Isolation, characterization and structure-elicitor activity relationships of hibernalin and its two oxidized forms from Phytophthora hibernalis Carne 1925.

Three alpha-elicitins, named hibernalin1, hibernalin2 and hibernalin3 (hib1, hib2 and hib3, respectively), were isolated by reverse phase-low-pressure liquid chromatography from culture filtrates of Phytophthora hibernalis Carne 1925, the causal agent of citrus lemon brown rot. Hib1 proved to be identical to syringicin previously isolated from culture filtrates of Phytophthora syringae. Hib2 and hib3 shared the same primary structure with hib1, but contained, at position 50, Met sulphoxide or sulphone, respectively. By SDS-PAGE, the three proteins showed the same electrophoretic mobility, corresponding to about 10 kDa. Exact M(r) values were obtained by MALDI-TOF-MS (10,194.82 for hib1, 10,209.33 for hib2 and 10,223.80 for hib3), while by ESI-MS an M(r) value of 10,194.90 was found for hib1 and no results for hib2 and hib3. The hibernalin forms showed a high propensity to self-association, after exposure to acetonitrile. Hib1 showed to be active in both the hypersensitivity response and electrolytes leakage assays; the sample containing hib1 and hib2 was only weakly active in the first assay and inactive in the second assay, while the sample containing all three hibernalin forms proved to be inactive in both tests. It is proposed that the different activities of the three hibernalin samples could be very likely attributed to both Met50 oxidation and aggregation.

Production of triacetylhydroxytyrosol from olive mill waste waters for use as stabilized bioantioxidant.

A hydroxytyrosol triacetyl derivative was very efficiently produced as a highly pure stabilized antioxidant compound by a short treatment of olive mill waste water (OMWW) organic extracts, rich in hydroxytyrosol, with an acetylating mixture composed of HClO4-SiO2 and Ac2O (Chakborti and Gulhane reaction), in mild and safe conditions. A successive single step of middle pressure liquid chromatography (MPLC) purification of the reaction product was performed, with an overall yield of 35.6%. (This process, including both the Chakborti and Gulhane reaction and the MPLC purification, is protected by an international patent under PCT/IT2005/000781.) The o-diphenol triacetyl derivative was also produced by direct reaction of hydroxytyrosol, previously purified by MPLC, with HClO4-SiO2 and Ac2O, with an overall yield of 29.5%. A further procedure for the production of the hydroxytyrosol triacetyl derivative was consistent with the direct treatment of raw OMWW with the acetylating agent and a single step of MPLC purification, with an overall yield of 27.6%. The purified natural triacetylhydroxytyrosol confirmed the same strong protective effects against the oxidative stress in human cells as the corresponding synthetic compound, likely because of the biochemical activation of the acetyl derivative into the active parent hydroxytyrosol by esterases. We therefore propose the utilization of OMWW for recovering hydroxytyrosol as a natural antioxidant in a chemically stabilized form, with a good yield, which can be potentially used as a nontoxic functional component in nutritional, pharmaceutical, and cosmetic preparations.

Protective effects of synthetic hydroxytyrosol acetyl derivatives against oxidative stress in human cells.

Chemically stable di- and triacetyl derivatives of the natural o-diphenol antioxidant hydroxytyrosol were synthesized, and their chemical and biological antioxidant activities were assessed in comparison with that of the native synthetic compound. The chemical antioxidant activity of the selected compounds was evaluated by measuring the ferric reducing antioxidant power (FRAP). The data clearly indicate that, as expected, the hydroxytyrosol analogues, modified in the o-diphenolic ring, are devoid of any chemical antioxidant activity. On the contrary, both acetyl derivatives, at micromolar concentrations, equally protect against tert-butylhydroperoxide-induced oxidative damages in Caco-2 cells and human erythrocytes. This paper for the first time reports that chemically stable hydroxytyrosol acetyl derivatives, although devoid of chemical antioxidant activity, are as effective as the parent compound in protecting human cells from oxidative stress-induced cytotoxicity, after metabolization by esterases at the intestinal level, suggesting their possible utilization in either nutritional (functional food), cosmetic, or pharmaceutical preparations.

Recovery and characterization of the metal polymeric organic fraction (polymerin) from olive oil mill wastewaters.

A dark and complex metal polymeric organic mixture, named polymerin, was recovered from olive oil mill wastewaters (OMWW) and characterized by chemical analysis, diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), and atomic absorption spectrometry (AAS). Polymerin proved to be composed of carbohydrates (52.40 mg 100(-1), w/w), melanin (26.14 mg 100(-1)), and proteins (10.40 mg 100(-1)), and the respective composition of monosaccharides, phenols, and amino acids was determined. It also contained metals (11.06 mg 100(-1)), mainly K(+) and, to a lesser extent, Na(+), Ca(2+), Mg(2+), Zn(2+), Fe(3+), and Cu(2+), which were naturally bound and chelated through carboxylate anions and other characteristic nucleophilic functional groups naturally occurring in polymerin. The distribution of polymerin relative molecular size was assessed to be approximately between 500.0 and 2.0 kDa by calibrated molecular weight gel filtration chromatography, indicating also that a fraction consisted of protein, melanin, and polysaccharide, strongly aggregated to each other in a supramolecular status by a combination of covalent and hydrogen bonds and CH/Pi interactions, and another fraction of only free polysaccharide. Polymerin was transformed into a potassium salt deglycosylated derivative, named KSDpolymerin, which was also characterized by chemical analysis, DRIFTS, and AAS. KSDpolymerin consisted of carbohydrates (6.00 mg 100(-1)), melanin (52.49 mg 100(-1)), and proteins (35.40 mg 100(-1)), and the composition of monosaccharides, phenols, and amino acids was determined. It also contained metals (6.11 mg 100(-1)), mainly K(+) and to a lesser extent Na(+), Ca(2+), Mg(2+), Zn(2+) and Fe(3+), bound as in polymerin. All the organic components were strongly linked in a supramolecular aggregate status and the relative average molecular size proved to be 6.3 kDa. Finally, we briefly discuss the possible use of such polymerins in agriculture as bioamendments and macro- and microelement biointegrators and as a biofilter for toxic metal removal, in light of their similarity with humic acids.

Production, characterization, and effects on tomato of humic acid-like polymerin metal derivatives from olive oil mill waste waters.

The dark polymeric organic fraction rich in potassium recovered from olive oil mill waste waters (OMWW) and named polymerin and the potassium salified deglycosylated polymerin derivative (K-SDpolymerin) were easily transformed into their metal derivatives by saturation with various metals, including Na, Cu, Zn, Mn, Fe, and Al. Saturated metal polymerins were characterized by diffuse reflectance infrared Fourier transform spectroscopy and atomic absorption spectrometry. Tests on tomato plants of the various polymerins showed that only the soluble polymerin, K-SDpolymerin, and the insoluble Mn-SDpolymerin were significantly toxic. The toxic effects of OMWW on tomato at the original concentration and diluted 1:10 were much stronger than those of any polymerin. The possible exploitation of polymerins as bioamendments and/or metal biointegrators as a functon of their phytotoxic effects, their humic acid-like nature, and their richness in macro- and micronutrient metals is also discussed.

Sustainable sorption strategies for removing Cr(3+) from tannery process wastewater.

Two different strategies for the removal of trivalent chromium (Cr(3+)) from contaminated water are reported. The first one is based on the sorption process on an organo-mineral complex, named LDH-HP, obtained in turn by sorption of polymerin, the humic acid-like fraction occurring in olive oil mill wastewater, on a layered double hydroxide (LDH) of magnesium and aluminium with carbonate in the interlayer. This sorption process is preliminary developed on simulated wastewater (SW) as theoretical model and successively applied on tannery process wastewaters (TPWs) natively containing Cr(3+). The removal of Cr(3+) from TPW is lower than that observed for SW, because of the large compositional variability of TPW. The second one is based on the direct production of a LDH of magnesium and chromium (Cr(3+)), using as starting material TPW, naming LDH-TPW the complex produced. This process allows the complete removal of Cr(3+) from TPW and also the abatement of chemical oxygen demand, indicating to be a very promising purification process for an industrial application.

Polymerin and lignimerin, as humic acid-like sorbents from vegetable waste, for the potential remediation of waters contaminated with heavy metals, herbicides, or polycyclic aromatic hydrocarbons.

Polymerin is a humic acid-like polymer, which we previously recovered for the first time from olive oil mill waste waters (OMWW) only, and chemically and physicochemically characterized. We also previously investigated its versatile sorption capacity for toxic inorganic and organic compounds. Therefore, a review is presented on the removal, from simulated polluted waters, of cationic heavy metals [Cu(II), Zn, Cr(III)] and anionic ones [Cr(VI)) and As(V)] by sorption on this natural organic sorbent in comparison with its synthetic derivatives, K-polymerin, a ferrihydrite-polymerin complex and with ferrihydrite. An overview is also performed of the removal of ionic herbicides (2,4-D, paraquat, MCPA, simazine, and cyhalofop) by sorption on polymerin, ferrihydrite, and their complex and of the removal of phenanthrene, as a representative of polycyclic aromatic hydrocarbons, by sorption on this sorbent and its complexes with micro- or nanoparticles of aluminum oxide, pointing out the employment of all these sorbents in biobed systems, which might allow the remediation of water and protection of surface and groundwater. In addition, a short review is also given on the removal of Cu(II) and Zn from simulated contaminated waters, by sorption on the humic acid-like organic fraction, named lignimerin, which we previously isolated for the first time, in collaboration with a Chilean group, from cellulose mill Kraft waste waters (KCMWW) only. More specifically, the production methods and the characterization of the two natural sorbents (polymerin and lignimerin) and their derivatives (K-polymerin ferrihydrite-polymerin, polymerin-microAl(2)O(3) and -nanoAl(2)O(3), and H-lignimerin, respectively) as well as their sorption data and mechanism are reviewed. Published and original results obtained by the cyclic sorption on all of the considered sorbents for the removal of the above-mentioned toxic compounds from simulated waste waters are also reported. Moreover, sorption capacity and mechanism of the considered compounds on polymerins and lignimerins are evaluated in comparison with other known natural sorbents, especially of humic acid nature and other organic matter. Some of their technical aspects and relative costs are also considered. Finally, the possible large-scale application of the considered sorption systems for water remediation is briefly discussed.

Synthesis, characterization, and sorption capacity of layered double hydroxides and their complexes with polymerin.

Mg-Al layered double hydroxides (LDHs) containing chloride (LDH-Cl) or carbonate (LDH-CO(3)) in the interlayer were obtained at room temperature and after calcitation at 450 degrees C (LDH-Cl-450 and LDH-CO(3)-450) and were characterized by X-ray diffraction and Fourier transform infrared analyses. Sorption isotherms of a humic acidlike fraction naturally occurring in olive oil mill waste waters, named polymerin, on these LDH minerals were carried out. Because LDH-CO(3) showed the highest capacity to sorb polymerin among the four LDH minerals synthesized, two organo-mineral complexes named LDH-CO(3)-LP (low polymerin) and LDH-CO(3)-HP (high polymerin) were prepared by coprecipitation (LDH-CO(3)-LP) and sorption onto a preformed LDH-CO(3) mineral (LDH-CO(3)-HP). These complexes were characterized chemically and physicochemically, and their stability to pH and after thermal treatment at 80 degrees C were evaluated. The diffuse reflectance infrared Fourier transform and X-ray analysis of the complexes indicated that polymerin was sorbed only on the external surfaces of LDH-CO(3) and no intercalation occurred. The LDH-polymerin complexes appeared to be more stable than LDH-CO(3) at pH 4.0 and showed that they were able to sorb both As(V) and Zn. Because the waste waters are usually contaminated with mixtures of pollutants in cationic and anionic forms, the LDH-polymerin complexes appear more suitable than the sorbents in a potential water remediation process.

Comparative study on the sorption capacity of cyhalofop Acid on polymerin, ferrihydrite, and on a ferrihydrite-polymerin complex.

A comparative study was performed on the sorption capacity of the phenoxy acid herbicide cyhalofop on polymerin (from olive oil mill effluents), ferrihydrite, and a ferrihydrite-polymerin complex, by using a batch equilibrium method. The most efficient sorbent showed to be ferrihydrite followed by the ferrihydrite-polymerin complex and polymerin. Cyhalofop acid bound to ferrihydrite by a combination of ionic and ion-dipole bonding, whereas the same herbicide bound to the ferrihydrite-polymerin complex by ionic bonding and to polymerin by hydrogen bonding. Simulated wastewaters contaminated with cyhalofop acid were completely purified by two sorption cycles on ferrihydrite and five cycles on the ferrihydrite-polymerin complex, whereas the same wastewaters maintained a constant residue even after five sorption cycles on polymerin. For the first time, the possible use of a mineral (ferrihydrite) and an organo-mineral complex (ferrihydrite-polymerin) as a filter for the control of the herbicide contamination in point sources is proposed and briefly discussed.

Sorption of phenanthrene by dissolved organic matter and its complex with aluminum oxide nanoparticles.

Intent of this study was to explore the potential application of polymerin, the polymeric, dissolved organic matter fraction from olive oil wastewaters, in technologies aimed at remediating hydrophobic organic compounds (HOCs) point-source pollution. Phenanthrene binding with polymerin was investigated. Moreover, the effect of addition of micro and nanoscale aluminum oxides (Al2O3) was studied, as well as sorption of polymerin on the oxides. Phenanthrene binding capacity by polymerin was notably higher than the sorption capacities for both types of Al2O3 particles. Polymerin sorption on nanoparticles was nearly 100 times higher than microparticles. In a three-phase system, using microparticles, higher phenanthrene sorption was found by adding into water polymerin, oxides and phenanthrene simultaneously. In contrast, using nanoparticles, a considerable enhancement of phenanthrene sorption was shown by adding phenanthrene to a pre-formed and dried polymerin-oxide complex. These findings support the application of polymerin, especially associated with Al2O3 nanoparticles, in remediation of water contaminated with HOCs. This work highlights the significant role of nanoparticles.

Potential remediation of waters contaminated with Cr(III), Cu, and Zn by sorption on the organic polymeric fraction of olive mill wastewater (polymerin) and its derivatives.

A study on the individual sorption of Cr(III), Cu, and Zn on polymerin, the humic-acid-like fraction of olive mill wastewater, and its derivatives, K-polymerin and an Fe(OH)x-polymerin complex, showed that these heavy metals were strongly sorbed on polymerin and K-polymerin in the order Cr(III) > Cu > Zn. The sorption on Fe(OH)x-polymerin was to a lower extent compared with that of the other two sorbents, but to a higher extent compared with ferrihydride [Fe(OH)x]. Combined atomic absorption spectrometry and diffuse reflectance infrared Fourier transform spectroscopy analyses showed that the selected heavy metals were individually sorbed on polymerin by means of a cation exchange mechanism, which was consistent with the replacement of Ca, Mg, K, and H bound to the carboxylate groups of the biosorbent and the concomitant chelation of the heavy metals by the OH groups of polymerin polysaccharide component. In binary combination and equimolar ratio, Cu was sorbed by polymerin more selectively than Zn. In ternary combination and equimolar ratio, Cr(III), Cu, and Zn were sorbed by polymerin in the order Cr(III) > Cu > Zn. The sorbing capacity of Zn and Cu was strongly influenced by Cr(III), whereas the sorbing capacity of Cr(III) was not affected bythe presence of the other two metals. The overall sorbing capacity of the binary and ternary mixtures of the three metals on polymerin proved to be considerable and much greater than that on Fe(OH)x-polymerin. Simulated wastewaters contaminated with Cu and Zn were purified after three sorption cycles by polymerin renewed at each cycle, whereas those containing a mixture of Cr(III), Cu, and Zn showed residues of Zn after five cycles. We briefly discuss environmental and industrial advantages for a possible exploitation of polymerin.

Analysis of carbohydrates and amino acids in vegetable waste waters by ion chromatography.

High-performance anion exchange chromatography coupled with pulsed amperometric detection was used for the quantitative determination of total and free sugars in olive oil mill waste waters (OMWW). Automated amino acid ion chromatography was employed to analyse total and free amino acids in the same OMWW. Sugars were analysed in samples pre-purified by means of a three-step purification procedure involving: (i) methanol precipitation of OMWW; (ii) dialysis of the obtained solid and liquid fractions; and (iii) chromatographic purification on RP18 phase followed by Amberlite resin. The amino acids were determined directly in samples obtained from the first two steps performed for sugar analysis. The analysis carried out with the reported methodologies allowed the quantitative determination of total sugars and amino acids and the differentiation between their free and bound forms. The sugars determined were arabinose, fructose, galactose, glucose, rhamnose, xylose, galacturonic and glucuronic acids, and the amino acids were Asp, Glu, Thr, Ser, Pro, Gly, Ala, Val, Met, Ile, Leu, Tyr, Phe, Lys, His, Arg and Cys. Asn, Gin, and Trp were not detected. The technological, biotechnological and environmental advantages arising from this analytical methodology applied to OMWW are briefly discussed.