Optimisation,characterisation and quantification of phenolic compounds in olive cake

The phenolic compounds in extracts from pressed olive cake were investigated. Free phenolic compounds were extracted from olive cake using methanol. To liberate bound phenolic compounds, the olive cake was subjected to basic and acidic hydrolysis followed by methanol extraction. The individual phenolic compounds and antioxidant activity of the extracts were determined. The highest total phenolic content and antioxidant activity were obtained using methanol extraction for 12 h at 70 °C. The RP-HPLC profiles for full-fat and defatted olive cake showed that protocatechuic acid, hydroxybenzoic acid, sinapic acid, p-coumaric acid, rutin and hesperidin were the predominant free phenolic compounds. Meanwhile, syringic acid, sinapic acid, caffeic acid and protocatechuic acid were the predominant bound phenolic acids. A positive correlation was observed between total phenolic content and antioxidant activity. The results indicated that most of the phenolic compounds in olive products were present in their free forms (75–90% of total phenolic content), while bound phenolic compounds were only a small proportion (10–25%) of total phenolic content.     

Characterisation of an oleuropein degrading strain of Lactobacillus plantarum. Combined effects of compounds present in olive fermenting brines (phenols,glucose and NaCl) on bacterial activity

This study aims to examine the effects of glucose and NaCl on the ability of an oleuropein degrading strain of Lactobacillus plantarum, strain B21, to grow in the presence of oleuropein, its degradation product, hydroxytyrosol, and p-coumaric acid. Oleuropein (10 g litre⁻¹) and 2 g litre⁻¹ hydroxytyrosol combined with NaCl did not inhibit bacterial growth, whereas 1 g litre⁻¹ p-coumaric acid showed low inhibitory activity. This study also presents that bacterial β-glucosidase and esterase are involved in the breakdown of oleuropein. Oleuropein (10 g litre⁻¹) incorporated in the cultivation medium without glucose was completely degraded to derivative products within 20 days, whereas in the presence of glucose, at concentrations up to 50 g litre⁻¹ β-glucosidase activity was partially inhibited, and 30–70% of oleuropein residual content remained in the cultivation medium. Esterase activity involved in the second step of biodegradation process, was not influenced by glucose. Incorporation of glucose in the growth medium adversely affected the ability of L plantarum to break-down oleuropein. The findings of this study are significant since it could lead to the development of a new biotechnology for removing the bitter principle, oleuropein, from olives replacing the traditional alkali treatment used for debittering olives. © 1998 SCI.     

Evaluation of table olive by‐product as a source of natural antioxidants

The main by‐product from the table olive canning industry is the stone with some residual olive flesh. The purpose of this study was to evaluate the composition – phenolic compounds (hydroxytyrosol, tyrosol and oleuropein) and tocopherol – and the antioxidant activity in different fractions (flesh, stone and seed) from the table olive by‐product and the whole by‐product. The highest amounts of phenolic compounds (1710.0 ± 33.8 mg kg^?1) as well as the highest antioxidant activity (8226.9 ± 9.9 hydroxytyrosol equivalents mg kg^?1) were obtained in the seed. The highest amounts of hydroxytyrosol (854.8 ± 66.0 mg kg^?1) and tyrosol (423.6 ± 56.9 mg kg^?1) were found in the whole by‐product from the pepper stuffed olives, while the stone without seed had the maximum oleuropein content (750.2 ± 85.3 mg kg^?1). α‐Tocopherol values were between 79.8 ± 20.8 mg kg^?1 in the seed of the olive stone and 6.2 ± 1.2 mg kg^?1 in the whole by‐product from the anchovy‐stuffed olives. In light of the results obtained, it would seem possible to use table olive by‐product as a source of natural antioxidants in foods, cosmetics or pharmaceutical products, thus contributing to diminishing the environmental impact of table olive by‐product and to its revalorisation.     

Human absorption and metabolism of oleuropein and hydroxytyrosol ingested as olive (Olea europaea L.) leaf extract

Phenolic compounds derived from the olive plant (Olea europaea L.), particularly hydroxytyrosol and oleuropein, have many beneficial effects in vitro. Olive leaves are the richest source of olive phenolic compounds, and olive leaf extract (OLE) is now a popular nutraceutical taken either as liquid or capsules. To quantify the bioavailability and metabolism of oleuropein and hydroxytyrosol when taken as OLE, nine volunteers (five males) aged 42.8 ± 7.4 years were randomized to receive either capsulated or liquid OLE as a single lower (51.1 mg oleuropein, 9.7 mg hydroxytyrosol) or higher (76.6 mg oleuropein, 14.5 mg hydroxytyrosol) dose, and then the opposite strength (but same formulation) a week later. Plasma and urine samples were collected at fixed intervals for 24 h post‐ingestion. Phenolic content was analyzed by LC‐ESI‐MS/MS. Conjugated metabolites of hydroxytyrosol were the primary metabolites recovered in plasma and urine after OLE ingestion. Peak oleuropein concentrations in plasma were greater following ingestion of liquid than capsule preparations (0.47 versus 2.74 ng/mL; p = 0.004), but no such effect was observed for peak concentrations of conjugated (sulfated and glucuronidated) hydroxytyrosol (p = 0.94). However, the latter peak was reached earlier with liquid preparation (93 versus 64 min; p = 0.031). There was a gender effect on the bioavailability of phenolic compounds, with males displaying greater plasma area under the curve for conjugated hydroxytyrosol (11 600 versus 2550 ng/mL; p = 0.048). All conjugated hydroxytyrosol metabolites were recovered in the urine within 8 h. There was wide inter‐individual variation. OLE effectively delivers oleuropein and hydroxytrosol metabolites to plasma in humans.     

Determination of Polyphenols in Commercial Extra Virgin Olive Oils from Different Origins (Mediterranean and South American Countries) by Liquid Chromatography–Electrospray Time-of-Flight Mass Spectrometry

The phenolic profiles of extra virgin olive oil samples from different origins and varieties have been studied and compared. The determination of 13 target polyphenols (gallic acid, hydroxytyrosol, tyrosol, vanillic acid, caffeic acid, syringic acid, gibberellic acid, p-coumaric acid, ferulic acid, oleuropein, resveratrol, luteolin, and apigenin) was carried out by liquid chromatography using a column (C₁₈, 4.6?×?100 mm) with small particle size (1.8 μm) and mass spectrometric detection using electrospray time-of-flight mass spectrometry (LC–TOFMS). Prior to LC–MS analysis, a solid-phase extraction procedure using SPE-Diol cartridges was used to isolate the polyphenol fraction. The full-scan mass spectra acquisition by TOF analyzers offered the possibility of searching for a large number of compounds by means of accurate mass information of the molecules and allowed the detection of other phenolic compounds not included in the initial target list, namely ligstroside aglycon, oleuropein aglycon, and elenolic acid. The proposed method was successfully applied to the analysis of 78 samples of extra virgin olive oil collected from ten different countries: Argentina (5), Chile (6), France (1), Greece (1), Italy (1), Morocco (4), Peru (32), Portugal (1), Spain (26), and Syria (1). No qualitative differences in the composition of the phenolic fraction were found between samples of different origins. Only quantitative differences were observed for some phenolic compounds. Therefore, further studies are still needed to exploit the possibility of using phenolic profile as a marker for olive oil geographical origin.     

Ability of Lactobacillus brevis strains to degrade food phenolic acids

The potential to degrade 15 food phenolic acids was investigated for several Lactobacillus brevis strains isolated from different sources. All the strains analysed in this study showed a similar metabolism on phenolic acids. Among the cinnamic acids assayed, only p-coumaric, ferulic and caffeic acids were metabolized by the L. brevis strains. These acids were decarboxylated to produce their corresponding vinyl derivatives. Contrarily to the results previously reported on Lactobacillus plantarum, the L. brevis strains analysed in this study were unable to subsequently reduce or metabolize these vinyl derivatives. In L. brevis, vinyl phenol, vinyl catechol, and vinyl guaiacol were the final metabolic products from p-coumaric, caffeic or ferulic acids, respectively. From the benzoic acids analysed, and similarly to L. plantarum strains, only gallic and protocatechuic acids were modified by L. brevis strains. Both acids were decarboxylated to pyrogallol and catechol, respectively. Currently, the enzymes involved in the metabolism of phenolic acids in L. brevis remain uncharacterized.     

Metabolism of food phenolic acids by Lactobacillus plantarum CECT 748

Phenolic acids account for almost one third of the dietary phenols and are associated with organoleptic, nutritional and antioxidant properties of foods. This study was undertaken to assess the ability of Lactobacillus plantarum CECT 748^T to metabolize 19 food phenolic acids. Among the hydroxycinnamic acids studied, only p-coumaric, caffeic, ferulic and m-coumaric acids were metabolized by L. plantarum. Cultures of L. plantarum produced ethyl and vinyl derivatives from p-coumaric and caffeic acids, 4-vinyl guaiacol from ferulic acid, and 3-(3-hydroxyphenyl) propionic acid from m-coumaric acid. Among the hydroxybenzoic acids analysed, gallic acid and protocatechuic acid were decarboxylated to pyrogallol and catechol, respectively. Inducible enzymes seem to be involved, at least in m-coumaric and ferulic acid metabolism, since cell-free extracts from cultures grown in the absence of these phenolic acids were unable to metabolize them. Further work is needed for the identification of the enzymes involved, since the knowledge of the metabolism of phenolic compounds is an important issue for the food industry.     

A Knowledge Base for The Recovery of Natural Phenols with Different Solvents

The current study denotes the prediction of activity coefficients of fifteen natural phenols (tyrosol, hydroxytyrosol, oleuropein, caffeic, cinnamic, p-coumaric, ferulic, gallic, p-hydroxybenzoic, p-hydroxyphenyl acetic, protocatechuic, rosmarinic, sinapic, syringic, and vanillic acid) in seven solvents (water, ethanol, methanol, acetone, dichloromethane, ethyl acetate, and diethyl ether), and three extraction temperatures (298.15, 313.15, and 333.15 K), using the universal functional-group activity coefficient model. Solvents were classified for their ability to dissolve phenols and were compared with experimental data of the literature in order to observe if the solvent extraction of phenols in practice matches with the authors’ theoretical approach. Results indicated the superiority of alcohols and acetone for the recovery of phenols in line with experimental data of previous studies. Furthermore, activity coefficients’ values were found to increase with the increase of temperature. This study provided a knowledge base for the selection of the most appropriate solvents for a given phenolic compound.     

Distribution of simple phenols,phenolic acids and flavonoids in Turkish monovarietal extra virgin olive oils for two harvest years

Monovarietal extra virgin olive oils extracted from six dominant and economically important Turkish olive cultivars (memecik, erkence, domat, nizip-yaglik, gemlik, ayvalik) were examined for their simple phenolics, phenolic acids and flavonoid compounds over 2005 and 2006 harvest years. Total phenol contents, oxidative stabilities and chromatic ordinates as colour parameters were also measured. The most typical phenolic compounds that were identified in both years are hydroxytyrosol, tyrosol, vanillic acid, p-coumaric acid, cinnamic acid, luteolin, and apigenin. Multivariate data were analysed by principal component and partial least square-discriminant analyses. It was observed that phenolic profiles of olive oils depended highly on harvest season. In addition, oils of different olive cultivars have different distribution of phenols. No significant correlation was observed between oxidative stability and phenolic compounds. Increase in peroxide value over an accelerated oxidation period of 11 days showed weak correlations with total phenol content, vanillin, syringic acid and colour parameter a^∗, as 0.56, 0.55, −0.42, and 0.51, respectively, in terms of correlation coefficient r.     

Phenolic compounds in natural black Spanish olive varieties

There is increasing interest in olive phenolic compounds because of their biological properties as well as their contribution to the colour, taste and shelf life of olive products. Phenolic compounds in natural black Spanish olives are characterised by HPLC-UV and HPLC-MS. Hydroxytyrosol-4-#-D-glucoside has been identified as the major phenolic compound in natural black olives. A qualitative and quantitative study of phenolic substances such as hydroxytyrosol, salidroside, tyrosol, verbascoside, luteolin-7-glucoside, rutin, demethyloleuropein and oleuropein has been carried out. Traces of other phenolic compounds such as catechol, vanillic acid, apigenin-7-rutinoside and ligustroside have also been found by mass spectroscopy. Secoiridoid aglycons, the main phenolic compounds in olive oil, were not detected in olive flesh except the dialdehydic form of elenolic acid linked to hydroxytyrosol (HyEDA), particularly in the Arbequina olive variety and only traces were detected in other varieties. Among anthocyanins, cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside were the only anthocyanin compounds detected in all the varieties studied. The UV and MS spectra of several unknown phenolic compounds have also been described.     

Antioxidant phenolic compounds loss during the fermentation of Chétoui olives

Evolution of phenolic compounds was studied during spontaneous and controlled fermentations of “Chétoui” cultivar olives at three degree of ripeness. Both spontaneous and controlled fermentations led to an important loss of total phenolic compounds with a reduction rate of 32–58%. Consequently, the antioxidant activity decreased by 50–72%. After fermentations, hydroxytyrosol and caffeic acid concentrations increased, whilst protocatechuic acid, ferulic acid and oleuropein concentrations decreased. The hydroxytyrosol concentration in black olives increased from 165 mg/100 g dry weight to 312 and 380 mg/100 g dry weight, respectively, after spontaneous and controlled fermentation. The oleuropein concentration in green olives decreased from 266 mg/100 g dry weight to 30.7 and 16.1 mg/100 g dry weight, respectively, after spontaneous and controlled fermentation. During olive fermentation, phenolic loss is essentially due to the diffusion of these compounds into the brine; the main phenolic compound identified and quantified in the different brines was hydroxytyrosol. To preserve antioxidant quality of table olives it is necessary to use a controlled process to minimise phenolic compound loss.     

HPLC analysis of oleuropein,hydroxytyrosol, and tyrosol in stems and roots of Olea europaea L. cv. Picual during ripening

BACKGROUND: Oleuropein (Ole), hydroxytyrosol (Htyr), and tyrosol (Tyr) are three of the main phenolic compounds present in the olive tree (Olea europaea L.) that have important antioxidant properties. To investigate the role of these phenolic compounds in the metabolism of stems and roots of Olea europaea L. cv. Picual during olive ripening, we identify and quantify the concentration of Htyr, Tyr, and Ole by reversed‐phase high‐performance liquid chromatography (RP‐HPLC). Rain‐fed olive trees, 30 years old, under traditional cultivation were studied in Jaén (Spain). From August to November, seven representative samples of the ripening process were taken. RESULTS: The concentration of these phenolic compounds proved higher in the stems than in the roots. From the middle of September to October the Htyr and Tyr concentration significantly increased in stems. The Ole concentration increased from the middle of September to the end of November. In the roots, the concentration of Htyr and Ole significantly declined during ripening. CONCLUSION: Ole, Htyr, and Tyr are present in the stems and roots of the olive tree and significantly change in concentration during ripening, demonstrating the involvement of these compounds in the metabolism of both organs during this phase. Copyright © 2010 Society of Chemical Industry     

Characterization and fractionation of phenolic compounds extracted from olive oil mill wastewaters

Liquid–liquid extraction was used in order to recover phenolic compounds from olive mill wastewater (OMWW), a polluting by-product of olive oil production process, and the extraction parameters have been optimized. HPLC analysis showed that hydroxytyrosol is the most abundant biophenol in ethyl acetate extracts from an acidified OMWW. Many other biophenols (tyrosol, caffeic acid, vanillic acid, verbascoside, luteolin-7-glucoside, dialdehydic form of decarboxymethyl oleuropein aglycon, ligstroside, luteolin) were identified.     

Involvement of oleuropein in (some) digestive metabolic pathways

Olive oil is the principal source of fats in the Mediterranean diet and it has been postulated that the components in olive oil can contribute to a lower incidence of coronary heart disease and cancers (prostate, colon, breast, and skin). The positive effects on human health can be attributed to the high level of phenolic compounds present in olive oil, the major ones being oleuropein, hydroxytyrosol and tyrosol. The aim of the present study was to evaluate the effect of oleuropein on enzymes involved in specific pathways of metabolism of proteins, carbohydrates and lipids.In particular, the effects of oleuropein on enzymes, such as trypsin, pepsin, lipase, glycerol dehydrogenase, glycerol-3-phosphate dehydrogenase, and glycerokinase, were investigated.Results demonstrate that oleuropein is able to activate pepsin and shows an inhibitory effect toward all the other enzymes tested, which suggests a new role for this polyphenol. In addition, a new method for lipase activity assay is presented.     

Lactobacillus pentosus dominates spontaneous fermentation of Italian table olives

Culture-dependent and -independent approaches were applied to identify the bacterial species involved in Italian table olive fermentation. Bacterial identification showed that Lactobacillus pentosus was the dominant species although the presence of Lactobacillus plantarum, Lactobacillus casei, Enterococcus durans, Lactobacillus fermentum and Lactobacillus helveticus was observed. Rep-PCR allowed to obtain strain-specific profiles and to establish a correlation with table olive environment. PCR-DGGE (Denaturing Gradient Gel Electrophoresis) confirmed the heterogeneity of bacterial community structure in fermented table olives as well as the prevalence of L. pentosus. The strains were characterized on the basis of technological properties (NaCl tolerance, β-glucosidase activity and the ability to grow in synthetic brine and in presence of 1 g/100 mL oleuropein). L. pentosus showed a high capacity of adaptation to the different conditions characterizing the olive ecosystem. This species showed the highest percentage of strains able to grow in presence of 10 g/100 mL NaCl, oleuropein and in the synthetic brine. Moreover, all the strains belonging to L. pentosus and L. plantarum species showed a β-glucosidase activity. This study allowed both to identify the main species and strains associated to Italian table olives and to obtain a lactic acid bacteria collection to apply as starter culture in the process of olive fermentation.     

Characterization of Bioactive Compounds from Monovarietal Virgin Olive Oils: Relationship Between Phenolic Compounds-Antioxidant Capacities

The characterization of bioactive components of virgin olive oil from nine varieties (Cuquillo, Empeltre, Manzanilla, Cornicabra, Picual, Arbequina, Lechin, Picudo, and Hojiblanca) has been carried out. Carotenoids, chlorophylls, α-tocopherol, fatty acids, total phenols, and individual phenols were determined. The antioxidant capacity was evaluated by 2,2′-diphenyl-1-picrylhydrazyl and 2,2′-azinobis (3-ethylbenzothiaziline-6-sulfonate) radical scavenging capacity assay, ferric reducing antioxidant potential assay, and oxygen radical absorbance capacity assay. Twelve phenolic compounds were detected and quantified by high-performance liquid chromatography with diode-array detection. Dialdehydic form of elenolic acid linked to tyrosol and hydroxytyrosol, oleuropein, and ligstroside aglycones were the major components in all the samples. Principal component analysis confirmed that Manzanilla, Lechin, Cuquillo, and Hojiblanca var. were the most similar. Furthermore, these results showed that the antioxidant capacity measured by different assays was highly influenced by the phenolic content, especially the dialdehydic form of elenolic acid linked to tyrosol and hydroxytyrosol.     

The production of hydrogen peroxide is not a common mechanism by which olive oil phenols induce apoptosis on HL60 cells

We have recently demonstrated that hydroxytyrosol (3,4-DHPEA), the most representative olive oil phenol, induces apoptosis on HL60 cells through the production of considerable amount of extracellular hydrogen peroxide (H₂O₂). The aims of the present investigation were first to assess the ability of different phenolic compounds to both produce extracellular H₂O₂ and induce apoptosis on HL60 cells, and second to elucidate whether the pro-apoptotic activity was mediated by the production of H₂O₂ in the cell culture medium. Based on the results phenols can be classified as follows: (1) those which were not able to induce both apoptosis and H₂O₂ accumulation (tyrosol, homovanillic alcohol and protocatechuic, o-coumaric, vanillic, homovanillic, ferulic and syringic acids); (2) those which showed a pro-apoptotic activity mediated, at least in part, by the production of H₂O₂, as evidenced by the ability of catalase to inhibit apoptosis (3,4-DHPEA, dopamine, 3,4-dihydroxyphenylacetic, 3,4-dihydroxy-hydrocinnamic, caffeic and gallic acids); and (3) those which induced apoptosis without the involvement of H₂O₂ (the secoiridoid derivatives of both hydroxytyrosol and tyrosol). Oleuropein showed a peculiar behaviour since, and although it caused an abundant production of H₂O₂ in the cell culture medium, it exerted a weak pro-apoptotic effect. From these results we may conclude that the cathecol moiety of the phenol molecule is necessary for the H₂O₂ producing activity, and that the 3,4-DHPEA metabolism to homovanillic alcohol and homovanillic acid may significantly reduce its pro-apoptotic potential. The real in vivo meaning of the phenol-induced H₂O₂ production remains to be investigated.     

Phenolic inhibitors involved in the natural fermentation of Gemlik cultivar black olives

Olive fruits of the Gemlik variety harvested from different regions of Turkey were placed in aseptic or non-aseptic brines containing 6 % NaCl. Olives of non-aseptic treatment were left to spontaneously ferment under anaerobic conditions. Samples for microbiological and chemical analysis were taken periodically during the course of the fermentation. No lactic acid bacteria growth was observed in three of the six samples, and yeasts were the prevailing microbial group in the other samples. Brines were analyzed for fermentable substrates (glucose, fructose, sucrose and mannitol), fermentation products (organic acids and ethanol) and phenolic–oleosidic compounds. Most of the unprocessed fruits had a low concentration of oleuropein. Hydroxytyrosol and oleoside 11-methyl ester were the main phenolic and oleosidic compounds in all brines. Likewise, the content of antimicrobials such as the dialdehyde form of decarboxymethyl elenolic acid, either free or linked to hydroxytyrosol, in brines was very low, which may permit the growth of lactic acid bacteria in these media. A growth test with two strains of Lactobacillus plantarum was applied to aseptic brines of all samples to determine whether these compounds inhibited lactic acid bacteria growth. The results of this study indicated that Gemlik olive is a “sweet” variety with a low antimicrobial compound content that can be fermented by lactic acid bacteria under favorable conditions.     

Transfer of phenolic compounds during olive oil extraction in relation to ripening stage of the fruit

The transfer of phenolic compounds of Olea europaea L. cv. Arbequina variety during olive oil extraction in relation to ripening stage was investigated. The parameters of oil extraction by the Abencor system are shown together with mass balances of the products and by products from the olive oil extraction in relation to olive paste. The phenolic compounds in olive paste, pomace, oil and wastewater were identified and measured by HPLC. Throughout the study, the concentrations of simple phenols, secoiridoids and flavonoids were higher in the olive paste and pomace phases than in oil and wastewater phases. High concentrations of 4‐(acetoxyethyl)‐1,2‐dihydroxybenzene (3,4‐DHPEA‐AC) and secoiridoid derivatives such as the dialdehydic form of elenolic acid linked to 3,4‐DHPEA (hydroxytyrosol) or p‐HPEA (tyrosol) (3,4‐DHPEA–EDA, p‐HPEA–EDA, where EDA is elenolic acid dialdehyde) and an isomer of oleuropein aglycone (3,4‐DHPEA–EA, where EA is elenolic acid aldehyde) were found in olive oil, together with lignan compounds. It was observed that 3,4‐DHPEA–EDA was the most abundant polyphenol present in the wastewater phase. This indicates that biotransformation occurred during olive extraction, especially in the crushing and malaxation operations, and reflects the possible chemical changes that lead to the formation of new compounds. Moreover, the distribution of compounds showed their affinities toward different phases. Copyright © 2005 Society of Chemical Industry     

Antitumor Perspectives of Oleuropein and Its Metabolite Hydroxytyrosol: Recent Updates

Olive fruit is a significant and promising source of potential bioactive compounds such as oleuropein and hydroxytyrosol. Oleuropein is the ester of elenolic acid and 3,4‐dihydroxyphenyl ethanol (HT). It is the main glycoside in olives, the degradation of which results in the formation of hydroxytyrosol in olive oil. Both plays a significant role in the reduction of coronary heart diseases and a certain type of cancers. Both olive oil phenols have an effective role counter to cell proliferation, cell growth, migration, invasion, and angiogenesis. They down regulate the expression of BCL‐2 and COX‐2 proteins, and reduced DNA damage. Hydroxytyrosol and oleuropein inhibited the multiple stages in colon carcinogenesis; initiation, promotion, and metastasis. They also provide protection against various human cancers including colorectal, skin, breast, thyroid, digestive, lung, brain, blood, and cervical. This review article discusses the anticancer perspectives and mechanisms of oleuropein and hydroxytyrosol in cell cultures and animal and human studies.