Coumarin Metabolism Research Articles

Metabolism and Antifungal Activity of Coumarin and its Derivatives

Metabolism and Antifungal Activity of Coumarin and its Derivatives

Selenium nanoparticles induce coumarin metabolism and essential oil production in Trachyspermum ammi under future climate CO2 conditions.

Research on nanoparticles (NPs) and future elevated CO2 (eCO2) is extensive, but the effects of SeNPs on plant growth and secondary metabolism under eCO2 remain uncertain. In this study, we explored the impact of SeNPs and/or eCO2 on the growth, physiology, chemical composition (primary metabolites, coumarins, and essential oils), and antioxidant capacity of Trachyspermum (T.) ammi. The treatment with SeNPs notably improved the biomass and photosynthesis of T. ammi plants, particularly under eCO2 conditions. Plant fresh and dry weights were improved by about 19, 33 and 36% in groups treated by SeNPs, eCO2, and SeNPs + eCO2, respectively. SeNPs + eCO2 induced photosynthesis, consequently enhancing sugar and amino acid levels. Similar to the increase in total sugars, amino acids showed variable enhancements ranging from 6 to 42% upon treatment with SeNPs + eCO2. At the level of the secondary metabolites, SeNPs + eCO2 substantially augmented coumarin biosynthesis and essential oil accumulation. Consistently, there were increases in coumarins and essential oil precursors (shikimic and cinnamic acids) and their biosynthetic enzymes. The enhanced accumulation of coumarins and essential oils resulted in increased overall antioxidant activity, as evidenced by improvements in FRAP, ORAC, TBARS, conjugated dienes, and inhibition % of hemolysis. Conclusively, the application of SeNPs demonstrates significant enhancements in plant growth and metabolism under future CO2 conditions, notably concerning coumarin metabolism and essential oil production of T. ammi.

Structural Impact Assessment of Cytochrome P450 2A13 Polymorphisms Using Molecular Dynamics Simulations.

One of the members of CYP, a monooxygenase, CYP2A13 is involved in the metabolism of nicotine, coumarin, and tobacco-specific nitrosamine. Genetic polymorphisms have been identified in CYP2A13, with reported loss or reduction in enzymatic activity in CYP2A13 allelic variants. This study aimed to unravel the mechanism underlying the diminished enzymatic activity of CYP2A13 variants by investigating their three-dimensional structures through molecular dynamics (MD) simulations. For each variant, MD simulations of 1000 ns were performed, and the obtained results were compared with those of the wild type. The findings indicated alterations in the interaction with heme in CYP2A13.4, .6, .8, and .9. In the case of CYP2A13.5, observable effects on the helix structure related to the interaction with the redox partner were identified. These conformational changes were sufficient to cause a decrease in enzyme activity in the variants. Our findings provide valuable insights into the molecular mechanisms associated with the diminished activity in the CYP2A13 polymorphisms.

Genome-wide analysis of the cytochrome P450 superfamily suggests its roles in coumarin biosynthesis and salt stress response in Melilotus albus

Coumarin, a secondary metabolite from the plant phenylpropane pathway, was certified as an important natural product for plants in response to abiotic stresses. Cytochrome P450s represent the largest monooxygenase superfamily in kingdoms, participating in various biochemical processes including coumarin biosynthesis. Here, we reported a total of 290 P450 genes identified from the Melilotus albus genome, and these MaP450s were grouped into 9 clans. These genes were unevenly distributed in eight chromosomes and all of them contain highly conserved feature motifs of P450s. MaCYP82L1 was differentially expressed in JiMa46 and JiMa49, meanwhile up-regulated in response to NaCl treatment. The heterologous expression in yeast showed that the MaCYP82L1 transformed yeast cells exhibited obvious resistance under drought and salt stresses. Furthermore, overexpression of MaCYP82L1 increased coumarin content in transgenic hairy roots. This study not only identified MaCYP82L1 as an important regulator that adjusts drought and salt resistance, but also provided a link between coumarin metabolism and abiotic stresses.

Anabolic metabolism of autotoxic substance coumarins in plants

Background Autotoxicity is an intraspecific manifestation of allelopathy in plant species. The specialized metabolites and their derivatives that cause intraspecific allelopathic inhibition in the plant are known as autotoxic substances. Consequently, autotoxic substances production seriously affects the renewal and stability of ecological communities. Methods This article systematically summarizes the types of autotoxic substances present in different plants. They mainly include phenolic compounds, terpenoids, and nitrogenous organic compounds. Phenolic coumarins are the main autotoxic substances in many plants. Therefore, we also discuss differences in coumarin types and content among plant varieties, developmental stages, and tissue parts, as well as their mechanisms of autotoxicity. In addition, we review the metabolic pathways involved in coumarin biosynthesis, the key enzymes, genes, and transcription factors, as well as factors affecting coumarin biosynthesis. Results Coumarin biosynthesis involves three stages: (1) the formation of the coumarin nucleus; (2) acylation, hydroxylation, and cyclization; (3) structural modification. The key enzymes involved in the coumarin nuclear formation stage include PAL, C4H, 4CL, HCT, CAOMT, COSY, F6’H, and CCoAOMT1, and the key genes involved include BGA, CYP450 and MDR, among others. Ortho-hydroxylation is a key step in coumarin biosynthesis and PS, COSY and S8H are the key enzymes involved in this process. Finally, UGTs are responsible for the glycosylation modification of coumarins, and the MaUGT gene may therefore be involved in coumarin biosynthesis. Conclusion It is important to elucidate the autotoxicity and anabolic mechanisms of coumarins to create new germplasms that produce fewer autotoxic substances.

Integrative analysis of metabolome, proteome, and transcriptome for identifying genes influencing total lignin content in Populus trichocarpa.

Lignin, a component of plant cell walls, possesses significant research potential as a renewable energy source to replace carbon-based products and as a notable pollutant in papermaking processes. The monolignol biosynthetic pathway has been elucidated and it is known that not all monolignol genes influence the total lignin content. However, it remains unclear which monolignol genes are more closely related to the total lignin content and which potential genes influence the total lignin content. In this study, we present a combination of t-test, differential gene expression analysis, correlation analysis, and weighted gene co-expression network analysis to identify genes that regulate the total lignin content by utilizing multi-omics data from transgenic knockdowns of the monolignol genes that includes data related to the transcriptome, proteome, and total lignin content. Firstly, it was discovered that enzymes from the PtrPAL, Ptr4CL, PtrC3H, and PtrC4H gene families are more strongly correlated with the total lignin content. Additionally, the co-downregulation of three genes, PtrC3H3, PtrC4H1, and PtrC4H2, had the greatest impact on the total lignin content. Secondly, GO and KEGG analysis of lignin-related modules revealed that the total lignin content is not only influenced by monolignol genes, but also closely related to genes involved in the "glutathione metabolic process", "cellular modified amino acid metabolic process" and "carbohydrate catabolic process" pathways. Finally, the cinnamyl alcohol dehydrogenase genes CAD1, CADL3, and CADL8 emerged as potential contributors to total lignin content. The genes HYR1 (UDP-glycosyltransferase superfamily protein) and UGT71B1 (UDP-glucosyltransferase), exhibiting a close relationship with coumarin, have the potential to influence total lignin content by regulating coumarin metabolism. Additionally, the monolignol genes PtrC3H3, PtrC4H1, and PtrC4H2, which belong to the cytochrome P450 genes, may have a significant impact on the total lignin content. Overall, this study establishes connections between gene expression levels and total lignin content, effectively identifying genes that have a significant impact on total lignin content and offering novel perspectives for future lignin research endeavours.

Growth Developmental Defects of Mitochondrial Iron Transporter 1 and 2 Mutants in Arabidopsis in Iron Sufficient Conditions.

Iron is the most abundant micronutrient in plant mitochondria, and it has a crucial role in biochemical reactions involving electron transfer. It has been described in Oryza sativa that Mitochondrial Iron Transporter (MIT) is an essential gene and that knockdown mutant rice plants have a decreased amount of iron in their mitochondria, strongly suggesting that OsMIT is involved in mitochondrial iron uptake. In Arabidopsis thaliana, two genes encode MIT homologues. In this study, we analyzed different AtMIT1 and AtMIT2 mutant alleles, and no phenotypic defects were observed in individual mutant plants grown in normal conditions, confirming that neither AtMIT1 nor AtMIT2 are individually essential. When we generated crosses between the Atmit1 and Atmit2 alleles, we were able to isolate homozygous double mutant plants. Interestingly, homozygous double mutant plants were obtained only when mutant alleles of Atmit2 with the T-DNA insertion in the intron region were used for crossings, and in these cases, a correctly spliced AtMIT2 mRNA was generated, although at a low level. Atmit1 Atmit2 double homozygous mutant plants, knockout for AtMIT1 and knockdown for AtMIT2, were grown and characterized in iron-sufficient conditions. Pleiotropic developmental defects were observed, including abnormal seeds, an increased number of cotyledons, a slow growth rate, pinoid stems, defects in flower structures, and reduced seed set. A RNA-Seq study was performed, and we could identify more than 760 genes differentially expressed in Atmit1 Atmit2. Our results show that Atmit1 Atmit2 double homozygous mutant plants misregulate genes involved in iron transport, coumarin metabolism, hormone metabolism, root development, and stress-related response. The phenotypes observed, such as pinoid stems and fused cotyledons, in Atmit1 Atmit2 double homozygous mutant plants may suggest defects in auxin homeostasis. Unexpectedly, we observed a possible phenomenon of T-DNA suppression in the next generation of Atmit1 Atmit2 double homozygous mutant plants, correlating with increased splicing of the AtMIT2 intron containing the T-DNA and the suppression of the phenotypes observed in the first generation of the double mutant plants. In these plants with a suppressed phenotype, no differences were observed in the oxygen consumption rate of isolated mitochondria; however, the molecular analysis of gene expression markers, AOX1a, UPOX, and MSM1, for mitochondrial and oxidative stress showed that these plants express a degree of mitochondrial perturbation. Finally, we could establish by a targeted proteomic analysis that a protein level of 30% of MIT2, in the absence of MIT1, is enough for normal plant growth under iron-sufficient conditions.

Interactive influence of elevated CO2 and arbuscular mycorrhizal fungi on sucrose and coumarin metabolism in Ammi majus

The elevated level of CO2 (eCO2) and arbuscular mycorrhizal fungi (AMF) have been known as successful eco-friendly agents for plant growth and development as well as quality enhancers. The current investigation was designed to study the influence of eCO2 (620 μmol CO2 mol−1 air) and AMF on sucrose and phenylpropanoid metabolism, including coumarins, the most important bioactive metabolite in Ammi majus. eCO2 and AMF were applied, and different parameters have been assessed in A. majus such as changes in mycorrhizal colonization, plant biomass production, photosynthesis, and levels of N, P, and Ca besides the key metabolites and enzymes in sucrose and coumarins metabolic pathways. The present outcomes revealed that eCO2 and AMF individually or combined enhanced the plant biomass and photosynthesis as well as nutrient concentrations. Furthermore, the levels of sucrose, soluble sugars, glucose, fructose, and the activities of some key enzymes in their metabolism besides phenylpropanoids metabolites in shoot and root of A. majus have been enhanced by eCO2 and AMF especially when combined. Moreover, upregulation of sucrose is linked to phenylpropanoids metabolic pathway via upregulation of phenylalanine ammonia-lyase activity suggesting high coumarin biosynthesis. Generally, the synergistic effect of both treatments was noted for most of the investigated parameters compared to the individual effect. It could be concluded that the combined application of eCO2 and AMF affects A. majus global metabolism and induces accumulation of phyto-molecules, coumarin, which might improve its medicinal and pharmacological applications.

Enhancing biomass and productivity of coumarins and essential oil in ajwain (Trachyspermum ammi) sprouts via laser light treatment

Ajwain, Trachyspermum (T.) ammi, is a coumarin-rich, highly nutritive, and medicinally important herb, however, there is not enough data on the efficiency of laser irradiation in inducing such properties. Herein, the effect of seed treatment with laser irradiations [helium-neon (He-Ne, 632 nm, 5 mW) and helium-cadmium (He-Cd, 460 nm, 16.2 mW)] at 500 mJ energy for 5 min on the biomass, photosynthesis, and the metabolism of coumarins, essential oil, and their precursor metabolites in two varieties of ajwain sprouts i.e., Desi and Chakwal was investigated for the first time. Laser irradiation increased sprout biomass accumulation by improving photosynthesis, chlorophyll content, and respiration. Additionally, laser treatment enhanced the metabolism of coumarins and essential oil, which was explained by the increased availability of their precursors (phenylalanine, cinnamic acid, and shikimic acid). There were also increases in the activity of the key enzymes involved in the biogenesis of phenylpropanoid and coumarins, i.e., 3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS), cinnamate-4-hydroxylase (C4H), 4-coumarate: coenzyme A ligase (4CL), and p-coumarate 3-hydroxylase (C3H). Furthermore, laser treatment induced the accumulation of total and individual phenolics and flavonoids. Consequently, laser treatment significantly improved the antibacterial properties of ajwain sprouts against four foodborne pathogens included Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, and Salmonella spp. Ajwain variety-specific responses to laser light treatment confirmed by principal component analysis (PCA) showed that Desi variety was more responsive. The efficiency of He-Ne was greater than He-Cd laser, which suggests He-Ne laser treatment as a promising approach to boost the production of valuable pigments, coumarins, and essential oils in ajwain sprouts.

Metabolic activation and deactivation of dietary-derived coumarin mediated by cytochrome P450 enzymes in rat and human liver preparations.

Dietary-derived coumarin is of clinical interest for its potential hepatotoxicity in humans because such toxicity is especially evident in rats. In this study, the oxidative metabolism of coumarin to active coumarin 3,4-epoxide (as judged by the formation rates of o-hydroxyphenylacetic acid) and excretable 7-hydroxycoumarin was investigated in liver fractions from rats and humans. In rat liver microsomes, the formation rate of o-hydroxyphenylacetic acid (~6 pmol/min/mg microsomal protein) from coumarin at 10 μM was dependent on the presence of liver cytosolic fractions. Rat hepatocytes mediated similar formation rates of o-hydroxyphenylacetic acid and 7-hydroxycoumarin (~0.1 nmol/hr/106 cells) at 0.20-20 μM coumarin. Human hepatocytes mediated the biotransformation of coumarin to o-hydroxyphenylacetic acid at roughly similar rates to those of rat hepatocytes. In contrast, the formation rates of 7-hydroxycoumarin by human hepatocytes were around 10-fold higher at ~1 nmol/hr/106 cells. In the presence of human liver cytosolic fractions, the oxidative formation rate of o-hydroxyphenylacetic acid was relatively high in cytochrome P450 (P450) 1A2-rich human liver microsomes. The inhibitory effects of furafylline/α-naphthoflavone and 8-methoxypsoralen, P450 1A2 and 2A6 inhibitors, respectively, were seen on the rates of o-hydroxyphenylacetic and 7-hydroxylation formations, respectively, in pooled human liver microsomes. Human liver microsomes selectively inactivated for P450 1A2 and 2A6 showed low rates of o-hydroxyphenylacetic acid and 7-hydroxylation formation (~20-30% of control), respectively. Among the P450 isoforms tested, recombinant human P450 1A2 predominantly mediated o-hydroxyphenylacetic formation. These results suggested that the metabolic activation and deactivation of coumarin were mediated mainly by P450 1A2 and 2A6 enzymes, respectively. The metabolic oxidation of coumarin via 3,4-epoxidation forming o-hydroxyphenylacetic acid could inform individual human risk assessments of dietary-derived coumarin, for which hepatotoxicity is especially evident in rats.

Toxicological research and safety consideration of coumarins

Coumarin is an important class of natural organic compounds, which widely exists in a variety of plants and microorganisms. Coumarins have many biological activities and wide clinical applications, such as anti-tumor, anti-HIV, anti-bacterial, anti-inflammatory, anti-oxidation, anti-coagulation, but they have obvious toxic effects in rodents. It was found that the toxicity of coumarins in different animals and organs was significantly different, and high dose oral administration was more likely to produce toxic reactions. Based on the research and analysis of domestic and foreign literatures in recent 60 years, this paper mainly summarized the hepatotoxicity and pulmonary toxicity induced by coumarins, and probed into their possible mechanisms. It was found that the toxicity of coumarins had metabolic differences and species differences. The liver of rats and lungs of mice were more susceptible to coumarins. Toxic reactions occurred mainly in the second metabolic pathway of coumarin metabolism in vivo. In order to put forward safety considerations and evaluate the impact of coumarin on human body, it was found that coumarin is unlikely to produce hepatotoxicity at normal exposure level. It was also suggested that species differences due to different metabolic patterns in model animals should be carefully considered when assessing coumarin toxicity, in order to provide reference for clinical research and rational use of coumarins and improve the rational use of coumarins.

From Genotype to Phenotype: Content and Activities of Cytochromes P450 2A6 in Human Liver In Vitro and Predicted In Vivo.

Unraveling the molecular mechanisms by which genetic variants of cytochrome P450 2A6 lead to different metabolic phenotypes remains a long-standing but important challenge. CYP2A6 is an enzyme involved in the metabolism of several clinical drugs as well as the metabolic activation of carcinogenic nitrosamines. Herein, CYP2A6 genotypes and phenotypes, as indicated by protein content [by liquid chromatography-mass spectrometry (MS)/MS] and metabolic activities [Vmax, clearance (CL)], were determined for 90 human liver samples. We determined the median, range, and interindividual and intraindividual variation of CYP2A6 content and activity at the microsomal, liver tissue, and whole liver level and predicted hepatic in vivo clearance by in vitro-in vivo extrapolation based on CYP2A6-mediated coumarin metabolism by each CYP2A6 genotype. These results reveal how different CYP2A6 genotypes yield different phenotypic traits in protein content and enzyme activity. For relative Vmax, CL, and protein content, the intraindividual percentage coefficients of variation (ICVs) were 41.0% (18.8%-125.1%), 28.5% (2.39%-133.5%), and 27.8% (2.68%-88.0%), respectively. The high ICVs implied large intraindividual variation at different levels, sometimes in a genotype-dependent manner. Intergenotype analysis revealed that the CYP2A6*4 allele demonstrated the most obvious effect on phenotypic outcomes, both in protein content and in metabolic activity. Indeed, decreased CYP2A6 protein content with the CYP2A6*4 genotype might explain the decreased metabolic activity from the molecular to the organismal level. These findings may allow useful predictions for CYP2A6-mediated drug metabolism on an individual patient basis in accord with the goal of achieving personalized medicine. SIGNIFICANCE STATEMENT: We provide the median, range, and interindividual and intraindividual variation in CYP2A6 content at the microsomal, liver tissue, and whole liver level by liquid chromatography-mass spectrometry (MS)/MS as well as activities at the protein, microsomal, liver tissue, and whole liver level both in vitro and at the organismal level based on CYP2A6-mediated coumarin metabolism with each CYP2A6 genotype, thereby allowing us to elucidate how different CYP2A6 genotypes yield differing phenotypic traits (protein content and enzyme activity), facilitating the development of personalized medicine.

Simultaneous Determination of Six Coumarins in Rat Plasma and Metabolites Identification of Bergapten in Vitro and in Vivo.

Coumarins are abundant in Umbelliferae and Rutaceae plants possessing varied pharmacological activities. The objectives of this study are to develop and validate the method for determination of six coumarins in rat plasma by liquid chromatography coupled with tandem mass spectrometry (LC-MS) and identify the metabolites of bergapten by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS), respectively. Data-dependent acquisition mode (DDA) was applied to trigger enhanced product ion (EPI) scans by analyzing multiple reaction monitoring (MRM) signals. An efficient data processing method "key product ions (KPIs)" was used for rapid detection and identification of metabolites as an assistant tool. The time to reach the maximum plasma concentration ( Tmax) for the six compounds ranged from 1 to 6 h. A total of 24 metabolites of bergapten were detected in vitro and in vivo. The results could provide a basis for absorption and metabolism of coumarins.

Kinetics of Letrozole Metabolism by Cytochrome P450 3A4: Modulation and Fitting to Non‐Michaelis Menten Models

Variable blood plasma concentration is an issue for many drugs, including letrozole, which can contribute to toxicity or sub‐effective dosing levels. There is evidence that letrozole is primarily metabolized by CYP2A6 however, CYP2A6 genetic variation only explains a portion (about 30%) of the variability in letrozole plasma concentrations. We hypothesized that CYP3A4 may play a larger role in letrozole metabolism than the current evidence suggests due to the influence of multiple ligand binding and the observation of non‐Michaelis‐Menten kinetic plots. The kinetics of letrozole metabolism by CYP3A4 were analyzed in the presence and absence of ketoconazole, a CYP3A4 modulator, and fitted to multiple ligand binding functions as well as the Michaelis‐Menten model. Further, the time‐dependent inhibition parameters of CYP2A6‐mediated letrozole metabolism were measured in the presence of the CYP2A6‐specific inactivator, trans‐cinnamic aldehyde (tCA). While the Eadie‐Hofstee plot for CYP3A4‐mediated letrozole metabolism was ambiguous, curve fitting showed the Michaelis‐Menten model appropriately characterized metabolism in the absence of ketoconazole (KM: 142+/−23.0 μM, Vmax: 13.0+/−1.44 pmol/nmol/min), suggesting a single ligand bound to the enzyme. A biphasic model most closely modeled letrozole metabolism in the presence of ketoconazole (KM1: 4.71+/−4.55 μM KM2: 513+/−164 μM, Vmax1:0.525+/−0.392 Vmax2: 30.9+/−7.60 pmol/nmol/min) and this was also reflected in the Eadie‐Hofstee plot, which was clearly biphasic; this supports a mechanism involving multiple‐ligand binding. TDI analysis of tCA revealed less effective inhibition of CYP2A6‐mediated letrozole metabolism by tCA (KI: 136 μM, kinact: 0.019 min−1) as compared to CYP2A6‐mediated coumarin metabolism. Together, these results suggest that CYP3A4 may contribute to variability in letrozole metabolism especially in the presence of an effector molecule (i.e., ketoconazole) since the value of KM1 is much closer to the letrozole plasma concentrations observed clinically than the KM value observed in the absence of an effector.Support or Funding InformationFunding from National Institute on Drug AbuseThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

New Metabolites of Coumarin Detected in Human Urine Using Ultra Performance Liquid Chromatography/Quadrupole-Time-of-Flight Tandem Mass Spectrometry.

Coumarin (1,2-benzopyrone) is a natural compound whose metabolism in humans was established in the 1970s. However, a new metabolite was recently identified in human plasma, indicating that the metabolism of coumarin has not been completely elucidated. To complement the knowledge of its metabolism, a rapid and sensitive method using UPLC-QTOF-MS was developed. A total of 12 metabolites was identified using MetaboLynxTM software, including eight metabolites not previously reported in human urine. The identified biotransformation included hydroxylation, glucuronidation, sulfation, methylation, and conjugation with N-acetylcysteine. The present work demonstrates that the metabolism study of coumarin was incomplete, possibly due to limitations of old techniques. The identification of eight inedited metabolites of such a simple molecule suggests that the information regarding the metabolism of other drugs may also be incomplete, and therefore, new investigations are necessary.

A kinetic study of the main guaco metabolites using syrup formulation and the identification of an alternative route of coumarin metabolism in humans.

For decades guaco species have been empirically used for the treatment of respiratory diseases. However, studies have shown that the toxic and therapeutic effects of the main guaco metabolites are dose-dependent, and none clinical study was done to evaluate the behavior of these substances in humans. In this work, a pilot study measuring the kinetic profile of the main guaco metabolites was performed leading to the knowledge of an alternative route of coumarin metabolism in humans. Initial screenings demonstrated that the administration of 60 mL of guaco syrup (single dose) did not provide sufficient levels of coumarin (COU), 7-hydroxycoumarin (7-HCOU), o-coumaric acid (OCA) and kaurenoic acid (KAU). The pharmacokinetic parameters were calculated by orally administering 60 mL of guaco syrup spiked with 1500 mg of COU. The kinetic study demonstrated that the plasmatic levels of 7-HCOU (considered the main metabolite of COU) were 10 times lower than the levels of COU, and the kinetic profile of 7-HCOU suggests sequential metabolism in the liver with low access of 7-HCOU to the systemic circulation. The study also demonstrated that OCA is one of the main bioavailable metabolites of COU. Therefore, the hydrolysis of the lactone ring forming a carboxylated compound is one of the possible routes of COU metabolism in humans. The half-lives of COU, 7-HCOU and OCA were approximately 4.0, 1.0 and 3.0 h, respectively and there was evidence that the recommended dosage of guaco syrup did not provide sufficient levels of COU, 7-HCOU or OCA to obtain a bronchodilation effect. Clinical studies are necessary to prove the efficacy and safety of products based on guaco.

Impact of age on hepatic cytochrome P450 of domestic male Camborough-29 pigs.

Swine is not only an important species in veterinary medicine research but also a popular animal model for human drug discovery. It is valuable to understand the impact of pig age on abundance and activity of porcine hepatic cytochrome P450 (CYP450). Liver microsomes were prepared from Camborough-29 intact male pigs at the age of 1day and 2weeks and the castrated male pigs at the age of 5, 10, and 20weeks. Hepatic CYP450 content in the liver microsomes was measured using a UV/visible spectroscopic method. The activities of CYP450s were evaluated by metabolism of phenacetin, coumarin, tolbutamide, bufuralol, chlorzoxazone, and midazolam. The porcine hepatic CYP450 content increased with age with a plateau between age 2 and 5weeks. Activities of all CYP450 enzymes increased with age of pigs too. The bufuralol 1'-hydroxylase showed the highest hepatic activities compared with other CYP enzymes at all ages of pigs. The average activities at the age of 20weeks were about five times higher than those at the age of 5weeks for most of the CYP enzymes. With compensation of the ratio of liver to body weights, the overall CYP450 metabolism capability of the pigs may be peaked around ages of 10 to 20weeks. Those findings suggest that metabolism can be significantly different in growing phase of pigs and that the age may be an important factor in porcine medicine evaluation and pig model development.

Effect of CYP2A6 genetic polymorphism on the metabolic conversion of tegafur to 5-fluorouracil and its enantioselectivity.

Tegafur (FT), a prodrug of 5-fluorouracil, is a chiral molecule, a racemate of R- and S-isomers, and CYP2A6 plays an important role in the enantioselective metabolism of FT in human liver microsomes (R-FT >> S-FT). This study examined the enantioselective metabolism of FT by microsomes prepared from Sf9 cells expressing wild-type CYP2A6 and its variants (CYP2A6*7, *8, *10, and *11) that are highly prevalent in the Asian population. We also investigated the metabolism of coumarin and nicotine, both CYP2A6 probe drugs, in these variants. Enzyme kinetic analyses showed that CYP2A6.7 (I471T) and CYP2A6.10 (I471T and R485L) had markedly lower Vmax values for both enantiomers than wild-type enzyme (CYP2A6.1) and other variant enzymes, whereas Km values were higher in most of the variant enzymes for both enantiomers than CYP2A6.1. The ratios of Vmax and Km values for R-FT to corresponding values for S-FT (R/S ratio) were similar among enzymes, indicating little difference in enantioselectivity among the wild-type and variant enzymes. Similarly, both CYP2A6.7 and CYP2A6.10 had markedly lower Vmax values for coumarin 7-hydroxylase and nicotine C-oxidase activities than CYP2A6.1 and other variant enzymes, whereas Km values were higher in most of the variant enzymes for both activities than CYP2A6.1. In conclusion, the amino acid substitutions in CYP2A6 variants generally resulted in lower affinity for substrates, while Vmax values were selectively reduced in CYP2A6.7 and CYP2A6.10. Consistent R/S ratios among CYP2A6.1 and variant enzymes indicated that the amino acid substitutions had little effect on enantioselectivity in the metabolism of FT.

Cytochrome P450 isoenzymes in rat and human liver microsomes associate with the metabolism of total coumarins in Fructus Cnidii.

Fructus Cnidii (Cnidium) is isolated from the dry and ripe fruit of Cnidium monnier (L.) Cuss (umbelifera), an annual herb. It is demonstrated that the active constituents of Fructus Cnidii are coumarins, known as Total Coumarins of Cnidium Monnier (TCCM). Osthole (Ost) and imperatorin (Imp) are the most active constituents of TCCM which are usually regarded as the quality indicators of medicinal Fructus Cnidii. The aim is to study the metabolism of Fructus Cnidii effective monomer osthole and imperatorin in vitro by liver microsomes. CYP3A4 inhibitor ketoconazole, CYP2D6 inhibitor qunidine, CYP2C8 inhibitor trimethoprim, CYP2C9 inhibitor sulfaphenazole, and CYP1A2 inhibitor α-naphthoflavone were used to investigate the metabolism from incubation time, substrate concentration and liver microsomal concentration, respectively. The concentration of liver microsomes was 0.2mg/ml. Ost (0.8/3.2/12.8uM) was incubated at 37°C for 20min while Imp (1.6/6.4/19.2uM) was incubated for 30min. Qunidine, trimethoprim and α-naphthoflavone could significantly inhibit the disappearance of Imp; meanwhile ketoconazole, sulfaphenazole and qunidine could inhibit the disappearance of Ost. CYP1A, CYP2C are involved in the metabolism of Imp and CYP3A mediates the metabolism of Ost in rat liver microsomes. In human liver microsomes, CYP1A2, CYP2C8, CYP2D6 are involved in the metabolism of Imp; CYP3A4 is involved in the metabolism of Ost at all the tested concentrations of Ost, while CYP2C9, CYP2D6 mediate the metabolism at high concentration of Ost.

Inter-individual Variability of Coumarin 7-hydroxylation (CYP2A6 activity) in an Iranian Population.

Coumarin hydroxylase (CYP2A6) is a polymorphic enzyme, and during the last decade has received a lot of attention because it is the principle human nicotine C-oxidase, which activates a number of procarcinogens, and metabolizes drugs. 150 healthy Iranian volunteers, (96 male, 54 female) aged 19 to 63 years old, were given 5 mg coumarin orally after an overnight fast. Urine samples were collected before drug administration and 2, 4 and 8 h after that. The extent and rate of the formation of 7-OH-coumarin (7OHC) was determined by the urinary excretion of the metabolite as measured by the fluorometric method. The proportion of 7OHC excreted during the first 2 hr compared to the 7OHC excretion at 8 h was the constant and stable individual characteristic for the rate of the formation of 7OHC (2 hr coumarin test). The total amount of 7OHC formed was 32.7±12.4% (mean±SD) of the given dose. On average, 66.0% of 7OHC formed was excreted in 2 hr. No clear-cut polymorphism in the rate of 7OHC formation was found. The total amounts of 7OHC excreted were significantly lower in females. Also, the rate and amount of coumarin metabolism in Iranian population were lower than Turkish and Finnish populations. Because of the importance of enzyme activity in drug metabolism, caution should be exercised in prescribing compounds metabolized by CYP2A6 enzyme in Iranian population.