The exposure of fatty products to environmental light can trigger lipid oxidation in food through a sensitized-photooxidation process, which involves the participation of the species singlet oxygen (O₂(¹Δ_g)). Therefore, preservation of food quality represents a subject of great economic interest to the food industry. In this sense, the phenolic compounds are natural antioxidants widely used in food industry. In this contribution we studied the interactions of phenolic derivatives (Phd), tyrosol and tyrosol derived isomers, with O₂(¹Δ_g) and their possible protective effect against the oxidative degradation of unsaturated fatty acids and amino acids. Besides, a potential synergistic interaction between Phd and antioxidants used in food industry were investigated. Phd substrates showed properties as antioxidant additives due to their high ability deactivating O₂(¹Δ_g) through a physical process and synergistic effect in the presence of commercial antioxidants. Phd presented an antioxidant protective effect toward O₂(¹Δ_g)-mediated degradation of methyl linoleate and tryptophan.

The effect of p-tyrosol on the spontaneous level of DNA damage in the cells of the bone marrow, liver, kidney, and rectum of mice (series I) and on the genotoxic effects of cytostatic drugs with different mechanisms of action in rat testicular cells (series II) was studied by DNA comet assay on C57BL/6 mice. p-Tyrosol was administered in a dose of 40 mg/kg once (series I) or for 5 days before and 5 days after cytostatic exposure (busulfan, paclitaxel, methotrexate; series II). It was found that p-tyrosol reduced spontaneous level of DNA damage in all studied organs. p-Tyrosol exhibited an antigenotoxic effect with respect to the DNA-damaging action of methotrexate and produced no genoprotective effect in case of busulfan and paclitaxel.

Keywords: DNA comets; genotoxicity; mice; p-tyrosol.

Tyrosol β-galactoside (TG) is a phenylethanoid glycoside with proven neuroprotective properties. This work deals with its biocatalytic production from tyrosol and lactose using Aspergillus oryzae β-galactosidase in immobilized form. Six commercial carriers were examined to find the optimal biocatalyst. Besides standard biocatalyst performance characteristics, adsorption of the hydrophobic substrate on immobilization carrier matrices was also investigated. The adsorption of tyrosol was significant, but it did not have adverse effects on TG production. On the contrary, TG yield was improved for some biocatalysts. A biocatalyst prepared by covalent binding of β-galactosidase on an epoxy-activated carrier was used for detailed investigation of the effect of reaction conditions on glycoside production. Temperature had a surprisingly weak effect on the overall process rate. A lactose concentration of 0.83 M was found to be optimal to enhance TG formation. The impact of tyrosol concentration was rather complex. This substrate caused inhibition of all reactions. Its concentration had a strong effect on the hydrolysis of lactose and all products. Higher tyrosol concentrations, 30-40 g/L, were favorable as pseudo-equilibrium concentrations of TG and galactooligosaccharide were reached. Repeated batch results revealed excellent operational stability of the biocatalyst.

Keywords: Immobilized β-galactosidase; Reaction network; Substrate adsorption; Transgalactosylation; Tyrosol β-galactoside.

A glycoside hydrolase from Penicillium oxalicum BL 3005 was purified to apparent homogeneity. Its molecular mass was estimated to be 90 kDa by SDS-PAGE. The enzyme was identified to be a new member of family-3 by peptide sequence. High transglycosylation activity was found in the hydrolytic reaction of cellobiose. In the reaction, salidroside (4-hydroxyphenethyl O-β-d-glucopyranoside) was formed by adding tyrosol as the glycosyl acceptor. The optimum reaction pH and temperature were pH 6.5 and 55 °C, respectively. The maximum yield of salidroside was almost 20 g/L. These results indicated that the β-glucosidase of P. oxalicum can be considered as a very promising catalyst for the synthesis of salidroside.

Although currently serving as the workhorse for metabolite characterization, one of the most challenging tasks for LC-MS is isomeric differentiation because isomers frequently yield identical quasi-molecular ions and fragmented ion species. Our previous studies have demonstrated that online energy-resolved MS (ER-MS) is an orthogonal technique for MS/MS experiments to facilitate isomeric identification. Herein, attempts were made for the in-depth characterization of the metabolic profiles of an effective natural product named salidroside (SA) in rats using LC coupled with three-dimensional mass spectrometry (LC-3D MS) that was configured by MS1, MS2 and online ER-MS as 1st, 2nd, and 3rd dimensions, respectively. Moreover, the metabolism characterization of its aglycone, namely, tyrosol (Try) was conducted in parallel to aid in proposing metabolic pathways. High-resolution MS1 and MS2 spectra were acquired by IT-TOF-MS, and subsequent data processing provided theoretical formula and sub-structures for each metabolite. Subsequently, online ER-MS was conducted for precursor > product ion transitions-of-interest to offer linkage information among the sub-structures via building breakdown graphs. As a result, ten (M1-10) and nine (M1, M2, and M5-11) metabolites were detected in SA- and Tyr-administrated biological samples, respectively, and their structures were qualitatively identified. Crucial metabolism occurred for either component. SA initially underwent hydrolysis to produce Tyr, and subsequently hydroxylation, oxidation, glucuronidation, and sulfation were observed as the primary metabolic pathways. To summarize, the metabolic fate of SA was understood in depth, and Tyr, as the hydrolytic product, was responsible for the occurrences of most metabolites (M1, M2, and M5-10). More importantly, identification confidences of the metabolites were significantly advanced by LC-3D MS, suggesting that it is eligible to serve as an integral part of the analyst's toolbox.

Organic components in olive mill wastewater (OMW) were analyzed by exhaustive solvent extraction of the lyophilisate followed by pre-chromatographic derivatization techniques and GC/MS-analysis of the extracts. Simple biophenols including tyrosol (Tyr), hydroxytyrosol (OH-Tyr) and homovanillic alcohol as well as complex biophenols including decarbomethoxy ligostride aglycon and decarbomethoxy oleuropein aglycon proved most abundant analytes. Hydroxylated benzoic and cinnamic acids are less abundant, which may indicate a humification process to have occurred. The pattern of organic components obtained from native OMW was compared with that obtained from hydrothermal carbonization (HTC) of the waste product. Former results provided strong evidence that HTC of OMW at 220°C for 14h results in an almost complete hydrolysis of complex aglycons. However, simple biophenols were not decomposed on hydrothermal treatment any further. Phenol and benzenediols as well as low molecular weight organic acids proved most abundant analytes which were generated due to HTC. Similarly to aglycons, lipids including most abundant acylglycerines and less abundant wax esters were subjected almost quantitatively to hydrolysis under hydrothermal conditions. Fatty acids (FAs) released from lipids were further decomposed. The pathways of volatile analytes in both native OMW and aqueous HTC solutions were studied by solventless headspace-Solid Phase Micro Extraction. Basically, a wide array low molecular alcohols and ketones occurring in native OMW survived the HTC process.

In this study, we investigated the effects of the different critical genes in the three modules on tyrosol production in Escherichia coli. Coexpression of the yahK and ARO10 genes increased the yield of tyrosol by 10% compared to that of the control. Tyrosol production by E. coli BFPT1 and E. coli BFPA1 was higher by 15.0% and 17.8% than that by the control, respectively, via coordinated expression of key genes from modules 2 and 3. The tyrosol yield of E. coli BFPE2 was 58.3% higher than that of the control (reaching 5.72 mM) when the expression levels of the key genes aroA and tyrA* from module 2 were balanced. The tyrosol yield of E. coli BFPG1 was increased by 52.6% (reaching 5.8 mM) compared to the control via coexpression of modules 1, 2, and 3. This work suggested that microbial production of tyrosol in E. coli has potential for industrial applications.

The present work proposed the preparation of triazolic analogues of tyrosol, a biophenol found in olive oil and whose wide range of bioactivities has been the target of many studies. We obtained fifteen novel tyrosol derivatives and the compounds of the series were later evaluated as acetylcholinesterase (AChE) inhibitors. The study of AChE inhibition is important for the development of new drugs and pesticides, and especially the research for managing Alzheimer's disease. The most active compound, namely 7-({1-[2-(4-hydroxyphenyl)ethyl]-1H-1,2,3-triazol-4-yl}methoxy)-4-methyl-2H-chromen-2-one (30), showed IC₅₀ value of 14.66 ± 2.29 μmol L^-1. Docking experiments corroborated by kinetic assay are suggestive of a competitive inhibition mechanism. Derivatives interacted with amino acids from the AChE active site associated to the development of Alzheimer's disease. The results indicate that the compounds synthesized have a high potential as prototypes for the development of new acetylcholinesterase inhibitors.

Keywords: 1,2,3-triazoles; Acetylcholinesterase; Alzheimer's disease; Docking; Tyrosol.

The main pharmacokinetic parameters of p-tyrosol after single (in 3 doses) and repeated intravenous injection were studied in rats. The content ofp-tyrosol in the blood plasma was determined by spectrofluorimetric method. The pharmacokinetic parameters of p-tyrosol are linear in the dose range from 50 to 200 mg/kg. Repeated administration leads to accelerated metabolic elimination of p-tyrosol.

The first total synthesis of the novel cyclohexylethanoid natural product clerobungin A has been achieved in six steps and 14% overall yield starting from commercially available tyrosol. Key steps in this sequence include a bioinspired oxidative dearomatization of a phenol and a hemiacetalization/oxa-Michael cascade to form the tricyclic ring system. Resolution of a late-stage intermediate via chiral HPLC allowed for the measurement of the chiroptical properties of both enantiomers of clerobungin A, supporting the scalemic nature of the natural product.

In this study, a recombinant Saccharomyces cerevisiae strain EKD13 overproducing mannoproteins has been used to obtain Albariño white wines. The inoculated strain prevailed and produced complete fermentation of the must, as also occurred in the case of spontaneous (non-inoculated) fermentation and in the must inoculated with the S. cerevisiae EC1118 strain. The analytical study of the wines obtained showed that the most important chemical differences among the wines produced with EKD-13, corresponded to the high concentration of mannoproteins, 2-phenyl ethanol and tyrosol. These differences were attributed to the expression, during must fermentation, of genes modified in the recombinant EKD-13 strain. The results obtained imply that this strain could be potentially useful to produce wines rich in mannoproteins that have distinctive characteristics compared to other similar wines, modifying the sensorial and technological parameters of the wines obtained.

A high-performance liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (HPLC-FT-ICR MS) method was developed to study the in vivo metabolism of salidroside for the first time. Plasma, urine, bile, and feces samples were collected from male rats after a single intragastric gavage of salidroside at a dose of 50 mg/kg. Besides the parent drug, a total of seven metabolites (three phase I and four phase II metabolites) were detected and tentatively identified by comparing their mass spectrometry profiles with those of salidroside. Results indicated that metabolic pathways of salidroside in male rats included hydroxylation, dehydrogenation, glucuronidation, and sulfate conjugation. Among them, glucuronidation and sulfate conjugation were the major metabolic reactions. And most important, the detection of the sulfation metabolite of p-tyrosol provides a clue for whether the deglycosylation of salidroside occurs in vivo after intragastric gavage. In summary, results obtained in this study may contribute to the better understanding of the safety and mechanism of action of salidroside.

The present study deals with the extraction of phenols from aqueous solutions by using the emulsion liquid membranes technique. Besides phenol, two derivatives of phenol, i.e., tyrosol (2-(4-hydroxyphenyl)ethanol) and p-coumaric acid (4-hydroxycinnamic acid), which are typical components of the effluents produced in olive oil plants, were selected as the target solutes. The effect of the composition of the organic phase on the removal of solutes was examined. The influence of pH of feed phase on the extraction of tyrosol and p-coumaric was tested for the membrane with Cyanex 923 as an extractant. The use of 2% Cyanex 923 allowed obtaining a very high extraction of phenols (97-99%) in 5-6 min of contact time for either single solute solutions or for their mixtures. The removal efficiency of phenol and p-coumaric acid attained equivalent values by using the system with 2% isodecanol, but the removal rate of tyrosol was found greatly reduced. The extraction of tyrosol and p-coumaric acid from their binary mixture was also analysed for different operating conditions like the volume ratio of feed phase to stripping phase (sodium hydroxide), the temperature and the initial concentration of solute in the feed phase.

A simple, fast and reliable method to quantify, simultaneously, 3,4-dihydroxyphenylglycol (DHPG), hydroxytyrosol (HT) and tyrosol (Ty) extracted and purified successfully from olive oil by-product, called alperujo, in animal plasma and tissues samples has been developed using a high-performance liquid chromatographic (HPLC) method with UV-Vis detection. Extraction of compounds is based on solid-phase extraction for plasma and homogenisation with zirconia beads and centrifugation for tissues. Calibration curves were linear for all three phenols at a relatively low concentration range (0.05-50μg/mL). This method has acceptable accuracy (91-95% in plasma and 63-100% in tissues), precision (1.11-8.26% intra-day and 0.32-9.5% inter-day) and sensitivity for detecting low concentrations of these phenols in small plasma volumes and several animal tissues such as liver, heart, kidney, muscle, testes, white adipose tissue (WAT) and brain.

Tyrosol hydroxylating Pseudomonas strain was previously isolated from olive mill wastewaters-irrigated soil. In the present work, experimental design was used to study the bioconversion of tyrosol in laboratory fermenters aiming at the recovery of the highest yields of hydroxytyrosol. The effects of biocatalyst loading and tyrosol concentration were studied. The bioconversion yield reached 86.9% (37.3 mM hydroxytyrosol) starting from a tyrosol concentration of 43 mM. Under these conditions, the specific productivity relative to the biocatalyst was 4.78 μM/min/g. The established model to predict bioconversion yield was validated in two bench-scale fermenters. At the downstream stage, the reaction product was recovered as a hydroxytyrosol rich solution after microfiltration and concentration under vacuum. Subsequent to this operation, hydroxytyrosol composition yielded 73.8% of the total dry matter.

Here we present new and original data on the endogenous conversion of tyrosol (Tyr) into hydroxytyrosol (OHTyr) in humans and its effects on the cardiovascular system. A randomized, crossover, controlled clinical trial was performed with individuals at cardiovascular risk (n = 33). They received white wine (WW) (females 1, males 2 standard drinks/day), WW plus Tyr capsules (WW + Tyr) (25mg Tyr capsule, one per WW drink), and water (control) ad libitum. Intervention periods were of 4 weeks preceded by three-week wash-out periods. We assessed the conversion of Tyr to OHTyr, its interaction with a polygenic activity score (PAS) from CYP2A6 and CYP2D6 genotypes, and the effects on cardiovascular risk markers. For further details and experimental findings please refer to the article "Cardiovascular benefits of tyrosol and its endogenous conversion into hydroxytyrosol in humans. A randomized, controlled trial" [1].

A novel gas chromatography-tandem mass spectrometry (GC-MS/MS) method was developed, using an ion trap mass spectrometer, for the simultaneous determination of olive oil bioactive components, elenolic acid, hydroxytyrosol, and tyrosol, in rat urine. Samples were analyzed by GC-MS/MS prior to and after enzymatic treatment. A solid phase extraction sample pretreatment step with greater than 80% analytical recoveries for all compounds was performed followed by a derivatization reaction prior to GC-MS/MS analysis. The calibration curves were linear for all compounds studied for a dynamic range between 1 and 500 ng. The limit of detection was in the mid picogram level for tyrosol and elenolic acid (300 pg) and in the low picogram level for hydroxytyrosol (2.5 pg). The method was applied to the analysis of rat urine samples after sustained oral intake of oleuropein or extra virgin olive oil as a diet supplement.

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.

Keywords: Candida albicans; Farnesol; Microextraction; Quorum sensing; Tyrosol; UHPLC-MS/MS.

OBJECTIVE

To investigate the effect of andrographolide (AG) on quroum sensing (QS) and relevant virulence genes of Candida albicans.

METHODS

Gas-chromatography-mass spectrometry (GC-MS) was applied to detect the changes in the content of farnesol and tyrosol in C. albicans intervened by AG. The real-time quantitative PCR (qRT-PCR) was adopted to inspect the expressions of relevant virulence genes such as CHK1, PBS2 and HOG1 regulated by QS.

RESULTS

At 2 h after the growth of C. albican, the farnesol and tyrosol secretions reduced, without notable change after intervention with AG. The secretions were highest at 12 h and decreased at 24 h. After the intervention with different concentrations of AG, the farnesol content reduces, whereas tyrosol increased, indicating a dose-dependence, particularly with 1 000 mg x L(-1) AG. qRT-PCR revealed that 1 000 mg x L(-1) AG could down-regulate CHK1 by 2.375, 3.330 and 4.043 times and PBS2 by 2.010, 4.210 and 4.760 times, with no significant change in HOG1.

CONCLUSIONS

AG could inhibit the farnesol secretion, promote the tyrosol secretion and down-regulate QS-related virulence genes CHK1 and PBS2 expressions.

Tri cluster is responsible for the biosynthesis of trichothecenes in Trichoderma spp. Tri6 gene present within the cluster encodes for a transcriptional regulator and is vital for the expression of all other tri genes of the cluster. Tri6 encodes for a 218-amino-acid residues long protein which contains three zinc finger motifs. Tri6 is able to regulate and bind within the GTGA/TCAC promoter region of tri genes. Here we highlight the binding of tri6 with the regulatory DNA element present at the upstream of tri3 gene and effect of two quorum-sensing molecules tyrosol and farnesol on its binding. Analysis showed that tyrosol binds at sequence GTGA/TCAC specific for tri6 binding and thus do not allow tri6 to bind on promoter region. Interactions of tyrosol with zinc finger motif of tri6 protein also resulted in structural changes making tri6 unsuitable for binding with the regulatory DNA element of tri3 gene promoter resulting in downregulation of the gene. Structural changes also resulted in loss of zinc from zinc finger 2 motif. In contrast to the tyrosol, tri6-DNA complex could easily accommodate farnesol molecule without showing any interference with the functional conformation of the tri6-DNA complex. Therefore, tri gene expression seems not to be negatively regulated by the farnesol.

The virulence of C. albicans is associated with the transitional evolution from yeast to filamentous forms. We were interested in the effects amphotericin B (AMB), ketoconazole (KTC) and gamma-radiations might have on these broadly defined phenotypes as determined by the CFU procedure. By using collagen gel as the 3-dimensional support of cell culture, diverse experimental conditions were contemplated in order to modulate the differentiation of Candida during sessile and planktonic growth. These conditions included the co-culture with human epithelial and endothelial cells and treatment with farnesol, tyrosol and conditioned medium from P. aeruginosa. The overall results were as follows: 1) The survival of Candida was inhibited by the exposure to gamma-radiations, but only after the organism was induced to progress into excess filamentation, while in normal growth conditions it proved to be radioresistant; 2) AMB inhibited the growth of yeast forms, while KTC was specifically toxic to filamentous forms and 3) the combined treatment of filamentous Candida with KTC and gamma-radiations resulted in the synergistic inhibition of the organism. These findings indicate that both the radiosensitivity of C. albicans and its response to the synergistic effects of gamma-radiations and KTC are filamentation-dependent pharmacological processes.

To establish high performance liquid chromatography(HPLC) fingerprints for crude and processed Ligustri Lucidi Fructus,and to evaluate their quality through the similarity calculation and chemical pattern recognition. The separation was performed with Syncronis C_(18) column(4.6 mm × 250 mm, 5 μm), with acetonitrile(A) and 0.1% phosphoric acid solution(B) as the mobile phase for gradient elution, and a detection wavelength of 280 nm. HPLC was used to detect 22 batches of crude and processed Ligustri Lucidi Fructus,and the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine(2012 Edition) was used to evaluate the similarity among 22 batches. The research on pattern recognition was conducted with cluster analysis(CA), principal component analysis(PCA), and partial least squares discriminate analysis(PLS-DA). HPLC fingerprints of crude and processed Ligustri Lucidi Fructus were established, with similarity ranging from 0.9 to 1.0. The crude and processed Ligustri Lucidi Fructus can be obviously distinguished by using CA, PCA and PLS-DA. According to the results of PLS-DA,11 constituents including hydroxytyrosol, tyrosol, specnuezhenide and oleuropein were the main marker components leading to the difference. The established fingerprint method is stable and reliable, and can provide method basis for quality control of crude and processed Ligustri Lucidi Fructus. Chemical pattern recognition is proved to be helpful in comprehensive quality control and evaluation of Ligustri Lucidi Fructus before and after the process.

Keywords: Ligustri Lucidi Fructus; chemical pattern recognition; fingerprint; quality control.