| Mycoplasma gallisepticum (MG) was a primary pathogen causing chronic respiratory disease (CRD) in chicken, and MG infection could lead to high unhealthy chicken rates, dropping egg production, lower weight, prolonged period of onset to market. So it may result in economic losses in the poultry industry. Compound Manhukang was a pharmaceutical preparation used for the treatment of upper respiratory infection disease caused by MG infection. The compound medicine was composed of five Chinese medicinal herbs i.e. Scutellariae Radix, Lonicerae japonicae Flos, Stemonae Radix. Asleris radix et rhizome and Glycyrrhiza Radix. The medicine had sufficient commercial potential because it had a definitely curative effect, without poison and side effect. As the complex chemical constituents in Manhukang and the unknown active substance in vivo, studying on the action mechanism, the process of biotransformation and metabolism would be aimLess, which also had severely constrainted the modern research of Manhukang, and resulted in the low export rate.The effective substances of Manhukang should be revealed by using the method of Serum pharmaco-chemistry and spectrum-effect combination. On this basis, a quality consistency assessment was developed based on quantitative determination of the effective substances and qualitative fingerprinting analysis. It has an actual sense of not only spreading the culture of traditional medicine, but also promoting the investigation and development, serving the human beings and transforming it into commodity’s advantages, which consequently advance economy development rapidly in local region.By using the method of Serum pharmaco-chemistry of Chinese Medicine, the analysis of constituents in blood was been carried out after oral administration of Manhukang extract. According to HPLC fingerprint and anti-mycoplasma activity test, grey relational analysis was applied to find the active compositions. On this basis, quantitative determination of active compositions was carried out with HPLC method as a supplement for quality control of Manhukang. And we have already achieved such achievement as follows:1. The establishment of Manhukang fingerprint and studies on pattern classification HPLC was introduced to analyze Manhukang samples from different batches. Mobile phase, chromatographic column and detected wavelength etc. were optimized to complete the systemic methodology and establish the HPLC fingerprint of Manhukang. Moreover, the similarity evaluation of chromatograms was processed by systematically comparing chromatograms with a professional analytical software recommended by State Food and Drug Administration. The quantitative fingerprint characteristic was treated with cluster analysis and principal component analysis with the help of SPSS11.5software, which proved to be consistent with the similarity evaluation. In order to identify the origins of these characteristic peaks from each herb, a comparative study was carried out between Manhukang and individual herbs. So the correlation between fingerprint of the preparations and that of their raw herbs was also examined in this study. Actually, the determined samples just need to be analyzed with the chromatographic conditions described above, and input the quantitative fingerprint characteristic of the samples into computer to calculate, then the classified results would be quickly acquired, which has both academic significance and applied value.2. Study on Serum pharmaco-chemistry of ManhukangBy using the established analysis method of HPLC-DAD, the analysis method of serum pharmaco-chemistry was established after optimizing the pretreatment condition of plasma. From a comprehensive analysis of the HPLC-DAD chromatography of Manhukang, controlled plasma and dosed plasma, the constituents in blood were analyzed at different time point. It can be seen that only No.11peak was more obvious at15min after oral administration. Then30min after oral administration, major essential components were detected. After an hour, six new components were detected and some components disappeared. The No.2peak appeared at120min after oral administration. By using the method of Serum pharmaco-chemistry of Chinese Medicine, the majority of common peaks in the fingerprint were absorbed into serum. They were the main material basis for the treatment of upper respiratory infection disease.3. Study on the correlation between characteristic of Manhukang fingerprint and pharmacodynamicsGlycyrrhiza Radix had no contribution to the common peaks of the fingerprint, so rounding it, the other herbs had been combined into nine groups of samples. At the premise of the acquired characteristic of fingerprint and pharmacodynamic data, grey relational analysis was selected to show what the constituents represented by fingerprint contributed to pharmaeodynamies. It could be concluded that effects were the total results of chemical constituents group in Manhukang. According to the relational grade, chlorogenic acid, baicalin, wogonoside and an unknown component had great contribution to anti-mycoplasma activity.4. Simultaneous determination of chlorogenic acid, baicalin and wogonoside in Manhukang by high performance liquid chromatography-DAD method A high performance liquid chromatographic method was developed to simultaneously determine chlorogenic acid, baicalin and wogonoside based on the chromatographic conditions of fingerprinting. The results show that RSD of the test results in the methodological study were less than3%, indicating that the instrument precision, reproducible, recovery and stability of the method were credible. The content of the three components were0.463±0.009,2.42±0.06,0.97±0.02, respectively. The establishment of content determination method could supply the deficiency of fingerprint in quantitative control. The efficient quality control method would be established combinative method using HPLC quantitative and qualitative analyses.5. Optimization of extraction of Manhukang by response surface methodologyThe extraction process of Manhukang was optimized with extraction temperature, extraction time and ratio of water to material as independent variables, and the overall desirability value of the yield of chlorogenic acid, baicalin and wogonoside as evaluation indexes; Quadratic polynomial fitting and depicting of3D-response surface were performed and from which the optimal extraction process was selected. The optimal extraction process was as follows:extraction temperature92.58℃, ratio of water to material25.23and extraction time2.42h. Under the optimized condition, the experimental value of0.738±0.015was well in close agreement with the value predicted by the model. Moreover, the overall desirability value in the condition was higher than that of the non-optimized condition. These results help in designing the process of optimal chlorogenic acid, baicalin and wogonoside from compound Manhukang.In this study, a new approach was provided for the research of Chinese medicine effective substance. The main material basis was identified, which were absorbed into serum by the method of Serum pharmaco-chemistry, then applying the combination of fingerprint and pharmacodynamics to identify the efficacy components. Meantime, advancing the quantitative analysis method of fingerprint combined with key efficacy component to fully control the quality of veterinary pharmaceutical in Chinese veterinary medicine. This study has theoretical significance and practical value as well as the modeling effect of the application of fingerprint, serum pharmaceutical chemistry and "spectrum-effect" relationships in Veterinary pharmaceutical. |
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