印楝油两性不育灭鼠颗粒剂的研制与质量标准及其药效学研究
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摘要
草原害鼠主要有高原鼠兔Ochotona curzoniae、高原酚鼠Myospalax baileyi、青海田鼠Microtus fuscces、苛岚绒鼠Eothenomys inez,玉龙绒鼠E.proditor、高原白腹鼠Niviventer coninga、喜马拉雅早獭Marmeta himalayana、高山姬鼠Apodemus chevieri、藏鼠兔Ochotana thibetana等10多种。它们喜食牧草,粮食等农作物,是造成我国“三江源区”草原严重退化、黑土滩、草地沙化的重要原因,严重影响和制约着青藏高原区的畜牧业生产和广大农牧民的生存和发展,因此加大害鼠灭治力度是十分必要的。长期以来,人们主要采用化学药物灭鼠,并取得较好成绩,但化学药物存在着污染环境、危害鼠类天敌并对人畜安全构成威胁等缺陷,且在灭治高原害鼠过程中,人们长期使用单一的饵料品种,鼠的拒食问题逐步显现,在一定程度上影响了灭治效果。因此,寻找新型灭治害鼠药物,特别是运用植物性不育灭鼠剂降低害鼠生育力,控制种群数量,从而在农业上对农作物起到减灾增产的实际效果。印楝(Azadirachta indicaA.Juss.)是楝科楝属乔木,原产于印度次大陆,印楝油(Neem oil)是印楝种子机械冷榨得到的植物油,由于其具有较强的杀虫效力,常作为农药防治虫害。现代研究表明,印楝油具有良好的抗生育作用。本研究拟将印楝油做成颗粒制剂应用于草原害鼠防治,通过筛选颗粒剂组方,从而提高药物适口性；在成功建立颗粒剂质量标准并对其做稳定性研究的基础上,对其进行摄食系数研究和药效学研究,从而筛选出最佳作用剂量及探明其作用机理,为生产中应用植物源不育剂控制鼠害提供依据。主要研究结果如下：
1.颗粒剂最佳处方筛选及制备工艺研究：以印楝油为主药,在对辅料做制剂处方的配伍前研究后,采用正交试验,以溶化性和摄食系数(Ingestion coefficient)为考察指标,对玉米粉(A)、牛肉香精(B)、花生香精(C)、黏合剂(D)和氧化镁(E)五因素的四个水平进行制剂处方考察,对结果进行综合评分并作方差分析,优选出最佳处方。而后对优选处方进行药效学、溶化性和稳定性验证。结果表明：配伍前研究中,玉米粉为最佳填充剂,增诱剂摄食系数以牛肉香精和花生香精最高,分别为1.66和1.87；正交优选结果为A4B4C3D1E4,即印楝油含量为40%的100g颗粒剂中含印楝油18ml,玉米粉15g,牛肉香精10g,花生香精10g,淀粉：糊精(7：1)5m1,氧化镁10g,叔丁基对苯二酚(TBHQ)0.01g。在优选处方的验证中,颗粒剂药效试验抗生育力为73.7%(P<0.01)；摄食系数为0.58；溶化性验证试验结果表明,水温37.5℃,搅拌5min,3批颗粒全部溶化,符合药典要求；稳定性验证中,薄层板上3批颗粒剂显色点RF相对标准偏差<2%,峰面积相对标准偏差RSD=7.48%,药品较稳定。
2.颗粒剂质量标准的研究：以实验室前期研究获得的体外杀精活性单体十八碳酸-四氢呋喃-3,4-二酯作为对照品,通过簿层色谱显色(Thin-layer chromatography colouration)和薄层色谱扫描法(Thin-layer chromatography scanning method)分别做定性鉴别和定量分析试验,并对定量分析测定方法做了方法验证,包括稳定性、精密度、重现性、加样回收率试验。最后对制剂做稳定性评价,包括高温试验(60℃)和高湿试验(25℃,RH92.5%±5%)以及加速试验(10℃±2℃,RH 75%±5%),加速试验包括含量测定、水分测定、溶化性、外观性状检验。结果表明：定性鉴别试验中,薄层板显色斑点清晰,分离度良好,阴性液无干扰；定量分析以TLC扫描法测定印楝油颗粒剂中十八碳酸-四氢呋喃-3,4-二酯的含量,单体平均含量1.95%；方法验证中,方法稳定性RSD=1.34%(n=5),精密度RSD同板=2.57%(n=5)、RSD异板=11.86%(n=5),重现性RSD=15.79%(n=5),平均加样回收率为90.82%、RSD=13.95%(n=5)；制剂稳定性评价中,制剂经过高温试验和高湿试验外观性状无显著变化,质量符合要求,加速试验中,含量测定试验中有效成分含量在第6周开始下降,水分测定试验6个月水分平均增加0.45%(7.68%-8.13%),溶化性符合药典规定,外观性状跟踪研究颗粒剂自第2月颜色开始变深。本试验所确定的质量分析方法稳定可靠,能作为本颗粒剂的质量控制标准；稳定性研究各项指标正常,颗粒剂稳定性较好,有效期暂定为一个月。
3.摄食系数研究和药效学研究：将前面已筛选的处方做成印楝油含量分别为30%、40%和50%的3组颗粒剂,并进行摄食系数和药效学研究,从而筛选出最佳剂量组。药效学试验包括抗生育力试验、雄雌生殖组织病理学研究、睾丸脏器指数试验、精子畸形率试验。研究结果表明,3个剂量组的颗粒剂摄食系数对雄鼠组分别为0.67、0.54和0.25；雌鼠组分别为0.55、0.50和0.22。将摄食系数符合要求(=0.3)的30%、40%印楝油颗粒剂进行抗生育药效学研究,结果表明：30%印楝油颗粒剂抗雄性生育力为60%；抗雌性生育力为46.7%；40%印楝油颗粒剂抗雄性生育力为80%；抗雌性生育力66.7%。综合考虑摄食系数和抗生育药效学研究结果,选择40%印楝油颗粒剂进行睾丸脏器指数、致畸试验和病理学研究。40%印楝油颗粒剂睾丸脏器指数1.56±0.54g／100g体重显著,与对照相比下降明显(P<0.05)；40%印楝油颗粒剂致精子畸形率45.22%(P<0.01)；病理学研究中,40%印楝油颗粒剂致雄雌生殖组织严重病变。研究结果表明,40%印楝油两性不育灭鼠颗粒剂有较好的适口性及抗生育效用,有望用于草原鼠害防治。
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 reducing
disaster 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 efficacy
of 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 granules
had 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.
1.颗粒剂最佳处方筛选及制备工艺研究：以印楝油为主药,在对辅料做制剂处方的配伍前研究后,采用正交试验,以溶化性和摄食系数(Ingestion coefficient)为考察指标,对玉米粉(A)、牛肉香精(B)、花生香精(C)、黏合剂(D)和氧化镁(E)五因素的四个水平进行制剂处方考察,对结果进行综合评分并作方差分析,优选出最佳处方。而后对优选处方进行药效学、溶化性和稳定性验证。结果表明：配伍前研究中,玉米粉为最佳填充剂,增诱剂摄食系数以牛肉香精和花生香精最高,分别为1.66和1.87；正交优选结果为A4B4C3D1E4,即印楝油含量为40%的100g颗粒剂中含印楝油18ml,玉米粉15g,牛肉香精10g,花生香精10g,淀粉：糊精(7：1)5m1,氧化镁10g,叔丁基对苯二酚(TBHQ)0.01g。在优选处方的验证中,颗粒剂药效试验抗生育力为73.7%(P<0.01)；摄食系数为0.58；溶化性验证试验结果表明,水温37.5℃,搅拌5min,3批颗粒全部溶化,符合药典要求；稳定性验证中,薄层板上3批颗粒剂显色点RF相对标准偏差<2%,峰面积相对标准偏差RSD=7.48%,药品较稳定。
2.颗粒剂质量标准的研究：以实验室前期研究获得的体外杀精活性单体十八碳酸-四氢呋喃-3,4-二酯作为对照品,通过簿层色谱显色(Thin-layer chromatography colouration)和薄层色谱扫描法(Thin-layer chromatography scanning method)分别做定性鉴别和定量分析试验,并对定量分析测定方法做了方法验证,包括稳定性、精密度、重现性、加样回收率试验。最后对制剂做稳定性评价,包括高温试验(60℃)和高湿试验(25℃,RH92.5%±5%)以及加速试验(10℃±2℃,RH 75%±5%),加速试验包括含量测定、水分测定、溶化性、外观性状检验。结果表明：定性鉴别试验中,薄层板显色斑点清晰,分离度良好,阴性液无干扰；定量分析以TLC扫描法测定印楝油颗粒剂中十八碳酸-四氢呋喃-3,4-二酯的含量,单体平均含量1.95%；方法验证中,方法稳定性RSD=1.34%(n=5),精密度RSD同板=2.57%(n=5)、RSD异板=11.86%(n=5),重现性RSD=15.79%(n=5),平均加样回收率为90.82%、RSD=13.95%(n=5)；制剂稳定性评价中,制剂经过高温试验和高湿试验外观性状无显著变化,质量符合要求,加速试验中,含量测定试验中有效成分含量在第6周开始下降,水分测定试验6个月水分平均增加0.45%(7.68%-8.13%),溶化性符合药典规定,外观性状跟踪研究颗粒剂自第2月颜色开始变深。本试验所确定的质量分析方法稳定可靠,能作为本颗粒剂的质量控制标准；稳定性研究各项指标正常,颗粒剂稳定性较好,有效期暂定为一个月。
3.摄食系数研究和药效学研究：将前面已筛选的处方做成印楝油含量分别为30%、40%和50%的3组颗粒剂,并进行摄食系数和药效学研究,从而筛选出最佳剂量组。药效学试验包括抗生育力试验、雄雌生殖组织病理学研究、睾丸脏器指数试验、精子畸形率试验。研究结果表明,3个剂量组的颗粒剂摄食系数对雄鼠组分别为0.67、0.54和0.25；雌鼠组分别为0.55、0.50和0.22。将摄食系数符合要求(=0.3)的30%、40%印楝油颗粒剂进行抗生育药效学研究,结果表明：30%印楝油颗粒剂抗雄性生育力为60%；抗雌性生育力为46.7%；40%印楝油颗粒剂抗雄性生育力为80%；抗雌性生育力66.7%。综合考虑摄食系数和抗生育药效学研究结果,选择40%印楝油颗粒剂进行睾丸脏器指数、致畸试验和病理学研究。40%印楝油颗粒剂睾丸脏器指数1.56±0.54g／100g体重显著,与对照相比下降明显(P<0.05)；40%印楝油颗粒剂致精子畸形率45.22%(P<0.01)；病理学研究中,40%印楝油颗粒剂致雄雌生殖组织严重病变。研究结果表明,40%印楝油两性不育灭鼠颗粒剂有较好的适口性及抗生育效用,有望用于草原鼠害防治。
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 reducing
disaster 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 efficacy
of 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 granules
had 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.
引文
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