奶牛子宫内膜炎病原菌分离鉴定及丹参抑菌活性成分研究
发布时间：2019-01-30 12:54
【摘要】：奶牛子宫内膜炎是临床上奶牛的常见多发疾病,主要由病原微生物感染引起,应用抗生素及化学合成类药物治疗有效,但日益严峻的病原耐药性、药物残留及环境灾害已引起社会各界的高度关注。中药多数来自天然,化学成分多样,临床疗效历经验证,业已成为新药研发的重要资源。丹参是唇形科植物丹参(Salvia miltiorrhiza Bge)的干燥根和根茎,具有活血祛瘀,通经止痛,凉血消痈的功效。现代药理研究表明,丹参具有显著的抗氧化,抗菌抗炎和改善微循环等作用。本试验进行了奶牛子宫内膜炎病原菌的分离鉴定、丹参抑菌活性部位的筛选、丹参抑菌活性部位成分的分离鉴定及其单体化合物的抑菌作用研究,取得以下结果:(1)奶牛子宫内膜炎病原菌的分离鉴定采用平板培养法从患有子宫内膜炎奶牛子宫分泌物中分离纯化病原菌,从形态学特征和分子生物学方面进行鉴定。结果表明,从患病奶牛子宫分泌物中分离纯化得到临床分离菌4种,经形态学及分子生物学鉴定为金黄色葡萄球菌、链球菌、枯草芽胞杆菌和大肠埃希氏菌O111:K58(B4)。(2)丹参抑菌活性部位的筛选采用试管预试法对丹参化学成分进行预试验,用石油醚、乙酸乙酯、正丁醇、水等溶剂制备丹参部位提取物,通过琼脂扩散法及微量肉汤法检测抑菌作用。结果表明,丹参中含有黄酮及其苷类、酚类、生物碱、氨基酸类、甾体、萜类和内酯类化合物;丹参石油醚及乙酸乙酯部位提取物对金黄色葡萄球菌、链球菌、沙门氏菌和枯草芽胞杆菌有不同程度的抑菌作用,正丁醇及水部位提取物抑菌作用不明显。说明石油醚和乙酸乙酯部位提取物是丹参的主要抑菌活性部位。(3)丹参抑菌活性部位成分的分离鉴定丹参石油醚和乙酸乙酯部位提取物,依次用不同比例的石油醚-乙酸乙酯梯度洗脱进行分离,并进行二次硅胶柱色谱和重结晶纯化,共得到6种单体化合物,经理化性质、13C-NMR和1H-NMR谱图测定分析进行鉴定结构为:丹参酮ⅡA、隐丹参酮、β-谷甾醇-D-葡萄糖苷、丹参酮Ⅰ、二氢丹参酮Ⅰ和β-谷甾醇。其中石油醚部位提取物中隐丹参酮、丹参酮ⅡA,乙酸乙酯部位提取物中丹参酮Ⅰ和二氢丹参酮Ⅰ的含量较高。(4)丹参单体化合物的抑菌作用采用琼脂扩散法和微量肉汤稀释法,对隐丹参酮、丹参酮Ⅰ、丹参酮ⅡA和二氢丹参酮Ⅰ等成分进行体外抑菌试验。结果表明:隐丹参酮、丹参酮Ⅰ、丹参酮ⅡA、二氢丹参酮Ⅰ对奶牛子宫内膜炎金黄色葡萄球菌、枯草杆菌临床分离株具有极强抑菌作用;对无乳链球菌标准株ATCC12386、大肠埃希氏菌标准株ATCC25922、大肠埃希氏菌临床分离株O111:K58(B4)及链球菌临床分离株等有不同程度的抑菌作用。
[Abstract]:Endometritis is a common disease in dairy cows, which is mainly caused by pathogenic microorganism infection. Antibiotics and chemosynthetic drugs are effective in treating dairy cows, but they are becoming more and more serious. Drug residues and environmental disasters have attracted great attention from all walks of life. Traditional Chinese medicine (TCM) has become an important resource for new drug research and development due to its natural and diverse chemical composition and proven clinical efficacy. Salvia miltiorrhiza is the dried root and rhizome of (Salvia miltiorrhiza Bge), which has the functions of activating blood and removing blood stasis, regulating menstruation and relieving pain, cooling blood and eliminating carbuncle. Modern pharmacological studies have shown that Salvia miltiorrhiza has significant antioxidant, anti-inflammatory and anti-inflammatory effects, and improve microcirculation. In this experiment, the isolation and identification of pathogenic bacteria from endometritis of dairy cows, the screening of active parts of Salvia miltiorrhiza, the isolation and identification of active components of active parts of Salvia miltiorrhiza and the bacteriostatic action of monomers were studied. The following results were obtained: (1) isolation and identification of pathogenic bacteria of dairy cow endometritis using flat plate culture method was used to isolate and purify the pathogen from the secretion of cow with endometritis, and to identify the pathogen in terms of morphological characteristics and molecular biology. The results showed that four clinical isolates were isolated from the uterine secretions of sick cows and identified as Staphylococcus aureus and Streptococcus aureus by morphological and molecular biology. Screening of active sites of Bacillus subtilis and Escherichia coli O111:K58 (B _ 4). (_ 2) Salvia miltiorrhiza. The chemical constituents of Salvia miltiorrhiza were pre-tested by the method of tube pre-test. The chemical constituents of Salvia miltiorrhiza were pretested with petroleum ether, ethyl acetate, n-butanol. The extracts of Salvia miltiorrhiza were prepared with water and the bacteriostatic effect was detected by Agar diffusion and broth method. The results showed that salvia miltiorrhiza contained flavonoids and its glycosides, phenols, alkaloids, amino acids, steroids, terpenoids and lactones. The extracts of petroleum ether and ethyl acetate of Salvia miltiorrhiza had different degree of bacteriostatic effect on Staphylococcus aureus, Streptococcus aureus, Salmonella and Bacillus subtilis, but the extracts of n-butanol and water had no obvious bacteriostatic effect. The results showed that the extracts of petroleum ether and ethyl acetate were the main antimicrobial active parts of Salvia miltiorrhiza. (3) the extracts of petroleum ether and ethyl acetate of Salvia miltiorrhiza were isolated and identified. In turn, six monomers were obtained by gradient elution of petroleum ether-ethyl acetate with different ratios, secondary silica gel column chromatography and recrystallization purification. The structures of tanshinone 鈪，
本文编号：2418150
[Abstract]:Endometritis is a common disease in dairy cows, which is mainly caused by pathogenic microorganism infection. Antibiotics and chemosynthetic drugs are effective in treating dairy cows, but they are becoming more and more serious. Drug residues and environmental disasters have attracted great attention from all walks of life. Traditional Chinese medicine (TCM) has become an important resource for new drug research and development due to its natural and diverse chemical composition and proven clinical efficacy. Salvia miltiorrhiza is the dried root and rhizome of (Salvia miltiorrhiza Bge), which has the functions of activating blood and removing blood stasis, regulating menstruation and relieving pain, cooling blood and eliminating carbuncle. Modern pharmacological studies have shown that Salvia miltiorrhiza has significant antioxidant, anti-inflammatory and anti-inflammatory effects, and improve microcirculation. In this experiment, the isolation and identification of pathogenic bacteria from endometritis of dairy cows, the screening of active parts of Salvia miltiorrhiza, the isolation and identification of active components of active parts of Salvia miltiorrhiza and the bacteriostatic action of monomers were studied. The following results were obtained: (1) isolation and identification of pathogenic bacteria of dairy cow endometritis using flat plate culture method was used to isolate and purify the pathogen from the secretion of cow with endometritis, and to identify the pathogen in terms of morphological characteristics and molecular biology. The results showed that four clinical isolates were isolated from the uterine secretions of sick cows and identified as Staphylococcus aureus and Streptococcus aureus by morphological and molecular biology. Screening of active sites of Bacillus subtilis and Escherichia coli O111:K58 (B _ 4). (_ 2) Salvia miltiorrhiza. The chemical constituents of Salvia miltiorrhiza were pre-tested by the method of tube pre-test. The chemical constituents of Salvia miltiorrhiza were pretested with petroleum ether, ethyl acetate, n-butanol. The extracts of Salvia miltiorrhiza were prepared with water and the bacteriostatic effect was detected by Agar diffusion and broth method. The results showed that salvia miltiorrhiza contained flavonoids and its glycosides, phenols, alkaloids, amino acids, steroids, terpenoids and lactones. The extracts of petroleum ether and ethyl acetate of Salvia miltiorrhiza had different degree of bacteriostatic effect on Staphylococcus aureus, Streptococcus aureus, Salmonella and Bacillus subtilis, but the extracts of n-butanol and water had no obvious bacteriostatic effect. The results showed that the extracts of petroleum ether and ethyl acetate were the main antimicrobial active parts of Salvia miltiorrhiza. (3) the extracts of petroleum ether and ethyl acetate of Salvia miltiorrhiza were isolated and identified. In turn, six monomers were obtained by gradient elution of petroleum ether-ethyl acetate with different ratios, secondary silica gel column chromatography and recrystallization purification. The structures of tanshinone 鈪，
本文编号：2418150
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