蜂胶渣提取物对青枯劳尔氏菌的抑制活性鉴定

Study on Inhibitory Activity of Propolis Residue Extracts Against Ralstonia solanacearum

青枯劳尔氏菌（Ralstonia solanacearum）是引发桑青枯病的病原细菌。为了寻求防治桑青枯病的天然药剂,以工业提取黄酮化合物剩余的蜂胶渣为原料,经甲醇、乙醇、乙酸乙酯、氯仿及石油醚等溶剂分级浸提获得除蜡蜂胶渣提取物。通过比浊法测试不同极性溶剂提取物对青枯劳尔氏菌5个生理小种菌株均有一定的抑制作用（抑菌率8.36%～99.73%）,其中乙醇提取物的抑菌活性显著高于其他溶剂的提取物（P〈0.05）。0.1 mg/m L除蜡蜂胶渣乙醇提取物对青枯劳尔氏菌RS-5菌株的抑制率达91.61%±4.75%,最低抑菌浓度为0.25 mg/m L,最小杀菌浓度为4 mg/m L,用扫描电子显微镜观察到部分病原细菌发生变形和细胞壁破损,用平板法监测到菌体的泳动性能受到不同程度抑制。基于负离子全扫描模式的LC-MS谱图鉴定除蜡蜂胶渣中主要含有咖啡酸及其酯类化合物（包括咖啡酸丙酯、咖啡酸丁酯、咖啡酸苯乙酯及咖啡酸戊酯）,采用选择性反应监测模式外标法检测出其中所含咖啡酸和咖啡酸苯乙酯的质量比为1∶3.3。研究结果表明,除蜡蜂胶渣的乙醇提取物对青枯劳尔氏菌具有良好的抑制活性,发挥抑菌活性的主要成分可能是咖啡酸及其酯类衍生物,该提取物有进一步开发为天然来源的桑青枯病防控药剂的潜力。

Ralstonia solanacearum is the pathogenic bacterium that causes mulberry bacterial wilt disease. In order to seek natural agents that can prevent and control this disease,dewaxed propolis residues after industrial extraction of flavonoids were chosen as the research objects. They were fractionally extracted with methanol,ethanol,ethyl acetate,chloroform and petroleum ether as solvent,respectively. The inhibitory effect of propolis residue extracts against plant bacterial wilt pathogen was determined by turbidimetry. Results indicated that the extracts of different polar solvents all have certain antibacterial effect（ inhibition rates 8. 36%-99. 73%）on five physiological strains of Rastonia solanacearum.The antibacterial effect of ethanol extract was significantly higher than that of other solvents（ P〈0. 05）. Inhibition rate of0. 1 mg/m L ethanol extract of propolis residues to Rastonia solanacearum RS-5 strain was about 91. 61%±4. 75%.The minimum inhibitory concentration（ MIC） and minimum bactericidal concentration（ MBC） were 0. 25 mg/m L and 4 mg/m L,respectively. Some of the treated bacteria were deformed and the cell walls were broken as observed under scanning electron microscope. Swimming mo-bility performance of bacteria was inhibited for some degree as reveled by plating method. Ingredients of de-waxed propolis residues identified by LC-MS based on negative ion full scan mode mainly contain caffeic acid and esters,including propyl caffeate,butyl caffeate,caffeic acid phenethyl ester and amyl caffeate. The mass ratio of caffeic acid to caffeic acid phenethyl ester was 1 ∶ 3. 3 as measured in the selective reaction monitoring mode using external standard method.The results show that caffeic acid and its ester derivatives from the ethanol extract of dewaxed propolis residues have good inhibitory activity against R. solanacearum,which provides an innovative way for further development of prevention and control pharmaceuticals against bacterial wilt from natural source.