Olive Oil Phenolic Compounds Research Articles

Analysis of Total Contents of Hydroxytyrosol and Tyrosol in Olive Oils

The most abundant phenolic compounds in olive oils are the phenethyl alcohols hydroxytyrosol and tyrosol. An optimized method to quantify the total concentration of these substances in olive oils has been described. It consists of the acid hydrolysis of the aglycons and the extraction of phenethyl alcohols with a 2 M HCl solution. Recovery of the phenethyl alcohols from oils was very high (<1% remained in the extracted oils), and the limits of quantification (LOQ) were 0.8 and 1.4 mg/kg for hydroxytyrosol and tyrosol, respectively. Precision values, both intraday and interday, remained below 3% for both compounds. The final optimized method allowed for the analysis of several types of commercial olive oils to evaluate their hydroxytyrosol and tyrosol contents. The results show that this method is simple, robust, and reliable for a routine analysis of the total concentration of these substances in olive oils.

Mechanisms of Action of Phenolic Compounds in Olive

ABSTRACTOlive oil, an oil rich in monounsaturated fatty acids (MUFCs) and minor constituents including phenolic compounds, is a major component of the Mediterranean diet. The potential health benefits of the Mediterranean diet were highlighted by the seminal Seven Countries Study, and more contemporary research has identified olive oil as a major element responsible for these effects. It is emerging that the phenolic compounds are the most likely candidates accounting for the cardioprotective and cancer preventative effects of extra virgin olive oil (EVOO). In particular, the phenolic compound, hydroxytyrosol has been identified as one of the most potent antioxidants found in olive oil. This review will briefly consider historical aspects of olive oil research and the biological properties of phenolic compounds in olive oil will be discussed. The focus of the discussion will be related to the mechanisms of action of hydroxytyrosol. Studies have demonstrated that hydroxytyrosol induces apoptosis and cell cycle arrest in cancer cells. Further, research has shown that hydroxytyrosol can prevent cardiovascular disease by reducing the expression of adhesion molecules on endothelial cells and preventing the oxidation of low-density lipoprotein (LDL). The molecular mechanisms accounting for these effects are reviewed.

Analysis of phenolic compounds in olive oil by solid-phase extraction and ultra high performance liquid chromatography–tandem mass spectrometry

In this study a simultaneous determination of several classes of polyphenolic compounds (benzoic acid derivates, cinnamic acid derivates, phenyl ethyl alcohols, flavones and other phenolic acids) in feed oils has been carried out by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Solid-phase extraction (SPE) with Diol and C(18) cartridges were compared in terms of recovery and number of extracted compounds, obtaining better results when Diol cartridges were used. The analytical procedure was validated, obtaining recoveries ranging from 70% (vanillic acid) to 111% (gallic acid) with repeatability values (expressed as relative standard deviations, RSDs) lower than 20% at two concentration levels (500 and 1000 μg/kg). Limits of quantification (LOQs) were always equal or lower than 50 μg/kg except for gentisic acid (100 μg/kg). Finally the method was applied to different types of feed oils, and it can be observed that higher concentrations of polyphenols were found in olive oil, whereas pomace olive oil and sunflower oil had the lowest level of these compounds.

The effects of a polyphenol present in olive oil, oleuropein aglycone, in an experimental model of spinal cord injury in mice

Several olive oil phenolic compounds, such us oleuropein have attracted considerable attention because of their antioxidant activity, anti-atherosclerotic and anti-inflammatory properties. The aim of this experimental study was to determine the effect of oleuropein aglycone, a hydrolysis product of oleuropein, in the inflammatory response, in particular in the secondary injury associated with the mouse model of spinal cord trauma. The injury was induced by application of vascular clips to the dura via a four-level T5–T8 laminectomy in mice. Oleuropein aglycone was administered in mice (100μg/kg, 40μg/kg, 20μg/kg, 10% ethanol, i.p.) 1h and 6h after the trauma. The treatment with oleuropein aglycone significantly decreased: (1) histological damage, (2) motor recovery, (3) nuclear factor (NF)-κB expression and IKB-α degradation, (4) protein kinase A (PKA) activity and expression, (5) pro-inflammatory cytokines production such as tumor necrosis factor (TNF-α) and interleukin-1β (IL-1β), 6) inducible nitric oxide synthase (iNOS) expression, (7) neutrophil infiltration, (8) lipid peroxidation, (9) nitrotyrosine and poly-ADP-ribose (PAR) formation, (10) glial cell-derived neurotrophic factor (GDNF) levels, (11) apoptosis (TUNEL staining, FAS ligand expression, Caspase 3, Bax and Bcl-2 expression). Thus, we propose that olive oil phenolic constituents such as oleuropein aglycone may be useful in the treatment of various inflammatory diseases.

Phenolic composition of virgin olive oils from cross breeding segregating populations

AbstractThe evaluation and characterization of segregating populations is a critical step in olive breeding programs. In this work, phenolic profiles of virgin olive oils (VOOs) from segregating populations obtained by cross breeding in Cordoba (Spain) have been evaluated. Genotypes obtained from open pollination of the cultivar Manzanilla de Sevilla, and from crosses between the cultivars Arbequina × Arbosana, Picual × Koroneiki and Sikitita × Arbosana were tested. The phenolic composition was determined after liquid–liquid extraction with 60:40 v/v methanol–water and subsequent chromatographic analysis with ultraviolet (UV) detection of both absorption and fluorescence in a sequential configuration. Results for all studied compounds showed high degree of variability between genotypes, with a higher range of variation than the observed for the genitors. Most of the observed variability was attributable to differences in genotypes within crosses rather than among crosses. Some issues related to breeding strategies are discussed.Practical applications: Phenolic compounds are considered to be of paramount importance for the assessment of virgin olive oil quality due to their contribution to the nutraceutical and sensory profile of this natural food. This study focuses on the evaluation of the content of phenolic compounds in olive oils originated from cross breeding in an olive breeding program (Cordoba, Spain). This step is crucial to determine the range of variation of phenolic compounds and the selection of interesting genotypes with higher composition in total phenols or in an individual phenol targeted at a breeding program.

Unsaturated Fatty Alcohol Derivatives of Olive Oil Phenolic Compounds with Potential Low-Density Lipoprotein (LDL) Antioxidant and Antiobesity Properties

A new route for the synthesis of fatty alcohol derivatives of hydroxytyrosol and other olive oil phenolic compounds was developed to allow the preparation of unsaturated derivatives. The biological activity of synthesized compounds was evaluated. Most of the compounds presented a significant antioxidant activity on low-density lipoprotein (LDL) particles. The activity of the tested products was significantly influenced by the number and position of unsaturations as well as modifications on the polar head of the synthesized compounds. Some of them presented modulation of food intake in rats and, due to their molecular similarity with CB(1) endogenous ligands, the endocannabinoid system and PPAR-α were also evaluated as potential targets. The pharmacodynamics could not be totally explained by CB(1) and PPAR-α receptor interactions because only two of the four compounds with biological activity showed a CB(1) activity and all of them presented low PPAR-α affinity, not justifying its whole in vivo activity. The hydroxytyrosol linoleylether (7) increased LDL resistance to oxidation with a capacity similar to that of hydroxytyrosol and was the most active in vivo compound with a hypophagic effect comparable to that of oleoylethanolamine. We consider that this compound could be a good lead compound for future drug development in obesity treatments.

Microchip electrophoresis with amperometric detection for a novel determination of phenolic compounds in olive oil

The relevance of the development of microchip electrophoresis applications in the field of food analysis is considered in this work. A novel method to determine important phenolic compounds in extra virgin olive oil samples using a miniaturized chemical analysis system is presented in this paper. Three interesting phenolic compounds in olive oil and fruit (tyrosol, hydroxytyrosol and oleuropein glucoside) were studied by end-channel amperometric detection using a 100 μm gold wire as working electrode in glass microchip electrophoresis. The electrochemical behavior of these compounds was studied and the medium to carry out their detection was selected (0.1 M aqueous sulfuric acid). The best conditions for the separation were achieved in sodium tetraborate (10% methanol, pH 9.50) with different concentrations for the sample and the running buffer in order to allow the sample stacking phenomenon. The injection was carried out using 600 V for 3 s and the separation voltage was set at 1000 V. The quality of the method was evaluated through its analytical figures of merit and by its performance on real extra virgin olive oil samples. Determination of these compounds was carried out using the standard addition calibration method with good recoveries.

Distribution of olive oil phenolic compounds in rat tissues after administration of a phenolic extract from olive cake

The distribution and accumulation of olive oil phenolic compounds in the body are topics lacked of information. The aim of this study was to evaluate the bioavailability, metabolism and distribution of phenolic compounds from olive cake. The metabolism and distribution of phenolic compounds were examined by UPLC-MS/MS after an acute intake of a phenolic extract from olive cake, analyzing plasma and tissues (heart, brain, liver, kidney, spleen, testicle and thymus) 1, 2 and 4 h after ingestion using Wistar rats as the in vivo model. The results showed a wide distribution of phenolic compounds and their metabolites in the tissues, with a main detoxification route through the kidneys. Highlighting the quantification of the free forms of some phenolic compounds, such as oleuropein derivative in plasma (Cmax 4 h: 24 nmol/L) and brain (Cmax 2 h: 2.8 nmol/g), luteolin in kidney (Cmax 1 h: 0.04 nmol/g), testicle (Cmax 2 h: 0.07 nmol/g) and heart (Cmax 1 h: 0.47 nmol/g); and hydroxytyrosol in plasma (Cmax 2 h: 5.2 nmol/L), kidney (Cmax 4 h: 3.8 nmol/g) and testicle (Cmax 2 h: 2.7 nmol/g). After a single ingestion of olive oil phenolic compounds, these were absorbed, metabolized and distributed through the blood stream to practically all parts of the body, even across the blood-brain barrier.

Interacción de los compuestos fenólicos del aceite de oliva virgen con las rutas de señalizacion celular

Introduction It has been speculated that the potential beneficial effects of olive oil could be due to modulation of genes involved in proliferative, antioxidant and inflammatory pathways. Research on the acting and mechanisms of phenolic compounds indicates that the beneficial effects associated with these compounds may not only be due to the elimination of free radicals, but could also modulate cell signalling processes, or could themselves act as signaling molecules. Methods Two virgin olive oil-based breakfasts with high (398 ppm) and low (70 ppm) content of phenolic compounds were administered to 20 patients with metabolic syndrome following a double-blinded random crossover design. Postprandial gene expression in peripheral blood mononuclear cells was analyzed at 4 hours by gene expression microarrays. Results We observed that the phenolic compounds of olive oil repressed the expression of 18 genes directly related to signalling processes, including 12 transcription factors involved in proliferation and cell growth. Conclusions Our results suggest that consumption of olive oil rich in phenolic compounds reduces the processes of cell proliferation in mononuclear cells in the postprandial state, which could reduce development of atherosclerosis.

Influence of Cultivar and Concentration of Selected Phenolic Constituents on the in Vitro Chemiopreventive Potential of Olive Oil Extracts

One of the main olive oil phenolic compounds, hydroxytyrosol (3,4-DHPEA), exerts in vitro chemopreventive activities (antiproliferative and pro-apoptotic) on tumor cells through the accumulation of H(2)O(2) in the culture medium. However, the phenol composition of virgin olive oil is complex, and 3,4-DHPEA is present at low concentrations when compared to other secoiridoids. In this study, the in vitro chemopreventive activities of complex virgin olive oil phenolic extracts (VOO-PE, derived from the four Italian cultivars Nocellara del Belice, Coratina, Ogliarola, and Taggiasca) were compared to each other and related to the amount of the single phenolic constituents. A great chemopreventive potential among the different VOO-PE was found following this order: Ogliarola > Coratina > Nocellara > Taggiasca. The antiproliferative and pro-apoptotic activities of VOO-PE were positively correlated to the secoiridoid content and negatively correlated to the concentration of both phenyl alcohols and lignans. All extracts induced H(2)O(2) accumulation in the culture medium, but this phenomenon was not responsible for their pro-apoptotic activity. When tested in a complex mixture, the olive oil phenols exerted a more potent chemopreventive effect compared to the isolated compounds, and this effect could be due either to a synergistic action of components or to any other unidentified extract constituent.

Hydroxytyrosol Induces Proliferation and Cytoprotection against Oxidative Injury in Vascular Endothelial Cells: Role of Nrf2 Activation and HO-1 Induction

Hydroxytyrosol (HT), a phenolic compound in olive oil and leaves, has been reported to prevent various human pathologies including cardiovascular diseases. This study investigated the effects of HT on proliferation and protection against oxidative stress-induced damage in vascular endothelial cells (VECs) and the molecular mechanism(s) involved. Treatment of VECs with HT increased cell proliferation, promoted wound repair, and protected cells against H(2)O(2) cytotoxicity through the activation of Akt and ERK1/2, but not p38 MAPK. HT increased the expression and nuclear translocation of nuclear factor-E2-related factor-2 (Nrf2). Nrf2 expression was attenuated by LY294002 and U0126, inhibitors of phosphatidylinositol-3-kinase and MEK1/2, respectively. Nrf2 siRNA decreased both proliferative and cytoprotective effects of HT and abrogated HO-1 induction. Moreover, HO-1 inhibition with HO-1 siRNA or zinc protoporphyrin IX significantly prevented HT-induced cell proliferation, cytoprotection, and reduction in intracellular reactive oxygen species (ROS), suggesting that HO-1 is involved in these HT functions. The findings demonstrate that HT positively regulates the antioxidant defense system in VECs through the activation of Nrf2 followed by cell proliferation and resistance to vascular injury. The present study provides a molecular basis for the contribution of HT in the Mediterranean diet to the prevention of cardiovascular diseases.

Hydroxytyrosol reduces intracellular reactive oxygen species levels in vascular endothelial cells by upregulating catalase expression through the AMPK–FOXO3a pathway

Reactive oxygen species are critically involved in the endothelial dysfunction that contributes to atherosclerosis development. Hydroxytyrosol (HT), a main phenolic compound in olive oil and leaves from Olea europaea L., has antiatherogenic properties with powerful antioxidant activity. The present study verifies the antioxidant activity of HT on H2O2-induced intracellular reactive oxygen species in porcine pulmonary artery endothelial cells (VECs) and the involved molecular mechanisms. Incubation of VECs with HT prevented the increase in intracellular reactive oxygen species levels in the presence of H2O2. HT increased catalase mRNA, protein and activity. Catalase siRNA suppressed HT-dependent reduction of intracellular reactive oxygen species. HT increased both cytosolic and nuclear protein levels of forkhead transcription factor 3a (FOXO3a), as well as the phosphorylation of AMP-activated protein kinase (AMPK) that translocates FOXO3a to the nucleus. AMPK siRNA and a specific inhibitor suppressed HT-induced FOXO3a upregulation and catalase expression. Moreover, FOXO3a siRNA blocked HT-dependent increase in catalase expression. Taken together, our findings strongly demonstrate that HT positively regulates the antioxidant defense system in VECs by inducing the phosphorylation of AMPK with subsequent activation of FOXO3a and catalase expression, and provides a molecular basis for the prevention of cardiovascular diseases by HT.

The effects of oleuropein aglycone, an olive oil compound, in a mouse model of carrageenan-induced pleurisy

Several olive oil phenolic compounds, such us oleuropein have attracted considerable attention because of their antioxidant activity, anti-atherosclerotic and anti-inflammatory properties. The aim of this study was to investigate the effects of oleuropein aglycone, a hydrolysis product of oleuropein, in a mouse model of carrageenan-induced pleurisy. Mice were anaesthetized and subjected to a skin incision at the level of the left sixth intercostals space. The underlying muscle was dissected and saline or saline containing 2% λ-carrageenan was injected into the pleural cavity. Injection of carrageenan elicited an acute inflammatory response characterized by: infiltration of neutrophils in lung tissues (P<0.01 versus sham. P<0.01 versus carrageenan) and subsequent lipid peroxidation (P<0.01 versus sham. P<0.01 versus carrageenan), increased production of tumor necrosis factor-α and interleukin-1β (P<0.01 versus sham. P<0.01 versus carrageenan), increased expression of adhesion molecules, increased synthesis of nitric oxide (P<0.01 versus sham. P<0.01 versus carrageenan), nitrotyrosine and poly-ADP-ribose (P<0.01 versus sham. P<0.01 versus carrageenan). Administration of oleuropein aglycone 30min after the challenge with carrageenan, caused a significant reduction of all the parameters of inflammation measured. Thus, we propose that olive oil phenolic constituents may be useful in the treatment of various inflammatory diseases.

Effects of Conventional Heating on the Stability of Major Olive Oil Phenolic Compounds by Tandem Mass Spectrometry and Isotope Dilution Assay

The quality of olive oils is sensorially tested by accurate and well established methods. It enables the classification of the pressed oils into the classes of extra virgin oil, virgin oil and lampant oil. Nonetheless, it would be convenient to have analytical methods for screening oils or supporting sensorial analysis using a reliable independent approach based on exploitation of mass spectrometric methodologies. A number of methods have been proposed to evaluate deficiencies of extra virgin olive oils resulting from inappropriate technological treatments, such as high or low temperature deodoration, and home cooking processes. The quality and nutraceutical value of extra virgin olive oil (EVOO) can be related to the antioxidant property of its phenolic compounds. Olive oil is a source of at least 30 phenolic compounds, such as oleuropein, oleocanthal, hydroxytyrosol, and tyrosol, all acting as strong antioxidants, radical scavengers and NSAI-like drugs. We now report the efficacy of MRM tandem mass spectrometry, assisted by the isotope dilution assay, in the evaluation of the thermal stability of selected active principles of extra virgin olive oil.

Characterization of antioxidant olive oil biophenols by spectroscopic methods

Olive oil contains numerous phenolic components with well-recognized health-beneficial activity. The major phenolic compounds present in olives and virgin olive oil-hydroxytyrosol, oleuropein and the oleuropein aglycones 3,4-DHPEA-EA and 3,4-DHPEA-EDA-as well as some of their metabolites were studied in the present work, regarding their main structural preferences. Vibrational spectroscopy (Raman) coupled to theoretical methods were used, aiming at fully characterizing the systems and therefore enabling their quick and reliable identification in food samples. The raman data, assisted by the theoretical simulations, allowed us to obtain the main geometrical and spectroscopic features of the olive oil constituents under study, which determine their known antioxidant and chemoprotective properties. In fact, it was verified that the spectra comprise distinctive bands for each compound, allowing their ready detection and differentiation. This is the first reported study on the structural behaviour of olive oil phenolic compounds, and it established Raman spectroscopy as a rapid, non-destructive and reliable analytical technique for identifying these bioactive components in dietary extracts. It can surpass other analytical methods currently used, once it allows the concomitant identification of several olive oil components in a particular sample.

Olive oil phenols modulate the triacylglycerol molecular species of human very low-density lipoprotein. A randomized, crossover, controlled trial

Virgin olive oil phenolic compounds have been revealed to be potent antioxidants as part of the Mediterranean diet. To test the hypothesis that these phenolics can modulate the serum and very low-density lipoprotein (VLDL) triacylglycerol concentrations in humans, a double-blind, randomized, crossover trial was designed. Thirty-three participants received 25 mL/d of refined olive oil (devoid of phenolic content [PC]), common olive oil (PC = 370 mmol/kg), and virgin olive oil (PC = 825 mmol/kg) in a Latin square design. The 3 olive oils were administered over 3 periods of 3 weeks, each one preceded by 2-week washout periods. All analyses were carried out on an intention-to-treat basis. The interventions did not modify the concentrations of serum and low-density lipoprotein cholesterol and triacylglycerol; but they exerted changes in the cholesterol, triacylglycerol, and phospholipid content of VLDL. The virgin olive oil consumption led to increased oleic and palmitic acids, as well as decreased linoleic acid, in VLDL. The main outcome was the significant dose-dependent linear trend between the PC in the olive oils and the palmitic (16:0) and linoleic (18:2 n-6) acid and their corresponding triacylglycerol molecular species in VLDL.

Use of multivariate statistical techniques to optimize the simultaneous separation of 13 phenolic compounds from extra-virgin olive oil by capillary electrophoresis

Characterization of phenolic compounds in olive oil has not been achieved as yet, owing to the complexities of their chemical structures and analytical matrix. The aim of this work is to optimize and validate a method for simultaneous separation and quantification of 13 phenolic compounds from extra-virgin olive oil: tyrosol, hydroxytyrosol, oleuropein glycoside, ferrulic acid, p-coumaric acid, cinnamic acid, p-hydroxybenzoic acid, gallic acid, caffeic acid, luteolin, apigenin, vanillic acid and 3,4-dihydroxybenzoic acid. A statistical central composite design, response surface analysis and the simultaneous optimization method of Derringer and Suich were used to separate all the peaks. These multivariate procedures were efficient in determining the optimal separation condition, using five peak-pair resolutions and runtime as responses. The optimized method employed a fused-silica capillary of 50μm i.d.×60cm effective length with extended light path, 50mmolL−1 boric acid electrolyte, 10.2 pH, 25°C, injection of 50mbar for 25s with application of reverse voltage (−30kV for 5s) before setting the running voltage (+30kV) with detection at 210nm and a run time of 12min. Peak resolutions are found to be very sensitive to pH values outside the 10.15–10.25 range but acceptable electropherograms can be obtained for a wide range of boric acid concentrations within this pH interval.

Nano and rapid resolution liquid chromatography–electrospray ionization–time of flight mass spectrometry to identify and quantify phenolic compounds in olive oil

The applicability of nanoLC-ESI-TOF MS for the analysis of phenolic compounds in olive oil was studied and compared with a HPLC method. After the injection, the compounds were focused on a short capillary trapping column (100 microm id, effective length 20 mm, 5 microm particle size) and then nanoLC analysis was carried out in a fused silica capillary column (75 microm id, effective length 10 microm, 3 microm particle size) packed with C18 stationary phase. The mobile phase was a mixture of water + 0.5% acetic acid and ACN eluting at 300 nL/min in a gradient mode. Phenolic compounds from different families were identified and quantified. The quality parameters of the nanoLC method (linearity, LODs and LOQs, repeatability) were evaluated and compared with those obtained with HPLC. The new methodology presents better sensitivity (reaching LOD values below 1 ppb) with less consumption of mobile phases, but worse repeatability, especially inter-day repeatability, resulting in more difficulties to get highly accurate quantification. The results described in this article open up the application fields of this technique to cover a larger variety of compounds and its advantages will make it especially useful for the analysis of samples containing low concentration of phenolic compounds, as for instance, in biological samples.

Exploratory analysis of human urine by LC–ESI-TOF MS after high intake of olive oil: understanding the metabolism of polyphenols

Olive oil polyphenols have important biological properties which closely depend on their bioavailability; it is, therefore, essential to understand how polyphenols are absorbed, metabolized, and eliminated from the body. An analytical method based on rapid-resolution liquid chromatography (RRLC) coupled with mass spectrometric detection with a time-of-flight analyzer (RRLC-ESI-TOF MS) has been developed for analysis of the main olive oil phenolic compounds and their metabolites in human urine. Urine samples from ten healthy volunteers were collected before and 2, 4, and 6 h after intake of 50 mL extra-virgin olive oil. The proposed method includes liquid-liquid extraction with ethyl acetate, which provides extraction recoveries of the phenolic compounds studied between 35 and 75% from spiked urine samples. Good repeatability was obtained--the relative standard deviations (RSDs) of peak areas in intra-day and inter-day studies were 4.3 and 6.5%, respectively. Statistical studies enabled us to discriminate between urine samples before and after intake, and facilitated the search for m/z values enabling this discrimination. Based on the very accurate mass information and the isotopic pattern provided by the TOF MS analyzer, together with other available information, ten of these biomarkers and more than 50 metabolites, obtained through phase I and phase II biotransformation reactions, were tentatively identified. Additionally, kinetic studies were conducted on the metabolites identified as possible biomarkers; for most of the compounds concentrations were maximum in the first two hours.

Impact of oregano and virgin olive oil phenolic compounds on acrylamide content in a model system and fresh potatoes

In this study the effect of olive oil and oregano phenolic compounds on antioxidant activity (AOA) and acrylamide formation in a model system and fresh potatoes was evaluated. The addition of oregano phenolic extract in the model system resulted in an increase of AOA and in a reduction of acrylamide content, up to 49%, after heating. On the contrary, addition of olive oil extract resulted in AOA reduction and a concentration-dependent effect on acrylamide formation was observed with a reduction up to 15% ( p < 0.05) followed by an increase up to 48%. The same trend, in acrylamide formation, was observed in fresh potatoes but to a lower extent. Finally, addition of tyrosol, oleuropein and p-hydroxyphenylacetic acid in the model system resulted in acrylamide reduction up to 50%. The discrepancy in acrylamide formation, caused by phenolic compounds, is ascribed to their structure and related to their terminal functional hydroxyl and aldehydic groups.