The new ultra-high performance liquid chromatography method with tandem mass spectrometry detection (UHPLC-MS/MS) has been optimized to allow fast, selective, and high-throughput analysis of two Candida albicans quorum sensing molecules (QSM), farnesol and tyrosol. The problem of the presence of the interference in the samples and system was successfully solved by careful optimization of chromatographic conditions. Charged hybrid stationary phase modified with pentafluorophenyl group and optimized gradient elution provided adequate separation selectivity and peak shapes. The impurity was identified as dibutyl phthalate and had the same m/z ions as farnesol leading to an important interference on selected reaction monitoring channel. Two different types of biological matrices originating from vaginal fluid, supernatant and sediment, were analysed. Micro-solid phase extraction in pipette tips was optimized for the selective isolation of QSM from the supernatant. The insufficient retention of farnesol on the extraction sorbent was improved when 1% of organic solvent was added prior to extraction, while the retention of tyrosol was only possible when using combined C8 and polymer sorbent type. Strong retention of farnesol had to be solved by increasing elution solvent strength and volume up to 600 μL. However, this approach did not allow the pretreatment of sediment samples due to the sorbent clogging. Therefore, our previously developed protein precipitation method was modified and validated to analyse the sediments. New developed UHPLC-MS/MS method provided suitable accuracy and precision for the determination of QSM in vaginal fluid while using only 50 μL sample volume and two different sample preparation methods.
- MeSH
- Candida albicans isolation & purification MeSH
- Adult MeSH
- Farnesol analysis MeSH
- Phenylethyl Alcohol analogs & derivatives analysis MeSH
- Middle Aged MeSH
- Humans MeSH
- Limit of Detection MeSH
- Young Adult MeSH
- Tandem Mass Spectrometry methods MeSH
- Vagina chemistry microbiology MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Validation Study MeSH
Evaluation of possible interactions with enzymes of drug metabolism is an important part of studies on safety and, in general, on the properties of any drug or biologically active compound. Here, focus is given on interactions of three sesquiterpenes (beta-caryophyllene oxide (CAO), trans-nerolidol (tNER) and farnesol (FAR)) with CYP3A4. To determine the CYP3A4 activity, specific substrates testosterone (TES) and midazolam (MDZ) were used. In human liver microsomes, the CAO inhibited the MDZ 1 ́-hydroxylation by mixed type inhibition and K(i) 46.6 microM; TES 6beta-hydroxylation was inhibited more strongly by tNER by the same mechanism and with K(i) of 32.5 microM. Results indicated a possibility of different mode of interaction of both compounds within the active site of CYP3A4 and this was why the molecular docking study was done. The docking experiments showed that the studied sesquiterpenes (CAO and tNER) bound to the CYP3A4 active site cause a significant decrease of binding affinity of substrates tested which corresponded well to the inhibition studies. The inhibition observed, however, most probably does not pose a real harm to microsomal drug metabolism as the levels of sesquiterpenes in plasma (assuming the use of these compounds as spices or flavoring additives) does not usually exceed micromolar range. Hence, the interaction of drugs metabolized by CYP3A4 with sesquiterpenes is less probable.
- MeSH
- Cytochrome P-450 CYP3A chemistry drug effects metabolism MeSH
- Farnesol chemistry pharmacology MeSH
- Cytochrome P-450 CYP3A Inhibitors pharmacology MeSH
- Microsomes, Liver enzymology MeSH
- Catalytic Domain MeSH
- Humans MeSH
- Models, Molecular MeSH
- Molecular Structure MeSH
- Polycyclic Sesquiterpenes chemistry pharmacology MeSH
- Sesquiterpenes chemistry pharmacology MeSH
- Molecular Docking Simulation MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Sesquiterpenes, the main components of plant essential oils, are bioactive compounds with numerous health-beneficial activities. Sesquiterpenes can interact with concomitantly administered drugs due to the modulation of drug-metabolizing enzymes (DMEs). The aim of this study was to evaluate the modulatory effects of six sesquiterpenes (farnesol, cis-nerolidol, trans-nerolidol, α-humulene, β-caryophyllene, and caryophyllene oxide) on the expression of four phase I DMEs (cytochrome P450 3A4 and 2C, carbonyl reductase 1, and aldo-keto reductase 1C) at both the mRNA and protein levels. For this purpose, human precision-cut liver slices (PCLS) prepared from 10 patients and transfected HepG2 cells were used. Western blotting, quantitative real-time PCR and reporter gene assays were employed in the analyses. In the reporter gene assays, all sesquiterpenes significantly induced cytochrome P450 3A4 expression via pregnane X receptor interaction. However in PCLS, their effects on the expression of all the tested DMEs at the mRNA and protein levels were mild or none. High inter-individual variabilities in the basal levels as well as in modulatory efficacy of the tested sesquiterpenes were observed, indicating a high probability of marked differences in the effects of these compounds among the general population. Nevertheless, it seems unlikely that the studied sesquiterpenes would remarkably influence the bioavailability and efficacy of concomitantly administered drugs.
- MeSH
- Aldo-Keto Reductases metabolism MeSH
- Hep G2 Cells MeSH
- Cytochrome P-450 CYP3A metabolism MeSH
- Farnesol pharmacology MeSH
- Hepatocytes metabolism MeSH
- Liver enzymology MeSH
- Carbonyl Reductase (NADPH) metabolism MeSH
- Middle Aged MeSH
- Humans MeSH
- RNA, Messenger metabolism MeSH
- Metabolic Clearance Rate MeSH
- Monocyclic Sesquiterpenes pharmacology MeSH
- Polycyclic Sesquiterpenes pharmacology MeSH
- Pregnane X Receptor agonists metabolism MeSH
- Receptors, Aryl Hydrocarbon agonists metabolism MeSH
- Cytochrome P450 Family 2 metabolism MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Sesquiterpenes pharmacology MeSH
- Cytochrome P-450 Enzyme System metabolism MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Aim: This work studied the impact of the quorum-sensing molecule, farnesol (FAR), on fluconazole (FLC)-resistant Candida albicans isolate CY 1123 compared with the susceptible standard strain C. albicans SC5314. The genes encoding efflux pumps belonging to the ATP-binding cassette (ABC) and major facilitator superfamilies, together with overexpression or point mutation of the ERG11 gene, are the main resistance mechanisms to azole antifungal drugs. Results: The upregulation of genes coding for CDR1, CDR2, and MDR1 were confirmed by qPCR with respect to the housekeeping gene ACT1 in the resistant strain. The contribution of the ERG11 gene was also observed. Markedly, increased pump activity (Cdr1 and/or Cdr2) in the CY 1123 strain was confirmed using diS-C3(3) assay. However, the addition of FAR to the yeasts diminished the difference in staining levels between the SC5314 and CY 1123 strains, demonstrating the concentration-dependent character that could be caused by an effective modulation of Cdr pumps. FAR (60 and 100 μM) was also able to decrease the minimal inhibitory concentrations (MIC50), denoting the inhibition of planktonic cells by 50%, from 8 to 4 μg/mL of FLC when the resistant strain CY 1123 was not cultivated with FLC. However, when it was exposed to 64 μg/mL of FLC, the MIC50 shifted from 64 to 8 μg/mL. Conclusion: Besides the many other effects of FAR on eukaryotic and prokaryotic cells, it also affects ABC efflux transporters, resulting in changes in resistance to azoles in C. albicans isolates. However, this effect is dependent on FAR concentrations.
- MeSH
- ATP-Binding Cassette Transporters metabolism MeSH
- Antifungal Agents pharmacology MeSH
- Biological Transport drug effects MeSH
- Candida albicans drug effects metabolism MeSH
- Farnesol pharmacology MeSH
- Fluconazole pharmacology MeSH
- Drug Resistance, Fungal drug effects MeSH
- Fungal Proteins metabolism MeSH
- Membrane Transport Proteins metabolism MeSH
- Microbial Sensitivity Tests methods MeSH
- Publication type
- Journal Article MeSH
Farnesol (FAR) has already demonstrated an inhibitory effect on Candida albicans biofilm. The aim of this work was to determine the effectiveness of externally added FAR in combination with fluconazole (FLC) on Candida albicans biofilm and on regulation of the ergosterol genes ERG20, ERG9, and ERG11. The effectiveness of compounds was determined by MTT assay and evaluated by the minimal inhibitory concentrations reducing a sessile biofilm to 50% activity (0.5 μg/mL and 200 μmol/L for FLC and FAR, respectively). These concentrations as well as 30 and 100 μmol/L FAR were selected for a study of the effectiveness of the FAR/FLC combination. The reduction in biofilm robustness mainly caused by the presence of 200 μmol/L FAR-alone or in combination with FLC-was accompanied by a significant inhibition of the yeast-to-hyphae transition that was observed by light microscopy and CLSM. Results from qRT-PCR indicated that while 30 μmol/L FAR only slightly regulated the expression of all 3 genes in the 48-h biofilm, the presence of 200 μmol/L FAR downregulated all the tested genes. However, the addition of 0.5 μg/mL of FLC to the samples with 200 μmol/L FAR restored the downregulation of the ERG20 and ERG11 genes to the control level. Moreover, the gene ERG9 was slightly upregulated. In summary, FAR acted via multiple effects on the C. albicans biofilm, but only a higher concentration of FAR proved to be effective.
- MeSH
- Antifungal Agents pharmacology MeSH
- Biofilms drug effects growth & development MeSH
- Candida albicans drug effects growth & development MeSH
- Ergosterol genetics metabolism MeSH
- Farnesol pharmacology MeSH
- Fluconazole pharmacology MeSH
- Genes, Fungal genetics MeSH
- Hyphae drug effects MeSH
- Metabolic Networks and Pathways drug effects MeSH
- Microbial Sensitivity Tests MeSH
- Gene Expression Regulation, Fungal drug effects MeSH
- Drug Synergism MeSH
- Dose-Response Relationship, Drug MeSH
- Publication type
- Journal Article MeSH
Sesquiterpenes, 15-carbon compounds formed from three isoprenoid units, are the main components of plant essential oils. Sesquiterpenes occur in human food, but they are principally taken as components of many folk medicines and dietary supplements. The aim of our study was to test and compare the potential inhibitory effect of acyclic sesquiterpenes, trans-nerolidol, cis-nerolidol and farnesol, on the activities of the main xenobiotic-metabolizing enzymes in rat and human liver in vitro. Rat and human subcellular fractions, relatively specific substrates, corresponding coenzymes and HPLC, spectrophotometric or spectrofluorometric analysis of product formation were used. The results showed significant inhibition of cytochromes P450 (namely CYP1A, CYP2B and CYP3A subfamilies) activities by all tested sesquiterpenes in rat as well as in human hepatic microsomes. On the other hand, all tested sesquiterpenes did not significantly affect the activities of carbonyl-reducing enzymes and conjugation enzymes. The results indicate that acyclic sesquiterpenes might affect CYP1A, CYP2B and CYP3A mediated metabolism of concurrently administered drugs and other xenobiotics. The possible drug-sesquiterpene interactions should be verified in in vivo experiments.
- MeSH
- Farnesol chemistry pharmacology MeSH
- Inhibitory Concentration 50 MeSH
- Cytochrome P-450 Enzyme Inhibitors chemistry pharmacology MeSH
- Liver enzymology MeSH
- Kinetics MeSH
- Rats MeSH
- Humans MeSH
- Sesquiterpenes chemistry pharmacology MeSH
- Subcellular Fractions enzymology MeSH
- Cytochrome P-450 Enzyme System metabolism MeSH
- Xenobiotics metabolism MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Velký počet oportunně patogenních bakterií využívá během nástupu infekce systém quorum sensing (QS). Pomocí signálních molekul (u gram -negativních bakterií N -acylhomoserinových laktonů) je řízeno chování celé populace bakterií (hlavně exprese faktorů virulence). Díky studiu QS systémů u gram -negativních bakterií byly již popsány látky se schopností interference s těmito systémy, představující nové léčebné strategie. Téhož by se dalo využít u kvasinek, kde by identifikace QS molekul a pochopení jejich mechanismů působení mohlo vést k efektivnější léčbě kvasinkových infekcí, např. vulvo -vaginální kandidózy způsobované převážně C. albicans. Doposud byly popsány struktury a funkce dvou hlavních signálních molekul účastnících se systému QS u C. albicans, a to farnesolu a tyrosolu. Vzhledem k tomu, že se rezistence mikroorganismů k antibiotikům a dezinfekčním látkám stává stále aktuálnější, je zapotřebí podrobnějšího popisu struktur, funkcí a metabolických drah dalších molekul účastnících se systému QS u kvasinek.
A large number of opportunistic pathogenic bacteria uses quorum sensing (QS) during the beginning of infection process. Signaling molecules (gram -negative bacteria used N -acyl -homoserine lactones) control the behavior of the whole population of bacteria (especially virulence factors expression). Due to the study of QS systems of gram -negative bacteria, compounds which have the ability to interfere with these systems have been described and they represent new treatment strategy. The same approach could be used in the case of yeast, wherein the identification of QS molecules and their mechanism of action could lead to developing much more effective treatment of yeast infections, e.g. vulvo -vaginal candidosis, which is caused mainly by C. albicans. Until now, the structure and function of two main signaling moleculs (farnesol and tyrosol) in the C. albicans QS system were described. Since the resistance of microorganisms to antibiotics and disinfectants becomes more current, detailed description of the structures, functions and the metabolic pathways involved in QS system in yeast is required.
- Keywords
- tyrosol,
- MeSH
- Candida albicans * physiology growth & development MeSH
- Virulence Factors MeSH
- Farnesol metabolism MeSH
- Phenylethyl Alcohol analogs & derivatives metabolism MeSH
- Candidiasis, Vulvovaginal MeSH
- Quorum Sensing * physiology MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
An original method based on capillary zone electrophoresis with fluorimetric detection has been developed for the determination of terpenic compounds. The method is based on the separation of a terpenes dynamically labeled by the non-ionogenic tenside poly(ethylene glycol) pyrenebutanoate, which was used previously for the labeling of biopolymers. The background electrolytes were composed of taurine-Tris buffer (pH 8.4). In addition to the non-ionogenic tenside aceton and poly(ethylene glycol) were used as the additives. The capillary zone electrophoresis with fluorometric detection at the excitation wavelength 335 nm and the emission wavelength 463 nm was successfully applied to the analysis of tonalid, cholesterol, vitamin A, ergosterol, estrone and farnesol at level of 10(-17) mol L(-1). Farnesol, is produced by Candida albicans as an extracellular quorum-sensing molecule that influences expression of a number of virulence factors, especially morphogenesis and biofilm formation. It enables this yeast to cause serious nosocomial infections. The sensitivity of this method was demonstrated on the separation of farnesol directly from the cultivation medium.
- MeSH
- Biofilms MeSH
- Butyrates chemistry MeSH
- Candida albicans chemistry metabolism MeSH
- Cholesterol chemistry MeSH
- Electrophoresis, Capillary methods MeSH
- Ergosterol chemistry MeSH
- Estrone chemistry MeSH
- Farnesol chemistry isolation & purification metabolism MeSH
- Fluorometry methods MeSH
- Polymers chemistry MeSH
- Quorum Sensing MeSH
- Sensitivity and Specificity MeSH
- Terpenes chemistry isolation & purification MeSH
- Tetrahydronaphthalenes chemistry MeSH
- Vitamin A chemistry MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Candida albicans is generally one of the most commonly isolated fungal pathogen from human body. It is a frequent cause of nosocomial infections, bloodstream infections, urinary infections and mucosal infections of oral cavity and vagina C. albicans can grow as hyphae, pseudohyphae, or budding yeast. Morphological conversion of a yeast form to pseudohyphal or hyphal one is often characterized by the change of commensal status to an invasive form. Farnesol and tyrosol can participate in these transformation processes as quorum sensing molecules together with some physical-chemical factors. A new analytical method for identification and quantification of biologically active substances farnesol and tyrosol using ultra high performance liquid chromatography (UHPLC) in connection with tandem mass spectrometry was developed. The analytes were separated on Acquity BEH C18 analytical column using binary mobile phase consisting of acetonitrile and formic acid 0.075% (75:25) at flow-rate 0.20 ml/min. SRM (selected reaction monitoring) mode was applied in order to ensure sufficient selectivity and sensitivity using the first most intensive transition as a quantitative (121>77 and 205>121) and second one for the confirmation purposes (121>93 and 205>109). The method was validated in terms of linearity (>0.9994), precision (0.5-3.8% RSD), accuracy (78.9-106.0%), LOD (limit of detection) and LOQ (limit of quantitation). The method can serve as an analytical tool for the detection and determination of quorum-sensing molecules in biological samples.
- MeSH
- Candida albicans chemistry MeSH
- Farnesol analysis chemistry MeSH
- Phenylethyl Alcohol analogs & derivatives analysis chemistry MeSH
- Spectrometry, Mass, Electrospray Ionization MeSH
- Calibration MeSH
- Humans MeSH
- Limit of Detection MeSH
- Quorum Sensing MeSH
- Tandem Mass Spectrometry methods MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Check Tag
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
