| Quality control and evaluation of traditional Chinese medicine (TCM) are the important means, which can ensure the safety, effectiveness. stability. controllability and clinical application.Because of the strong toxicity. the quality of toxic TCM will definitely influence its safety and effectiveness in clinical application. However, the current pattern of quality control and evaluation, which is mainly based on the qualitative and quantitative determination of chemical components, is difficult to reflect the safety and efficacy of toxic TCMs.In this paper, we studied the Aconiti Lateralis Radix (Fuzi) in order to enrich and improve the quality control methodology of toxic TCMs in view of bioassay. The toxic-potency assay of Fuzi was established based on the minimum lethal dose to the whole animal. We also established the determination method for the content of6kinds of alkaloids by the relative correction factor. The21kinds of diterpenoid alkaloids were detected by the HPLC-MS/MS. Based on the toxic-potency assay and chemical composition analysis, we consider the specifications of aconite product and the quality testing method should be modified. The quality standards were revised in draft. Our study also provides a reference for the clinical application of toxic TCMs. The main results are as follows:1. The toxic-potency assay method was established and verified for the quality control of Fuzi.Based on the minimum lethal dose, hemolysis reaction, toxic effect ingredient transfer rate, osmotic pressure and relevant physical indicators, this paper studied some critical factors such as drug-delivery way, sample preparation process and the drug concentration in relationship with the minimum lethal dose. And the verification results with different processed products showed that the method can be applied to all kinds of lateral root of aconite processing varieties (FL%<15%).2. The determination method for the content of6kinds of alkaloids by the relative correction factor was established for the first time.Benzoyl hypaconitine was used as the reference substance to calculate the contents of other5aconitine. The relative correction factors were1.039(RSD=1.6%),1.026(RSD=2.2%),0.948(RSD=3.7%),0.957(RSD=1.7%),0.876(RSD=1.8%) which indicated the good reproducibility. Validated with the15batches of samples and the results indicated that the multi-components quantities by one marker were no significant difference with the conventional external standard method (P>0.05).3. The scientific basis of toxic reduction with processed Fuzi was investigated by combination of toxic potency assay, chemical determination and HPLC-MS/MS fingerprinting results.The results of toxicity studies and HPLC-MS/MS detection showed that the toxicity sequence of7alkaloids:3-acetyl aconitine,mesaconitine,aconitine,hypaconitine, deoxyaconitine,10-hydroxy aconitine mesaconitine. The above7kinds of alkaloids decreased significantly in the processed products (P<0.01), while epoxy alkaloids such as dehydration benzoylaconine alkaloids, MAs such as benzoyl hypacoitine, and new aconine increased significantly (P<0.01or P<0.05).Content determination results show that the DAs in processed products was only1.8%-2.6%of non-processed products. While the content of MAs increased4.5～4.6times (steaming or stir-frying without Danba, P<0.01) or no insignificant different (steaming or stir-frying with Danba, P>0.05).These results suggested that3-acetyl aconitine and DAs are toxic components. While the epoxy alkaloids, MAs and amino alcohol alkaloids may be active ingredients. Processing transformed the former into the latter, which is the reason of keeping effectiveness and eliminating toxicity.4. The "conserving property" effect of different processed method was discussed by comparing the toxic potency, toxic/effective substance and inorganic impurity content.It was found that the contents of DAs were no insignificant different between "Wu dan fu pian"(steaming without Danba) and "Dan fu pian"(steaming or stir-frying with Danba). Most active substance content (12/14) was significantly higher than "Dan fu pian"(P<0.01or P<0.05), in which3kinds of MAs content increased about6times. The result suggested that the abilities of "eliminating toxicity" of different processing methods were equivalent at all. In contrast, the processing method by steaming or stir-frying without Danba might have better effect for "conserving property" than the method processed with Danba stipulated by China Pharmacopoeia. We believe that the new processing method without Danba can be recommended in further application due to it offers a simple procedure and it will not introduce inorganic impurities in the products.But, for "Wu dan fu pian"(stir-frying without Danba), the toxicity of and toxic substances content are significantly higher than traditional prepared products (P<0.01or P<0.05). The toxicity of the former is twice as much as that of the latter.5. Preliminary constructed the toxicity index of Fuzi.In order to improve the convenient and practical of safety evaluation and control, this paper proposed to explore the biological poison assay estimates,"toxicity index" based on toxic components content comprehensive weighted. And constructed the "aconite toxicity index" prediction model. After verification, the model can be used to predict biological poison of aconite.6. The revised quality standard draft of Fuzi was proposed.Based on the results of quality assessment, scientific basis and rationality of processed method, we proposed amendments to product specifications, quality inspection method, i.e.:1) increasing the specifications of "Wu dan fu pian"(steaming without Danba);2) revising the DAs limit test;3) revising the MAs determination method;4) increasing the toxic potency assay.In summary, the biological poison assay method and "toxic effect index model", which can make up the shortage of chemical analysis methods, enrich and develop the TCM quality control and evaluation model. This paper provided the mentality for the objective toxicity evaluation of TCM. Also, constructed the research model and supplied methodology example for quality control standard of TCM toxicity safety, which will lay the foundation for the clinical rational drug use. |
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