A New Pattern For Toxicity Constituents Recognition And Quality Control Of Traditional Chinese Drug(Xanthium) By Component “Knock-out/Knock-in” Stratage

1 Objective In this study, we made a toxic herbal Xanthium which commonly used in clinical medicine as a model drug, based on the toxicity constituents recognition and quality control of traditional Chinese drug by the target component knock-out/knock-in, to identified and confirm the toxic substances of Xanthium,and its quantitative control of toxic components and evaluation method were preliminary studied.2 Method 1. The main active component such as chlorogenic acid, neochlorogenic acid these five kinds of phenolic acids and toxic components of atractyloside and carboxylatractyloside content in 10 batches of Xanthium were determined by ultra performanceliquid chromatography; and chemical fingerprint as an indicator of water-soluble glycosides based on uplc-dad was established. 2. Through acute toxicity test in mice, liver toxicity index such as ALT, AST and organ index were determined, combining with chemical fingerprint information, Pearson correlation analysis and stepwise regression analysis was established to related the “spectrum – poison” through SPSS 22.0, identified and confirmed the toxic ingredients of Xanthium. 3. The sample of Xanthium were isolated by thin layer chromatography(TLC), "knocked out" two target components--atractyloside and carboxyl atractyloside, using UPLC method to identify the target component and negative samples to evaluate the method of "knock out". The in vitro cytotoxicity test with MTT assay, trypan blue staining and LDH release assay were used to determine cell toxicity effects of the target component, further recognition and identification of the major toxic components in Xanthium.4. Through the "knock in" strategy, different concentration of target samples were gradually added into the negative samples. The cytotoxicity of the obtained samples was evaluated, then we set up the “dose-toxicity” relationship.Based on the “dose-toxicity” relationship, get the limits of the highest content of “knock-out” components, provide the basis for the formulation of quality control in the upper limit.3 Results 1. The main active component, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 1,3 coffee acyl quinic acid and 3,5 coffee caffeoylquinic acid content are in line with the Pharmacopoeia limited of 10 batches of Xanthium materials. water-soluble glycoside component atractyloside and carboxylatractyloside of Xanthium determination results show that, carboxylatractyloside content generally higher in 10 batches of Xanthium, atractyloside and carboxylatractyloside content of Xanthium with different habitats and sources were within the acceptable range. Experiment set up the water-solution glycoside components of chemical fingerprint, similarity results show that the selected 10 batches of Xanthium materials quality is uniform. 2. Mouse acute toxicity test results showed that the 10 batches of Xanthium aqueous extraction on mice produced different degrees of liver toxicity, serum AST values were greater than 73.16±1.02 IU / L, ALT values were greater than 22.65±0.89 IU / L, organ index showed a similar trend with ALT and AST values, indicating that different Xanthium on mice acute toxicity is not the same. S9 and S10 samples had weak toxicity, S7 sample had strong toxicity. “spectrum- poison” relationship showed that, peak No. 8(carboxyl Atractyloside) and 11 peaks(Atractyloside) are most significant positively associated with the toxicity, confirmed the main toxic components in Xanthium are atractyloside and carboxylatractyloside. 3. The "knocked out" chemical composition mixtures from TLC separation are recorded as target components X+, the residual component is negative samples X-. The target components are identificated as atractyloside and carboxylatractyloside mixture, the toxic effects of cytotoxicity test was used to evaluate “knock-out” components,MTT method, trypan blue staining and LDH release assay results showed that target components have significant cytotoxicity, toxicity increased with the increase of drug action time and concentration, toxic effect reached the highest at 48 h. 4. Based on the “dose-toxicity” relationship, the limits content of atractyloside and carboxylatractyloside is: the upper limit of the total contents of both is not more than 0.35%.4 Conclusion This study is based on the target composition "knock-out / knock-in 'toxicity of Chinese herbal medicine material identification and quality control mode, established the hepatotoxicity in mice experiment evaluation method relatated to toxic components in Xanthium, we make "spectrum-toxicity "relations as the breakthrough point, on the toxicity assessment of the composition target of, a preliminary identification and confirmation of the main toxicity constituents of Xanthium, and provided experimental basis for the establishment of toxicity mechanism and quality control method of Xanthium.