| ObjectiveOctahydrocurcumin,the food-derived antioxidants,are the final hydrogenated metabolite of curcumin(a popular cooking spice,food pigment,and flavouring agent derived from the rhizome of Curcuma longa used for treatment of liver disorders).Nowadays,attention has been focused on octahydrocurcumin due to its similar but superior beneficial properties to curcumin,including anti-inflammatory,anti-tumor and especially anti-oxidant activities.However,ascribed to its poor availability,the pharmacological activities of octahydrocurcumin is rarely explored especially hepato-protective effect.Octahydrocurcumin is chiral,consisting of two stereoisomers.One is(3S,5S)-octahydrocurcumin((3S,5S)-OHC)and the other is(3R,5S)-octahydrocurcumin(also called Meso-octahydrocurcumin,Meso-OHC).However,there were rare reports about whether curcumin could be metabolized into octahydrocurcumin stereoisomers.It is well recognized that enantiomers of a chiral drug may differ in their pharmacological activities and pharmacokinetic properties due to the selective interaction with biomacromolecule such as enzymes.While,there was none report about whether octahydrocurcumin stereoisomers exhibited different effects on enzymes,thus leading to different pharmacological activities.Firstly,the present study was performed to investigate the potential and mechanism of octahydrocurcumin against acetaminophen(APAP)-induced hepatotoxicity in parallel to curcumin or tetrahydrocurcumin.Secondly,to investigate whether the identified curcumin could be metabolized into octahydrocurcumin stereoisomers,different samples such as plasma,liver,urine and feces from rats were collected after oral administration with curcumin,then analyzed using HPLC.Thirdly,the octahydrocurcumin stereoisomers were isolated and purified through flash column chromatography combined with solvent extraction;after that,the structure was individually identified through LC-MS chromatogram,Nuclear magnetic resonance(NMR)spectroscopy,specific rotation and single-crystal X-ray diffraction analysis.Fourthly,the different effects of octahydrocurcumin stereoisomers on CYPs and UGT enzymes activities and mRNA expression were investigated using an in vitro cocktail of probe substrates and reverse transcription-polymerase chain reaction(RT-PCR).Finally,the different potential and mechanisms of octahydrocurcumin stereoisomers against APAP-induced hepatotoxicity were investigated.Methods and results1.Hepato-protective effect of octahydrocurcumin against acetaminophen-induced hepatotoxicityThe present study was performed to investigate the potential and mechanism of octahydrocurcumin against APAP-induced hepatotoxicity in parallel to CUR and THC.All mice were subjected to the APAP,curcumin,tetrahydrocurcumin,octahydrocurcumin and N-acetyl cysteine pretreatment before overdosed with APAP,and liver tissues and serum were collected and examined.Our results showed that octahydrocurcumin dose-dependently enhanced the liver function(ALT and AST levels)and alleviated the histopathological deterioration.Besides,octahydrocurcumin significantly restored the hepatic antioxidant status by miring levels of MDA and ROS,and elevated levels of GSH,SOD,CAT and T-AOC.In addition,octahydrocurcumin suppressed the activity and expressions of CYP2E1,and bound to the active sites of CYP2E1.Moreover,octahydrocurcumin activated the Keapl-Nrf2 pathway and enormously enhanced the transcriptional and translational activation of Nrf2-targeted genes(GCLC,GCLM,NQO1 and HO-1)against oxidative stress,via inhibiting the expression of Keapl and blocking the interaction between Keapl and Nrf2.Particularly,octahydrocurcumin exerted superior hepato-protective and antioxidant activities to curcumin and tetrahydrocurcumin.2.Metabolism of curcumin in ratThe present study was conducted with male Sprague-Dawley rats.After oral administration with curcumin(100 mg/kg),the octahydrocurcumin stereoisomers in different samples such as plasma,liver,urine and feces from rats were collected after oral administration then analyzed using HPLC.Our results showed that curcumin could be metabolized into octahydrocurcumin stereoisomers,which were disproportionate in different samples:the ratio of M2 and M1 were 0.34,0.73,4.59 and 0.81 in plasma,liver,urine and feces respectively.3.Isolation and purification of octahydrocurcumin stereoisomersIn the present study,the octahydrocurcumin stereoisomers were isolated and purified through flash column chromatography combined with solvent extraction and preparative liquid chromatography,which were named as M1 and M2 in accordance with their retention time.After that,the structures were individually identified through LC-MS chromatogram,Nuclear magnetic resonance(NMR)spectroscopy,specific rotation and single-crystal X-ray diffraction analysis.After isolation by flash column chromatography combined with solvent extraction,the purity of stereoisomers M1 and M2 were over 95%;after purified by preparative liquid chromatography,the purity of stereoisomers M1 and M2 were over 98%.The structures of M1 and M2 were identified as octahydrocurcumin through LC-MS and 1H-NMR.The structures of M1 were identified as Meso-OHC through 13C-NMR,single-crystal X-ray diffraction analysis and specific rotation;M2 were identified as(3S,5S)-OHC through 13C-NMR and specific rotation.4.Different effect of octahydrocurcumin stereoisomers on CYPs and UGT enzymes activitiesIn the present study,the different effects of octahydrocurcumin stereoisomers on CYP(CYP1A2、CYP2A6、CYP2C9、CYP2C8 and CYP2E1 and UGTs enzymes activities and mRNA expression were investigated using an in vitro cocktail of probe substrates and reverse transcription-polymerase chain reaction(RT-PCR).Our results showed that Meso-OHC and(3S,5S)-OHC had none significant influence on activities and mRNA expression of CYP1A2、CYP2A6、CYP2C9、CYP2C8 and UGTs.However,Meso-OHC and(3S,5S)-OHC did exhibit stereoselective inhibition effect on activities and mRNA expression of CYP2E1:the inhibition effect of Meso-OHC was more powerful than(3S,5S)-OHC,which may result from different binding energy and binding sites.5.Hepato-protective effect of octahydrocurcumin stereoisomers against acetaminophen-induced hepatotoxicityThe present study was performed to investigate the potential and mechanism of octahydrocurcumin stereoisomers against APAP-induced hepatotoxicity.All mice were subjected to the APAP,Meso-OHC,(3S,5S)-OHC and N-acetyl cysteine pretreatment before overdosed APAP,and liver tissues and serum were collected and examined.Our results showed that Meso-OHC and(3S,5S)-OHC dose-dependently enhanced the liver function(ALT and AST levels)and alleviated the histopathological deterioration.Besides,OHC significantly restored the hepatic antioxidant status by miring levels of MDA,3-NT and 4-HNE,and elevated levels of GSH,SOD,CAT and T-AOC.In addition,Meso-OHC and(3S,5S)-OHC markedly suppressed the activity and expressions of CYP2E1,induced the activity and expressions of UGT1A1.Moreover,Meso-OHC and(3S,5S)-OHC activated the Keapl-Nrf2 pathway and enormously enhanced the transcriptional and translational activation of Nrf2-targeted genes(GCLC,GCLM,NQO1 and HO-1)against oxidative stress,via inhibiting the expression of Keapl and blocking the interaction between Keap1 and Nrf2.Besides,Meso-OHC and(3S,5S)-OHC also inhibited the NF K B(p65)pathway and enormously reduced the transcriptional and translational activation of NF κ B(p65)-targeted genes(TNF-α and IL-1β)to against inflammatory response.Particularly,Meso-OHC exerted superior hepato-protective and antioxidant activities to(3S,5S)-OHC;(3S,5S)-OHC exerted superior anti-inflammatory activities to Meso-OHC.ConclusionFirstly,octahydrocurcumin possessed superior hepato-protective effect against APAP-induced acute liver injury through restoring liver antioxidant status,suppressing CYP2E1 expression,activating Keap1-Nrf2 pathway,and up-regulating expressions of Nrf2-target genes.Secondly,curcumin could be metabolized into octahydrocurcumin stereoisomers with different proportions in different samples.Lastly,Meso-OHC and(3S,5S)-OHC,OHC stereoisomers,exhibited stereoselective inhibition effect on activities and mRNA expression of CYP2E1 in L-02 cell and stereoselective hepato-protective effect against APAP-induced hepatotoxicity... |
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