Investigation And Application Of The Quality Evaluation & Control Methods For Radix Isatidis Based On Bioassay

Quality control and evaluation is an important guarantee for the safety and validity of traditional Chinese drugs (TCD). However, the current pattern of quality control for TCD, which mainly refers to the chemical pattern—focusing on the qualitative and quantitative determination of target compound, has little relevance with the safety and efficacy and is difficult to intrinsically evaluate and control the quality of TCD. This "chemical pattern" has restricted the development of TCD modernization.A novel pattern and method for quality control of TCD based on orthodox & superior medicinal material and bioassay, which may be used to make up the current quality control and evaluation system of "component theory" for TCD, was initially proposed by our research team on the foundation of overviewing the development processes of model amd criteria of quality control for drugs (including chemical medicine, biological medicine and TCD) and comparatively analyzing the differences and rationality of production quality management model for these three kinds of drugs. Radix isatidis (Banlangen in Chinese), as a commonly used drug in clinic with definite pharmacodynamic actions but unclarifed components, was studied in this paper. The bioassay method and criteria for Radix isatidis and its granula prepapration were established based on the assay of antiviral and antibacterial activities using various biological tools. Also, the method and criteria were further applied for the bioassay of Radix isatidis of different metropolis and commercial specifications, and its granula prepapration of different manufacturers and batches. The main findings were as follows:(1) The estimation method of bioassay based on fluorescence detection was established for measuring the antiviral activities of Radix isatidis on influenza virus according to the theory of testing the activity of influenza virus neuraminidase (NA). In this study, the method was designed and optimized with good repeatability (RSD=5.78%) on the basis of the "parallel line of qualitative response" theory. The assay on pharmacological action showed that Radix isatidis had the inhibitory effect on NA activity. IC₅₀ of the representative samples was 0.90±0.20 mg (crude drug)·mL^-1. The results of potency determination for many samples showed that this method could be used to evaluate and distinct the quality of different batches of Radix isatidis samples. As a bioassay method for Radix isatidis, this method had high sensitivity and was easy to quantitate and related to antiviral activity.(2) The assay method for determing the limiting value of antiviral activity of Radix isatidis based on the red blood cell agglutination was established according to the positive correlation (P<0.01, R=0.83) between the antiviral activity of Radix isatidis and the agglutination activity of red blood cell. The thrombotest on the microdosis panel was implemented for this method. The specificity, precision, stability and toleration of this established method was to be in line with the requirements for TCD standardization. The results of potency determination for many samples showed that this method could be used to evaluate and distinct the quality of Radix isatidis of different metropolis and commercial specifications. As a bioassay method for Radix isatidis, this method was safe, convenient and related to antiviral activity. So it could be used as a preferred method.(3) Staphylococcus aureus (SA) was selected as the biological model, the bioassay method of antibacterial potency for Radix isatidis was built based on the cup-plate method. The results of methodology study showed that both of these methods with good reproducibility and intermedial precision (RSD<10%) and low confidence interval rate of less than 30% were to be in line with the requirements for biassay. The measured results of different samples suggested that cup-plate method with antibacterial activity as index could be used for quality control and evaluation of Radix isatidis.(4) SA was chosen as a model living being, a bioassay method based on biothermokinetics was built to evaluate the quality of Radix isatidis.There is a significant linear correlation (r=0.983, P<0.01) in the definitive range between the logarithmic concentration of Radix isatidis and the parameter of biothermokinetics lg (T_{max sample}/T_{max blank}), This bioassay was also satisfied in confidence interval rate and precision. Meanwhile, the good correlation (R=0.94) between the two antibacterial potency results assay by biothermokinetics method and cup-plate method respectively suggested that the two methods had good consistency. The biothermokinetics method could be used as a preferred technology for antibacterial potency biassay of Radix isatidis because of the superiority of its fingerprinting, specificity, universality, holography and conveniency.(5) The results of the appliaction of the preferred bioassay methods on Radix isatidis from various regions showed that the differences of potency values of Radix isatidis samples from the same region of GAP depot were least (RSD for antibacterial potency was 10.2%, for antiviral potency was 11.5%). The potency value of samples from GAP depot (including different regions) was high and the differences between of various batches were smallest (RSD<16.1%). There are large differences between casual or non-GAP depot samples (RSD> 25%). The results of determination the limiting value of antiviral activities using the detection method of erythrocyte agglutination activity showed that the products from different manufacturers had some differences, and the products of different batches from the same manufacturer (some large-scale enterprises) have less fluctuations (RSD=7.8%～11.1%). The bioassay results of the samples with sugar were indistinctively different from that without sugar (P>0.05). All the results suggested that the established method with good adaptivity could be used for quality control and evaluation of Radix isatidis and its granulation preparation.(6) The method of UPLC was applied to determine the contents of adenosine and uridine and method of phenol-sulfuric acid colorimetric assay was used for measuring the contents of polysaccharide in Radix isatidis. The results showed that the average contents of polysaccharide was 242.48 mg·g^-1 (RSD=10.4%), uridine was 30.9μg·g^-1(RSD=9.8%), adenosine was 24.3μg·g^-1, (RSD=10.5%). The changes of the contents of polysaccharide, uridine and adenosine were not obvious. Whether these three indexes could be used for the quality control of Radix isatidis need further study.(7) The results of multivariate correlation analysis on the chemical composition and the bioassay for antivirual and antibacterial activities of Radix isatidis indicated that each variable was to be separated from one another and there were no correlations among them (the model of multivariate correlation analysis was not established, P>0.05). There were no significant correlation between the bioassay for antiviral and antibacterial activities (R<0.70). All the results suggested that it was difficult to establish the correlation between the quality control based on the determination of adenosine, uridine and polysaccharide and the pharmacodynamic actions of antiviral and antibacterial activities. The established methods for bioassay of antivirual and antibacterial activities could not be replaced each other. They could be used for quality control of Radix isatidis from antiviral and antibacterial activities, respectively.(8) The quality criteria of bioassay for Radix isatidis and corresponding operation procedures were worked out according to the study of determing the limiting value of antiviral activity based on the red blood cell agglutination and the antibacterial potency based on bio thermokinetics. All these investigations provided some basises for the practical application of bioassay methods and criteria on Radix isatidis.The results of this study showed that the established method for the bioassay of antibacterial and antiviral activities of Radix isatidis had much application values. The combination of these methods and the existing ChP methods of conventional and chemical assay used for controlling the quality of Radix isatidis could improve the quality control level, as well as provided references for the study of quality control of TCD.