【作者】 杨永峰；

【导师】 彭镇华；

【作者基本信息】 中国林业科学研究院 ， 森林培育， 2010， 博士

【摘要】 植物抑螺相对于传统的物理与化学药物抑螺而言可具有经济上高效、生态上环保、效用上持续等显著优点,利用植物抑螺技术防治血吸虫病有着极其广阔的应用前景。为了深入挖掘我国抑螺植物资源,加大血吸虫病植物防治力度,本文选择了我国重要经济树种、传统土农药——乌桕为对象,对其抑螺效果、抑螺有效活性成分的提取、分离与鉴定,以及基于生物化学和超微结构角度下的抑螺机理等方面,开展了全面系统的分析研究,为科学评价与合理利用抑螺植物提供方法及依据。具体内容与结论如下:1、通过对乌桕根、枝、叶和种子等不同器官的四种不同溶剂粗提物的抑螺效果分析,发现乌桕不同器官均具有良好的抑螺效果,显示乌桕树种是一种优良的抑螺植物材料。比较得出,不同器官中以新叶的粗提物抑螺效果最佳,同时,对于前一年的落叶,也仍表现出良好的抑螺效果,说明乌桕叶具有持续稳定的抑螺特性;其最佳溶剂为石油醚。新叶石油醚提取物浸杀3d、4d、5d的LC50分别为20.327,7.125,5.724mg/L,落叶石油醚提取物分别为37.570,18.248,9.446mg/L。新叶石油醚提取物浸杀5d的LC90为24.409 mg/L,落叶石油醚提取物为69.922mg/L。新叶石油醚提取物相较落叶石油醚提取物,表现出更好的抑螺活性,但新叶与落叶石油醚提取物的抑螺效果均达到WHO规定的有效灭螺剂标准,即粗提物浓度低于100 mg/L时应具有有效杀螺活性的要求。2、应用色谱层析与薄层层析法分离纯化石油醚新叶提取物,结果显示弗罗里硅土填充柱洗脱的分离效果最佳,并可将石油醚提取物中的抑螺物质分离集中在较窄的区域,即实验中的496-526收集管中(浓缩后记为A12)。对A12分离进一步发现在相同的浓度和环境条件下,54-56管归类的一组抑螺活性最强,30mg/L的浓度浸杀3天,钉螺的死亡率达到了100%;其次为100-250管,达90%;再次为41-50管,为80%。表明这些管中含有抑螺活性物质。3、采用GC-MS方法对具有抑螺活性的样品浓缩物中极性较小、沸点较低的物质进行了研究,分离与鉴定了相关化合物,确定3,7,11,15-四甲基-2-烯-1-十六醇、棕榈酸、9,12,15-十八烯酸甲酯、1,2-苯二甲酸双( 2-乙基己酯)、8,11,14-烯-二十酸(8,11,14-Eicosatrienoic acid)等可能是主要的抑螺物质。4、采用HPLC-TOFMS法对具有抑螺活性的四个样品中极性较大、沸点较高的物质进行了分析鉴定,从化合物的共性方面考虑, C44H58N2O3、C24H42N2O8、C25H44N6O13、C22H43NO和C20H36N4O5几个成分出现的几率较高,其中C44H58N2O3在四个样品中均发现,C24H42N2O8在三个样品中存在,C25H44N6O13、C22H43NO和C20H36N4O5在两个样品中存在。这些成分可能具有抑螺活性。5、抑螺的生物化学机理研究表明:①糖元方面,随着处理液的浓度增加和处理时间的延长,钉螺体内糖元的含量显著降低。浓度为10mg/L、20mg/L、30mg/L、40mg/L、50mg/L的A12-2处理液浸杀钉螺96小时后,其肌糖元含量分别比对照降低了40.9%、49.8%、61.5%、63.3%、72.5%。分析认为:A12-2影响了钉螺的肝功能,使局部肝细胞坏死,直接影响糖元合成;激活或钝化糖元代谢过程中的某些酶,促进糖元分解和抑制糖元合成,导致糖元含量下降;影响消化道功能,引起摄食量减少,细胞摄取葡萄糖减少,糖元合成下降是主要原因。②蛋白质方面,钉螺头足部与肝脏部总蛋白含量经24h～96h处理后,都表现出降低→增高→降低的趋势。处理液开始作用时,钉螺体内总蛋白含量逐步下降,至48h后呈上升趋势分别达到1.726 g/L与1.997 g/L,这可能是由于钉螺受到A12-2处理后刺激了体内代谢,从而产生大量特定蛋白质以克服这种逆境胁迫的结果。随着时间的延长,钉螺体内总蛋白含量又不断下降直至超过钉螺的生理阈值,影响其能量代谢而使个体活性降低,渐至死亡。③相关酶的活性方面,碱性磷酸酶、琥珀酸脱氢酶、谷草转氨酶的活性在A12-2处理液的作用下,随着处理时间的延长和浓度的增加,都表现出显著降低→缓慢升高→显著降低的变化趋势。三种酶的这一变化趋势与有机体基于应激性的正常病理反应完全一致。而谷丙转氨酶活性随着时间的增加呈现不断降低的单一变化趋势,说明处理液对谷丙转氨酶有着更为直接的抑制作用。6、抑螺的超微结构机理研究表明:①钉螺头、触角以及足部等外部结构方面,应用扫描电镜,对经过15mg/L、30mg/L两个浓度处理24h、72h后的钉螺进行测定显示,随着浸杀时间延长和浓度增加,钉螺头和触角都由表面皱褶非常明显到结构遭到破坏,趋向平坦,并出现糜烂物且表面明显变形,严重溃损。足部开始肿胀,继而出现糜烂物,溃烂变形,并可见浸蚀孔洞,表面绒毛脱落,最终严重变形,裂变溃伤现象明显。②钉螺内部肝脏结构方面,应用透射电镜,对经过15mg/L浓度A12-2处理液处理24h后的钉螺进行测定显示,肝细胞呈现肿胀,细胞核核膜变形,出现大量溶酶体,且部分溶酶体呈空泡状。部分线粒体表现出肿胀,呈狭长状,小部分已呈空泡,嵴模糊病损。粗面内质网杂乱分布且肿胀。随着处理时间的增加,出现大量中晚期溶酶体,以及大量小型空泡,细胞核肿胀,核内物质散乱分布,线粒体已经变形,由于自溶作用,核膜消失,渐成空泡等现象较为明显。③经过处理液浸杀后的钉螺,其肝脏细胞线粒体出现嵴模糊的病损,说明钉螺的氧化磷酸化过程受到极大影响。同时粗面内质网受到破坏,会造成整个机体的代谢紊乱。这与经过处理后的钉螺体内碱性磷酸酶、谷草转氨酶、谷丙转氨酶、琥珀酸脱氢酶及糖元含量的变化相吻合。钉螺经A12-2处理后,其肝细胞内细胞器、膜结构的破坏,均意味着肝功能的下降、肝器质的损坏,细胞结构和功能的破坏最终导致钉螺死亡。7、本项研究,分析鉴定了乌桕的抑螺物质,即3,7,11,15-四甲基-2-烯-1-十六醇、C44H58N2O3等可能的抑螺化合物,深入揭示了乌桕的抑螺机理,即乌桕中的抑螺物质对钉螺的软体组织、肝细胞结构以及新陈代谢、酶的活性等具有严重的损伤或抑制作用,研究结果充分表明乌桕是一种极有价值的优良植物抑螺资源。更多还原

【Abstract】 It has a great pospective using plant molluscicide technique to control schistosomiasis, because of its high efficiency in ecnomic, environment friendly in ecology and duration in effectiveness, compared to the traditional physo-chemical drugs of molluscicidal materials. In order to quest the snail-inhibiting plant resources, and also to enhance schistosomiasis-controlling potential, in this work, we selected the important economic species - the traditional pesticides - Sapium sebiferun as an target object. the molluscicidal effectiveness, and the the extraction, separation and identification of the active compounds were systematically investigated, and the mechanism based on biochemical and ultrastructural areas were studied. This work can provide method for scientific evaluation and rational use of molluscicidal plants. The main results were as follows:1. It was found that there were good effectiveness to control snail in different organs of Sapium sebiferun through the investigation of the snail-inhibiting effect by extraction of effective components in the roots, branches, leaves and seeds of tube Sapium using four solvents, indicating that Sapium sebiferun is a great plant for snail– inhibiting material. By compared, we think that the new leaves were best one for controlling snail, and secondly, the old leaves falled the year before, showing a persisted characteristics of controlling snail. It also showed the petroleum ether was best solvent for extraction of active components among the fours.LC50 of snail, by immersed in the extract of petroleum ether of new leaves for 3d, 4d, 5d were 20.327, 7.125, and 5.724 mg/L respectively, and of ols leaves, 37.570, 18.248, 9.446 mg/L, respectively. LC90 of snail, by immersed in the extract of petroleum ether of new leaves for 5d was 24.409 mg/L, while of old lesves, 69.922 mg/L. New leaves has better active snail-inhibiting potential than that of old ones, however, the both ones had attained the requirement of WHO for effective snail-inhibiting standard, which is that there is an effective snail-inhibiting activity when the concentration of crude extracts were lower than 100 mg/L. 2. It showed Florisil-packed column was the best one for separation of active components by using ether chromatography or thin layer chromatography to purify the extract of petroleum ether of the new leaves, and that can concentrate the target components to a narrow area, namely the 496-526 tube (A12). After further separation of the A12 sample, we found the activity of the classifiction of 54-56 tube was most stronger for controlling snail. Mortal rate of snail was 100% in the concentration of 30mg/L, immersed for 3 days, and followed by 100-250 tube, to 90%, and by 41-50 tube, to 80%. This result showed these tubes containing the active substance to control snail.3. GC-MS was applied to separate and identify the nonpolar and low-boiling-point active components in the concentrated extracts. It confirmed that the compounds of 3,7,11,15 - tetramethyl -2 - en -1 - hexadecadiene alcohol, palmitic acid, 9,12,15 - 18 allyl methyl, 1,2 - terephthalic acid bis (2 - ethylhexyl), and 8,11,14 - en - 20 acid (8,11,14 -Eicosatrienoic acid) et al. were potential snail-inhibiting substance.4. Apliction of HPLC-TOFMS to analyse the polar and high-boiling-point active components in the four concentrated extracts, we found that the appearing rate of the compounds C44H58N2O3, C24H42N2O8, C25H44N6O13, C22H43NO and C20H36N4O5 were very high, in which the C44H58N2O3 were found in four samples, C24H42N2O8 existed in three samples, and C25H44N6O13, C22H43NO and C20H36N4O5 existed in the two samples. It showed these compound were potential snail-inhibiting components.5. Snail-inhibiting mechanism of biochemistry showed that: 1) with increasement of solution concentration and prelonging of treatment time, the snail body glycogen levels were significantly lower. After immersed for 96 hours in 10, 20, 30, 40, and 50mg/L of treatment concentration, the levels of the muscle glycogen content in snail were lower of 40.9%, 49.8%, 61.5%, 63.3%, 72.5% than those of the control test. We think that A12-2 affected the snails liver function, and that lead to the necrosis of partial liver cell, and also impact the synthesis of glycogen. It activate or passivate certain enzymes in the process of glycogen metabolism, and promote glycogen breakdown and inhibition of glycogen synthesis, leading to glycogen content decreased. And it also affect gastrointestinal function, reducing of food intake, and decreasing glucose uptake. The decreasement of glycogen synthesis may be the main reason.2) For total protein investigation, it have shown the trend of decreasement– increasement– decreasement after 24-96h treatment for heed and liver of snail. When the treatment solution activiated, the total protein content in the oncomelania body was decreased step by step, until to 48 h, increased, and up to 1.726 g/L and 1.997 g/L respectively. this may be activated by A12-2, snails accelerated the metabolism in its body, and produces a large number of specific proteins to overcome this stress condition. With prolonging of time, the snail body began to decline until the total protein content was more than snail’s physiological threshold, and that affected the energy metabolism, leading to the activity-decrease of individual snail until to death.3) for investigation of enzymes: activity of alkaline phosphatase, succinate dehydrogenase, aspartate aminotransferase showed the trend of significant decreasement– slow increasement–significant decreasement with prolonging of treatment time and increasement of concentration in the action of A12-2 treatment solution. The change of the three enzymes was correlated with the change of organic pathology based on the normal stress response. The ALT activity was decreased with increasing of time, indicating direct inhibition of treatment solution to alanine aminotransferase.6. Snail-inhibiting mechanism of ultrastracture showed that: 1) Application of scanning electromicroscopy to observe the head and antennae of snail, we found that the structure of the surface folds in the head and antennae were destructed significantly, and tended to flat, under the concentration of 15 and 30 mg/L for 24 and 72 h. There also has appeared the marked erosion material to the surface deformation, leading to a serious collapse damage. Foot began to swell, inflammate, and erode, deform, resulting to visible holes of erosion, loss of surface hair, severe deformation in the end. there is clearly the phenomenon of erosions.2) Application of TEM to observe the liver of snail, it found that the liver cells were swollen, the nucleus membrane shape was as a large number of lysosomes, vacuolization and some lysosomes, when immersed in 15 mg/L of A12-2 treatment solution for 24 h. Some mitochondria swelled, appearing a long and narrow shape, a small part of vacuolization, and a cristae fuzzy lesion. Rough endoplasmic reticulum chaotically distributed, and swelled. When the treatment concentration and treatment time increased, a large number of middle and late lysosomes and numerous small vacuoles appeared, and nuclear swelled and material in nuclear scattered, mitochondria deformed. Due to autolysis reaction, the membrane of nuclear disappears, and gradually became empty bubbles and other obvious phenomena.3) Treated snails after the liquid immersion, the liver cell mitochondria cristae was ambiguous lesions, indicating the process of oxidative phosphorylation of snails had been impacted. While rough endoplasmic reticulum is damaged, leading to disorder of metabolism in the body. This is consistent with the change of alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, succinate dehydrogenase and glycogen content in the snail body after treatment.Treated by A12-2 solution, the liver cell organelles and membrane structure in smail were damaged, those meaned the decline of liver function, the damaging of the liver devices. The damage of cell structure and function was eventually leaded to the death of snail.7. The snail-inhibiting materials in Sapium sebiferun were analysed and identified, 3,7,11,15 - tetramethyl -2 - en -1 - hexadecadiene alcohol and C44H58N2O3 et al. were potential snail-inhibiting compounds. In this work, the snail-inhibiting mechanism was widely expositted, it was found that the compounds in Sapium sebiferun has a potential damage or inhibition to soft tissue, liver structure, metabolism, and activity of enzyme. The results showed that Sapium sebiferun is a relatively new type of molluscicidal plant resources, their active components can inhibit the survival of snails through the following channels:更多还原