Research On The Preparation Of The Neem Oil Granules For Rodents' Ambisexual Sterility And Study On Its Quality Standard And Pharmacodynamics

The crossland rodents are mainly included the Ochotona curzoniae,Myospalax baileyi, Microtus fuscces,Eothenomys inez,E.proditor,Niviventer coninga,Marmeta himalayana, Apodemus chevieri,Ochotana thibetana and etc, which like eating grass, grain and other crops.So those are an important reason for the serious degeneration of the grassland,the cause of black beach and the desertification of grassland,all of the above happened in China's "Three Rivers Area", which has seriously affected and restricted the livestock's production and the farmers and herdsmen'life and development in Qinghai-Tibet Plateau district. Therefore, it is necessary for the government to make good efforts to eliminate the rodents.For a long time, people mainly use chemicals drugs for rodent control, and have good results, but they always pollute the environment,have a threat to the rodents' predators and endanger the safety of people and livestock and also other defects, at the same time, in the process of eliminating the crossland rodents,people have always used one single bait species for a long time, as a result,the rodents'antifeedant problems have gradually emerged, which affected the eradication to a certain extent.Therefore, the search of new type rodents drugs, particularly to use the plant sterile rodenticide to reduce rodent fertility and control its population, which can make practical results for reducingdisaster and increasing production in agriculture. The Neem (Azadirachta indica A. Juss.) is Meliaceae Melia trees, native to the Indian subcontinent, The Neem oil is a vegetable oil obtained from the neem seed by cold pressing on the machines. Because of its strong insecticidal effect,it is often used as a pesticide for pest control.The modern researches show that the neem oil has a good anti-fertility effect. This study intends to make neem oil into granules and to use it in crossland rodents control, by screening prescriptions for the granules, thereby enhancing the palatability of drug; On the basis of successful establishment of quality standards and do the research on the stability studies, in order to screen out the best doses and prove its mechanism,we research the coefficient of absorb food and pharmacodynamics studies of this granule, so that we can provide evidences for the application of this plant origin sterile agent in the rodent control.The main study results are as follows:1. The screening of the best preparation for the granule and the study on its Preparation Process:To take the neem oil as the principal agent, after the compatibility study of adjuvants,we did the orthogonal test which take the dissolvation and Ingestion coefficient as the evaluation index. The five-factor such as the corn flour (A), beef flavor (B), peanut flavor (C), adhesive (D) and magnesium (E) were taken to do the prescription investigation of preparation,all of the above was investigated in four levels, a comprehensive evaluation of the results and the analysis of variance was taken to optimize the prescription of preparation.Then we did researches on its dissolvaton, pharmacodynamics, and the verification of stability. The results showed as follows:In pre-study, corn meal is the best filler,in Ingestion Coefficient(IC) study, the beef flavor and the peanut flavor had the highest level, the IC is 1.66 and 1.87, respectively; The orthogonal optimization results is A4B4C3D1E4, that is to say,the 40% of the principal agent concentration of the neem oil granules which weighs 100g containing neem oil 18ml, corn flour 15g, beef flavor 10g, Peanut Flavor 10g, starch:dextrin (7:1) 5ml, magnesium oxide 10g, tert-butyl hydroquinone (TBHQ) 0.01g. In the optimized prescription validation, anti-fertility efficacyof granule was 73.7%(P<0.01); The IC was 0.58; The validation results of dissolvation test showed that while the water temperature was 37.5℃, stirred 5min,3 batches of the granuleshad dissolved, which was fit with the requirements of Pharmacopoeia; In the stability verification, the relative standard deviation(RSD) of RF of the color points on the plate of 3 batches of granules was less than 2%, the RSD of peak area was7.48%, so the drugs were stable. 2. The study of the granules'quality standard:Take the spermicidal active monomer in vitro called Octadecanoic acid-tetrahydrofuran-3,4-diyl ester,which was acquired from 'preliminary studies in laboratory as the reference standard, did the qualitative identification and quantitative analysis test by Thin-layer chromatography colouration and the Thin-layer chromatography scanning method(TLC) respectively, and did method verification of a quantitative analysis method, including the stability, precision, reproducibility, and recovery test. Finally, agents were been done the stability evaluation, including high-temperature test (60℃) and high humidity test (25℃, RH92.5%±5%) and the accelerated test (40℃±2℃, RH 75%±5%), the accelerate test included the content mensuration, moisture determination, dissolvation, appearance and character tests. The results showed as follows:In the qualitative identification test, the point of thin layer plate showed clear, separate, negative fluid without interference; The quantitative analysis for the content test of Octadecanoic acid- tetrahydrofuran-3,4-diyl ester in neem oil granules,the average monomer content was 1.95%; In the method validation, the RSD of method of stability was 1.34%(n=5), with the RSD of same board precision was 2.57% (n=5), RSD of different board precision is 11.86%(n=5), the RSD of reproducibility was 15.79%(n=5), the average recovery was 90.82%, RSD=13.95%(n=5); In the evaluation of preparation stability,after the high temperature and high humidity test of preparation, the preparation meet the requirements of quality, with no significant change in appearance and characters,In the accelerated test, the active ingredient content in content test began to decrease in the first 6 weeks, afer 6 months,the average water increased 0.45%(7.68%-8.13%)in moisture determination test, and the dissolvation test was consistent with the Pharmacopoeia,in the tracking study of granules appearance, the granules'color began to change in second month. The test's quality analysis methods were stable and reliable, it can be used as the quality control standards; the indicators of stability study were proper, the stability of granules is good,the validity duration were tentatively estimated for one month.3. The study on the granules'Ingestion Coefficient and pharmacodynamics:30%,40% and 50% of the principal agent concentration of neem oil granules had been made under the prescription to do researches on the Ingestion Coefficient and pharmacodynamics in order to find out the best principal agent dose group. The pharmacodynamic tests included anti-fertility tests, the male and female reproductive tissue pathology,the testis index test and the sperm abnormality test. The results showed as follows:Three dose groups' Ingestion Coefficient on the male were 0.67,0.54 and 0.25,respectively; the female group were 0.55,0.50 and 0.22, respectively. As a result, the granules which contained 30% and 40% neem oil met the lowest requirements of Ingestion Coefficient,which was greater than or equal to 0.3.So they were been taken to pharmacodynamics study which the result is that the 30% neem oil granules'anti-male fertility was 60%; the anti-female fertility was 46.7%; 40% neem oil granules'anti-male fertility was 80%; the anti-female fertility was 66.7%. Considering the Ingestion Coefficient and the anti-fertility efficacy study results, the 40% neem oil granules were taken to further studies such as testis index test, teratogenicity testing and pathology study. The 40% neem oil granules'testis weight index was 1.56±0.54g/100g, decreased significantly compared with the control group(P<0.05); 40% neem oil granules induced the test groups'sperm abnormality rate into 45.22%(P<0.01); In the study of pathology,40% neem oil granules caused the serious disease in male and female reproductive tissues. The results showed that 40% of the principle agent concentration of neem oil granules for rodents'ambisexual sterility had good palatability and significant anti-fertility effect, so it could be prospective in rodents'control on the grassland.