Safety Evaluation Of Psoralen Based On Metabolomics And In Vivo Metabolism Studies Of Bakuchiol

OBJECTIVE:An ultra performance liquid chromatography/quadrupole time of flight mass spectrometry(UPLC-PDA-Q-TOF-MS)instrument has been used,with its high-separation and high-resolution,to study the complex components of traditional Chinese medicine “Fructus psoralen”.Metabonomics based on UPLC-Q-TOF-MS technology was applied to analyze endogenous metabolites from rat serum after two days’ administration of ethanol extract(EEFP)and aqueous extract(AEFP)of Fructus Psoraleae,and find the relative endogenous marker.Then combined with the results of hematological parameters,biochemical parameters and histological in rats,to clarify the efficacy and toxicity mechanisms of Fructus Psoraleae from the molecular level.Using UPLC-PDA-QTOF-MS to study bakuchiol’s metabolites in plasma,bile,urine,and feces after oral administration in rats,Peak area-time curves in 24 h of bakuchiol’s major metabolites in plasma were plotted to reflect the trends of the change of their blood concentrations,then to elaborate Bakuchiol’s metabolite profiling and metabolic characteristics.METHODS:(1)Percolation process with 70% ethanol was chosen to prepare EEFP,the percolation fluid was filtered and evaporated under vacuum,the fingerprints and major ingredients of EEFP were determined by UPLC-PDA-Q-TOF-MS.(2)Sprague-Dawley male rats were randomly divided into four groups(n = 6).Dosages were administered with EEFP and AEFP once daily for two continuous weeks,Collection of whole blood,serum and tissue.hematological parameters obtained with automatic blood analyzers,biochemical parameters obtained with automatic biochemical analyzer,pathological results obtained using HE staining.Then serum was analyzed by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS).Principal component analysis(PCA)and partial least squares-discriminant analysis(PLS-DA)were used to analysis the difference of endogenous metabolites in different groups,then potential biomarkers were found through orthogonal partial least squares discriminant analysis(OPLS-DA),variable importance in the projection(VIP)and t test and their relative intensity were determined.(3)16 rats were divided into three groups at random.Rats in the first group were maintained in metabolic cages for urine and feces collection.Before the experiment,the blank urine and feces samples from each rat were collected for 12 h using the metabolic cage.Then the rats were given a single dose of bakuchiol at 500 mg/kg of body weight by gavage into the stomach and the drug-containing urine and feces samples were collected for 24 h.Rats in the second group for plasma collection were fasted for 12 h with free access to water before oral administration.The retro-orbital blood samples(about 500 μL)were collected prior to dosing(t=0)and at 0.5,1,2,4,8,12,24 h post-dosing of bakuchiol(500 mg/kg).Plasma was obtained by centrifuging the blood at 4000 rpm for 10 min.Rats in the third group for bile collection were fasted for 12 h with free access to water before oral administration.4 rats were given doses of bakuchiol(500 mg/kg),another 4 rats were given an equivalent solution.Bile samples were collected for 12 h via PE-10 tube.Bakuchiol’s metabolites in urine,bile,plasma and feces of rat were discovered using Waters Metabolynx XS software,the potential metabolites were screened out based on well-defined representative structure with mass defect filter(MDF)technology.Then,their structures were further identified based on MSMS fragmentation,UV absorption features and retention time.RESULTS:(1)From the UV spectrum,14 kinds of ingredients have a higher content,then choose the 14 ingredients for MSMS spectrometry analysis,by comparing MS and MSMS spectrometry data and retention time sequence from the reference literature,12 compounds have been identified,they are psoralen,psoralen,neobavaisoflavone,bavachin,corylin,bavchromanol,psoralidin,isobavachalcone,bavachinin,corylifol,bakuchalcone and bakuchiol.(2)After 2 week’s administration of EEFP,Compared with the control group,WBC,NEUT,and MON were significantly increased in the high dosage treatment group,PCT was significantly increased in the moderate and high dosage treatment groups,platelet(PLT)was decreased in all treatment groups,but not at statistically significant levels of reduction,ALT,TBIL and CRE were significantly increased in the high dosage treatment group,the liver samples from the high dose treatment group showed mild focal necrosis,steatosis,and inflammation,no pathological changes were observed in heart or kidney tissue samples from any of the groups.In the study of serum metabonomics based on UPLC/QTOF-MS,10 potential biomarkers were identified,they are Lyso PC(20:4),p-Cresol,ascorbic acid,p-Cresol sulfate,IDP,PC(14:1/20:5),PC(14:1/16:1),PE(22:1/18:1),PC(22:2/18:1),and PGP(18:1/22:6),compared with the control group,the contents of Lyso PC(20:4)in EEFP high dose and PGP(18:1/22:6)in EEFP medium,high dose significantly increased,the contents of p-Cresol,PC(14:1/20:5),PC(14:1/16:1),PC(22:2/18:1),and p-Cresol sulfate,IDP in EEFP low,medium,high dose and ascorbic acid,PE(22:1/18:1)in EEFP medium,high dose significantly decreased.(3)After 2 week’s administration of AEFP,compared with the control group,WBC was increased with a dose-dependent manner,but has no significantly difference,PLT and PCT were significantly decreased in the medium dosage treatment group,LYM,MON and NEUT were significantly increased in AEFP medium and high dose,AST and UREA were significantly decreased in AEFP medium and high dose.Through serum metabonomics,5 potential biomarkers were identified,they are indole,L-phenylalanine,phytosphingosine,lactic acid and ecgonine methyl,compared with the control group,indole,the contents of L-phenylalanine and phytosphingosine was increased in AEFP low,medium,high dose with a dose-dependent manner,the contents of lactic acid and ecgonine methyl ester was decreased in AEFP low,medium,high dose with a dose-dependent manner.(4)A total of 11 metabolites were identified after a single oral administration of bakuchiol,including 6 in plasma,10 in bile,8 in urine and 2 in feces,the metabolic transformation pathways of bakuchiol in rats included oxidation,hydroxylation,methylation,O-glucuronide conjugation and O-sulphate conjugation,the level of M1 reached a peak at 12 h and increased throughout a 24 h period,whereas,the level of M5 reached a peak at 4 h and then slowly decreased.CONCLUSION:(1)In this study,an ultra performance liquid chromatography quadrupole time of flight mass spectrometry(UPLC-PDA-Q-TOF-MS)technology has been used to achieve rapid qualitative analysis of multi-component of Fructus psoralen,12 components were identified,they are coumarins,such as psoralen,isopsoralen,flavonoids,such as corylin,chalcones,such as isobavachalcone,and meroterpenoid,such as bakuchiol.(2)After 2 week’s administration of EEFP,the change of hematological parameters,biochemical parameters and histological results show that long term EEFP exposure at high dose could induce liver toxicity in rats.10 potential biomarkers contribute to liver toxicity like Lyso PC,p-Cresol,ascorbic acid,p-Cresol sulfate and PC have been found,through analysis metabolism pathway of each biomarkers,the results were consistent with hematological parameters,biochemical parameters and histological results in explaining the toxic effects mechanism of EEFP in rats.(3)After 2 week’s administration of AEFP,the change of hematological parameters,biochemical parameters show that Fructus psoralen has significant anti-clotting,improve renal function and potential toxic effects.5 potential biomarkers like indole,L-phenylalanine,phytosphingosine,lactic acid and ecgonine methyl have been found,through analysis metabolism pathway of each biomarkers,Fructus psoralen’s mechanism of pharmacological and toxicity effects may be relate to amino acid metabolism,phospholipid metabolism,and glycolysis.(4)In the present study,UPLC-PDA-QTOF-MS coupled with Metabo Lynx XS software was successfully utilized to identify the metabolites of bakuchiol in rat plasma,bile,urine and feces.A total of 11 metabolites were detected and tentatively identified,the metabolic pathways and dynamic profiles of main metabolites in plasma from bakuchiol were studied.Among the 11 metabolites,the oxidation product and glucuronidated conjugates(M1,2,5)were major metabolites of bakuchiol in rats,M1 was mainly existed in serum and urine,M2 was mainly existed in urine and bile,M5 was mainly existed in bile and serum.M1 showed prolonged elimination in plasma.