治疗奶牛乳房炎的抑菌、抗炎免疫蒙兽药成分的筛选及其作用机理研究

Based on the previous study, this study is an original innovation on the Mongolian veterinary drugs. The antibacterial and curative effect of traditional Mongolian compound medicine was equal to that of Ciprofloxacin in the treatment of mastitis; and the antibacterial effect of orthogonal designed Mongolian compound medicine in vitro was equal to that of Ciprofloxacin. Compound 2 could improve the immunity better than that of Levamisole and vaccine; and the milk ingredient and milk production was significantly improved; it was non-toxic. To determine the active components and obtain a potent antibacterial and anti-inflammatory Mongolian compound of components medicine, and its mechanism of action, the following study is designed. Four kinds of organic solvent will be extracted after ethanol extraction and to test and determine the antibacterial and anti-inflammatory effect of each of them; silica gel column chromatography will be used to separate and purify petroleum ether extracts and mass spectrum and NMR spectroscopy will be used to determine the main components. Synergy test and orthogonal design, the protection rate of antibacterial action（include biotic resistant bacterial ） in vitro and anti-inflammation in vivo and anti-inflammatory immune tests will be processed with the antibacterial active ingredients and anti-inflammatory active ingredients of these two kinds of compound mentioned above, respectively，also their mechanism. To obtain the antibacterial and anti-inflammatory immune compounds and the best effect compound of ingredients by orthogonal design. We will study the efficacy of the ingredients compound, antibacterial and anti-inflammatory，enhance-immune mechanism, recovery of milk ingredient and milk production, dosage form of compound in the case of cow mastitis to get the best antibacterial, and potent anti-inflammatory immune Mongolian compound of ingredients medicine, the substitutes of antibiotics.

这是前期研究基础上的蒙兽药原始创新研究。用传统蒙药学组成的复方在治疗乳房炎中抑菌、疗效与环丙沙星相当，而正交设计蒙药复方体外抑菌与环丙沙星相当；2复方提高免疫均优于左旋咪唑及疫苗；牛奶成分和产奶量极显著改善恢复；无毒。为确定其有效成分，获得强效抑菌抗炎免疫蒙药成分复方及其作用机理，进行以下研究。乙醇提取后用4种有机溶剂萃取，各萃取物做抑菌抗炎免疫试验、确定其作用；有效萃取物用硅胶色谱和反相高效液相色谱等分离纯化，质谱和核磁波谱确定主要成分。对上两种复方的各抑菌和抗炎免疫有效成分，分别做协同试验和正交设计后作体外抑菌和体内抗菌保护率（包括耐药菌）、抗炎免疫试验等，分别得到抑菌、抗炎免疫成分复方；再正交设计得到最佳疗效复方；研究机理。在奶牛乳房炎，研究成分复方的疗效、抑菌抗炎免疫作用；对患牛产奶量及乳成分恢复；复方的剂型研究；筛选出最佳抑菌和强效抗炎免疫的绿色蒙药成分复方，替代抗生素。

为了解前期筛选的治疗奶牛乳房炎的蒙药复方的作用机理以及筛选出最佳抑菌、抗炎免疫的绿色蒙药成分复方，分别提取其有效成分，用正交设计法分别筛选抑菌有效成分复方进行抑菌作用和机理研究、抗炎免疫有效成分复方进行抗炎免疫作用和机理研究；并将这两种成分复方的成分进一步正交设计筛选出最佳疗效的抑菌抗炎免疫成分复方进行小鼠模型乳腺炎疗效研究，毒理学研究，在乳房炎奶牛研究成分复方的疗效、抑菌抗炎免疫作用；对患牛产奶量及乳成分恢复；复方的剂型研究。结果：成功提取了有效成分复方的各成分；成功筛选出由没食子酸、盐酸小檗碱等组成的体内外抑菌有效成分复方，其作用与环丙沙星相当，作用机理是破环革兰氏阴性和阳性菌细胞壁、细胞膜而使电导率增加；成功筛选出由栀子苷、姜黄素等组成的抗炎免疫有效成分复方，其疗效接近环丙沙星，能显著提高小鼠非特异性和特异性免疫力，抗炎作用与地塞米松相近，其作用机理可能通过抑制TLR4表达而干预TLR4/NF-κB信号通路的激活发挥作用；并且上述3种成分复方和草药复方对革兰氏阴性和阳性6种病原菌均有较好的抑菌效果，抗菌谱广。成功筛选出抑菌抗炎免疫成分复方，疗效接近环丙沙星，经奶牛乳头管注射，可通过自身的抑菌特性直接抑制乳腺内细菌繁殖，可显著提高奶牛非特异性和特异性免疫力，乳腺炎症减轻，进而减少体细胞数达到对奶牛乳房炎的治疗。可刺激HPA产生一系列免疫反应，防止过度的炎症反应；极显著的提高ACTH、E、COR、T4的含量，但是抗生素抑制甲状腺素的合成，会对机体的生长发育和产奶造成一定影响，反之蒙药成分复方不仅可以促进机体生长和产奶，对抗外界不良条件时都有着积极作用。说明，蒙药成分复方可以通过神经-内分泌系统的活动提高奶牛的免疫力与生产能力。上述所有成分复方和草药急性、亚急性、慢性毒理学研究表明，使用剂量在安全范围，无毒。该项研究为蒙药及其成分复方替代抗生素，治疗奶牛乳房炎提供了依据。