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.     

The response of phenylalanine ammonia‐lyase,polyphenol oxidase and phenols to cold stress in the olive tree (Olea europaea L. cv. Picual)

BACKGROUND: In this study the activities of phenylalanine ammonia‐lyase (PAL) and polyphenol oxidase (PPO) and the concentrations of hydroxytyrosol, tyrosol and oleuropein in olive tree (Olea europaea L. cv. Picual) leaves were investigated before and after cold stress by freezing. The air temperature fell to below ?7 °C and, according to the specific field conditions, four categories of orchard were selected: not cold stressed (NS), lightly cold stressed (LS), moderately cold stressed (MS) and heavily cold stressed (HS). RESULTS: In LS and MS samples the PAL specific activity at saturated substrate concentration rose 4.8‐ and 1.9‐fold respectively compared with NS samples. In HS samples the PAL activity declined by 47% compared with NS samples. A low level of PAL protein was detected in all samples affected by cold stress. In LS, MS and HS samples the PPO specific activity at saturated substrate concentration was 1.9‐, 4.4‐ and 9.8‐fold higher respectively than in NS samples. K_m values also increased after cold stress. In MS and HS samples the concentration of oleuropein was 69 and 82% higher respectively than in NS samples, while the concentrations of hydroxytyrosol and tyrosol decreased. CONCLUSION: The response of PAL activity may be part of a recovery process of the olive leaf against cold stress, while the response of PPO and oleuropein may be part of an antioxidant protection mechanism. Copyright © 2009 Society of Chemical Industry     

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.     

Characterisation of phenolic extracts from olive pulp and olive pomace by electrospray mass spectrometry

Methanol extracts of olive pomace (two‐phase olive oil extraction) and olive pulp were analysed by reverse phase HPLC and the eluted fractions were characterised by electrospray ionisation mass spectrometry. This technique allowed the identification of some common phenolic compounds, namely, verbascoside, rutin, caffeoyl‐quinic acid, luteolin‐4‐glucoside and 11‐methyl‐oleoside. Hydroxytyrosol‐1′‐β‐glucoside, luteolin‐7‐rutinoside and oleoside were also detected. Moreover, this technique enabled the identification, for the first time in Olea europaea tissues, of two oleoside derivatives, 6′‐β‐glucopyranosyl‐oleoside and 6′‐β‐rhamnopyranosyl‐oleoside, and of 10‐hydroxy‐oleuropein. Also, an oleuropein glucoside that had previously been identified in olive leaves was now detected in olive fruit, both in olive pulp and olive pomace. With the exception of oleoside and oleuropein, the majority of phenolic compounds were found to occur in equivalent amounts in olive pulp and olive pomace. Oleoside was the main phenolic compound in olive pulp (31.6 mg g^?1) but was reduced to 3.6 mg g^?1 in olive pomace, and oleuropein (2.7 mg g^?1 in the pulp) almost disappeared (<0.1 mg g^?1 in the pomace). Both these phenolic compounds were degraded during the olive oil extraction process. Copyright © 2004 Society of Chemical Industry     

Oil content,phenolic profiling and antioxidant potential of Tunisian olive drupes

BACKGROUND: The aim of this work was to investigate the effect of the maturation process of the olive fruit on oil content, phenolic profile and antioxidant activity of four Tunisian cultivars (Zelmati, Chemchali, Chemlali and Chétoui). RESULTS: The average oil content of the studied varieties ranged between 17.50% and 20.25% at the first stage of maturation and from 30.20% to 35% in the last harvest. Qualitative and quantitative analysis of phenolic compounds were carried out using HPLC and LC‐MS/MS. Twenty‐six biophenolic compounds were identified. In all samples, hydroxytyrosol and oleuropein were the major compounds identified while rutin and luteolin 7‐O‐glucoside were the two main flavonoids. The total phenolic content varied from 3.46 to 4.30 g kg^?1 at the first stage of maturation and from 8.71 to 11.52 g kg^?1 of fruit fresh weight at the last maturation phase. Total flavonoid content reached 432.80 mg kg^?1. The antioxidant activity of the extract was evaluated by DPPH and ABTS assays. The IC₅₀ values of the olive extracts ranged from 2.69 to 10.96 µg L^?1 and from 2.15 to 3.03 mmol L^?1 trolox equivalent at the last stage of maturation. CONCLUSION: A relationship between the changes in phenolic content and the physicochemical changes in Tunisian olive fruit during maturation was established. These findings could be used for controlling the production processes and correlating the oil sensorial characteristics to the polyphenolic pattern. Copyright © 2010 Society of Chemical Industry     

Phenylalanine ammonia‐lyase and phenolic compounds in leaves and fruits of Olea europaea L. cv. Picual during ripening

BACKGROUND: The kinetic and molecular properties of phenylalanine ammonia‐lyase (PAL) in leaves and fruit of the olive tree (Picual variety) have been studied during the seasonal process of fruit maturation. The concentrations of total phenolic compounds, oleuropein, hydroxytyrosol and tyrosol, have also been determined. This study has been made in rainfed 30‐year‐old olive trees in Jaén, Spain, cultivated by the traditional method. RESULTS: PAL specific activity was assayed and hyperbolic kinetics were observed in both organs. The K_m value for L ‐Phe was 0.22 mmol L^?1 in leaf and 0.26 mmol L^?1 in fruit. In leaf, the highest PAL specific activity was found in the stage prior to veraison. By immunoblot, a PAL‐immunoreactive 75 kDa polypeptide was detected in leaf and fruit. In leaf, the level of this protein progressively rose until the last stages of ripening at the same time that total phenols increased. In fruit, PAL activity and protein change as in two series coinciding with different fruit‐maturation period. By immunohistochemistry under light microscopy, PAL was located in the epidermis and parenchyma cells of leaf and fruit. CONCLUSION: These results demonstrate the involvement and regulation of PAL during fruit ripening of olive, cv. Picual. Copyright © 2008 Society of Chemical Industry     

New genotypes of table olives: profile of bioactive compounds

New table olive genotypes (48) coming from a cross‐breeding programme were evaluated. Most of the fruit traits covered a wide range of variability on the set of genotypes, fruit weight (1.1–9.7 g), pulp‐to‐pit ratio (1.7–10.0), fruit shape (1.0–1.6) and oil content (1.3–15.2%). This is the first time that healthy compounds such as triterpenic acids and phenolic compounds have also been evaluated in olive progenies. Genotypes were stored for 2 months in sterilised brine (5% NaCl and 0.5% acetic acid). A high amount of maslinic (685.0–1394.2 mg kg^?1 olive flesh) and oleanolic acids (275.3–817.9 mg kg^?1 olive flesh) was found in the flesh of olives stored. The main oleosidic and phenolic compounds evaluated in brines were hydroxytyrosol (1.9–8.4 mmol L^?1), hydroxytyrosol glucosides (0.4–19.8 mmol L^?1), oleuropein (0.0–4.7 mmol L^?1) and the antimicrobial compounds, dialdehydic form of decarboxymethyl elenolic acid linked to hydroxytyrosol (0.0–3.4 mmol L^?1) and decarboxymethyl elenolic acid (0.0–1.7 mmol L^?1), the latter two being observed in only ten genotypes. The wide range of variation observed for most compounds indicates that the contents of these healthy compounds may be used as selection criteria in table olive breeding programmes.     

Influence of Eriophyid mites (Aculus olearius Castagnoli and Aceria oleae (Nalepa) (Acarina: Eriophyidae)) on some physical and chemical characteristics of Ayvalık variety olive fruit

BACKGROUND: Aculus olearius Castagnoli is a recently recorded species that damages olive fruits in the Mediterranean basin of Turkey. Thus, the effects of Eriophyid mites (Aculus olearius Castagnoli and Aceria oleae (Nalepa) (Acarina: Eriophyidae) on the olive fruits from Ayval?k variety in southern Turkey were studied for the first time in terms of some physical parameters and chemical constituents including some individual phenolics. RESULTS: The Eriophyid damaged fruits had higher L* values (lighter colour) and tyrosol level (37.53 mg kg^?1) than the undamaged fruits (28.51 mg kg^?1) in August. In contrast, Eriophyid damaged fruits were darker in colour and had lower levels (25.77 mg kg^?1) of tyrosol than those of undamaged fruits (79.14 mg kg^?1) in October. Eriophyid damaged samples had higher values of vanillic acid than the undamaged samples. An increase in the average concentrations of hydroxytyrosol and p‐coumaric acid was observed in the fruits harvested in August, whilst the oleuropein content decreased. CONCLUSION: The harvest in October can be recommended regarding the higher dimensional values, total oil, dry matter and oleuropein contents. But the interaction between harvest time and Eriophyid damage was found effective in terms of tyrosol content and skin colour; as tyrosol values were lower in the fruits harvested in October and the fruits were darker. The resistance of undamaged fruits against Eriophyid damage can be linked to high tyrosol content of these fruits. Copyright © 2010 Society of Chemical Industry     

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     

Quality attributes and their relations in fresh black ripe ‘Kalamon’ olives (Olea europaea L.) for table use – phenolic compounds and total antioxidant capacity

Quality attributes were investigated in fresh Greek black table ‘Kalamon’ olives prior to processing. Fruit weight, dimensions, respiration and ethylene production rates, firmness, peel colour, moisture, oil content, total antioxidant capacity (TAC), the concentration of total phenolics (TP) and phenolic compounds (hydroxytyrosol, oleuropein, tyrosol, verbascoside, luteolin‐7‐O‐glucoside, luteolin, rutin) were determined in olives from different orchards. There was a significant effect of orchard on most attributes, but not on fruit firmness. Verbascoside, oleuropein and hydroxytyrosol were the major phenolics, and the presence of verbascoside in ‘Kalamon’ olives is revealed for the first time. Positive correlations were found among fruit weight, dimensions, respiration and ethylene. TAC was positively related mainly to TP, hydroxytyrosol, verbascoside and rutin, but inversely to oleuropein. Luteolin was inversely related to luteolin‐7‐O‐glucoside. Colour darkening was directly related to TAC, while colour parameters were positively and moderately affected by oil and moisture.     

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.     

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.     

Syringa oblata Lindl var. alba as a source of oleuropein and related compounds

The leaf methanol extract of Syringa oblata Lindl var. alba was investigated as a source of oleuropein and related compounds. The extract had a high total phenol content and a radical scavenging activity similar to that of the respective extract from Olea europaea leaves. HPLC‐DAD characterisation of the two most abundant phenolic compounds in the extract of S. oblata indicated that both had UV spectra matching that of oleuropein. The presence of oleuropein was verified by using LC‐MS. Identification of the second compound was only feasible after isolation (preparative HPLC) and spectroscopic characterisation [LC‐MS, ¹H NMR and homonuclear two‐dimensional correlated spectroscopy (COSY)]. The compound identified was the known bioactive syringopicroside. On the basis of MS data other peaks were assigned to oleuropein aglycone, verbascoside, ligstroside and syringopicroside derivatives, as well as to a luteolin rutinoside. The findings are promising for the potential exploitation of S. oblata leaf extract as a source for oleuropein and other bioactive ingredients. Copyright © 2006 Society of Chemical Industry     

Valorisation of olive mill wastewater by enhancement of natural hydroxytyrosol recovery

Recovery of natural and simple phenolic compounds with high added value was performed successfully from olive mill wastewater. Three types of olive mill wastewater were assayed in bioconversion at 15 and 40 L in mechanically stirred systems for 2 h at 50 °C. Maximum hydroxytyrosol concentrations of about 1.53, 0.83 and 0.46 g L^?1 were obtained in the presence of 5 IU Aspergillus niger β‐glucosidase per milliliter of OMW in North OMW, MSP and MCC, which were 2.70‐, 1.38‐ and 1.77‐fold higher than the controls, respectively. Enzymatic pretreatment was followed by two tangential flow membrane separation steps, microfiltration (MF) and ultrafiltration (UF), and concentration procedures for the ultrafiltration permeate. The latter exhibited a COD level of 48.44 g L^?1. The UF permeate concentration increased the hydroxytyrosol concentration to 7.2 g L^?1. Hence, this innovative work described an environmently friendly process; pilot‐scale has allowed producing a new and natural product which obeyed the dietary requirements; it contains some minerals beneficial to health and does not contain heavy metals or chemicals. Overall, this chemical‐free large‐scale process proved efficient for recovery of natural hydroxytyrosol.     

Enrichment of table olives with polyphenols extracted from olive leaves

The possibility to increase the nutritional value of table olives using polyphenols extracted from olive leaves was studied. Leaves were subjected to extraction using water with proportions of 1%, 5% and 10% of leaves and various temperatures and times (room temperature/24 h, 40 °C/10 min and 70 °C/5 min). The antioxidant activity of extracts was determined using the Rancimat method and their content in oleuropein and hydroxytyrosol was determined by HPLC. The extract with the highest antioxidant activity and polyphenol content (10% ratio of olive leaves extracted at room temperature for 24 h) was used for the treatment of debittered table olives. The treated and untreated olives were subjected to determination of their α-tocopherol, oleuropein and hydroxytyrosol content. A 457% increase was observed for oleuropein and 109% for hydroxytyrosol content after treatment. Sensory evaluation of treated table olives showed an increase in bitterness. However, treated and untreated table olives showed equal overall acceptability and overall preference.     

Oleuropein/ligstroside isomers and their derivatives in Portuguese olive mill wastewaters

Olive mill wastewaters (OMW) are a potential source of biophenols, but they have a complex composition with many unknown phenolics. The analysis of purified methanol extracts from two Portuguese OMW by electrospray mass spectrometry in the negative mode showed [M−H]⁻ ions at m/z 539 and m/z 523, corresponding respectively to oleuropein and ligstroside isomers which contain the glucose unit linked to its aromatic moiety. Also, the fragmentation pathway of the [M−H]⁻ ions at m/z 863, 685 and 847 indicated the presence of a diglucoside derivative of the oleuropein isomer and of mono- and diglucosides of the ligstroside isomer, respectively. Moreover, the two OMW samples contained an elenoic derivative of the ion at m/z 685 and a degradation product (m/z 453) of the [M−H]⁻ ion at m/z 523. Future studies focusing on the abundance of these compounds on OMW, as well as their bioactivities, will determine their possible industrial exploitation.     

Browning reactions in olives: Mechanism and polyphenols involved

The mechanism of the browning reaction in olives has been disclosed by following the polyphenol changes in healthy and bruised fruits and using “in vitro” models. It was later validated in independent experiments with active and denatured enzymatic extracts, treated or not with ascorbic acid to prevent oxidation. The proposed mechanism would consist of two steps. First, there is an enzymatic release of hydroxytyrosol, due to the action of the fruits’ β-glucosidases and esterases on oleuropein and hydroxytyrosol glucoside; additional hydroxytyrosol can also be produced (in a markedly lower proportion) by the chemical hydrolysis of oleuropein. In a second phase, hydroxytyrosol and verbascoside are oxidized by the fruits’ polyphenoloxidase (mainly) and by a chemical reaction, which occurs to a limited extent due to the olive flesh pH, ≈5.0.     

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.     

Use of a lactic acid bacteria starter culture during green olive (Olea europaea L cv Ascolana tenera) processing

Among the Italian olive germplasm, ‘Ascolana tenera’ is one of the best varieties for table olive production. This research addressed the impact of different processing types (Greek‐style and Spanish‐style) on the fermentation and phenolic composition of olive fruit. In particular, the effects of a lactic acid bacteria (LAB) starter culture on the fermentation of naturally green olives processed according to the traditional Greek method were studied. Results revealed that Spanish‐style processing produced a dramatic loss of total phenolics, while natural olive processing favoured a higher retention of biophenols. Oleoside 11‐methylester, a phenol‐related compound, and hydroxytyrosol, tyrosol, vanillic acid, 3,4‐dihydroxyphenylglycol, oleuropein and oleuropein aglycons, as the main phenols, were detected in olive fruit. More interestingly, this research indicated that inoculation with LAB affected the pH, total acidity, microbial profile and palatability of olives. Olives fermented with the LAB starter culture were perceived by panellists to be less bitter and more aromatic than those spontaneously fermented. Thus the use of LAB inoculants during olive fermentation could be applied with the currently available technology. Copyright © 2005 Society of Chemical Industry