【作者】 刘勇；

【导师】 陈进军；

【作者基本信息】 广东海洋大学 ， 动物营养与饲料科学， 2010， 硕士

【摘要】 苦地胆(Elephantopus scaber Linn)是一种分布广泛、来源丰富的民间传统中草药。相关研究发现,苦地胆内酯是苦地胆的主要有效成分。本文作者所在实验室曾根据苦地胆内酯的相关化学性质,试制了苦地胆注射液,发现其具有很好的抗鸡体内大肠埃希氏菌感染的效果。然而作为苦地胆有效成分,苦地胆内酯的抗菌效果及其抗菌机理研究,尚未见报道。本文从苦地胆的化学成分分析着手,采用80%乙醇热回流浸提,制备苦地胆内酯粗提物,经硅胶柱层析分离、纯化,再结晶,制得苦地胆内酯类化合物,用薄层层析法进行苦地胆内酯检测与鉴定,并进行了苦地胆内酯类化合物的体外抑菌效果及机理研究。主要研究结果如下:1.采用试管分项提取法和圆形纸层析法进行了苦地胆全草植物化学预试,结果显示,苦地胆全草含有内酯、三萜、黄酮及其甙类、氨基酸、多糖及甙类、皂甙、酚类、挥发油、有机酸、甾醇、强心甙、香豆素及其甙类等物质,不含生物碱、蒽醌及其甙类等物质。2.采用薄层层析法,以石油醚(60℃～90℃)︰氯仿︰乙醚(3︰5︰2)作为展开剂,异羟肟酸铁试剂作为显色剂,可以很好的分离、鉴定苦地胆内酯类化合物中的四个内酯成分,Rf值分别为0.16、0.33、0.64和0.85。3.体外抗菌试验表明:当苦地胆内酯类化合物浓度为2mg/mL时,对巴氏杆菌(Pasteurella multocida, PM)、蜡状芽孢杆菌(Bacillus cereus, BC)具有明显的抑制作用(P<0.05),对金黄色葡萄球菌(Staphylococcus aureus, SA)、肠炎沙门氏菌(Salmonella enteritidis, SE)和鸡大肠埃希氏菌(Colibacillosis in chicken, CC)呈现极明显的抑制作用(P<0.01);浓度分别为10mg/mL、50mg/mL时,苦地胆内酯类化合物对5种供试菌均有极明显的抑制作用(P<0.01)。苦地胆内酯类化合物对供试菌SA、SE、PM、CC、BC的最小抑菌浓度(minimal inhibitory concentration, MIC)分别为0.313mg/mL、0.313mg/mL、0.156mg/mL、0.625mg/mL和1.25mg/mL;对供试菌BC的最小杀菌浓度(minimal bactericidal concentration, MBC)为2.5mg/mL,对其它4种供试菌的MBC均为0.625mg/mL。4.温度对苦地胆内酯类化合物抗菌活性的影响试验表明:当环境温度超过60℃时,苦地胆内酯类化合物的抗菌活性明显降低。5.苦地胆内酯类化合物对细菌作用的时间、浓度与抑(杀)菌效果的关系试验表明:苦地胆内酯类化合物对细菌作用效果的关键因素是浓度。0.5倍MIC浓度的苦地胆内酯类化合物对供试菌的抑菌作用维持时间较短;1倍MIC浓度和2倍MIC浓度的苦地胆内酯类化合物能更长时间地维持较高抑菌率;2倍MIC浓度的苦地胆内酯类化合物具有杀菌作用。6.苦地胆内酯类化合物对细菌生长曲线的影响试验表明:苦地胆内酯类化合物主要在细菌的对数生长期发挥抑制作用,能抑制细菌分裂增殖。7.通过扫描电镜和透射电镜观察苦地胆内酯类化合物对细菌超微结构的影响,发现苦地胆内酯类化合物对细菌的抑杀机理是破坏细菌的部分细胞壁,改变细胞膜的通透性,导致细胞组分丧失、蛋白质变性,释放出溶菌酶导致细菌自溶。更多还原

【Abstract】 Elephantopus scaber Linn is a kind of traditional Chinese herbal medicine which is widespread and abundant. The present studies found that lactones are the primary pharmacological effective compounds of E. scaber. Our laboratory has preparaed injection from E. scaber according to the chemical properties of the lactones, and found that the E. scaber injection showed markedly curative effects in chickens infected with Escherichia coli. Nevertheless, there has not been reported about the antibacterial effects and the mechanism of the purified extract of lactones from E.scaber (PELES) so far.Taking the analyses of chemical constituents of E.scaber as the first step, the crude extract from E.scaber was prepared with 80% (v/v) ethanol and followed by purification with silica gel column chromatography and crystallization in this thesis. More importantly, the PELES was detemined and identified by a novel way of thin layer chromatography (TLC) and the antibacterial effects in vitro and the antibacterial mechanism were studied, respectively. The main findings of the thesis were as follows:1. It was found by subentry extracting trial in cuvette and round paper chromatographic trial, that the whole plant of E. scaber herb contained lactone, amino acids, polysaccharide, saponins, naphtha, organic acid, sterol, triterpene, cardiac glycoside, coumarin, flavone, naphthol but no alkaloid and anthraquinone.2. The PELES was determined and identified by TLC, with the developing solvent of petroleum ether (60℃～90℃)︰chloroform︰ether = 3︰5︰2 and the colour developing agent of Ferrum hydroxamic acid, and four specific spots were found with the volumes of Rf at 0.16, 0.33, 0.64 and 0.85, respectively.3. The bacteriostatic test in vitro of PELES was conducted by agar diffusion test, and the results showed that 2.0 mg/mL of PELES inhibited the growthes of Pasteurella multocida (PM) and Bacillus cereus (BC) (P<0.05) and significantly inhibited the growthes of Staphylococcus aureus (SA), Salmonella enteritidis (SE) and Colibacillosis in chicken (CC) (P<0.01), respectively, that PELES at 10 mg/mL and 50 mg/mL significantly inhibited the growthes of all the five strains of bacteria used (P<0.01), that the minimum inhibitory concentrations (MIC) of PELES was 0.313 mg/mL on SA and SE and 0.156 mg/mL on PM, 0.625 mg/mL on CC, and 1.25 mg/mL on BC, and that the minimum bactericidal concentrations (MBC) of PELES on BC was 2.5 mg/mL while the MBC on the other four bacteria used was 0.625 mg/mL.4. As to the effect of temperature to the antibacterial activity of PELES, it was found that PELES was a kind of low heat-resistent substance when the ambient temperature was above 60℃, the antibacterial activity became rather poor.5. With regard to the relationship among the concentration of PELES-time-antibacterial effect, it was found that the key factor influencing the antibacterial effect of PELES was the concentration of PELES. I.e., half MIC of PELES showed antimicrobial effect but maintained a shorter period. And 1 or 2 times of MIC of PELES kept longer inhibition effects, respectively, while 2 times of MIC of PELES sterilized the bacteria used.6. It was found by the test of the impact of PELES on the bacterial growth curve, that PELES suppressed the logarithmic growth of the bacteria, respectively, which means that the bacterial proliferation was inhibited.7. The observation of the affects of PELES to the bacterial ultrastructure by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed that the killing mechanism of PELES onto bacteria was to destroy part of the cell wall and the membrane permeability of the bacteria, leading to the loss of the bacterial cellular components, proten denaturalization and bacterial autolysis induced by release of lysozymei更多还原