Analysis Of Metabolic Components And Hepatic Cytotoxicity Of Water Extracts From Polygonum Multiflorum Thunb

Objective:To isolate and identify chemical constituents of n-butanol fraction of aqueous aextract of Polygoni Multiflori Radix（AEPMR）.To detect the hepatotoxicity of AEPMR and its ethyl acetate extract and n-butanol extract.To detect the hepatotoxicity of AEPMR incubated with or without the rat liver microsomes.To investigate the optimal incubation conditions and metabolites of emodin-8-O-D-β-glucopyranoside（EG）in rat liver microsomes,and analyze the effect of EG on CYP450 enzymes in rat liver microsomes.Meaning:To clarify the chemical components of AEPMR,and to establish the material basis for the study of hepatotoxicity of Polygoni Multiflori Radix.To analyze the contents of the main components,detect the cytotoxicity of aqueous extract and other two kinds of extract of Polygoni Multiflori Radix.To analyze the relationship between changes of components contents and hepatotoxicity of AEPMR.Emodin-8-O-D-β-glucopyranoside is the main combinational anthraquinone in AEPMR,but lack of metabolic research,and the liver microsomes metabolism was simple and easy to operate.We used rat liver microsomes to do the metabolic research about emodin-8-O-D-β-glucopyranoside.We investigated the effect of emodin-8-O-D-β-glucopyranoside on CYP450 in rat liver microsomes to reveal reaction of medicines.Methods:A series of column chromatographic techniques（silic gel,polyamide,Sephadex LH-20）were used to isolate the n-butyl alcohol extract of AEPMR,different spectral analysis was used to identify the compounds.The main components contents of the three kinds of extracts of Polygoni Multiflori Radix were determined by HPLC.Meanwhile,MTT assay was used to determine the cell toxicity of three kinds of extract of Polygoni Multiflori Radix on L02 and HepG2.And then we analyzed the relationship between the components and toxicity of the AEPMR according to the results.AEPMR was incubated with or without rat liver microsome in vitro and the incubated solutions of AEPMR were dried by freeze drying,then interacted with liver L02 and HepG2 cells with different concentrations.Contents of three main components（trans-2,3,5,4’-tetrahydroxystibene-2-O-β-D-glucopyranoside,emodin-8-O-β-glucopyranoside,emodin）of AEPMR in incubated solutions were quantitated by the established HPLC conditions.And the relationship between components and toxicity was analyzed.The metabolic elimination of emodin-8-O-D-β-glucopyranoside in rat liver microsomes was determined by HPLC to determine the optimal incubation conditions.The effect of emodin-8-O-D-β-glucopyranoside on the subtypes of CYP450 enzyme was investigated by using the cock-tail method to detect the metabolic elimination rate of specific substrate of CYP450 enzyme subtypes.Results:10 compounds were isolated from the n-butanol sites of Polygoni Multiflori Radix,nine compounds were identified:physcion-8-O-β-D-glucopyranoside,physcion,emodin,emodin-8-O-β-D-glucoside,trans-2,3,5,4’-tetrahydroxystibene-2-O-p-β-glucopyra-noside,kaempferol,questin,5-carboxymethy1₇-hydroxyl-2-methylchromone,2,5-dimethyl-7-hydroxychromone.MTT cell assay revealed that three kinds of extract inhibit the two kinds of liver cells in dose-dependent manner,the toxicity of three kinds of extract:aqueous extract>ethyl acetate extract>n-butanol extract.The IC50of AEPMR on L02 cells was 4.187 mg/mL and HepG2 cells was 3.786 mg/mL,HepG2 cells were detected more sensitivity than L02 cells.The solution of AEPMR not incubated with rat liver microsomes showed more toxic than the group which incubated with rat liver microsomes at the concentration of 12 mg（crude drug）/mL（P<0.01）.Contents of the three main components of AEPMR in incubated with rat liver microsomes group were detected significantly lower than those in incubated without rat liver microsomes group.Studies on the metabolism of emodin-8-O-D-β-glucopyranoside in rat liver microsomes showed that the optimal incubation system in rat liver microsomes for emodin-8-O-D-β-glucopyranoside was 1 mg/mL liver microsomes,0.05 mM emodin-8-O-D-β-glucopyranoside concentration,and 60 min incubation time.One related metabolic peak of emodin-8-O-D-β-glucopyranoside was found.The IC50 of emodin-8-O-D-β-glucopyranoside on the activity of CYP1A2 was 130 μM and CYP2C9 was 0.887 μM.Emodin-8-O-D-β-glucopyranoside activated the CYP2D1（similar to CYP2D6 of humanbeing）and its EC50 was 1.504 μM.The EC50/IC50 of CYP3A1（similar to CYP3A4 of humanbeing）and CYP2E1 could not be fitted.Conclusions:2,5-dimethyl-7-hydroxychromone was isolated from Polygoni Multitlori Radix for the first time.The hepatotoxicity of AEPMR was decreased with the metabolism of rat liver microsomes,the contents of three main components in AEPMR were decreased.Emodin-8-O-D-β-glucopyranoside could activate or inhibit the CYP450 enzyme subtypes in rat liver microsomes.