Enrichment,Analysis And Hepatotoxicity Evaluation Of Dianthrone Compounds In Polygoni Multiflori Caulis

Polygoni Multiflori Caulis,a traditional Chinese medicine,is the overground part stem of Polygonum multiflorum Thunb.,which mainly contains anthraquinones,flavonoids and stilbene glycosides.Recently,hepatotoxicity of Polygoni Multiflori Caulis in clinical application has been raised more attention,but the potential toxic compounds have remained unclear.Anthrone dimers called dianthrones,often exist widely in Rheum(Polygonaceae)and other terrestrial plants.and its structures and activities are diverse.In recent study,minor dianthrones isolated from Polygoni Multiflori Radix via phytochemical methods have been proved obvious toxicity related to liver damage.Polygoni Multiflori Caulis and Polygoni Multiflori Radix come from the different parts of Polygonum multiflorum Thunb.,and phytochemical investigations indicated that Polygoni Multiflori Caulis had similar main constituents with Polygoni Multiflori Radix.However,there has been lack of study on dianthrones in Polygoni Multiflori Caulis.In this study,the qualitative and semiquantitative analysis of the dianthrones in Polygoni Multiflori Caulis were conducted by UPLC-Q-TOF MS.The dianthrones had been further isolated and identified,and meanwhile the toxicity of the dianthrones was evaluated by cell toxicity tests.This study would provide basis for safety evaluation and further study of hepatotoxic components in Polygoni Multiflori Caulis.Methods: The dianthrones in Polygoni Multiflori Caulis were extracted by water bath reflux extraction method and enriched by macroporous resin.The dianthrones were qualitatively analyzed by UPLCQ-TOF MS/MS-IDA technology,and the aglycone of dianthrone derivatives were verified by acid hydrolysis.The relative contents of the dianthrones in Polygoni Multiflori Caulis were calculated by area normalization method.Dianthrone monomers in Polygoni Multiflori Caulis were obtained by phytochemical methods such as extraction,D101 macroporous adsorption resin,Rp-C18 and so on.The structural information such as functional groups,molecular weight and absolute configuration of sugars were analyzed by physicochemical properties and spectrum technology(UPLC/Q-TOF-MS,NMR,FT-IR,UV,etc.).Finally,MTT assay was used to evaluate the hepatotoxicity of the dianthrones on L02 and Hep G2.Results: Seventy-two dianthrone glycosides and their five dianthrone aglycones were found in Polygoni Multiflori Caulis for the first time,including 29 dianthrone compounds regarded as potential new compounds.The dianthrones were divided into three types,and they were emodin dianthrones(type I),physcion-emodin dianthrones(type II)and physcion dianthrones(type III).Among them,type I and II dianthrones had diagnostic ions at m/z 254 416 and 458,while type III dianthrones had diagnostic ions at m/z 268 537.The semiquantitative results showed that the relative contents proportion of the type I and type II dianthrones were higher,while that of the type III were less.Two novel dianthrones PMC-a-1 and PMC-a-2 were isolated and characterized as emodin-8-O-β-D-glucopyranoside-emodin-8’-O-β-Dglucopyranoside dianthrone(type I).They were tentatively identified as cis-trans isomers.The hepatic cytotoxic tests showed that PMC-a-1 and PMC-a-2 were potential hepatotoxic substances,but their activities were different.PMCa-1 had certain cytotoxic effects on Hep G2 and L02,and its IC50 value within 48 h was 45.25 μM,72.78 μM,respectively.PMC-a-2 only had cytotoxic effect on L02 and IC50 within 48 h was 61.11 μM.Conclusion: The dianthrones in Polygoni Multiflori Caulis were systematically analyzed for the first time.Two novel dianthrones PMC-a-1 and PMC-a-2 were isolated from Polygoni Multiflori Caulis for the first time and showed potential hepatotoxicity.