【摘要】：奶牛子宮內(nèi)膜炎是臨床上奶牛的常見多發(fā)疾病,主要由病原微生物感染引起,應(yīng)用抗生素及化學合成類藥物治療有效,但日益嚴峻的病原耐藥性、藥物殘留及環(huán)境災(zāi)害已引起社會各界的高度關(guān)注。中藥多數(shù)來自天然,化學成分多樣,臨床療效歷經(jīng)驗證,業(yè)已成為新藥研發(fā)的重要資源。丹參是唇形科植物丹參(Salvia miltiorrhiza Bge)的干燥根和根莖,具有活血祛瘀,通經(jīng)止痛,涼血消癰的功效�，F(xiàn)代藥理研究表明,丹參具有顯著的抗氧化,抗菌抗炎和改善微循環(huán)等作用。本試驗進行了奶牛子宮內(nèi)膜炎病原菌的分離鑒定、丹參抑菌活性部位的篩選、丹參抑菌活性部位成分的分離鑒定及其單體化合物的抑菌作用研究,取得以下結(jié)果:(1)奶牛子宮內(nèi)膜炎病原菌的分離鑒定采用平板培養(yǎng)法從患有子宮內(nèi)膜炎奶牛子宮分泌物中分離純化病原菌,從形態(tài)學特征和分子生物學方面進行鑒定。結(jié)果表明,從患病奶牛子宮分泌物中分離純化得到臨床分離菌4種,經(jīng)形態(tài)學及分子生物學鑒定為金黃色葡萄球菌、鏈球菌、枯草芽胞桿菌和大腸埃希氏菌O111:K58(B4)。(2)丹參抑菌活性部位的篩選采用試管預試法對丹參化學成分進行預試驗,用石油醚、乙酸乙酯、正丁醇、水等溶劑制備丹參部位提取物,通過瓊脂擴散法及微量肉湯法檢測抑菌作用。結(jié)果表明,丹參中含有黃酮及其苷類、酚類、生物堿、氨基酸類、甾體、萜類和內(nèi)酯類化合物;丹參石油醚及乙酸乙酯部位提取物對金黃色葡萄球菌、鏈球菌、沙門氏菌和枯草芽胞桿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顯。說明石油醚和乙酸乙酯部位提取物是丹參的主要抑菌活性部位。(3)丹參抑菌活性部位成分的分離鑒定丹參石油醚和乙酸乙酯部位提取物,依次用不同比例的石油醚-乙酸乙酯梯度洗脫進行分離,并進行二次硅膠柱色譜和重結(jié)晶純化,共得到6種單體化合物,經(jīng)理化性質(zhì)、13C-NMR和1H-NMR譜圖測定分析進行鑒定結(jié)構(gòu)為:丹參酮ⅡA、隱丹參酮、β-谷甾醇-D-葡萄糖苷、丹參酮Ⅰ、二氫丹參酮Ⅰ和β-谷甾醇。其中石油醚部位提取物中隱丹參酮、丹參酮ⅡA,乙酸乙酯部位提取物中丹參酮Ⅰ和二氫丹參酮Ⅰ的含量較高。(4)丹參單體化合物的抑菌作用采用瓊脂擴散法和微量肉湯稀釋法,對隱丹參酮、丹參酮Ⅰ、丹參酮ⅡA和二氫丹參酮Ⅰ等成分進行體外抑菌試驗。結(jié)果表明:隱丹參酮、丹參酮Ⅰ、丹參酮ⅡA、二氫丹參酮Ⅰ對奶牛子宮內(nèi)膜炎金黃色葡萄球菌、枯草桿菌臨床分離株具有極強抑菌作用;對無乳鏈球菌標準株ATCC12386、大腸埃希氏菌標準株ATCC25922、大腸埃希氏菌臨床分離株O111:K58(B4)及鏈球菌臨床分離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，

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