體外抗鴿源禽毛滴蟲中藥的篩選及其作用機理研究
發(fā)布時間：2018-09-04 08:29
【摘要】：鴿毛滴蟲病是由鞭毛蟲禽毛滴蟲(Trichomonas gallinae)引起的,是危害鴿子最重要的疾病之一,給世界養(yǎng)鴿業(yè)造成了嚴重的經(jīng)濟損失。T.gallinae通常寄生于各種鳥類上消化道導(dǎo)致黏膜炎癥、潰瘍,甚至因肉芽腫阻塞食道而造成宿主饑餓死亡。甲硝唑、地美硝唑、替硝唑等硝基咪唑類藥物被認為是預(yù)防和治療鴿毛滴蟲病最主要的藥物。然而隨著化學(xué)藥物的使用,藥物殘留、藥物毒副作用及耐藥株的產(chǎn)生等問題不斷出現(xiàn),關(guān)于硝基咪唑藥物的使用也引起了更多關(guān)注和質(zhì)疑。因此,篩選具有抗T.gallinae活性的新型低毒藥物來減少或替代合成藥物的使用成為研究熱點之一。目前,基于自然界天然產(chǎn)物及其衍生物的藥物已占醫(yī)藥市場的30%以上,其中,中藥以天然植物居多,因具有品種繁多、價格低廉、無污染、毒副作用小、低殘留、不易產(chǎn)生抗藥性等諸多優(yōu)點而受到更多青睞。金蓮花是我國常見中草藥之一,其在臨床上多用于治療上呼吸道感染、咽炎、泌尿系統(tǒng)感染、細菌性痢疾等疾病,且對細菌、病毒、陰道毛滴蟲等均有一定抑制和殺滅作用,是一種具有多種醫(yī)藥功效的中藥。研究表明,雞冠花不僅可用于肝損傷如肝纖維化、肝硬化和肝癌的治療,而且,對治療陰道毛滴蟲和提高動物機能也有顯著效果,是一味良好的中藥。研究表明,利用單味中藥之間發(fā)揮的協(xié)同作用組成中藥復(fù)方對鴿毛滴蟲病的治療有顯著效果。本研究首先對分離的T.gallinae體外培養(yǎng)體系進行建立。隨后從具有殺蟲、抑菌、清熱解毒等作用的中藥中篩選了具有抗T.gallinae活性的中藥金蓮花、雞冠花及兩個中藥復(fù)方。然后通過活蟲體計數(shù)法測定藥物對T.gallinae的IC50、通過MTT法測定藥物對CEF的TC50,從而確定藥物的選擇指數(shù)。最后對藥物引起T.gallinae死亡的機理進行研究。試驗具體分為以下4個部分:1.T.gallinae的分離、鑒定及體外培養(yǎng)本研究自患病鴿口腔分離到疑似T.gallinae的蟲體,通過臨床癥狀、病理變化、病原學(xué)診斷及分子生物學(xué)診斷確診為T.gallinae,在含抗生素的HF培養(yǎng)基中傳代培養(yǎng)后獲得純培養(yǎng)的T.gallinae,并對其生長曲線進行了測定,結(jié)果顯示,蟲體轉(zhuǎn)種時以1.5×105個/m L為初始密度在HF培養(yǎng)基中生長良好,培養(yǎng)36h~60h時處于對數(shù)增長期。2.篩選具有體外抗T.gallinae活性的中藥試驗證明,在試驗中藥中,金蓮花、雞冠花、花椒、香薷水煎劑(MLCs=100mg/mL)相對其他試驗藥物有較高的抗T.gallinae活性,青蒿、蛇床子、白頭翁水煎劑(MLCs=200mg/mL)對T.gallinae有一定抑制或殺滅作用,而苦參、千里光、荊芥、貫眾、仙鶴草、常山水煎劑(MLCs"g400mg/mL)需要較大劑量才能殺滅全部蟲體,對T.gallinae的作用較小。3.T.gallinae體外藥敏試驗及選擇指數(shù)的確定利用活蟲體計數(shù)法,分別測定了甲硝唑、地美硝唑、雞冠花、金蓮花及兩個中藥復(fù)方A和B體外不同時間、濃度對T.gallinae的相對抑制率。結(jié)果表明,本試驗分離的T.gallinae對甲硝唑、地美硝唑均具有耐藥性,但對地美硝唑相對更敏感。金蓮花、雞冠花、復(fù)方A和復(fù)方B體外作用48h對蟲體的IC50分別為0.91、0.34、0.26、0.39mg/m L,四種藥物的抗蟲活性復(fù)方A㧐雞冠花㧐復(fù)方B㧐金蓮花。利用MTT法對金蓮花、雞冠花、復(fù)方A和復(fù)方B水提物體外作用48h對CEF的TC50進行測定,分別為1.91、1.80、7.06、9.09mg/mL,通過IC50和TC50確定藥物的SI(TC50/IC50)。結(jié)果顯示:金蓮花水提物具有低選擇性(SI=2.10)、雞冠花水提物具有顯著選擇性(SI=5.34)、復(fù)方A(SI=27.37)和復(fù)方B水提物(SI=23.30)不具有體外細胞毒性。4.各藥物造成T.gallinae死亡的機理通過藥物對蟲體形態(tài)、核、散射光、線粒體膜電位及凋亡的影響判斷藥物造成T.gallinae死亡的機理。結(jié)果表明,甲硝唑和金蓮花水提物作用的T.gallinae變圓,蟲體核被破壞產(chǎn)生凋亡小體,但膜仍保持完整,內(nèi)容物不發(fā)生外溢,氫化酶體膜電位在短時間內(nèi)大量變化,在死亡蟲體中早期凋亡蟲體占大多數(shù),由以上結(jié)果推測兩藥物主要導(dǎo)致T.gallinae凋亡;而雞冠花、復(fù)方A和B水提物作用的T.gallinae迅速破裂死亡、內(nèi)容物外溢,蟲體核變圓變大,氫化酶體膜電位在24h才開始大量變化,在大量死亡蟲體中,僅少數(shù)處于早期凋亡,由以上結(jié)果推測三藥物可導(dǎo)致T.gallinae凋亡、壞死、脹亡。
[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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.39
本文編號：2221548
[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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.39
【參考文獻】
相關(guān)期刊論文 前3條
1 張瓊;黃慧聰;梁韶暉;潘長旺;;苦參對陰道毛滴蟲蛋白質(zhì)差異表達的影響[J];中國病原生物學(xué)雜志;2009年03期
2 蘆殿梅,胡孝素,馬瑩,王雅靜,陳建平;雙氫青蒿素對杜氏利什曼原蟲前鞭毛體作用體外實驗研究[J];中國寄生蟲病防治雜志;1999年01期
3 趙立子;魏建和;;中藥荊芥最新研究進展[J];中國農(nóng)學(xué)通報;2013年04期
，本文編號：2221548
本文鏈接：http://sikaile.net/yixuelunwen/dongwuyixue/2221548.html
最近更新
教材專著
