Evaluation Of (Processed) Polygonum Multiflorum And Its Main Components On CYP450 Activity By Cocktail Probe Drugs

BackgroundPolygonum multiflorum belong to tonic herbs with tonifying the liver and kidney,essence and blood.In our previous research,(processed)Polygonum multiflorum have differences in the gene expression of hepatobiliary transporter.The liver is the major site of drug metabolism and transformation in vivo,including phase I and phase II drug metabolizing enzymes whichdetermine the level of drug metabolism and transport and have intimate contact about the occurrence or development pharmacodynamics.This study was to identify the connection between Polygonum multiflorum with different processed products or its main component and hepatic enzyme CYP450 through the metabolism of polygonum multiflorum and black bean extract in human liver microsomes(HLMs).To enrich the processing theory and the understanding of main effective component.To identify the isoforms of CYP450 and evaluate theinteraction between drugs and enzymes.To provide experimental basis for further research of the metabolism of(processed)Polygonum multiflorum in vivo and clinical rational drug using.Objectives1 To establish and verify the simultaneous determination of five metabolites of cytochrome P450 probe substrates by liquid chromatography tandem mass spectrometry.2 To study the effect of polygonum multiflorum with different processed products and black bean extract on CYP450.3 To study the effects of major components in polygonum multiflorum and black beans on CYP450.Methods1 There were established Cocktail method coupled with liquid chromatography tandem mass spectrometry method for simultaneous determination of six metabolites of CYP450 probe drugs including:6-hydroxychlorzoxazone(CYP2E1),4’-hydroxydiclofenac(CYP2C9),paracetamol(CYP1A2),4’-hydroxylmephe nytoin(CYP2C19),dextrorphan(CYP2D6),oxidized nifedipine(CYP3A4).The specific inhibitors of probe substrates were used for validation the in vitro cocktail method and determination the inhibition potency.2 To study the effects of polygonum multiflorum with different processed products and black bean extracts on CYP450.Detection the metabolites generated by subtypes of probe substrates through comparing the experimental group contained different concentrations of extracts with the control group.The effects were analyzed from the extract on CYP450.3 To study the components extracted from polygonum multiflorum,Anthraquinone compounds:emodin,physcion,emodin-8-O-B-D-pyran indicant,rhein,chrysophanol,Anthraquinone compounds:gallic acid,Styrene glycoside,5-hydroxymethyl furfural and isoflavones:daidzi,daidzein,genistin,genistein extracted from black beans on CYP450.Detection the metabolites generated by subtypes of probe substrates through comparing the experimental group contained monomer compositions with the control group.The effects were analyzed from the extract and its compatibility on CYP450.Result1 An efficient,fast and reliable LC-MS/MS method has been established for monitoring six metabolites of CYP450 probe substrates in HLMs.The method was formally validated and showed good performances in terms of sensitivity,precision and accuracy.The specific inhibitors of probe substrates were used for validation simultaneously.This cocktail probe substrate method can be utilized for the rapid simultaneous determination of the influences of compounds on the six CYP450 enzymes.2 Polygonum multiflorum with different processed products had inhibited CYP2E1,CYP2C9,CYP1A2,CYP2D6 activities and the inhibition increased with the concentrations.Polygonum multiflorum had little effect on CYP3A4 at 10 u g/mL and 100μg/mL,while there had obvious inhibitory actions at 300μg/mL.Polygonum Multiflori Radix Praeparata(processed with water)and Polygonum Multiflori Radix Praeparata(processed with black beans)were less than Polygonum Multiflori Radix in inhibitory.Black bean extracts had no significant impact on CYP2C9 and CYP3A4.There had inhibitory on CYP2E1 and CYP2D6.At 1μg/ml,black bean extracts reflected inhibition on CYP1A2 while had promotion at 10μg/mL and 200μg/mL.3 Anthraquinone compounds had inhibition on CYP2E1 and had inconsistent results on other enzymes.But the emodin had strong inhibitory on CYP2C19 with the IC5O value 1.43μmol/L.The other compounds showed no influences on CYP2D6 and CYP3A4.Styrene glycoside had inhibition on CYP2E1,CYP1A2 and CYP2C19 while reflected no inhibition on the else.Gallic acid processed CYP450 activities at certain concentrations.5-hydroxymethyl furfural had promotion on CYP2D6 and had no obvious effects on the other enzymes.CYP450 enzymes were promoted by daidzi and genistin in black beans at certain concentrations.Daidzein and Genistein had inhibitory trend on CYP450.ConclusionThe cocktail probe substrate method can be utilized for the rapid simultaneous determination of the influences of Polygonum Multiflori Radix and black bean extracts and its main compounds on the six CYP450 enzymes.Polygonum Multiflori Radix,Polygonum Multiflori Radix Praeparata(processed with water),Polygonum Multiflori Radix Praeparata(processed with black beans)and black bean extracts had inhibitory on CYP450 to a certain extent.Radix Praeparata(processed with water)and Polygonum Multiflori Radix Praeparata(processed with black beans)were less than Polygonum Multiflori Radix in inhibitory.It showed that processed Polygonum Multiflori could reduce the effects on CYP450 enzymes,which it could be the mechanism of weakening the toxity.Polygonum Multiflori Radix Praeparata(processed with black beans)weakened the inhibitory trend compared with Radix Praeparata(processed with water)hinting that the reasonableness of Polygonum Multiflori Radix processed by black beans.The compounds had inhibitory on CYP2E1,CYP2C9,CYP1A2,CYP2C19 and CYP2D6,and had no influence on CYP3A4.The increased free Anthraquinone,Gallic acid,5-hydroxymethyl furfural and the decreased Styrene glycoside in the processing affected the CYP450 activities.The different of pharmacology and medical efficacy between Radix Polygonum Multiflori and processed Polygonum Multiflori related to the composition changes.