Polygonum Multiflorum Hepatotoxic Substance Basis And Quality Control Research

Polygonum multiflorum is a traditional Chinese medicine in China.Its hepatotoxicity reports have been increased in recent years.In this paper,we took Polygonum multiflorum as the research object,explored its main toxic material basis through immunological stress simulating experiments in vitro and in vivo and set up fingerprint of each fractions for defining quality control standards to decrease adverse effects and ensure security in clinic.The main research contents are as follows:1.Screening for main components associated with hepatotoxicity of Polygonum multiflorumObjective To identify the main hepatotoxic constituents of Polygonum multiflorumMethods LPS-stimulated immunological stress L-02 cells model were applied to test the proliferation inhibition ratio by different drugs including raw Polygonum multiflorum,its macroporous resin column eluted water washed fraction,50%ethanol fraction and 95%ethanol fraction and processed products through MTT method,detect apoptosis by Flow cytometry and observe expressions of ALT and AST by Elisa assay in vitro.Immunological stress rats induced by LPS were divided into 18 groups and received gavage using different doses of drugs.The expressions of ALT,AST and LDH in serum were tested,liver index and pathological changes of liver were observed.Results The 50%ethanol fraction possessed of the highest inhibition ratio,apoptotic activity,and expressions of ALT and AST in vitro Model when the concentration was 400 μg·mL-1.Moreover,the 50%ethanol fraction remarkably increased the liver index and the expressions of ALT,AST and LDH,also changed the pathologic state in vivo.Conclusion The hepatotoxicity of Polygonum multiflorum can induce an immunological stress model.The largest toxicity was showed at the 50%ethanol fraction.2.Verification of Hepatic Toxic Material Basis of Polygonum MultiflorumObjective To screen the main toxic compounds of Polygonum multiflorum and verify its toxic material basisMethods The main compound called stilbene glycoside(TSG)was extracted and isolated in the 50%ethanol fraction.LPS-stimulated immunological stress L-02 cells model were applied to test the proliferation inhibition ratio by TSG through MTT method,detect apoptosis by Flow cytometry and observe expressions of ALT and AST by Elisa assay in vitro.Immunological stress rats induced by LPS were divided into 16 groups and received gavage using different doses of drugs.The expressions of ALT,AST and LDH in serum were tested,liver index and pathological changes of liver were observed.Results The proliferation inhibition ratio,apoptotic activity and expressions of ALT and AST were increased,presenting dose dependence in vitro.The liver index and the expressions of ALT,AST and LDH also increased when the rats were treated with TSG.We observed that drug deposited in liver cells,the expressions of inflammatory factors increased,hepatic metabolism decreased,and hepatocytes exhibited a tendency to necrosis.Conclusion TSG can induce hepatotoxicity in the immunological stress conditions.However,due to the existence of cis-trans geometric isomerism of stilbene glycosides the main toxic basis remains to be further verified.3.Study on quality control of Polygonum multiflorumObjective Establishing a fingerprint to examine the differences between crude and processed Polygonum multiflorum and their different parts.Methods To establish a content determination method of the 50%ethanol extract from crude/processed root of Polygonum multiflorum from different habitats in China and set up the fingerprint by using UPLC.The UPLC analysis was performed on Waters ACQUITY UPLC@BEH C18 chromatographic column(2.1×50 mm,1.7 μm)at(25±5)℃.A binary gradient elution system was composed of acetonitrile(phase A)and 0.5%acetic acid solution(phase B).Detection was performed at the wavelength of 254nm,and the mobile flow rate was set at 0.3 mL·min-1.To establish a content determination method of the 95%ethanol extract from crude/processed root of Polygonummultiflorum from different habitats in China and set up the fingerprint by using HPLC.The HPLC analysis was performed on Agilent ZORBAX SB-C18 chromatographic column(250mm×4.6mm,5μm)at(25±5)℃.A binary gradient elution system was composed of methanol acetonitrile(phase A)and 0.05%acetic acid solution(phase B).Detection was performed at the wavelength of 280nm,and the mobile flow rate was set at 1 mL·min-1.Similarity Evaluation System for Chromatographic Fingerprint of TCMs(Ver.2012)and SPSS 22.0,SIMCA13.0 were used in cluster analysis and principal component analysis(PCA),find the main differences and determine the content.Results The fingerprints of 35 batches,50%and 95%ethanol extracted fractions from crude/processed,established by UPLC and HPLC.The evaluation of similarity shows that there is less difference in raw product among all parts,the similarity is above 0.8,and the difference between artefacts processed by processed products is quite different.The cluster analysis can basically divide two parts into two categories:raw and processed.The main component analysis showed that the main difference component in the 50%ethanol fraction was stilbene glycoside,and the difference components in the 95%ethanol fraction were emodin and emodin.Some production areas processed products were assigned to the raw group due to the high content of stilbene glycosides and emodin.From the chemical composition,it reflects the similarities and differences in the processing methods of different producing areas.Conclusion We have established fingerprints and determination methods for different fractions,and clarified that the main differences between different fractions were stilbene glucoside,emodin and emodin.Chemical constituents of processed products varied greatly,suggested that the processing technology of Polygonum multiflorum should be standardized.The determination method is simple,sensitive,reliable and can be used in fast identifying the crude/processed root of P.multiflorum or as a method for overall quality control of root of P.multiflorum.