain, and used to treat the arthritis,toothache, neuradynamia, and mice sarcoma etc. diseases. On the other hand, Oxytropisglabra DC. could lead to livestocks poisoned，inducing locoism.The primary toxicant thatOxytropis glabra DC.causes livestock poisoning is alkaloid.To further determine the toxicityand pharmacological activity of Oxytropis glabra DC.and select its effective compositions，the alkaloids of Oxytropis glabra DC. were systematic analysed, and its toxicity andanticancer activity were examined in this study, and achieving the following results. 1. The extraction of alkaloid from Oxytropis glabra DC. The gum residues wereextracted by 95% hot ethanol from 15kg Oxytropis glabra DC.，acidified with 1%HCl andfiltrated to give acid liquid. The acid liquid was extracted with chloroform to remove impurity,Then its pH was adjusted to 9～10 with NaOH to give basic liquid. The basic liquid wasextracted with chloroform,ethyl actate, and n-butanol in succession. And the chloroform part,ethyl actate part, and n-butanol part were attained respectively after the solvents beingdistilled. The alkaloidal composition from the chloroform part, ethyl actate part and n-butanolpart was analysed by TLC. The content of swainsonine in these parts was monitored with gaschromatograph .Results showed that the extraction rate of the gum residues by hot methanolwas 14.7% . The quality of total alkaloids in chloroform part, ethyl actate part and n-butanolpart was 3.01g,5.15g and 119.57g respectively and the extraction rate of total alkaloids was0.85%. The result of TLC analysis indicated that Oxytropis glabra DC. contained at least 9indolizidine alkaloids, and the variety of alkaloids was more in chloroform part and ethylactate part than in n-butanol part . Compared to the criterion swainsonine, three polarity partsall had the stain similar to that of swainsonine. The content of swainsonine in chloroformpart 、ethyl actate part and n-butanolpart was 5.03%,8.34% and 11.08% respectively. 2．The isolation and analyses by GC-MS of minimum polarity alkaloid fromOxytropis glabra DC. The chloroform part and ethyl actate part was merged into one part,then it was isolated into several polarity sections with silica gel column chromatography. Thepolarity sections that alkaloids was focusing on by the examination of TLC was analysed withGC-MS. The analysis of GC-MS indicated that Oxytropis glabra DC. contained at least 9minimum polarity alkaloids.<WP=8>iv 　　西北农林科技大学 ２００４ 届硕士学位论文：小花棘豆生物碱成分系统分析及其活性研究　　　 3．Isolation ,purification, and identification of swainsonine from Oxytropis glabraDC. The n-butanol part from Oxytropis glabra DC. was isolated and purified by silica gelcolum chromatography and get one monomer compound that was a white crystal. Thechemical structures of the monomer compound were identified by means of MP and spectraldata (IR, MS, HNMR, CNMR). The result showed that the white crystal was swainsonine. 1 13And its extraction rate was 0.002%. 4．The study on toxicity of the n-butanol part from Oxytropis glabra DC. Themice were poured with 1600mg/kg, 800mg/kg, and 400mg/kg of n-butanol part fromOxytropis glabra DC. respectively per day. In the 60th day, the test of histopathology andultrastructure revealed that the main changes included cellular vacuoles in liver, kidney andother solid organs cytoplasmic vacuolation. The pathological changes were similar to that ofthe toxicity caused by swainsonine. So, the main toxicity composition is swainsonine. 5．The study of swainsonine on antitumor activities The inhibitory effects ofswainsonine on S180 cells was analyzed by drawing up更多还原