It is established that the chronic administration of Rhodiola rosea extract (RRE) in a single daily dose of 1 ml/kg (p.o.) during 8 days increased the resistance of myocardium with respect to the cardiotoxic action of isoproterenol and the arrhythmogenic action of epinephrine in rats. Pretreatment with RRE prevented the stressor cardiac damages, as measured by 99mTc-pyrophosphate accumulation in the heart. The cardioprotective action of RRE was maximum after 5-day administration. The antiarrhythmic effect of the adaptogen was maximum after 8-day administration. It was found that p-tyrosol also exhibited antiarrhythmic and cardioprotective properties. Pretreatment with RRE decreased the infarction size/risk area ratio during the coronary artery occlusion and reperfusion in vivo. The chronic administration of RRE increased th e tolerance of the isolated perfused rat heart to the pathogenic action of global ischemia and reperfusion. Pretreatment with RRE not only prevented the occurrence of arrhythmias, but also abolished cardiac electrical instability in rats with postinfarction cardiac sclerosis. It has been found that the chronic administration of RRE (1 ml/kg, p.o., over 8 days) increased the level beta-endorphin in rat blood plasma and the content of leu-enkephalin in myocardial tissue. Naloxone (2 mg/kg) abolished cardioprotective and antiarrhythmic effect of the adaptogen. It was suggested that both RRE effects depend on the occupancy of opioid receptors by endogenous opioid peptides. It has been found that the sympathetic nervous system is involved in the development of antiarrhythmic effect of RRE, while HSP-70 is not involved in the cardioprotective and antiarrhythmic effect of adaptogen. It is concluded that the mechanism of cardioprotective and antiarrhythmic action of RRE needs further investigation.

Tyrosol and its glycosylated product salidroside are important ingredients in pharmaceuticals, nutraceuticals and cosmetics. Despite the ability of Saccharomyces cerevisiae to naturally synthesize tyrosol, high yield from de novo synthesis remains a challenge. Here, we used metabolic engineering strategies to construct S. cerevisiae strains for high-level production of tyrosol and salidroside from glucose. First, tyrosol production was unlocked from feedback inhibition. Then, transketolase and ribose-5-phosphate ketol-isomerase were overexpressed to balance the supply of precursors. Next, chorismate synthase and chorismate mutase were overexpressed to maximize the aromatic amino acid flux towards tyrosol synthesis. Finally, the competing pathway was knocked out to further direct the carbon flux into tyrosol synthesis. Through a combination of these interventions, tyrosol titres reached 702.30 ± 0.41 mg l^-1 in shake flasks, which were approximately 26-fold greater than that of the WT strain. RrU8GT33 from Rhodiola rosea was also applied to cells and maximized salidroside production from tyrosol in S. cerevisiae. Salidroside titres of 1575.45 ± 19.35 mg l^-1 were accomplished in shake flasks. Furthermore, titres of 9.90 ± 0.06 g l^-1 of tyrosol and 26.55 ± 0.43 g l^-1 of salidroside were achieved in 5 l bioreactors, both are the highest titres reported to date. The synergistic engineering strategies presented in this study could be further applied to increase the production of high value-added aromatic compounds derived from the aromatic amino acid biosynthesis pathway in S. cerevisiae.

'Megaritiki' is an olive cultivar widely used in Greece for the production of low polyphenol olive oil and table olives. To investigate possible metabolic differentiation in comparison with other varieties, the composition of 'Megaritiki' olive fruits and wastewaters from the debittering procedure was studied. Moreover, the recovery of bioactive metabolites from wastewater using adsorption resin was studied to exploit this byproduct. Metabolites in fruits and wastewaters were monitored using NMR spectroscopy. The major constituents of wastewater were hydroxytyrosol-4-O-glucoside, 11-methyl-oleoside, hydroxytyrosol, and tyrosol but not oleuropein. Furthermore, wastewater afforded rengyoxide and rengyoside B, which are for the first time isolated from olives. The final edible olives, besides hydroxytyrosol and tyrosol, contained rengyoxide and cleroindicin C, which are the first isolated from the species, haleridone for the first time isolated from edible olives, and four metabolites, which are the first reported as natural products, megaritodilactone, megaritolactonic acid, methyl ester of megaritolactonic acid B, and megaritolactonol.

Olive oil and table olives are rich sources of biophenols, which provides a unique taste, aroma and potential health benefits. Specifically, green olive drupes are enriched with oleuropein, a bioactive biophenol secoiridoid. Olive oil contains hydrolytic derivatives such as hydroxytyrosol, oleacein and elenolate from oleuropein as well as tyrosol and oleocanthal from ligstroside. Biophenol secoiridoids are categorized by the presence of elenoic acid or its derivatives in their molecular structure. Medical studies suggest that olive biophenol secoiridoids could prevent cancer, obesity, osteoporosis, and neurodegeneration. Therefore, understanding the biomolecular dynamics of oleuropein can potentially improve olive-based functional foods and nutraceuticals. This review provides a critical assessment of oleuropein biomolecular mechanism and computational mapping that could contribute to nutrigenomics.

A novel approach is presented to determine hydrophilic phenols in olive oil samples, employing vortex-assisted reversed-phase dispersive liquid-liquid microextraction (RP-DLLME) for sample preparation and screen-printed carbon electrodes for voltammetric analysis. The oxidation of oleuropein, hydroxytyrosol, caffeic acid, ferulic acid and tyrosol was investigated, being caffeic acid and tyrosol selected for quantification. A matrix-matching calibration using sunflower oil as analyte-free sample diluted with hexane was employed to compensate matrix effects. Samples were analyzed under optimized RP-DLLME conditions, i.e., extractant phase, 1M HCl; extractant volume, 100µL; extraction time, 2min; centrifugation time, 10min; centrifugation speed, 4000rpm. The working range showed a good linearity between 0.075 and 2.5mgL-1 (r = 0.998, N = 7) for caffeic acid, and between 0.075 and 3mgL-1 (r = 0.999, N = 8) for tyrosol. The methodological limit of detection was empirically established at 0.022mgL-1 for both analytes, which is significantly lower than average contents found in olive oil samples. The repeatability was evaluated at two different spiking levels (i.e., 0.5mgL-1 and 2mgL-1) and coefficients of variation ranged from 8% to 11% (n = 5). The applicability of the proposed method was tested in olive oil samples of different quality (i.e., refined olive oil, virgin olive oil and extra virgin olive oil). Relative recoveries varied between 83% and 108% showing negligible matrix effects. Finally, fifteen samples were analyzed by the proposed method and a high correlation with the traditional Folin-Ciocalteu spectrophotometric method was obtained. Thereafter, the concentrations of the fifteen oil samples were employed as input variables in linear discriminant analysis in order to distinguish between olive oils of different quality.

Genes with the potential to code for enzymes involved in phenolic compound metabolism were detected in the genome of Lactobacillus pentosus IG1, isolated from a green olive fermentation. Based on homology, these genes could code for a 6-P-β Glucosidase, two different Tannases, a Gallate decarboxylase and a p-Coumaric decarboxylase. Expression of up to seven of these genes was studied in L. pentosus IG1 (olive fermentation) and CECT4023T (corn silage), including responses upon exposure to relevant phenolic compounds and different olive extracts. Genes potentially coding Tannase, Gallate decarboxylase and p-Coumaric acid decarboxylase significatively increased their expression upon exposure to such compounds and extracts, although it was strain dependent. In general, both the genetic organization and the characteristics of gene expression resembled very much those described for Lactobacillus plantarum. In accordance to the observed induced gene expression, metabolism of specific phenolic compounds was achieved by L. pentosus. Thus, methyl gallate, gallic acid and the hydroxycinamic acids p-coumaric, caffeic and ferulic were metabolized. In addition, the amount of phenolics such as tyrosol, oleuropein, rutin and verbascoside included in a minimal culture medium was noticeably reduced, again dependent on the strain considered.

A moderately halotolerant, Gram-positive, aerobic, motile, spore-forming bacterium, designated as strain YAS1, was isolated from an olive-brine fermentation rich in aromatic compounds, after enrichment on tyrosol. Strain YAS1 grew between 25 and 45 degrees C and optimally at 37 degrees C. It grew in the presence of 0-15% (v/w) NaCl, with an optimum of 3-6% (v/w) NaCl. The DNA G+C content was found to be 49.9 mol%. Phylogenetic analysis of the 16S rRNA gene revealed that this isolate was a member of the genus Bacillus. The newly isolated strain YAS1 represents the first moderately halotolerant bacterium transforming tyrosol to p-hydroxyphenylacetic acid (PHPA) in the presence of yeast extract.

Camellia oil is widely used in some parts of the world partly because of its high oxidative stability. The effect of heating a refined camellia oil for 1 h at 120 degrees C or 2 h at 170 degrees C with exogenous antioxidant, namely, caffeic acid and tyrosol, was studied. Parameters used to assess the effect of heating were peroxide and K values, volatile formation, and fatty acid profile. Of these, volatile formation was the most sensitive index of change as seen in the number of volatiles and the total area count of volatiles in gas chromatograms. Hexanal was generally the dominant volatile in treated and untreated samples with a concentration of 2.13 and 5.34 mg kg(-1) in untreated oils heated at 120 and 170 degrees C, respectively. The hexanal content was significantly reduced in heated oils to which tyrosol and/or caffeic acid had been added. Using volatile formation as an index of oxidation, tyrosol was the more effective antioxidant of these compounds. This is contradictory to generally accepted antioxidant structure-activity relationships. Changes in fatty acid profiles after heating for up to 24 h at 180 degrees C were not significant.

Activity-guided isolation of compounds from the fruits of Amomum xanthioides resulted in the purification of fourteen phenolic compounds, 4-hydroxy-benzaldehyde (1), 3,4-dihydroxybenzaldehyde (2), 3,5-dimethoxy-4-methylbenzaldehyde (3), syringic aldehyde (4), benzoic acid (5), 3,4-dihydroxy benzoic acid (6), vanillic acid (7), 3-hydroxy-2-methoxybenzoic acid (8), o-vanillic acid (9), phenylacetic acid (10), tyrosol (11), pyrocatechol (12), 1,2,4,5-tetramethoxybenzene (13), and 3,3',5,5'-tetramethoxybiphenyl-4,4'-diol (14). To evaluate the anti-allergic inflammatory activities of these compounds, we examined the inhibitory effects of the isolates (1-14) on histamine release and on the expressions of tumor necrosis factor (TNF)-ca and interleukin (IL)-6 genes by using human mast cells. Of the tested compounds, 9, 11, and 13 suppressed histamine release from mast cells, and all isolates attenuated the expressions of the pro-inflammatory cytokines, TNF-α and IL-6 genes in human mast cells.

The potential of fluorescence spectroscopy for characterizing the deterioration of extra virgin olive oil (EVOO) during heating was investigated. Two commercial EVOO were analysed by HPLC to determine changes in EVOO vitamin E and polyphenols as a result of heating at 170 degrees C for 3 h. This thermal oxidation of EVOO caused an exponential decrease in hydroxytyrosol and vitamin E (R(2)=0.90 and 0.93, respectively) whereas the tyrosol content was relatively stable. At the same time, amounts of preformed hydroperoxides (ROOH), analysed by an indirect colorimetric method, decreased exponentially during the heating process (R(2)=0.94), as a result of their degradation into secondary peroxidation products. Fluorescence excitation spectra with emission at 330 and 450 nm were recorded to monitor polyphenols and vitamin E evolution and ROOH degradation, respectively. Partial least-squares calibration models were built to predict these indicators of EVOO quality from oil fluorescence spectra. A global approach was then proposed to monitor the heat charge from the overall fluorescence fingerprint. Different data pretreatment methods were tested. This study indicates that fluorescence spectroscopy is a promising, rapid, and cost-effective approach for evaluating the quality of heat-treated EVOO, and is an alternative to time-consuming conventional analyses. In future work, calibration models will be developed using a wide range of EVOO samples.

Phenylethanoids, including 2-phenylethanol, tyrosol, and salidroside are a group of phenolic compounds with a C6-C2 carbon skeleton synthesized by plants. Phenylethanoids display a variety of biological activities, including antibacterial, anticancer, anti-inflammatory, neuroprotective, and anti-asthmatic activities. Recently, successful microbial synthesis of phenylethanoids through metabolic engineering and synthetic biology approaches has been reported and could allow phenylethanoid production from alternative microbial sources. Here, we review the recent achievements in the synthesis of phenylethanoids by microorganisms. The work done so far will contribute to the production of diverse phenylethanoids using various microbial systems and facilitate exploration of further diverse biological activities of phenylethanoids.

Analyte-responsive chromo-fluorogenic reactions under accessible conditions are important for designing small-molecule spectroscopic probes. We describe a series of newly-constructed motifs based on the chromo-fluorogenic reaction between catechol derivatives (typically hydroxytyrosol, dopamine, and levodopa) and naphthoresorcin (NR) in aqueous solution under ambient conditions. The weakly absorptive and fluorogenic catechols/naphthoresorcin was converted to products having visible absorption and bright fluorescence within several minutes. The chromo-fluorophores produced from this reac-tion had a maximum absorbance at 458 nm and emission at 480 nm with high fluorescence quantum yields (30-84%). Inspired by the tyrosinase-catalyzed hydroxylation of monophenols to catechols, the tyrosinase-enabled chromo-fluorogenic reaction was verified by using monophenol (typically tyrosol) as the substrate. In this regard, a dual-readout tyrosinase ac-tivity assay was developed by virtue of the in-situ "turn-on" optical signals. Furthermore, a test of tyrosinase inhibition, by using a common inhibitor kojic acid, demonstrated the potential of the chromo-fluorogenic reaction for developing other tyrosinase related assays and signal transduction.

Background: olive pomace extract (OPE) is a rich source of health promoting polyphenols (hydroxytyrosol (HTS) and tyrosol (TS)) and can be used as a nutraceutical ingredient of dietary supplements and functional foods. Its adequate bioavailability is a prerequisite for excreting biological activity and can be significantly and specifically affected by different food matrices.

Methods: in order to investigate food effects on polyphenol bioaccessibility, OPE was co-digested with different foods according to internationally harmonized in vitro digestibility method. Impact of particular nutrients on HTS and TS permeability was assessed on Caco-2 cell monolayer.

Results: HTS and TS bioaccessibility and transepithelial permeability can be significantly affected by foods (nutrients), especially by casein and certain types of dietary fiber. Those effects are polyphenol-and nutrient-specific and are achieved either through complexation in gastrointestinal lumen and/or through direct effects of nutrients on intestinal monolayer.

Conclusions: obtained results emphasize the significance and complexity of polyphenol interactions within the food matrix and the necessity of individual investigational approaches with respect to particular food/nutrient and interacting phenolic compounds.

Keywords: Caco-2 monolayer; hydroxytyrosol; in vitro digestion; olive pomace; polyphenol-food interaction; tyrosol.

Some constituents of the Mediterranean diet, such as extra-virgin olive oil (EVOO) contain substances such as hydroxytyrosol (HT) and its metabolite homovanillic alcohol (HA). HT has aroused much interest due to its antioxidant activity as a radical scavenger, whereas only a few studies have been made on the HA molecule. Both chemical synthesis and extraction techniques have been developed to obtain these molecules, with each method having its advantages and drawbacks. In this study, we report the use of tyrosol from olive mill wastewaters as a starting molecule to synthesize HT and HA, using a sustainable procedure characterized by high efficiency and low cost. The effects of HT and HA were evaluated on two cell lines, THP-1 human leukemic monocytes and L-6 myoblasts from rat skeletal muscle, after treating the cells with a radical generator. Both HT and HA efficiently inhibited ROS production. In particular, HT inhibited the proliferation of the THP-1 leukemic monocytes, while HA protected L-6 myoblasts from cytotoxicity.

Keywords: antioxidant; homovanillic alcohol; hydroxytyrosol; olive mill wastewaters; sustainable; tyrosol.

In this work, a new eco-friendly procedure for the synthesis of hydroxytyrosol and tyrosol α-glycosidic derivatives was proposed by using the marine α-glucosidase from Aplysia fasciata, and a commercial tyrosinase from mushroom for the bioconversion of tyrosol glycosidic derivatives into the corresponding hydroxytyrosol products. New hydroxytyrosol mono- and di-saccharide derivatives were synthesized at final concentrations of 9.35 and 10.8 g/l of reaction, respectively, and their antioxidant activity was evaluated by DPPH test. The best antioxidant agent resulted the (3,4-dihydroxyphenyl) ethyl-α-D-glucopyranoside; it showed a radical scavenging activity similar to that of the hydroxytyrosol, together with an increased hydrosolubility. This molecule could be a good response to many food industry demands, always in search of cheap antioxidants with nutritional properties to improve the nutritional value and the quality of foods.

The aim of this study was to investigate the ability of the sulfate metabolites of hydroxytyrosol (HT) and tyrosol (TYR) to act as antioxidants counteracting the pro-oxidant effect of oxidized cholesterol in intestinal cells. For this purpose, we synthesized sulfate metabolites of HT and TYR using a chemical methodology and examined their antioxidant activity in Caco-2 monolayers in comparison with the parent compounds. Exposure to oxidized cholesterol led to ROS production, oxidative damage, as indicated by the MDA increase, a decrease of reduced glutathione concentration and an enhancement of glutathione peroxidase activity. All the tested compounds were able to counteract the oxidizing action of oxidized cholesterol; HT and TYR sulfate metabolites showed an efficiency in protecting intestinal cells comparable to that of the parent compounds, strengthening the assumption that the potential beneficial effect of the parent compounds is retained, although extensive metabolisation occurs, the resulting metabolites being able to exert a biological action themselves.

Hydroxytyrosol and tyrosol phospholipids were enzymatically synthesized and investigated for their bilayer properties. Dynamic light scattering demonstrated that hand extrusion at 100nm consistently resulted in liposomes of nearly 85nm diameter for both phosphatidyl-hydroxytyrosol (DOPHT) and phosphatidyl-tyrosol (DOPT). Transmission electron microscopy showed DOPT and DOPHT liposomes extruded at 100-nm to be spherical and non-distinctive from one another. Zeta potential measurements resulted in surface charges<-25mV, demonstrating both DOPT and DOPHT form highly stable liposomes. Quartz crystal microbalance with dissipation monitoring measurements demonstrated that liposomal adsorption was dependent on a combination of DOPT (or DOPHT) mole-percent and calcium ions concentration. Fluorescence anisotropy measurements indicated that melting temperatures of DOPT and DOPHT were below 4°C, suggesting that adsorption behavior and liposome formation was limited by electrostatic interactions and not gel-state formation.

BACKGROUND

Hydnora abyssinica (HA) A. Braun is an endemic Sudanese medicinal plant traditionally used as anti-inflammatory and against many infectious diseases. However, it proved to be very rich in phenols and tannins, so the present study was undertaken to investigate the immunomodulatory potential of the whole plant ethanolic extract and its isolated compounds.

METHODS

Lymphocyte proliferation, chemiluminescence and superoxide reduction assays were used for immunomodulatory evaluation. While, MTT (3-(4, 5-dimethylthazol-2-yl)-2, 5-diphenyl tetrazonium bromide) test was performed on 3 T3 cell line clone in order to evaluate the cytoxicity effect of the extracts and isolated compounds of phenolic derivatives which were carried out by chromotographic techniques.

RESULTS

Catechin, (1), tyrosol (2) and benzoic acid, 3, 4, dihydroxy-, ethyl ester (3) compounds were isolated from HA ethanolic extract which revealed potent immunosuppressive activity against reactive oxygen species from both polymorph nuclear cells (PMNs) (45-90 % inhibition) and mononuclear cells (MNCs) (30 -65 % inhibition), T lymphocyte proliferation assay (70-93 % inhibition) as well as potent inhibitory effect against superoxide production (42-71 % inhibition) at concentrations of 6.25-100 μg/mL. Catechin (1) was found the most potent immunosuppressive agent among all constituents examined.

CONCLUSIONS

These results can support the traditional uses of H. abyssinica extracts as anti-inflammatory and immunosuppressive and further investigations of the mode of action and other pharmacological studies are highly desirable.

The aim of the study was to identify new potential chemical markers of extra virgin olive oil (EVOO) quality by using a multicomponent analysis approach. Sixty-six EVOOs were purchased from the Italian market and classified according to their price as low price EVOOs (LEVOOs) and high price EVOOs (HEVOOs) costing 3.60-5.90euro/L and 7.49-29.80euro/L respectively. Sensory and chemical parameters strictly related to olive oil quality have been investigated, like volatile substances, polar phenolic substances, antioxidant activity, fatty acid composition, and α-tocopherol. Significant differences in terms of chemical composition and sensory features have been highlighted between the two EVOOs classes investigated, proving a generally lower level of quality of LEVOOs, clearly showed also by means of principal component analysis. Among the most interesting outcomes, R ratio (free tyrosol and hydroxytyrosol over total free and bound forms), measuring the extent of secoiridoids hydrolysis, resulted to be significantly higher in LEVOOs than in HEVOOs. Other key differences were found in the volatile substances composition, in the stearic acid percentage and in p-coumaric acid content.

A library of hybrid and dimer compounds based on the natural scaffold of artemisinin was synthesized. These derivatives were obtained by coupling of artemisinin derivatives, artesunate, and dihydroartemisinin with a panel of phytochemical compounds. The novel artemisinin-based hybrids and dimers were evaluated for their anticancer activity on a cervical cancer cell line (HeLa) and on three complementary metastatic melanoma cancer cell lines (SK-MEL3, SK-MEL24, and RPMI-7951). Two hybrid compounds obtained by coupling of artesunate with eugenol and tyrosol, and one of the dimer compounds containing curcumin, emerged as the most active and cancer-selective derivatives.

At high cell density or under low nutrient conditions, yeasts collectively adapt their metabolism by secreting aromatic alcohols in what is known as quorum sensing. However, the mechanisms and role of quorum sensing in yeast are poorly understood, and the methodology behind this process is not well established. This paper describes an effective approach to study quorum sensing in yeast fermentations. The separation, detection, and quantification of the putative quorum-sensing molecules 2-phenylethanol, tryptophol, and tyrosol have been optimized on a simple HPLC-based system. With the use of a phenyl HPLC column and a fluorescence detector, the sensitivity of the system was significantly increased. This allowed extraction and concentration procedures to be eliminated and the process to be scaled down to 2 mL minifermentations. Additionally, an innovative method for rapid viable-cell counting is presented. This study forms the basis for detailed studies in kinetics and regulation of quorum sensing in yeast fermentation.

Drug discovery for a vigorous and feasible lead candidate is a challenging scientific mission as it requires expertise, experience, and huge investment. Natural products and their derivatives having structural diversity are renowned source of therapeutic agents since many years. Tyrosol (a natural phenylethanoid) has been extracted from olive oil, and its structure was confirmed by elemental analysis, FT-IR, FT-NMR, and single crystal X-ray crystallography. The conformational analysis for tyrosol geometry was performed by Gaussian 09 in terms of density functional theory. Validation of bond lengths and bond angles obtained experimentally as well as theoretically were performed with the help of curve fitting analysis, and values of correlation coefficient (R) obtained as 0.988 and 0.984, respectively. The charge transfer within the tyrosol molecule was confirmed by analysis of HOMO→LUMO molecular orbitals. In molecular docking with COX-2 (PDB ID: 5F1A), tyrosol was found to possess satisfactory binding affinity as compared to other NSAIDs (Aspirin, Ibuprofen, and Naproxen) and a COX-2 selective drug (Celecoxib). ADMET prediction, drug-likeness and bioactivity score altogether confirm the lead/drug like potential of tyrosol. Further investigation of simulation quality plot, RMSD and RMSF plots, ligands behavior plot as well as post simulation analysis manifest the consistency of 5F1A-tyrosol complex throughout the 20 ns molecular simulation process that signifies its compactness and stability within the receptor pocket. Abbreviations ADMET Absorption, Distribution, Metabolism, Excretion and Toxicity Å Angstrom COX-2 Cyclooxygenase-2 DFT Density Functional Theory DMF Dimethylformamide FMO Frontier Molecular Orbital FT-IR Fourier-transform Infrared Spectroscopy FT-NMR Nuclear Magnetic Resonance Spectroscopy HOMO Highest Occupied Molecular Orbital LUMO Lowest Unoccupied Molecular Orbital MD Molecular Dynamics NS Nanosecond NSAIDs Non-steroidal anti-inflammatory drugs OPE Osiris Property Explorer RMSD Root-Mean-Square Deviation RMSF Root Sean Square Fluctuation Communicated by Ramaswamy H. Sarma.

Olive waste was used as a sustainable resource because it contained a variety of valuable compounds. The polyphenols active fraction from enrichment by microporous resin and extraction with ethyl acetate were analysed by different chromatographic methods. A total of 14 polyphenolic compounds were isolated and identified by structure elucidation. Based on the above obtained compounds, tyrosol was selected as a characteristic polyphenol and participated in transesterification reaction to synthesise β-ketoester using Yb(OTf)₃. Then the Biginelli reaction with benzaldehyde, urea and ketoester (1:1.2:1.2) was performed at 90 °C for 3.0 h under the acidic condition. In addition, the β-ketoester prepared using tyrosol with benzyl had a greater inhibitory effect on α-glucosidase and α-amylase, and the inhibition of enzyme activity for 3, 4-dihydropyrimidinone derivatives prepared using abovementioned β-ketoester was improved significantly. Meanwhile, fluorine-containing dihydropyrimidinone derivatives were considerable inhibitors for both enzymes.

In this work, the phytochemical profile and the biological properties of Colchicum triphyllum (an unexplored Turkish cultivar belonging to Colchicaceae) have been comprehensively investigated for the first time. Herein, we focused on the evaluation of the in vitro antioxidant and enzyme inhibitory effects of flower, tuber, and leaf extracts, obtained using different extraction methods, namely maceration (both aqueous and methanolic), infusion, and Soxhlet. Besides, the complete phenolic and alkaloid untargeted metabolomic profiling of the different extracts was investigated. In this regard, ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) allowed us to putatively annotate 285 compounds when considering the different matrix extracts, including mainly alkaloids, flavonoids, lignans, phenolic acids, and tyrosol equivalents. The most abundant polyphenols were flavonoids (119 compounds), while colchicine, demecolcine, and lumicolchicine isomers were some of the most widespread alkaloids in each extract analyzed. In addition, our findings showed that C. triphyllum tuber extracts were a superior source of both total alkaloids and total polyphenols, being on average 2.89 and 10.41 mg/g, respectively. Multivariate statistics following metabolomics allowed for the detection of those compounds most affected by the different extraction methods. Overall, C. triphyllum leaf extracts showed a strong in vitro antioxidant capacity, in terms of cupric reducing antioxidant power (CUPRAC; on average 96.45 mg Trolox Equivalents (TE)/g) and ferric reducing antioxidant power (FRAP) reducing power (on average 66.86 mg TE/g). Interestingly, each C. triphyllum methanolic extract analyzed (i.e., from tuber, leaf, and flower) was active against the tyrosinase in terms of inhibition, recording the higher values for methanolic macerated leaves (i.e., 125.78 mg kojic acid equivalent (KAE)/g). On the other hand, moderate inhibitory activities were observed against AChE and α-amylase. Strong correlations (p < 0.01) were also observed between the phytochemical profiles and the biological activities determined. Therefore, our findings highlighted, for the first time, the potential of C. triphhyllum extracts in food and pharmaceutical applications.