Dihydrotanshinone I-Induced CYP1 Enzyme Inhibition and Alteration of Estradiol Metabolism.

Dihydrotanshinone I (DHTI) is a pharmacologically active component occurring in the roots of the herbal medicine Salvia miltiorrhiza Bunge. This study investigated DHTI-induced inhibition of CYP1A1, CYP1A2, and CYP1B1 with the aim to determine the potential effects of DHTI on the bioactivation of estradiol (E2), possibly related to preventive/therapeutic strategy for E2-associated breast cancer. Ethoxyresorufin as a specific substrate for CYP1s was incubated with human recombinant CYP1A1, CYP1A2, or CYP1B1 in the presence of DHTI at various concentrations. Enzymatic inhibition and kinetic behaviors were examined by monitoring the formation of the corresponding product. Molecular docking was further conducted to define the interactions between DHTI and the three CYP1s. The same method and procedure were employed to examine the DHTI-induced alteration of E2 metabolism. DHTI showed significant inhibition of ethoxyresorufin O-deethylation activity catalyzed by CYP1A1, CYP1A2 and CYP1B1 in a concentration-dependent manner (IC50 = 0.56, 0.44, and 0.11 µM, respectively). Kinetic analysis showed that DHTI acted as a competitive type of inhibitor of CYP1A1 and CYP1B1, whereas it noncompetitively inhibited CYP1A2. The observed enzyme inhibition was independent of NADPH and time. Molecular docking analysis revealed hydrogen bonding interactions between DHTI and Asp-326 of CYP1B1. Moreover, DHTI displayed preferential activity to inhibit 4-hydroxylation of E2 (a genotoxic pathway) mediated by CYP1B1. Exposure to DHTI could reduce the risk of genotoxicity induced by E2. SIGNIFICANCE STATEMENT: CYP1A1, CYP1A2, and CYP1B1 enzymes are involved in the conversion of estradiol (E2) into 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2) through oxidation. 2-OHE2 is negatively correlated with breast cancer risk, and 4-OHE2 may be a significant initiator and promoter of breast cancer. The present study revealed that dihydrotanshinone I (DHTI) competitively inhibits CYP1A1/CYP1B1 and noncompetitively inhibits CYP1A2. DHTI exhibits a preference for inhibiting the genotoxicity associated with E2 4-hydroxylation pathway mediated by CYP1B1, potentially reducing the risk of 4-OHE2-induced genotoxicity.

Evidence for the Metabolic Activation of Deferasirox In Vitro and In Vivo.

Deferasirox (DFS) is used for the treatment of iron accumulation caused by the need for long-term blood transfusions, such as thalassemia or other rare anemia. Liver injury due to exposure to DFS has been documented, and the toxic mechanisms of DFS are unknown. The present study aimed to investigate the reactive metabolites of DFS in vitro and in vivo to help us understand the mechanisms of DFS hepatotoxicity. Two hydroxylated metabolites (5-OH and 5'-OH) were identified during incubation of DFS-supplemented rat liver microsomes. Such microsomal incubations fortified with glutathione (GSH) or N-acetylcysteine (NAC) as capture agents offered two GSH conjugates and two NAC conjugates. These GSH conjugates and NAC conjugates were also detected in bile and urine of rats given DFS. CYP1A2 and CYP3A4 were found to dominate the metabolic activation of DFS. Administration of DFS induced decreased cell survival in cultured primary hepatocytes. Pretreatment with ketoconazole and 1-aminobenzotrizole made hepatocytes less susceptible to the cytotoxicity of DFS.

Reduction of emodin-8-O-ß-D-glucoside content participates in processing-based detoxification of polygoni multiflori radix.

BACKGROUND: The occurrence of severe liver injury by the herbal medicine Polygoni Multiflori Radix (PMR) has drawn significant attention. The fact that processing attenuates PMR-induced hepatotoxicity has been well accepted, but the mechanisms are still ambiguous. PURPOSE: This study aimed to illuminate the mechanism of processing-based attenuation of PMR hepatotoxicity. METHODS: The contents of emodin-8-O-ß-d-glucoside (EG) and emodin (EMD) in raw and processed PMR were quantified. The difference in toxicokinetic behaviors of EG and EMD was determined in vivo, and the disposition properties of EG were investigated in vitro and in vivo. RESULTS: Decreased EG content was found in processed (black bean) PMR. Processed PMR showed reduced adverse effects relative to raw PMR. In addition, less hepatic protein adduction derived from EMD was produced in mice after exposure to processed PMR than that in animals receiving raw PMR. Glucose transporters SGLT1 and GLUT2 participated in the absorption of EG, and effective hydrolysis of EG to EMD took place in the intestinal epithelial cells during the process of absorption. Cytosolic broad-specificity ß-glucosidase and lactase phlorizin hydrolase, as well as intestinal flora, participated in the hydrolysis of EG. The circulated EMD resulting from the deglycosylation of EG executed the hepatotoxic action. CONCLUSION: EG is a pre-toxin and can be metabolically activated to EMD participating in the hepatotoxic event. The reduction of EG content due to processing is a key mechanistic factor that initiates the detoxification of PMR.

Difference in hepatotoxicity of furan-containing components in cortex dictamni correlates the efficiency of their metabolic activation.

BACKGROUND: Cortex Dictamni (CD) has been associated with an increased risk of liver injury, which may be attributable to the metabolic activation of its furan-containing components (FCC). However, the hepatotoxic potencies of these FCCs and the mechanisms behind the differences in their toxicity intensity remain unknown. METHODS: The constituents of CD extract were determined by LC-MS/MS. Potentially toxic FCCs were screened by a previously published method. Hepatotoxicity of potentially toxic FCCs was evaluated in cultured mouse primary hepatocytes and mice. The ability to deplete hepatic glutathione (GSH), along with the formation of the corresponding GSH conjugates, resulting from the metabolic activation was determined ex vivo in mice. Intrinsic clearance rates (CLint,Vmax/Km) were assessed by a microsome-bases assay. RESULTS: A total of 18 FCCs were detected in CD extract. Among them, four FCCs, including rutaevin (RUT), limonin (LIM), obacunone (OBA) and fraxinellone (FRA) were found to be bioactivated in microsomal incubations. Only FRA displayed significant hepatotoxicity in vitro and in vivo. Similarly, FRA caused GSH depletion and GSH conjugation the most in vivo. The order of CLint for the four FCCs was FRA>>OBA>LIM>RUT. CONCLUSION: FRA is the major toxic FCC component of hepatotoxic CD extract. The hepatotoxicity of FCCs is closely related to the efficiency of their metabolic activation.

DNA damage and up-regulation of PARP-1 induced by columbin in vitro and in vivo.

Columbin (CLB) is the most abundant (>1.0%) furan-containing diterpenoid lactone in herbal medicine Tinospora sagittate (Oliv.) Gagnep. The furano-terpenoid was found to be hepatotoxic, but the exact mechanisms remain unknown. The present study demonstrated that administration of CLB at 50 mg/kg induced hepatotoxicity, DNA damage and up-regulation of PARP-1 in vivo. Exposure to CLB (10 µM) induced GSH depletion, over-production of ROS, DNA damage, up-regulation of PARP-1 and cell death in cultured mouse primary hepatocytes in vitro. Co-treatment of mouse primary hepatocytes with ketoconazole (10 µM) or glutathione ethyl ester (200 µM) attenuated the GSH depletion, over-production of ROS, DNA damage, up-regulation of PARP-1, and cell death induced by CLB, while co-exposure to L-buthionine sulfoximine (BSO, 1000 µM) intensified such adverse effects resulting from CLB exposure. These results suggest that the metabolic activation of CLB by CYP3A resulted in the depletion of GSH and increase of ROS formation. The resultant over-production of ROS subsequently disrupted the DNA integrity and up-regulated the expression of PARP-1 in response to DNA damage, and ROS-induced DNA damage was involved in the hepatotoxicity of CLB.

In Vitro and In Vivo Metabolic Activation of Tolterodine Mediated by CYP3A.

Tolterodine (TOL) is an antimuscarinic drug used for the treatment of patients with overactive bladder presenting urinary frequency, urgency, and urge incontinence. During the clinical use of TOL, adverse events such as liver injury took place. The present study aimed at the investigation of the metabolic activation of TOL possibly associated with its hepatotoxicity. One GSH conjugate, two NAC conjugates, and two cysteine conjugates were found in both mouse and human liver microsomal incubations supplemented with TOL, GSH/NAC/cysteine, and NADPH. The detected conjugates suggest the production of a quinone methide intermediate. The same GSH conjugate was also observed in mouse primary hepatocytes and in the bile of rats receiving TOL. One of the urinary NAC conjugates was observed in rats administered TOL. One of the cysteine conjugates was found in a digestion mixture containing hepatic proteins from animals administered TOL. The observed protein modification was dose-dependent. CYP3A primarily catalyzes the metabolic activation of TOL. Ketoconazole (KTC) pretreatment reduced the generation of the GSH conjugate in mouse liver and cultured primary hepatocytes after TOL treatment. In addition, KTC reduced the susceptibility of primary hepatocytes to TOL cytotoxicity. The quinone methide metabolite may be involved in TOL-induced hepatotoxicity and cytotoxicity.

[Main spicy components, mechanism and masking technology for spicy flavor of Chinese medicine: a review].

Many Chinese medicinal materials, vegetable oils and extracts, and even Chinese patent medicines are spicy, which influences the medication compliance of patients, especially children. Different from the sour, sweet, bitter, salty, and umami tastes, it is a painful sensation formed when the spicy substances stimulate the nerve endings. At the moment, there are a few studies on the spicy components and mechanism and masking technology for the spicy flavor of Chinese medicine in the pharmaceutical industry, and the findings in food science are usually taken as a reference, which fail to guide the masking of the spicy flavor in Chinese medicine preparations. According to literature research, the exterior-releasing medicine, dampness-resolving medicine, and interior-warming medicine are spicy, especially some vegetable oils and extracts. Taking Zingiberis Rhizoma and prescriptions containing this medicinal as an example, the spicy components in Chinese medicine and the structure-activity characteristics were analyzed to reveal the mechanism for the spicy flavor: spicy components activate the transient receptor potential vanilloid subfamily member 1(TRPV1). The advantages and disadvantages of separation, neutralization with sugar, and inclusion for the masking of the spicy flavor were summarized and the applicability in Chinese medicine was analyzed. Moreover, the future development direction was put forward. This study is expected to promote the development of spicy masking technology for Chinese medicine prescriptions for children.

2,3,5,4'-Tetrahydroxy stilbene-2-Ο-ß-D-glucoside, a mechanism-based inactivator of CYP2C19 and CYP3A4, potentiates hepatic protein adduction and hepatotoxicity induced by emodin in vivo.

2,3,5,4'-Tetrahydroxy stilbene-2-Ο-ß-D-glucoside (TSG) and emodin (EMD) are two main components of Polygonum multiflorum Thunb. (PMT). Its root is widely used as herbal medicine and supplement. However, PMT-induced liver injury has drawn increasing attention. The purpose of this study was to investigate the interaction of TSG with EMD in the aspects of enzymology, pharmacokinetics, and hepatotoxicity. Co-administration with TSG increased internal exposure of EMD, EMD-derived hepatic protein adduction, and EMD-induced liver injury in mice. Mouse and human liver microsomal incubation study demonstrated that co-incubation with TSG decreased the formation of hydroxylation metabolites of EMD. Human recombinant cytochrome P450 enzyme incubation study showed that TSG induced time-, concentration-, NADPH-dependent and irreversible inhibition of CYP2C19 and CYP3A4. An epoxide metabolite derived from TSG was responsible for the observed enzyme inactivations. The findings allow us to better understand the mechanisms by which herbal processing detoxifies raw PMT.

Diverse sesquiterpenoids from Litsea lancilimba Merr. with potential neuroprotective effects against H2O2-induced SH-SY5Y cell injury.

Five undescribed sesquiterpenoids (1-5), and nine known sesquiterpenoids (6-14) were obtained from the fruits of Litsea lancilimba Merr. by LC-MS/MS molecular networking strategies. Litsemene A (1) possessed a unique 8-member ring through unexpected cyclization of the methyl group on C-10 of guaiane. Their structures were elucidated by spectroscopic techniques including IR, UV, NMR, HR-ESI-MS, and their absolute configurations were assigned by ECD calculations. All isolated sesquiterpenoids were analyzed by bioinformatics and evaluated for their neuroprotective effects against H2O2-induced injury in human neuroblastoma SH-SY5Y cells.

Asparagine and Glutamine Residues Participate in Protein Covalent Binding by Epoxide Metabolite of 8-Epidiosbulbin E Acetate In Vitro and In Vivo.

Dioscorea Bulbifera L. (DBL), an effective traditional Chinese medicine, has been restricted because of multiple reports that it can cause severe hepatotoxicity. 8-Epidiosbulbin E acetate (EEA), one of the main components of DBL, can induce severe liver injury. It has been reported that EEA can be metabolized by CYP3A to the corresponding cis-enedial intermediate which alkylates the lysine residues of proteins to form pyrroline derivatives. The present study unexpectedly found that the reactive intermediate reacted with the amide groups of asparagine (Asn) and glutamine (Gln) residues of hepatic proteins of mice treated with EEA. The amide-derived protein modification increased with the increase in the dose administered. Like the adduction of the primary amine of lysine residues, the electrophilic metabolite reacted with the amide groups of Asn and Gln residues to offer the corresponding pyrrolines. The structures of the pyrrolines were confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy.

Diosbulbin B: An important component responsible for hepatotoxicity and protein covalent binding induced by Dioscorea bulbifera L.

BACKGROUND: Dioscorea bulbifera L. (DBL) is an herbal medicine used for the treatment of thyroid diseases and tumors in China. However, the hepatotoxicity of DBL limits its wide safe use. Diosbulbin B (DSB) is the most abundant diterpene lactone occurring in DBL. Numbers of studies showed that this furanoterpenoid plays an important role in DBL-induced liver injury and that DSB is metabolized to a cis-enedial intermediate which reacts with protein to form protein covalent binding and induces hepatotoxicity. PURPOSE: The present study aimed to define the association of DSB content in DBL with the severity of DBL hepatotoxicity to ensure the safe use of the herbal medicine in clinical practice and to determine the role of DSB in DBL-induced liver injury. METHODS: Chemical chromatographic fingerprints of DBL were established by UPLC-MS/MS. Their hepatotoxicity potencies were evaluated in vitro and in vivo. Metabolic activation of DSB was evaluated by liver microsomal incubation. Protein modification was assessed by mass spectrometry and immunostaining. RESULTS: The contents of DSB in DBL herbs collected from 11 locations in China varied dramatically with as much as 47-fold difference. The hepatotoxicity potencies of DBL herbs were found to be proportional to the contents of DSB. Intensified protein adduction derived from the reactive metabolite of DSB was observed in mice administered DBL with high contents of DSB. CONCLUSION: The findings not only demonstrated that contents of DSB can be quite different depending on harvest location and special attention needs to pay for quality control of DBL but also suggest DSB is a key contributor for DBL-induced hepatotoxicity.

Evidence for adduction of biologic amines with reactive metabolite of 8-epidiosbulbin E acetate in vitro and in vivo.

Dioscorea bulbifera L. (DBL) is one of traditional Chinese medicines and has been used for the treatment of goiter, tumor and carbuncles. However, clinic application of the herbal medicine has been limited, due to reported severe hepatotoxicity. 8-Epidiosbulbin E acetate (EEA), one of the major components of DBL, can cause severe liver damage. The furan ring of EEA is metabolized by CYP3A4 to a cis-enedial reactive intermediate prone to react amino and/or thiol groups of amino acid residues. In this study, we investigated the interaction of the reactive intermediate with biologic amines. EEA-derived biologic amine adducts, including spermidine, spermine, putrescine, ornithine, lysine and glutamine were detected in cultured mouse primary hepatocytes treated with EEA. Only spermidine adduct was observed in bile of mice given EEA. The detection of the adducts was established by labeling with bromobenzyl mercaptan and LC-MS/MS analysis. Exposure of EEA resulted in concentration dependent cytotoxicity in hepatocytes. Pretreatment with spermidine attenuated the susceptibility of cells to the cytotoxicity of EEA, because of the compensation of the depleted spermidine.

Development of a mechanism-based biomarker for Dioscorea bulbifera L. exposure and hepatotoxicity in rats.

BACKGROUND: Dioscorea bulbifera L. (DBL) is a common herbal medicine where furanoterpenoid diosbulbin B (DSB) is a major component responsible for its hepatotoxicity. The metabolic oxidation of the furan moiety of DSB, resulting in covalent binding to hepatic protein, is considered to initiate its liver injury. PURPOSE: We aimed to develop a mechanism-based plasma protein adduction-based biomarker to determine DBL exposure and to predict the onset of hepatotoxicity induced by DBL. METHODS: Rats were intragastrically treated with DBL extract, and the plasma samples were collected. Plasma ALT and AST were measured with commercial kits. Plasma protein modification was determined by immunoblot assay. Assessment of DSB-induced protein adduction was achieved by LC-MS/MS analysis of complete proteolytic digestion of adducted protein to pyrroline derivative A4 using pronase enzyme. The structure of the resulting pyrroline derivatives was confirmed by NMR. RESULTS: Plasma protein of rats treated with DBL extract was covalently modified by the metabolite of DSB. Pyrroline derivative A4 was detected in proteolytic digestion of plasma obtained from rats administered DBL extract. The protein adduction elevated with the increase in the dosage of DBL extract. A detectable level of plasma was observed 10 days after withdrawal of DBL extract post 30-day continuous administration. In addition, the elevation trend of plasma ALT was found to be proportional to the accumulation trend of pyrroline derivative A4. CONCLUSION: DSB-derived plasma protein adduction correlated well with the exposure of DBL in rats. The protein adduction may be used as a good biomarker for diagnosis of DBL-induced liver injury and a useful indicator for DBL medication plans.

Phytochemistry, synthesis, analytical methods, pharmacological activity, and pharmacokinetics of loganin: A comprehensive review.

Iridoid glycosides (IGs) are found in many medicinal and edible plants, such as Gardenia jasminoides, Cistanche tubulosa, Eucommia ulmoides, Rehmanniae Radix, Lonicera japonica, and Cornus officinalis. Loganin, an IG, is one of the main active ingredient of Cornus officinalis Sieb. et Zucc., which approved as a medicinal and edible plant in China. Loganin has been widely concerned due to its extensive pharmacological effects, including anti-diabetic, antiinflammatory, neuroprotective, and anti-tumor activities, etc. Studies have shown that these underlying mechanisms include anti-oxidation, antiinflammation and anti-apoptosis by regulating a variety of signaling pathways, such as STAT3/NF-κB, JAK/STAT3, TLR4/NF-κB, PI3K/Akt, MCP-1/CCR2, and RAGE/Nox4/p65 NF-κB signaling pathways. In order to better understand the research status of loganin and promote its application in human health, this paper systematically summarized the phytochemistry, analysis methods, synthesis, pharmacological properties and related mechanisms, and pharmacokinetics based on the research in the past decades.

Metabolic Activation of Militarine In Vitro and In Vivo.

Bletilla striata is consumed as food and herbal medicine. Militarine (MLT) is a major ingredient in B. striata. Previous studies demonstrated that MLT showed teratogenic toxicity to zebrafish embryos. The present study aimed to identify reactive metabolites possibly involved in the cytotoxicity of MLT and determine the metabolic pathways involved. MLT was found to be hydrolyzed to p-hydroxybenzyl alcohol (HBA) by ß-glucosidase and esterases. The resulting HBA further underwent spontaneous dehydration to form quinone methide. HBA was also metabolized to the corresponding sulfate, followed by departure of the sulfate to generate a quinone methide. The resultant quinone methide reacted with hepatic glutathione (GSH) and protein to form the corresponding GSH conjugate and protein adduction. Additionally, inhibition of sulfotransferases (SULTs) attenuated the susceptibility of hepatocytes to the toxicity of MLT. This study provides that the hydrolytic enzymes ß-glucosidase, esterases, and SULTs participate in the metabolic activation of MLT.

Small Molecule Accurate Recognition Technology accelerated isolation of structurally diverse sesquiterpenes from Litsea lancilimba Merr.

Four undescribed sesquiterpenes (1-4) and 20 known sesquiterpenes (5-24) were obtained from the fruits of Litsea lancilimba Merr. by Small Molecule Accurate Recognition Technology (SMART). The gross structures and the relative configurations of the new compounds were deduced by spectroscopic data analysis. The absolute configurations were defined by comparison of their experimental and calculated electronic circular dichroism (ECD) spectra data. All compounds were screened for their neuroprotective effects against H2O2-induced SH-SY5Y cells injury. Compounds 2, 3, 7, 8 and 13 exhibited comparable neuroprotective activity to the positive trolox at 50 µM.

Metabolic Activation of Perampanel Mediated by CYP1A2.

Perampanel (PRP), a noncompetitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropanoic acid (AMPA) receptor antagonist with high selectivity, has been used as a new adjuvant for the treatment of fractional seizures with or without primary generalized tonic-clonic seizures and secondary generalized seizures in epilepsy patients over the age of 12. Adverse events such as liver injury have been reported during the clinical application of PRP. The purpose of the study is to explore the in vitro and in vivo metabolic activation of PRP. Two GSH conjugates were detected in rat liver microsomal incubations containing PRP, GSH, and NADPH. The two GSH conjugates were both obtained from the bile of rats and rat primary hepatocytes after exposure to PRP. Similar microsomal incubations complemented with N-acetylcysteine (NAC) in place of GSH offered two NAC conjugates. As expected, the NAC conjugates were detected in the urine of PRP-treated rats. One of the two NAC conjugates was identified as NAC conjugate 12 verified by chemical synthesis. The individual human recombinant P450 enzyme incubation assay demonstrated that CYP1A2 dominated the catalysis for the metabolic activation of PRP. Pretreatment with α-naphthoflavone (NTF) decreased the formation of PRP-derived GSH conjugates in both livers of rats and cultured primary hepatocytes after being treated with PRP. Additionally, NTF was found to decrease the susceptibility of primary hepatocytes to the cytotoxicity of PRP. The findings indicate that PRP was metabolized to the corresponding epoxide, which could participate in PRP-induced cytotoxicity.

Shenxiong glucose injection inhibits H2O2-induced H9c2 cell apoptosis by activating the ERK signaling pathway.

BACKGROUND: Shenxiong glucose injection (SGI) is a traditional Chinese medicine injection composed of water extract of Salvia miltiorrhiza and Ligustrazine hydrochloride. SGI has shown strong antioxidant and anti-apoptotic properties. However, the mechanisms underlying its anti-apoptotic effect need to be addressed. METHODS: H9c2 cell apoptosis model was established by treatment of hydrogen peroxide (H2O2). Cell survival rates were examined by MTS assay, cell apoptosis rates were determined by flow cytometry, levels of intracellular ROS were assessed by ROS kit, proteome phosphorylation was determined by phosphoproteomic analysis, and extracellular signal-regulated kinase (ERK), phosphorylated ERK, phosphorylated c-Jun, B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), Bcl-2, and cleaved caspase-3 were examined by Western blot. RESULT: SGI showed protective effects against H2O2-induced reduced cell viability, elevated ROS, and increased apoptosis in H9c2 cells. Phosphorylation proteomics detected a total of 3369 proteins with 78 protein of upregulated phosphorylation and 104 protein of downregulated phosphorylation. Kyoto Encyclopedia Genes and Genomes pathway analyses of differentially phosphorylated proteins showed that the ERK pathway, the downstream pathway of the focal adhesion pathway related to apoptosis, was highly enriched, and the phosphorylation levels of ERK and c-Jun were confirmed by Western blot. In addition, the ERK pathway inhibitor PD98059 significantly inhibited the anti-apoptotic effect of SGI. CONCLUSION: SGI antagonizes H2O2-induced cell apoptosis by activating the ERK pathway.

New bisabolane-type phenolic sesquiterpenoids from the marine sponge Plakortis simplex.

Six new bisabolane-type phenolic sesquiterpenoids, including plakordiols A-D (1-4), (7R, 10R)-hydroxycurcudiol (5) and (7R, 10S)-hydroxycurcudiol (6) were isolated from the marine sponge Plakortis simplex collected from the South China Sea. Their structures were determined based on extensive analysis of spectroscopic data. Their configurations were assigned by coupling constant analysis, NOESY correlations, and the modified Mosher's method. Furthermore, their cytotoxic and antibacterial activities were evaluated.

Oxypeucedanin is a Mechanism-based Inactivator of CYP2B6 and CYP2D6.

BACKGROUND: Herbal medicine Angelica dahurica is widely employed for the treatment of rheumatism and pain relief in China. Oxypeucedanin is a major component in the herb. OBJECTIVES: The objectives of this study are aimed at the investigation of mechanism-based inactivation of CYP2B6 and CYP2D6 by oxypeucedanin, characterization of the reactive metabolites associated with the enzyme inactivation, and identification of the P450s participating in the bioactivation of oxypeucedanin. METHODS: Oxypeucedanin was incubated with liver microsomes or recombinant CYPs2B6 and 2D6 under designed conditions, and the enzyme activities were measured by monitoring the generation of the corresponding products. The resulting reactive intermediates were trapped with GSH and analyzed by LC-MS/MS. RESULTS: Microsomal incubation with oxypeucedanin induced a time-, concentration-, and NADPH-dependent inhibition of CYPs2B6 and 2D6 with kinetic values of KI/kinact 1.82 µM/0.07 min-1 (CYP2B6) and 8.47 µM/0.044 min-1 (CYP2D6), respectively. Ticlopidine and quinidine attenuated the observed time-dependent enzyme inhibitions. An epoxide and/or γ-ketoenal intermediate(s) derived from oxypeucedanin was/were trapped in microsomal incubations. CYP3A4 was the primary enzyme involved in the bioactivation of oxypeucedanin. CONCLUSION: Oxypeucedanin was a mechanism-based inactivator of CYP2B6 and CYP2D6. An epoxide and/or γ- ketoenal intermediate(s) may be responsible for the inactivation of the two enzymes.