| Chicken colibacillosis is a general term for various infections diseases, which is caused by some serotypes of pathogenic Escherichiacoli. Its clinical manifestations are variety of symptoms, commonly including acute septicemia, airsacculitis, perihepatitis, pericarditis, yolk, peritonitis, enteritis, omphalitis and swollen head syndrome. It is accountable for huge economic losses for the poultry industry every year due to high morbility and mortality. Now people often use antibacterial agents, Chemical antibiotics and vaccines to cure or prevent this disease, which has highlighted problems such as serious drug residues, antibiotic resistance and a variety of pathogenic Escherichia coli serotype restriction vaccines commonly used. In order to develop new drugs with low cost, good curative effect and low residue, under the guidance of the basic theories of TCM, the study tried to make reasonable adjustments and modified, then grouped formation "modified Pulsatilla ". Then use modern methods of extraction to extract active ingredient in the prescription In this process, extact yield of effctive parts, minimal inhibitory concentration and minimum bactericidal concentration of clinical isolates of Chicken Escherichia coli, contents of aesculetin and berberine hydrochloride were selected as indexes. At last, the best combination was obtained. On the basis of this, the effctive parts of Compound Modified Pulsatilla was used for the preparation of granules. Then, the quality standards of the granules were established. And finally the clinical efficacy of the granules of chicken colibacillosis was investigated. The main results are as follows:1. Single factor test was used to take extraction solvent as single-factor study. Extact yield of effctive parts, minimal inhibitory concentration and minimum bactericidal concentration of clinical isolates of Chicken Escherichia coli, contents of aesculetin and berberine hydrochloride were investigated by reflux condense, agar dilution method and reverse phase high-performance liquid chromatography. Results showed that, while the solvent of concentration of ethanol was30%,50%,60%,70%and95%respectively. Extraction ratio was20.93%、23.02%、22.08%、22.44%and18.34%respectively; MIC of clinical isolates of Chicken Escherichia coli was3.270mg·mL-1,3.597mg·mL3.450mg·mL-1,3.506mg·mL-1and5.731mg·mL-1respectively; MBC of clinical isolates of Chicken Escherichia coli was13.080mg·mL-1,7.194mg·mL-1,6.900mg·mL-1,7.012mg·mL-1and5.731mg·mL-1respectively; Contents of aesculetin was0.04%,0.08%,0.10%,0.11%and0.07%respectively; Contents berberine hydrochloride was0.19%,0.45%,0.37%,0.55%and0.38%respectively. The results showed that all indexes were obvious better than30%and95%of concentration of ethanol, when taking50%,60%and70%of concentration of ethanol as extraction solvent. So50%,60%and70%of concentration of ethanol were selected to screen the best extraction techonogy by orthogonal design in the next step.2. In order to screen the best extraction techonogy, the orthogonal design table L9(34) was used. Soild-liquid ratio (A), extraction time(B), each time of extraction (C) and concentration of ethanol (D) were selected as the four factors and extact yield of effctive parts, MIC and MBC of clinical isolates of Chicken Escherichia coli, contents of aesculetin and berberine hydrochloride were used as idexes by reflux condense, agar dilution method and reverse phase high-performance liquid chromatography. The best extraction techonogy A3B3C1D2, which meaned that soild-liquid ratio was1:10, extraction time was3, spent half an hour each time and the concentration of ethanol was60%, was identified by analysis of orthogonal experiment. Nuder the optimum technology, the rate of complex prescription extraction was24%, MIC and MBC of clinical isolates of Chicken Escherichia coli both were3.750mg·mL-1, and the contents of aesculetin and erberine hydrochloride was0.13%and0.57%respectively.3. Adjuvant material and the nature of various aspects of Modified Pulsatilla granule were evaluated. In order to screen the appropriate proportion of liquid(the concentration was2gram crude drug each milliliter) and accessories, the preparation method of granule was used. The results showed that the quality of the soft material and the appearance of the granule were both meet the standards of Pharmacopoeia,when the proportion of liquid and accessories was1:5. Then, taking the appearance, clarity and water content as idexes and the amount of soluble starch, a-lactse, dextin and the different concentration of HPMC as factors, to evaluate the optimum formula. The results showed that The optimal preparation condition of "Modified Pulsatilla" granule as follow:content of soluble starch, a-lactse and dextin was60%,20%and20%respectively, when0.3%HPMC was used as adhesives. Under the optimum formula, the nature of various aspects of Modified Pulsatilla as follow:the granule color was uniform, unqualified was less, clarity was good and the water content was2.90%. Indexes above were all comply with the quality requirements of ganule of chinese medcine.4. Established the method of determination contents of aesculetin and berberine hydrochloride in "Modified Pulsatilla" granule by SH MADZU LC-2010C HT (CLASS-VP6.13multi).of reverse phase high-performance liquid chromatography. Methanol was used as solvent of the granule of "Modified Pulsatilla" Chromatographic conditions of aesculetin were these that Ture Kromasil C18column(4.6mm×250mm,5μm) with acetonitrile-0.1%phosphoric acid (12:88, v/v) mixture as the mobile phase was adopted, flow rate was1.1mL·min-1, the detecting wavelength was set at334nm, the column temperature was25℃and the sample size was10μL. Results showed that a good linear relationship emerged between the range of21.60μg·mL-1～129.60μg·mL-1of the concentration of esculetin (r=0.9997, n=6), the average recovery rate was98.27%and RSD was1.16%(n=6); Chromatographic conditions of berberine hydrochloride were these that Ture Kromasil C18column(4.6mm×250mm,5μm) with acetonitrile-0.03mol·L-1patassium dihydrogen phosphate (30:70, v/v) mixture as the mobile phase was adopted, flow rate was1.0mL·min-1, the detecting wavelength was set at265nm, the column temperature was30℃and the sample size was10μL.Results showed that a good linear relationship emerged between the range of93.00μg·L-1～186.0μg·mL-1of the concentration of berberine hydrochloride (r=0.9997, n=6), the average recovery rate was99.49%and RSD was0.76%(n=6). Though measuring, results showed that average aontents of aesculetin and berberine hydrochloride in "Modified Pulsatilla" was0.36mg·g-1and2.90mg·g-1respectively According to these above, Results indicate that the established approache is simple, sensitive and applicable.5. Studied on the therapeutic effect on chicken colibacillosis of "Modified Pulsatilla" granule. Selected some sick chickens, which were suspected of being infected colibacillosis, after diagnose, randomly selected150sick chickens which were divided into5groups, each group had30sick chickens. Ⅰ group was low-dose group (the proportion of granule mass and feed mass was2%); Ⅱ group was middle-dose group (the proportion of granule mass and feed mass was4%); Ⅲ group was high-dose group (the proportion of granule mass and feed mass was8%); Ⅳ group was control group which had1%sulfamonomethoxine; Ⅴ group was the blank group which had no drugs but only feed. After7days, the effect was preliminary assessed. The results showed that the ratio of no treatment was76.7%of the blank group, the ratio of no treatment was43.3%of the sulfamonomethoxine group, the ratio of no treatment was56.7%of the low-dose group, the ratio of no treatment was40%of the middle-dose group and the ratio of no treatment was36.7%of the high-dose group. Those above meaned that condition of the blank group had no improvement and condition of all other group had improvement. Results indicate that the granule of "Modified Pulsatilla" has effect on chicken colibacillosis and the best addition is4%in the feed. |
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