Establishment Of Quality Evaluation And Draft Quality Standards For Bufonis Venenum

Traditional Chinese medicine(TCM)has complex chemical composition and multiple pharmacological activities.Its inherent "multi-component,multi-target" characteristics determine that TCM need to establish a characteristic and specific quality and biological activity evaluation system.The research team members have conducted research on the material basis and quality control of Toad Venom in the early stage.Toad Venom is dried to make Bufonis venenum(tablet),which is then smashed into a piece(powder).Due to the high price of glutinous rice cakes,the commercial products are often adulterated,resulting in uneven quality of Bufonis venenum,affecting quality stability and efficacy and safety.Therefore,this study focuses on the quality control marker screening,quality evaluation and quality standards establishment of the product Bufonis venenum.The details are as follows:1.Literature researchCheck out the relevant literature of Bufonis venenum and learn about the main role of Bufonis venenum.Bufonis venenum is mainly used for anti-inflammatory in traditional Chinese medicine,but it has been gradually applied to anti-tumor treatment since modern times.Bufonis venenum is complex in composition,The Small molecule metabolite mainly include bufadienolides,alkaloids,sterols and other compounds and the macromolecular compounds are mainly polypeptides and proteins.For the detection of components in Bufonis venenum,the commonly used detection methods are mainly liquid chromatography-mass spectrometry.The first part of this chapter mainly introduces the detection and identification of TCM based on mass spectrometry and the screening methods of active markers.The second part introduces the current methods and status of quality control and evaluation of TCM.2.Screening of anti-tumor cell activity markers by PLS multidimensional statistical analysis-two-dimensional high-resolution mass spectrometry,and confirming the binding of Bufonis venenum markers to macromolecular proteins by centrifugal ultrafiltration-mass spectrometry and non-denaturing chromatography-mass spectrometry based on affinity principle active.(1)Reanalysis of Bufonis venenum methanol extract AB SCIEX Triple-TOF 5600 high resolution mass spectrometry fileThe reanalysis of Bufonis venenum methanol extract AB SCIEX Triple-TOF 5600 high-resolution mass spectrometry showed that Bufonis venenum is rich in bufadienolides,proteins,peptides,etc.,which are basically consistent with the previous research results of the research group.The small molecule bufadienolides and macromolecular peptides in Bufonis venenum methanol extract were identified by AB SCIEX Triple-TOF 5600 high resolution mass spectrometry.The results showed that the buckwheat is rich in bufadienolides,including free and arginine organic acid ester-bound bufadienolides,proteins,peptides,etc.,which are basically consistent with the previous research results,according to the characteristics of the secondary mass spectrometry and the cleavage rules.Can be effectively identified and identified.(3)Screening of active markers based on centrifugation-ultrafiltration mass spectrometry and non-denaturing ion exchange chromatography(2)Screening of anti-tumor cell-related markers based on PLS(partial least squares)multidimensional analysis-two-dimensional high resolution mass spectrometryIn the early stage,the research team established a marker for anti-tumor cell activity based on pearson correlation coefficient method-two-dimensional chromatography mass spectrometry.The method is simple and easy,but the pearson correlation analysis is a one-factor correlation analysis,without considering the complex multivariate chemical composition of Bufonis venenum,and the pearson correlation fails to consider the interaction between the multiple components.To this end,this section uses PLS multivariate statistical analysis to solve the above problems,and then to find and identify the anti-tumor cell activity-related markers from the complex secondary metabolites of Bufonis venenum for the quality control of Bufonis venenum.The results showed that the anti-tumor cell activity markers screened by PLS multivariate statistical analysis had an 80%coincidence rate with the markers selected by the research group using pearson correlation,indicating that the method is feasible.(3)Study on active markers based on centrifugation-ultrafiltration mass spectrometry and ion exchange chromatographyThe binding of bufadienolides to total protein of tumor cells was further studied.DHFR/MTX was used as a positive control to investigate the binding relationship between bufadienolides and total protein of SMMC-7721 liver cancer cells.The target drug was incubated with SMMC-7721 total protein by ion exchange liquid chromatography,and the fractions were collected.The collected fractions were subjected to mass spectrometry.The results showed that the MTX in the drug control group peaked at Fr.19,while the MTX-DHFR peaked at Fr.55 and Fr.62,indicating that chromatographic migration occurred after MTX bound to its target protein.The results demonstrate the effectiveness of the method.The binding of SMMC-7721 total protein to 8 kinds of bufadienolides showed that the control group was mainly concentrated in Fr.4,and a small part was in Fr.11 and 14;The drug that binding to protein is mainly concentrated in the peak of Fr.56.Based on the above results,it was preliminarily determined that the bufadienolides marker can bind to the tumor cell SMMC-7721 protein.In addition,our study also showed that bufadienolides inhibited lipopolysaccharide-stimulated lymphocyte proliferation and had anti-inflammatory activity in addition to anti-tumor cells.In view of the contribution of this component to the activity of Bufonis venenum,we used it as a marker to evaluate the quality of Bufonis venenum products.Based on the above studies,the binding of the target protein to the active ligand small molecule was screened by centrifugation-ultrafiltration mass spectrometry based on the affinity principle.Centrifugal-ultrafiltration mass spectrometry was established using three sets of known proteins and their ligands as positive controls.The results indicate that the method is capable of recognizing known target proteins and active ligands,and is also suitable for screening or confirming active small molecule ligands against a target protein of interest.We used this method to initially demonstrate that the quality control marker bufalin binds to two potential anti-myeloma targets.This ingredient is an active substance of Bufonis venenum.3.Quality evaluation of different batches of Bufonis venenum productsThis part of the study optimizes the detection method of bufadienolides in Bufonis venenum,and determines the content of different batches of Bufonis venenum products and Leiyun base samples.The correlation between the in vitro antitumor activity of different batches of Bufonis venenum samples and the content before and after the correction of the weight coefficient was analyzed,which indicated that the established quality evaluation method can reflect the quality of Bufonis venenum.(1)Optimization of qualitative and quantitative detection methods for Bufonis venenumBased on the current 2015 Chinese Pharmacopoeia method,the Bufonis venenum thin layer qualitative detection and high performance liquid chromatography quantitative detection method were optimized.The qualitative identification method investigated and optimized the indicators including sample concentration,spotting amount,thin plate type,and development time.Quantitative testing examined and optimized indicators including column,mobile phase composition,gradient,and flow rate.(2)The establishment of Bufonis venenum quality evaluation method and the quality evaluation of the productsThe content of 43 batches of Bufonis venenum was evaluated and the anti-tumor cell activity was evaluated.PCA analysis showed that there were large differences in the content of Bufanis venenum bufadienolides from different sources and batches,and the batch-to-batch consistency was poor.Considering that the two bufadienolides components(the pharmacopoeia method)do not fully reflect the quality of Bufonis venenum,the total content of the eight bufadienolides may be more suitable for Bufonis venenum quality evaluation.Analysis of the above results revealed that the correlation coefficient between eight bufadienolides of diffierent batches its anti-tumor cells was 0.4,indicating that there was no correlation;we further designed the weight coefficient correction based on the activity contribution and found the corrected bufadienolides.The correlation coefficient increased to 0.56,which was significantly correlated.Therefore,using the corrected total content of bufadienolides as an indicator can better reflect the quality of Bufonis venenum.(3)Comparison of one-test multi-evaluation method and direct measurement methodIn order to reduce the consumption of bufadienolides in large quantities,we used cinobufagin as an internal reference material to achieve relative quantitative detection of the other 5 bufadienolides in Bufonis venenum by a multi-evaluation method.The results were accurate and reliable.Appendix:draft quality standards and drafting instructions for Bufonis venenumIn summary,in order to carry out quality control and evaluation of Bufonis venenum,we carried out research on the selection and quality control of markers.Eight bioactive markers were screened,and the quality evaluation method and quality standard of Bufonis venenum were established,which is of great significance for the quality control of Bufonis venenum and its safety effectiveness.