【作者】 房春林；

【导师】 杨光友；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2010， 博士

【摘要】 杨树花(Flos popul)为杨柳科(Salicaceae)植物毛白杨(Populus tomentosa)、加拿大杨(Populus canadensis)或同属树种植物干燥雄花序，性味苦寒,具有清热解毒,化湿止痢之功效。但因目前缺乏对杨树花药理、药效及毒理学等方面的系统研究,其主要活性物质的化学成分也尚不清楚,因此局限了杨树花在兽医临床上的开发利用。本研究将杨树花(毛白杨雄花序)与黄芩复方,研制了杨树花复方注射液,对其制备工艺、药理、毒理、质量标准及临床应用等方面进行了系统研究。主要研究内容和结果如下：1、采用试管法和薄层层析方法对杨树花水提液和水提液的乙酸乙酯及正丁醇萃取物的化学成分进行初步检识,结果表明,杨树花水提液中主要化学成分包括多糖、黄酮类、有机酸、强心甙、内酯及香豆素,蒽醌类化合物、酚类或鞣质；可能含有生物碱,不含有皂苷、甾醇、三萜类;乙酸乙酯萃取液中含有化学成分与水提液中基本一致；而正丁醇萃取液含有黄酮、内酯及香豆素、有机酸和鞣质。以总黄酮含量为考察指标,采用水提醇沉法和醇提法对杨树花提取工艺进行研究,并按L9(43)正交试验设计对杨树花水提醇沉工艺进行优化,优化后的提取工艺条件为：提取溶液PH为7,煎煮提取2次,提取时间为2h,醇沉过程中乙醇浓度为65%,超滤液浓度小于1.5g／mL(按原生药计),超滤温度为15-45℃,压强大于0.05Mpa。采用大孔吸附树脂和硅胶层析方法对杨树花中水杨苷进行了提取分离和纯化,结果表明,经过大孔吸附树脂和硅胶柱层析后,所得水杨苷纯度分别为24.17%和96.23%并建立了HPLC方法检测其含量,以C18色谱柱,乙腈-水(7：93)为流动相,检测波长为270nm,柱温25℃,水杨苷在0.025μg-4.0μg(r=0.9994)之间呈现良好线性关系。2、采用药敏实验、联合抗菌作用实验、抗细菌感染实验及抗炎实验方法,对杨树花提取物及复方制剂的药理作用进行了研究。结果显示,大肠杆菌、沙门氏菌的标准菌和致病菌对杨树花水提液及其复方制剂均表现出了中度敏感,大肠杆菌、沙门氏菌标准菌对杨树花水提液乙酸乙酯及正丁醇萃取物也表现出了中度敏感,但沙门氏菌致病菌对杨树花水提液乙酸乙酯及正丁醇萃取物表现为低敏；杨树花与黄芩联合抗菌具有相加作用。抗细菌感染及抗炎实验结果证实,杨树花水提液及复方制剂在体内对大肠杆菌具有抑菌作用,高、中、低剂量的杨树花水提液均可使小鼠耳肿胀度明显降低,其抑制率分别为65.0%、58.9%和51.6%,与对照组相比,差异极显著(P<0.01);同时高、中剂量组对小鼠腹腔毛细血管通透性即化学性腹膜炎具有较好的抑制作用,其抑制率分别为57.02%和53.84%,与对照组相比,差异极显著(P<0.01),而低剂量组效果不明显,其抑制率仅为5.06%。对小鼠免疫器官影响试验结果表明,杨树花单方制剂高、中、低剂量组可显著提高小鼠的脾脏及胸腺指数,与对照组相比,差异极显著(P<0.01)；表明,杨树花提取物及复方制剂具有明显的抑菌、抗炎作用。3、参照农业部新兽药一般毒性实验技术要求,对杨树花复方制剂进行了急性毒性和亚慢性毒性试验,结果表明,杨树花复方注射液LD50为46.4g/kg体重,95%可信区间为46.40±10.22g/kg,亚慢性毒性试验结果表明,高、中、低剂量三组小鼠连续28天腹腔注射给药后,一般情况、体重增长速度、血液学指标、血液生化指标与正常对照组比较均无明显差异,说明在上述剂量下(相当于临床给药剂量的50倍),杨树花注射液连续腹腔注射28天无明显毒性反应,兽医临床用药是安全的。4、通过对杨树花复方注射液制剂助溶剂、抗氧化剂的筛选及PH值范围的确定来探讨杨树花复方制剂的制备工艺,并采用留样观察法对制剂的稳定性进行考察。结果表明,以二甲基乙酰胺为助溶剂,亚硫酸钠或亚硫酸氢钠为抗氧化剂,PH范围为6.5-7.5时,制备的复方制剂稳定性好,常温(25℃)条件下可保存1年。同时对杨树花复方制剂的质量标准进行了研究,采用HPLC方法检测复方制剂中黄芩苷的含量,结果显示,此检测方法简单,结果准确。5、采用药敏试验检测从临床分离的猪源大肠杆菌的耐药性,并同时采用二重PCR方法检测从临床分离的猪源大肠杆菌的耐喹诺酮类和磺胺类药物的主要耐药基因GyrA和Sul2基因,并以此临床分离耐药细菌为受试菌,以杨树花复方制剂为耐药性消除剂进行体外耐药性消除试验,并观察消除前后细菌生长情况、细菌的最小抑菌浓度的变化。同时通过分析大肠杆菌耐药菌消除前后质粒电泳图谱的变化及耐药基因GyrA和Sul2基因的碱基和氨基酸序列变化来探讨耐药性消除机理。结果表明,临床分离的猪源大肠杆菌对恩诺沙星及磺胺嘧啶具有很强的耐药性,二重PCR方法也检测到了耐喹诺酮类和磺胺类药物的主要耐药基因GyrA和Sul2基因,表明所分离菌为耐药菌。耐药消除试验结果表明,杨树花复方制剂对耐恩诺沙星和磺胺嘧啶大肠杆菌的耐药性具有一定的消除作用,其消除率分别为19.5%和18.18%,而对照组消除率为0,经消除后的细菌生长速度明显减慢,对恩诺沙星和磺胺嘧啶的MIC值也明显降低。耐药性消除前后细菌质粒电泳图谱条带数无变化,同时GyrA和Sul2基因序列分析表明,消除前后两耐药基因的碱基及氨基酸序列均无明显变化。6、为验证杨树花复方注射液对仔猪大肠杆菌病的临床治疗效果,对人工感染仔猪大肠杆菌进行临床治疗试验。结果表明,高、中、低剂量的杨树花复方注射液对人工感染的仔猪大肠杆菌病均有较好的治疗效果,其治愈率分别为90%、90%、80%。穿心莲注射液对照组治愈率为60%。临床扩大试验结果表明,杨树花复方注射液对仔猪大肠杆菌病有效率为97.14%,治愈率为84..28%,死亡率为2.86%。表明杨树花复方注射液对仔猪大肠杆菌病具有较好的临床治疗效果。更多还原

【Abstract】 Flos populi is the dry male inflorescence of Populus canadensis,populus tomentosa or same species plant. It’s character is bitter-cold, and the pharmacological effect is clearing heat and detoxicate, dissipating dampness and zhili. But the system researches of Flos populi in pharmacology, pharmacodynamics, toxicology and the main active chemical composition are deficient. So it limits the exploitation in veterinary clinical. In this study, Flos populi compound injection composed of Flos populi and Scutellaria baicalensi was developed, and it was studied on preparation technology, pharmacology, toxicology, quality standards and clinical application. The results of research are summarized as following:1、Chemical composition of Flos populi aqueous extract、Ethyl acetate and n-butanol extract of Flos populi aqueous extract was tested by the method of test tube and thin-layer chromatography. The result showed that aqueous extract and it’s Ethyl acetate extract contained polysaccharide, flavonoids, organic acid, quinones,lactones and coumarins, phenols and tanin, it may contain alkaloids, have no terpenoid and steroid, saponins, n-butanol extract contained flavonoids, actones and coumarins, organic acid and tanin.The content of flavonoids extracted were regarded as the assessing index, the extraction technology of Flos populi was studied by water-extraction and alcohol-precipitation and alcohol extracting method. The water-extraction and alcohol-precipitation technology was optimized by orthogonal design, the optimum extraction conditions are concluded as follow:Extraction solvent PH 7, extracted 2 times,2 hours for each time,65% alcohol precipitation,ultrafiltration solvent concentration less than 1.5g/mL, ultrafiltration temperature 15-45℃, ultrafiltration pressure exceed 0.05Mp.Salicin extarcted from Flos populi could be effctively purified by macroporous adsorption resin(MAR)AB-8 and silica gel column chromatography, In result, the purity of salicin after these steps was 24.17% and 96.23% respectively. The determination method of salicin was developed by HPLC on a C18 column, The methyl cuanide-water(7:93) as mobile phase. detection wavelength was 270 nm with column temperature 25℃and flowrate 1.OmL/min. The results showed that had a goodlinearity within the range 0.025μg-4.0μg (r=0.9994).2、The pharmacological effect of Flos populi extracts and the compound preparation were studied in the drug sensitivity test, the combined antibacterial activity test, the antibacterial infection test and anti-inflammatory effect test. The results showed that Flos populi aqueous extract and the compound preparation had moderate antibacterial activity against four tested bacteria (pathogenic and standard strains of E.coli and salmonella), Ethyl acetate and n-butanol extracts of Flos populi aqueous extract also showed a moderate antibacterial activity against pathogenic and standard strains of E.coli and standard strains of salmonella.In contrast, the antibacterial activity against the pathogenic salmonella was low, Flos populi and Scutellaria baicalensis had an additive effect on the antibacterial activity against E.coli.In the antibacterial infection and anti-inflammatory effect tests,the Flos populi aqueous extract and compound preparation had antibacterial activity against E.coli in vivo, and high,moderate and low dose groups of Flos populi aqueous extract can reduced the auricular swelling in mice, and the inhibition rates were 65.0%,58.9% and 51.6%, respectively,compared with cotrol group, the different was highly significant (P<0.01). High and moderate dose groups of Flos populi aqueous extract could significantly reduce the capillary permeability of mice, the inhibition rates were 57.02% and 53.84%, respectively, compared with control group, the difference was highly significant(P<0.01). The Flos populi aqueous extract could increase the immune organ indexes of mice. In conclution, the Flos populi aqueous extract and compound preparation possessed the significant antibacterial and anti-inflammatory effects.3、Acute toxicity and chronic toxicity of the Flos populi compound preparation were studied. In result, The LD50 Of the Flos populi compound preparation in mic ewas 46.4g/kg, and the 95% confidence interval was 46.40±10.22g/kg. The mice of the high,moderate and low dose groups were intraperitoneal injection administered for 28 days continuously with Flos populi compound preparation, The results show that these differences of indexes of blood rutine and biochemical, velocit of increase of weight, organ/body coefficients and histopathological examination were no significant compared with control group. The results showed that there is no obvious toxicity to mice after 28 days intraperitoneal injection administered, Indicating that it is safe to veterinary clinical administration.4、The preparation technology and stability of Flos populi compound injection were studied by the screening of auxiliary solvent, antioxidant, the investigation of the PH extent and roomtemperature retained sample observation test.In result, The expiration time of the Flos populi compound injection developed with auxiliary solvent(dimethyl-acetylamide),antioxidant(soduium or sodium hydrogen sulfite) was tentatively set on 1 years at 25℃.,the PH extent of injection is 6.5-7.5.The quality Standard of Flos populi compound injection was also studied in the test, establishing a method of HPLC to determinate the content of Baicalin in the injection. The results showed that these methods established was accurate and reliable for the quality control5、To study the eliminating effect and mechanism of drug resistance of Flos populi compound preparation against E.coli, the drug resistance E.coli isolated from clinical samples were detected by the drug sensitivity test and duplex PCR to detected the Quinolones and Sulfonamides-resistance genes (GyrA and Sul2), And the experiment of drug resistance elimination was performed in vitro with the drug resistance E.coli isolated from clinical samples as the target bacteria and Flos populi compound preparation as eliminating agent,Meanwhile the mechanism of the drug resistance elimination was detected by the analysis of the plasmid profile of E.coli, the sequence of gene (GyrA and Sul2) before and after eliminating action. Minimum Inhibitory Concentration (MIC) of Enrofloxacin and Sulfadiazine against E.coil was detected before and after eliminating action.The results showed that the E.coli isolated from clinical samples were high-level resistance, the drug resistance elimination rate of enrofloxacin、sulfadiazine-resistance and the control group was 19.5%、18.18% and 0, respectively. The growth rate of E.coli eliminated was step down. The counts of plasmid profile strap of E.coli and the genes (GyrA and Sul2) sequence were no change before and after eliminating test of drug resistant. In concluded, Flos populi compound preparation has the eliminating effect on the drug resistant of E.coli. The mechanism of eliminating action on enrofloxacin、sulfadiazine-resistance may not be the change in the drug resistance genes.6、To evaluate the clinical therapeutic effect of Flos populi compoud injection on swine infected with Escherichia coli artificially. The cure rate of Flos populi compoud injection of high,moderate and low dose groups were 90%、90%、80%, respectively. The cure rate of Audrographini control group was 60%.The results of clinical scale-up test showed that the total effective rate of Flos populi compoud injection was 97.14%, The cure rate was 84..28%, the Mortality was 2.86%.It was concluded that the clinical therapeutic effect Flos populi compoud injection was obvious.更多还原