Biochemical warfare on the reef : the role of glutathione transferases in consumer tolerance of dietary prostaglandins

© 2010 The Authors. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS ONE 5 (2010): e8537, doi:10.1371/journal.pone.0008537.Despite the profound variation among marine consumers in tolerance for allelochemically-rich foods, few studies have examined the biochemical adaptations underlying diet choice. Here we examine the role of glutathione S-transferases (GSTs) in the detoxification of dietary allelochemicals in the digestive gland of the predatory gastropod Cyphoma gibbosum, a generalist consumer of gorgonian corals. Controlled laboratory feeding experiments were used to investigate the influence of gorgonian diet on Cyphoma GST activity and isoform expression. Gorgonian extracts and semi-purified fractions were also screened to identify inhibitors and possible substrates of Cyphoma GSTs. In addition, we investigated the inhibitory properties of prostaglandins (PGs) structurally similar to antipredatory PGs found in high concentrations in the Caribbean gorgonian Plexaura homomalla. Cyphoma GST subunit composition was invariant and activity was constitutively high regardless of gorgonian diet. Bioassay-guided fractionation of gorgonian extracts revealed that moderately hydrophobic fractions from all eight gorgonian species examined contained putative GST substrates/inhibitors. LC-MS and NMR spectral analysis of the most inhibitory fraction from P. homomalla subsequently identified prostaglandin A2 (PGA2) as the dominant component. A similar screening of commercially available prostaglandins in series A, E, and F revealed that those prostaglandins most abundant in gorgonian tissues (e.g., PGA2) were also the most potent inhibitors. In vivo estimates of PGA2 concentration in digestive gland tissues calculated from snail grazing rates revealed that Cyphoma GSTs would be saturated with respect to PGA2 and operating at or near physiological capacity. The high, constitutive activity of Cyphoma GSTs is likely necessitated by the ubiquitous presence of GST substrates and/or inhibitors in this consumer's gorgonian diet. This generalist's GSTs may operate as ‘all-purpose’ detoxification enzymes, capable of conjugating or sequestering a broad range of lipophilic gorgonian compounds, thereby allowing this predator to exploit a range of chemically-defended prey, resulting in a competitive dietary advantage for this species.Financial support for this work was provided by the Ocean Life Institute Tropical Research Initiative Grant (WHOI) to KEW and MEH; the Robert H. Cole Endowed Ocean Ventures Fund (WHOI) to KEW; the National Undersea Research Center - Program Development Proposal (CMRC-03PRMN0103A) to KEW; Walter A. and Hope Noyes Smith, and a National Science Foundation Graduate Research Fellowship to KEW

Phytochemical study of products and by-products of the olive tree. An investigation on their biological role

Olea europaea leaves and olive mill waste waters (OMWW) were studied phytochemically, 20 natural products were isolated. The analytical courses and results were presented in the present PhD Thesis. A brief description of the results follows. Olive mill wate waters were subjected to elaboration with adsorption resins XAD16 and XAD7HP, resulting in the production of 2 fractions, one enriched with polyphenols and one containing the colorants of the olive tree, respectively. The methanol fraction that resulted from the elaboration with resins XAD16 of the olive mill waste waters was studied phytochemically. In total 11 secondary metabolites were isolated and identified, which can be categorized to 3 natural products categories. Specifically, they belong to iridoids, flavonoids and phenolic derivatives (phenolic acids, phenylethanols). From the isolated products, 2 are new natural products. These are the 2-[5- eth-(Ζ)-ylidene-2-oxo-tetrahydro-pyran-4-yl] acrylic acid methyl ester και τον 1-methyl-7-oxo-6,7,9,10-tetrahydro-1Η, 5Η-pyrano[3,4-c]pyran-4- carboxylic acid methyl ester The acetone extract of the leaves of Olea europaea family Oleacea was studied. In total 9 secondary metabolites were isolated and identified, which can be categorized to 4 natural products categories. Specifically, they belong to iridoids, flavonoids, triterpenoids and phenolic derivatives (phenolic acids, phenylethanols). From the isolated products 1 is encountered for the first time in Olea europaea. This was 8-epi-kingiside. Conclusively, a high technology procedure of exploitation of the olive mill waste water was developed, during which natural polyphenols are recovered and at the same time thousands tones of olive mill waste waters, which are a source of pollution, are removed from the environment. A procedure was developed for the isolation of oleuropein, hydroxytyrosol, tyrosol and lactone in large quantities Quantitation of oleuropein and related metabolites in decoctions of Olea europaea leaves from ten Greek cultivated varities by HPLC was performed The anti-ischemic, antioxidative and hypolipidemic effect of oleuropein in anesthetized rabbits was studied In vivo experiments were conducted in order to confirm if oleuropein prevents inflammation-induced bone loss in the ovarictomised rat and for the dose-response study of its effectΣτην παρούσα διατριβή μελετήθηκαν φυτοχημικά τα φύλλα της Olea europaea cv. koroneiki και τα υγρά απόβλητα ελαιοτριβείων, απομονώθηκαν συνολικά 20 φυσικά προϊόντα και παρουσιάστηκαν αναλυτικά οι πορείες και τα αποτελέσματα. Η επιλογή των φύλλων και των υγρών αποβλήτων έγινε διότι αποτελούν παραπροϊόντα της ελαιουργίας αλλά ταυτόχρονα και πλούσια πηγή πολυφαινολικών ενώσεων. Επιγραμματικά στην παρούσα εργασία: Τα υγρά απόβλητα ελαιοτριβείων (κατσίγαρος) υποβλήθηκαν σε κατεργασία με ρητίνες προσρόφησης XAD16 και XAD7ΗΡ, με αποτέλεσμα την παραγωγή 2 κλασμάτων, ενός που ήταν εμπλουτισμένο σε πολυφαινόλες και ενός που περιείχε τις χρωστικές της ελιάς αντίστοιχα. Το κλάσμα που προέκυψε από την επεξεργασία των υγρών αποβλήτων με ρητίνη προσρόφησης XAD16 εισήλθε στους χρωματογραφικούς διαχωρισμούς. Απομονώθηκαν και ταυτοποιήθηκαν συνολικά 11 δευτερογενείς μεταβολίτες που κατατάσσονται σε 3 κατηγορίες φυσικών προϊόντων. Συγκεκριμένα, ανήκουν στα ιριδοειδή, φλαβονοειδή και φαινολικά παράγωγα Από τα απομονωθέντα μόρια, 2 αποτελούν νέα φυσικά προϊόντα. Πρόκειται για τον μεθυλεστέρα του 2-[5-αιθ-(Ε)-υλιδενο-2-οξο- τετραϋδροπυραν-4-υλ] ακρυλικού οξέος και τον μεθυλεστέρα του 1- μέθυλο-7-οξο-6,7,9,10-τετραϋδρο-1Η, 5Η-πυρανο[3,4-c]πυρανο-4- καρβοξυλικού οξέος Μελετήθηκε φυτοχημικά το ακετονικό εκχύλισμα των φύλλων της Olea europaea cv. koroneiki. Απομονώθηκαν και ταυτοποιήθηκαν συνολικά 9 δευτερογενείς μεταβολίτες που κατατάσσονται σε 4 κατηγορίες φυσικών προϊόντων. Συγκεκριμένα, ανήκουν στα ιριδοειδή, φλαβονοειδή, τριτερπένια και φαινολικά παράγωγα (φαινολικά οξέα, φαινυλαιθανόλες). Από τα απομονωθέντα μόρια, 1 απαντάται για πρώτη φορά στην Olea europaea. Πρόκειται για τον 8-επι-κινγκισίδη. Αναπτύχθηκε συνολικά μία διαδικασία υψηλής τεχνολογίας, αξιοποίησης των υγρών αποβλήτων των ελαιοτριβείων, κατά την οποία ανακτώνται οι φυσικές πολυφαινόλες και παράλληλα απομακρύνονται από το περιβάλλον χιλιάδες τόνοι υγρών αποβλήτων Αναπτύχθηκε κατάλληλη μεθοδολογία για την απομόνωση ολευρωπεΐνης, υδροξυτυροσόλης, λακτόνης και τυροσόλης σε βιομηχανικά κλίμακα. Μελετήθηκε η περιέκτικότητα των αφεψημάτων που προέρχονταν από τα φύλλα 10 διαφορετικών Ελληνικών καλλιεργούμενων ποικιλιών ελιάς σε ολευρωπεΐνη, ελενολικό οξύ, τυροσόλη και 5-(αιθυλιδενο-2-οξο- τετραϋδρο-πυρανυλ-4-) οξικό οξύ (Λακτόνη) και παρατηρήθηκαν σημαντικές διαφοροποιήσεις ανάμεσά τους. Πραγματοποιήθηκαν πειράματα σε κουνέλια in vivo όπου η ολευρωπεΐνη εμφάνισε σημαντική αντι-ισχαιμική, αντιοξειδωτική και υπολιπιδαιμική δράση Πραγματοποιήθηκαν πειράματα σε αρουραίους in vivo στα οποία παρατηρήθηκε δράση της ολευρωπεΐνης κατά της οστεοπόρωσης, ενώ εξετάστηκε και η πιθανότητα η δράση αυτή να είναι δοσοεξαρτόμενη

Quantitation of oleuropein and related metabolites in decoctions of Olea europaea leaves from ten greek cultivated varieties by HPLC with diode array detection (HPLC-DAD)

An extraction procedure and chromatographic methodology for the simultaneous quantitation of four major constituents in the boiling water extracts (decoctions) of Olea europaea leaves has been developed. The four studied constituents were oleuropein, elenolic acid, hydroxytyrosol, and tyrosol. The quantitation was performed using HPLC-DAD, whereas qualitative data were acquired using LC-MS. The developed methodology was applied in the study of ten Olea europaea varieties commonly cultivated in Greece. The chromatographic analysis revealed important differences among the varieties. The decoction of variety gaidouroelia was identified as the best source of oleuropein, but it was completely lacking of elenolic acid. The decoction of variety koronaiiki was the best source of hydroxytyrosol, whereas the variety mastoides was the best source of tyrosol and elenolic acid. In addition, the methanol and acetone extracts of one of the studied varieties (koranaiiki) were investigated, in order to compare the concentration of oleuropein in the extracts and the decoction. Interestingly, only a very low percent of the total oleuropein is present in the traditionally prepared decoction, while elenolic acid, which is a minor constituent of the extracts, was found to be one of the major constituents of the decoction. Copyright © Taylor & Francis, Inc

DNA protecting and genotoxic effects of olive oil related components in cells exposed to hydrogen peroxide

In search for compounds, able to protect nuclear DNA in cells exposed to oxidative stress, extracts from olive leaves, olive fruits, olive oil and olive mill waste water were tested by using the "single cell gel electrophoresis" methodology (comet assay). Jurkat cells in culture were exposed to continuously generated hydrogen peroxide (11.8 ± 1.5 μM per min) by direct addition into the growth medium of the appropriate amount of the enzyme "glucose oxidase" in the presence or absence of the tested total extracts. The protective effects of the tested extracts or isolated compounds were evaluated from their ability to decrease hydrogen peroxide-induced formation of single strand breaks in the nuclear DNA, while the toxic effects were estimated from the increase of DNA damage when the extracts or isolated compounds were incubated directly with the cells. Significant protection was observed in extracts from olive oil and olive mill waste water. However, above a concentration of 100 μg/ml olive oil extracts exerted DNA damaging effects by themselves in the absence of any H2O2. Extracts from olive leaves and olive fruits although protective, were also able to induce DNA damage by themselves. Main compounds isolated from the above described total extracts, like oleuropein glucoside, tyrosol, hydroxytyrosol and caffeic acid, were tested in the same experimental system and found to exert cytotoxic (oleuropein glucoside), no effect (tyrosol) or protective effects (hydroxytyrosol and caffeic acid). In conclusion, cytoprotective as well as cytotoxic compounds with potential pharmaceutical properties were detected in extracts from olive oil related sources by using the comet assay methodology. © 2005 Taylor & Francis Group Ltd

New prostaglandins from the chemically defended soft coral Plexaura nina

Three new prostaglandins were isolated as minor constituents of the organic extract from the Caribbean soft coral Plexaura nina. The structures of the new natural products were established by means of spectral data analysis, including 2D NMR experiments. The unpalatability of the lipid extract of the coral and the defensive role of the major prostaglandin metabolites were determined by laboratory and field fish-feeding assays

A new process for the management of olive oil mill waste water and recovery of natural antioxidants

The high polyphenol content of the wastewater is the major environmental problem caused by the olive mills. A pilot scale system for the treatment of the olive oil mills wastewater was developed aiming at the recovery of high added value-contained polyphenols and the reduction of the environmental problems. The treatment system consists of three main successive sections: The first one includes successive filtration stages aiming at the gradual reduction of the wastewater suspended solids up to a limit of 25 μm. The second section includes passing of the filtered wastewater through a series of adsorbent resins (XAD16 and XAD7HP) in order to achieve the deodoring and decolorization of the wastewater and the removal/ recovery of the polyphenol and lactone content. The third section of the procedure includes the thermal evaporation and recovery of the organic solvents mixture, which has been used in the resin regeneration process, and finally the separation of the polyphenols and other organic substance contents using fast centrifuge partition chromatography. The final outcome of the whole procedure is (i) an odorless yellowish wastewater with a 99.99% reduced content in polyphenols and 98% reduced COD, (ii) an extract rich in polyphenols and lactones with high antioxidant activity and high added value, (iii) an extract containing the coloring substances of the olive fruit, and (iv) pure hydroxytyrosol. © 2007 American Chemical Society

Olive oil and its main phenolic micronutrient (oleuropein) prevent inflammation-induced bone loss in the ovariectomised rat

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Olive oil and its main phenolic micronutrient (oleuropein) prevent inflammation-induced bone loss in the ovariectomised rat

The present study was designed to evaluate the effect of olive oil and its main polyphenol (oleuropein) in ovariectomised rats with or without inflammation. Rats (6 months old) were ovariectomised or sham-operated as control. Ovariectomised rats were separated into three groups receiving different diets for 3 months: a control diet with 25g peanut oil and 25g rapeseed oil/kg (OVX), the control diet with 50g olive oil/kg or the control diet with 0.15 g oleuropein/kg. The sham-operated group was given the same control diet as OVX. Inflammation was induced 3 weeks before the end of the experiment by subcutaneous injections of talc (magnesium silicate) in one-half of each group. The sucess of ovariectomy was verified at necropsy by the atrophy of uterine horns. Inflammation, oleuropein or olive oil intakes did not have any uterotrophic activity, as they had had no effect on uterus weight. The plasma concentration of α-1-acid glycoprotein (an indicator of inflammation) was increased in OVX rats with inflammation. With regard to bone variables, osteopenia in OVX was exacerbated by inflammation, as shown by a decrease in metaphyseal and total femoral mineral density. Both oleuropein and olive oil prevented this bone loss in OVX rats with inflammation. At necropsy, oleuropein and olive oil consumption had had no effect on plasma osteocalcin concentrations (marker of bone formation) or on urinary deoxypyridinoline excretion (marker of bone resorption). In conclusion, oleuropein and olive-oil feeding can prevent inflammation-induced osteopenia in OVX rats. © The Authors 2004

The olive constituent oleuropein exhibits anti-ischemic, antioxidative, and hypolipidemic effects in anesthetized rabbits

The goal of this study was to evaluate the efficacy of the antioxidant olive constituent, oleuropein, on infarct size, oxidative damage, and the metabolic profile in rabbits subjected to ischemia. Oleuropein, 10 or 20 mg/(kg·d), was administered to 8 groups that consumed a normal or hypercholesterolemic diet for 6 wk or only the higher dose for 3 wk. Circulating levels of malondialdehyde, protein carbonyl, nitrite+nitrate, cholesterol, triglycerides, SOD activity, and the metabolic profile were measured using 1H NMR spectra. In rabbits that consumed the normal diet, the infarct size (percentage of infarct to risk areas) was reduced by the administration of 10 mg oleuropein/(kg·d) (16.1 ± 2.9%) or 20 mg oleuropein/(kg·d) for 3 wk (21.7 ± 2.2%) or for 6 wk (24.3 ± 1.3%) compared with the control group (48.05 ± 2.0%, P < 0.05). Only the higher dose of 20 mg/(kg·d) reduced the infarct size in hypercholesterolemic rabbits (34.7 ± 4.4% for 6 wk and 34.8 ± 6.1% for 3 wk) compared with the cholesterol-fed control group (52.8 ± 2.4%, P < 0.05). Oleuropein decreased the plasma lipid peroxidation product and protein carbonyl concentrations compared with the control groups, in which these factors increased relative to baseline due to ischemia and reperfusion. Furthermore, in rabbits administered oleuropein, RBC superoxide dismutase activity did not change during ischemia and reperfusion. This activity was significantly higher than in both control groups in which it was reduced by ischemia and reperfusion compared with baseline. Treatment for 6 wk with both doses of oleuropein reduced total cholesterol and triglyceride concentrations. 1H NMR spectra revealed a different profile of glycolysis metabolites in the oleuropein-treated groups compared with the controls. Oleuropein, for 3 or 6 wk, reduced the infarct size, conferred strong antioxidant protection and reduced the circulating lipids. This is the first experimental study in vivo that suggests the possibility of using an olive constituent in the treatment of ischemia. © 2006 American Society for Nutrition

Major phenolic compounds in olive oil modulate bone loss in an ovariectomy/inflammation experimental model

This study was conducted to determine whether the daily consumption for 84 days of tyrosol and hydroxytyrosol, the main olive oil phenolic compounds, and olive oil mill wastewater (OMWW), a byproduct of olive oil production, rich in micronutrients, may improve bone loss in ovariectomized rats (an experimental model of postmenopausal osteoporosis) and in ovariectomized rats with granulomatosis inflammation (a model set up for senile osteoporosis). As expected, an induced chronic inflammation provoked further bone loss at total, metaphyseal, and diaphyseal sites in ovariectomized rats. Tyrosol and hydroxytyrosol prevented this osteopenia by increasing bone formation (p < 0.05), probably because of their antioxidant properties. The two doses of OMWW extracts had the same protective effect on bone (p < 0.05), whereas OMWW did not reverse established osteopenia. In conclusion, polyphenol consumption seems to be an interesting way to prevent bone loss. © 2008 American Chemical Society