体外抗鸽源禽毛滴虫中药的筛选及其作用机理研究

发布时间：2018-09-04 08:29

【摘要】：鸽毛滴虫病是由鞭毛虫禽毛滴虫(Trichomonas gallinae)引起的,是危害鸽子最重要的疾病之一,给世界养鸽业造成了严重的经济损失。T.gallinae通常寄生于各种鸟类上消化道导致黏膜炎症、溃疡,甚至因肉芽肿阻塞食道而造成宿主饥饿死亡。甲硝唑、地美硝唑、替硝唑等硝基咪唑类药物被认为是预防和治疗鸽毛滴虫病最主要的药物。然而随着化学药物的使用,药物残留、药物毒副作用及耐药株的产生等问题不断出现,关于硝基咪唑药物的使用也引起了更多关注和质疑。因此,筛选具有抗T.gallinae活性的新型低毒药物来减少或替代合成药物的使用成为研究热点之一。目前,基于自然界天然产物及其衍生物的药物已占医药市场的30%以上,其中,中药以天然植物居多,因具有品种繁多、价格低廉、无污染、毒副作用小、低残留、不易产生抗药性等诸多优点而受到更多青睐。金莲花是我国常见中草药之一,其在临床上多用于治疗上呼吸道感染、咽炎、泌尿系统感染、细菌性痢疾等疾病,且对细菌、病毒、阴道毛滴虫等均有一定抑制和杀灭作用,是一种具有多种医药功效的中药。研究表明,鸡冠花不仅可用于肝损伤如肝纤维化、肝硬化和肝癌的治疗,而且,对治疗阴道毛滴虫和提高动物机能也有显著效果,是一味良好的中药。研究表明,利用单味中药之间发挥的协同作用组成中药复方对鸽毛滴虫病的治疗有显著效果。本研究首先对分离的T.gallinae体外培养体系进行建立。随后从具有杀虫、抑菌、清热解毒等作用的中药中筛选了具有抗T.gallinae活性的中药金莲花、鸡冠花及两个中药复方。然后通过活虫体计数法测定药物对T.gallinae的IC50、通过MTT法测定药物对CEF的TC50,从而确定药物的选择指数。最后对药物引起T.gallinae死亡的机理进行研究。试验具体分为以下4个部分:1.T.gallinae的分离、鉴定及体外培养本研究自患病鸽口腔分离到疑似T.gallinae的虫体,通过临床症状、病理变化、病原学诊断及分子生物学诊断确诊为T.gallinae,在含抗生素的HF培养基中传代培养后获得纯培养的T.gallinae,并对其生长曲线进行了测定,结果显示,虫体转种时以1.5×105个/m L为初始密度在HF培养基中生长良好,培养36h~60h时处于对数增长期。2.筛选具有体外抗T.gallinae活性的中药试验证明,在试验中药中,金莲花、鸡冠花、花椒、香薷水煎剂(MLCs=100mg/mL)相对其他试验药物有较高的抗T.gallinae活性,青蒿、蛇床子、白头翁水煎剂(MLCs=200mg/mL)对T.gallinae有一定抑制或杀灭作用,而苦参、千里光、荆芥、贯众、仙鹤草、常山水煎剂(MLCs"g400mg/mL)需要较大剂量才能杀灭全部虫体,对T.gallinae的作用较小。3.T.gallinae体外药敏试验及选择指数的确定利用活虫体计数法,分别测定了甲硝唑、地美硝唑、鸡冠花、金莲花及两个中药复方A和B体外不同时间、浓度对T.gallinae的相对抑制率。结果表明,本试验分离的T.gallinae对甲硝唑、地美硝唑均具有耐药性,但对地美硝唑相对更敏感。金莲花、鸡冠花、复方A和复方B体外作用48h对虫体的IC50分别为0.91、0.34、0.26、0.39mg/m L,四种药物的抗虫活性复方A㧐鸡冠花㧐复方B㧐金莲花。利用MTT法对金莲花、鸡冠花、复方A和复方B水提物体外作用48h对CEF的TC50进行测定,分别为1.91、1.80、7.06、9.09mg/mL,通过IC50和TC50确定药物的SI(TC50/IC50)。结果显示:金莲花水提物具有低选择性(SI=2.10)、鸡冠花水提物具有显著选择性(SI=5.34)、复方A(SI=27.37)和复方B水提物(SI=23.30)不具有体外细胞毒性。4.各药物造成T.gallinae死亡的机理通过药物对虫体形态、核、散射光、线粒体膜电位及凋亡的影响判断药物造成T.gallinae死亡的机理。结果表明,甲硝唑和金莲花水提物作用的T.gallinae变圆,虫体核被破坏产生凋亡小体,但膜仍保持完整,内容物不发生外溢,氢化酶体膜电位在短时间内大量变化,在死亡虫体中早期凋亡虫体占大多数,由以上结果推测两药物主要导致T.gallinae凋亡;而鸡冠花、复方A和B水提物作用的T.gallinae迅速破裂死亡、内容物外溢,虫体核变圆变大,氢化酶体膜电位在24h才开始大量变化,在大量死亡虫体中,仅少数处于早期凋亡,由以上结果推测三药物可导致T.gallinae凋亡、坏死、胀亡。
[Abstract]:Trichomonas gallinae is caused by Trichomonas gallinae. It is one of the most important diseases harming pigeons, causing serious economic losses to the pigeon industry in the world. Nitroimidazole, dimetronidazole, tinidazole and other nitroimidazoles are considered to be the most important drugs for the prevention and treatment of trichomoniasis in pigeons. However, with the use of chemical drugs, drug residues, drug side effects and the emergence of drug-resistant strains, the use of nitroimidazole drugs has also aroused more concern and doubt. Selection of new low toxic drugs with anti-T.gallinae activity to reduce or replace the use of synthetic drugs has become one of the research hotspots.At present, drugs based on natural products and their derivatives have accounted for more than 30% of the pharmaceutical market.Among them, most of the traditional Chinese medicines are natural plants, because of their variety, low price, no pollution, little toxicity and side effects. Golden Lotus is one of the most common Chinese herbal medicines in China. It is widely used in the treatment of upper respiratory tract infections, pharyngitis, urinary tract infections, bacterial dysentery and other diseases. It has certain inhibitory and killing effects on bacteria, viruses, vaginal trichomonas and so on. Studies have shown that Cockscomb can be used not only for the treatment of liver injury such as liver fibrosis, liver cirrhosis and liver cancer, but also for the treatment of Trichomonas vaginalis and the improvement of animal function. It is a kind of good Chinese medicine. Studies have shown that using the synergistic effect of single Chinese medicine to compose Chinese medicine compound prescription for pigeon hair drops. In this study, the T. gallinae isolate culture system in vitro was established, and then from the Chinese herbal medicine with insecticidal, bacteriostatic, antipyretic and detoxicating effects screened with anti-T. gallinae activity of the Chinese herbal medicine Golden Lotus, Cockscomb Flower and two Chinese herbal prescriptions. Finally, the mechanism of T. gallinae death was studied. The experiment was divided into the following four parts: 1. T. gallinae isolation, identification and in vitro culture of T. gallinae suspected from the sick pigeon oral isolates, through clinical symptoms, Pathological changes, pathogenic diagnosis and molecular biology diagnosis were confirmed as T. gallinae. T. gallinae was cultured in HF medium containing antibiotics. The growth curve of T. gallinae was measured. The results showed that the initial density of 1.5 Screening of Chinese herbal medicine with anti-T.gallinae activity in vitro showed that the decoction of Trollius chinensis, Cockscomb, Zanthoxylum bungeanum and Herba Elsholtziae (MLCs=100mg/mL) had higher anti-T.gallinae activity than other experimental drugs. Artemisia annuae, Fructus cni, Radix Pulsatillae (MLCs=200mg/mL) had certain inhibition or killing effect on T.gallinae. Metronidazole, dimetronidazole, cockscomb flower, golden lotus flower and two Chinese medicinal herbs were determined by in vitro susceptibility test and selection index of T. gallinae. The results showed that the isolated T. gallinae was resistant to metronidazole and dimetronidazole, but was more sensitive to dimetronidazole. The IC50 of Trollius chinensis, Cockscomb, Compound A and Compound B were 0.91, 0.34, 0.26, 0.39 mg/m L, respectively. Anti-insect activity of compound A? Cockscomb flower? Compound B? Golden Lotus flower. MTT method was used to determine the TC50 of CEF after 48 hours of in vitro action of water extracts from Flos Trollii, Flos Cockscomb, Compound A and Compound B, respectively, 1.91, 1.80, 7.06, 9.09 mg/mL. The SI (TC50/IC50) of the drug was determined by IC50 and TC50. The water extract of Cornus cristata had significant selectivity (SI=5.34), compound A (SI=27.37) and compound B (SI=23.30) had no cytotoxicity in vitro. 4. The mechanism of T. gallinae death was determined by the effects of drugs on the morphology, nucleus, scattered light, mitochondrial membrane potential and apoptosis of T. gallinae. Metronidazole and water extract of Trollius chinensis turned round, the nucleus of the worm was destroyed and apoptotic bodies were produced, but the membrane remained intact and the contents did not spill out. The membrane potential of hydrogenase changed a lot in a short time, and the early apoptotic bodies of the dead worms accounted for the majority. However, T. gallinae treated with water extract A and B of Compound Cockscomb and Cockscomb had a rapid rupture and death, the contents spilled out, the nucleus rounded and enlarged, and the membrane potential of hydrogenase began to change dramatically after 24 hours. Only a few of the dead insects were in the early stage of apoptosis.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.39

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