| 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:... |
PDF Full Text Request