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The presence of pharmaceutical compounds (PhCs) in the effluents of wastewater treatment plants (WWTPs) is an ecological concern. The issue could be alleviated by trapping those substances by cyclodextrin (CD) polymers or photolyzing them by pulsed light (PL). Consequently, a sequential CD polymer/PL system was tested for the removal of PhCs. Firstly, a survey detected the presence of recurrent PhCs in the effluents of local WWTPs. Then, pure water was spiked with 21 PhCs, 100 µg/L each one. The three-dimensional network provides amphiphilic features to the CD polymer that reduced the pollutant concentration by 77 %. Sorption involves a plead of physical and chemical mechanisms hindering the establishment of a general removal model for all compounds. The performed simulations hint that the retention capacity mainly correlates with the computed binding energies, so that theoretical models are revealed as valuable tools for further improvements. The complementary action of PL rose the elimination to 91 %. The polymer can be reused at least 10 times for ibuprofen (model compound) removal, and was able to eliminate the ecotoxicity of an ibuprofen solution. Therefore, this novel sequential CD polymer/PL process seems to be an efficient alternative to eliminate PhCs from wastewater.

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The present study evaluates the removal capacity of microalgae photobioreactors of environmental pollutants present in wastewater from the dry riverbed El Albujón, as a way to minimize the eutrophication process of the Mar Menor. Particularly, the capacity of four autochthonous microalgae consortia collected from different locations of the salty lagoon to remove emerging contaminants (simazine, atrazine, terbuthylazine, adenosine and ibuprofen), nitrates, and phosphates, was evaluated. Among the four microalgae consortia, consortium 1 was the best in terms of biomass productivity (0.11 g L-1 d-1) and specific growth rate (0.14 d-1), providing 100% removal of emerging contaminants (simazine, atrazine, terbuthylazine, adenosine and ibuprofen), and a maximal reduction and consumption of macronutrients, especially nitrates and phosphates, reaching levels below 28 mg L-1, that is, a decrease of 89.90 and 99.70% of nitrates and phosphates, respectively. Therefore, this consortium (Monoraphidium sp., Desmodesmus subspicatus, Nannochloris sp.) could be selected as a green filter for successful large-scale applications. This study is the first one that combines the successful removal of herbicides, ibuprofen and adenosine as emerging contaminants, and nitrate removal.

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Total phenolics (TP), vitamin C, antioxidant activity and colour of preserved peppers were evaluated at 4, 25 and 50 ℃ storage during 30-day intervals. Except for 4 ℃, TP decreased during storage at 25 ℃ and 50 ℃, being softer for fortified samples with ß-CDs. The protective effect was evident, since 50 ℃ samples containing ß-CDs exhibited lower TP loss (19%) than control samples (38%) for 5 months storage. A decrease in the vitamin C content was observed for both samples as time and temperature progressed. In samples stored at 50 ℃ the protective effect of ß-CD only was evident at the first month, since fortified samples showed lower vitamin C loss (10%) than control samples. The fortified samples with ß-CDs exhibited lowest antioxidant activity loss (40%) during 90-day storage at 50 ℃, than control samples (64%). The colour changes were in line with those observed for total phenolics and at the end of study, the presence of 1% ß-CDs delayed the darkening of samples at both (25 and 50 ℃) storage conditions.

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The inclusion complex of sulphathiazole in ß-cyclodextrin has been investigated. A 1:2 stoichiometry of the complex was established and formation constants K2 (42.83 ± 3.27 M(-1)) and K1 (4.98 ± 0.36 M(-1)) were calculated by using the changes produced on the native fluorescence of the drug, when included on the hydrophobic cyclodextrin cavity. An enhancement in the fluorescence emission of sulphathiazole and protection of the drug against photochemical reactions has been attained upon inclusion. In solutions of ß-CD dual emission (458 nm) was noticed in STZ. Formation of the inclusion complex of STZ should result in dual emission, which is due to a twisted intramolecular charge transfer band (TICT). A fluorimetric method for the determination of sulphathiazole has been proposed and applied in honey without sample treatment. The optimized fluorimetric method showed detection and quantitation limits of 9.74 ng/g and 32.48 ng/g, respectively. Selectivity is high, showing no cross-reactivity to other chemically related antibiotics. The results obtained for blind honey samples (mean recovery 97%), were in good agreement with those obtained by liquid chromatography separation and mass spectrometry detection (LC-MS) (mean recovery 102%), showing that the proposed method might be used for the determination of sulphathiazole residues without expensive equipment.

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A rapid duplex ELISA for the simultaneous determination of two of the most widely used organophosphorous insecticides in tangerine juices is described. To accomplish this aim, two individual enzyme-linked immunosorbent assays for chlorpyrifos and fenthion pesticides were integrated into one ELISA test. The strategy uses 96-well plates with specific wells coated with the corresponding haptenized conjugate. The optimized duplex ELISA was accomplished within 40 min achieving a detection limit of 0.20±0.04 µg/L and 0.50±0.06 µg/L, for chlorpyrifos and fenthion, respectively in tangerine juice samples. The determination of residues of both pesticides was carried out by simple sample dilution, without any extra sample clean-up procedure. Results of testing precision, stability, and selectivity demonstrated that the assay provided reliable analytical performances for the simultaneous determination of residues of chlorpyrifos and fenthion in fruit juice samples below the established European maximum residue limits (MRL). In addition, the accuracy and reliability of this duplex bioanalytical method is demonstrated by analyzing blind spiked juice samples and the results, correlated well with those achieved using a well-established GC/MS method (recoveries between 95% and 106%).

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Soluble and membrane-bound peroxidases (PODs) were extracted from red cabbage using Triton X-114. Optimum activity was obtained at pH 4.0 for both enzymes, and both were inactivated by sodium dodecyl sulfate (SDS). The K(M) and V(m) values for H(2)O(2) were found to be 0.98 mM and 8.1 µM/min, respectively, for soluble POD and 0.82 mM and 6.1 µM/min, respectively, for membrane-bound POD. When the 2,2'-azinobis(3-ethylbenzothiazolinesulfonic acid (ABTS) concentration was increased, maintaining a steady concentration of H(2)O(2), the activity was inhibited at the highest ABTS concentrations in soluble POD. Ascorbic acid was found to be the most active modulator of POD activity. The effect of cyclodextrins was also studied, and the complexation constant between ABTS and hydroxypropyl-ß-cyclodextrins (HP-ß-CDs) was calculated (K(c) = 312 M(-1)). Membrane-bound POD is more thermostable than soluble POD, losing >90% of relative activity after 5 min of incubation at 76.6 and 30.2 °C, respectively.

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The antioxidant activity of resveratrol in the absence and presence of increasing concentrations of HP-ß-CDs was determined using three different methods: ORAC, ABTS and DPPH. The three methods were validated and compared for their linearity, precision and accuracy in measuring resveratrol antioxidant activity. The results indicated that the most sensitive method is the ORAC assay, which can measure the lowest resveratrol concentration (0.15-2 µM) with the highest precision. In the presence of increasing concentrations of HP-ß-CDs, the antioxidant activity of resveratrol was seen to increase when it was measured by the ORAC and ABTS assays. However, no increase was observed when the DPPH assay was used. With the ORAC assay, the antioxidant activity increased until all the resveratrol had been included in HP-ß-CDs (0.4 mM CDs), whereas in the case of ABTS assay the plateau in antioxidant activity was reached after 2 mM HP-ß-CDs, suggesting that the CDs interferences in the measurement method. When the DPPH assay was used, no effect was observed when increasing concentrations of HP-ß-CDs, indicating that in a methanolic medium resveratrol is free. Therefore, so this method cannot be used to measure the effect of resveratrol complexation with CDs on its antioxidant activity.

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The evolution of °Brix, protein content, polyphenoloxidase activity and peroxidase activity during the ripening of Crimson Seedless table grape was studied in three consecutive years (2006, 2007 and 2008). The total protein content was determined according to Bradford's dye binding method, and polyphenoloxidase (PPO) and peroxidase (POD) were extracted using Triton X-114 and characterised using spectrophotometric methods. The year had a statistically significant effect on all the studied parameters and there was an interannual correlation in the evolution of protein, PPO, POD and °Brix. All the studied parameters were statistically correlated, except POD activity with protein content. Weather conditions during the ripening period had a greater effect on protein content than PPO and POD activity.

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A lateral flow immunoassay (LFIA) was developed in the competitive reaction format and applied to test sulphathiazole (STZ) residues in honey samples. To prepare the assay test, a hapten conjugate and goat antirabbit antiserum as capture and control reagent, respectively, were dispensed on nitrocellulose membrane. Polyclonal antiserum against sulphathiazole was conjugated to colloidal gold nanoparticles and used as the detection reagent. The visual limit of detection (cut-off value) of the sulphathiazole LFIA was 15ng/g, reaching qualitative results within 10min. The assay was evaluated with STZ spiked honey samples from different geographical origins (n=25). The results were in good agreement with those obtained from liquid chromatography separation and mass spectroscopy detection (LC-MS), indicating that the LFIA test might be used as a qualitative method for the determination of sulphathiazole residues without expensive equipment. The test was also highly specific, showing no cross-reactivity to other chemically similar antibiotics. To our knowledge, this is the only work where a development of LFIA tests for the detection of sulphathiazole residues is performed.

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Peroxidase (POD) was extracted from red alga (Mastocarpus stellatus) using Triton X-114 and characterised by UV-spectrophotometry. Optimum activity using 2,2´-azinobis(3-ethylbenzothiazolinesulphonic acid) (ABTS) as the H-donor was obtained at pH 5.0. In the presence of the anionic detergent, sodium dodecyl sulphate (SDS), however, POD was inactivated at all the pH values studied and totally inactivated at 1mM SDS. When the enzyme was kinetically characterised, the KM and Vm values for ABTS were found to be 13mM and 40µM/min, respectively. In addition, when the H2O2 concentration was increased, at a fixed concentration of ABTS, the activity was inhibited at the highest H2O2 concentrations. In a study of the effect of several reducing agents, l-cysteine was found to be the most active. A thermal inactivation study showed a first-order inactivation kinetic, and the Arrhenius plot yielded a straight line with a slope equivalent to an activation energy of 121.6kJ/mol. Significant inactivation occurred at temperatures of>35°C, with>90% of the relative activity being lost after only 5min of incubation at 48.4°C.

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The chemical control of crops by organophosphate insecticide treatment is usually limited because the insecticides do not maintain their efficiency for long periods for several reasons, including environmental conditions or rapid degradation of the active ingredient. Chlorpyrifos is an organophosphate insecticide used worldwide to control a variety of soil insects and arthropods in a wide range of crops. It is easily soluble in organic solvents but shows poor water solubility. The inclusion of chrorpyrifos in cyclodextrins (CDs) improves its water solubility, bioavailability, and insecticidal activity and helps prevent overdosing, leading to more cost-effective and more environmentally friendly agricultural practices. Solubility studies of chlorpyrifos in the presence of different types of CDs show G2-beta-CDs to be the most effective CDs in the complexation process, giving 1:2 complexes, with complexation constant (Kc) values of 12.34 +/- 3.1 M(-1) for K1 and 3895 +/- 183 M(-1) for K2. These complexation constant values were corroborated by applying a fluorimetric method.

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The effect of the complexation of resveratrol with hydroxypropyl-beta-cyclodextrins (HP-beta-CDs) on the antioxidant capacity of the polyphenol is studied for the first time by means of the oxygen radical absorbance capacity (ORAC) method, using fluorescein (FL) as the fluorescent probe. The method is validated through its linearity, precision, and accuracy for measuring the ORAC of resveratrol in the absence or presence of cyclodextrins (CDs). The complexation of resveratrol in CDs increased the net area under the FL decay curve (net AUC) of resveratrol up to its saturation level, at which the polyphenol showed almost double the antioxidant activity it shows in the absence of CDs. The complexation constant ( K c) between resveratrol and HP-beta-CDs was calculated by linear regression of the phase solubility diagram ( K c = 18048 M (-1)). The antioxidant activity of resveratrol was dependent on the complexed resveratrol because CDs acts as a controlled dosage reservoir that protects resveratrol against rapid oxidation by free radicals. In this way, its antioxidant activity is prolonged and only reaches its maximum when all the resveratrol is complexed.

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The kinetics of the activation process of latent peach PPO by trypsin was studied. By coupling this activation process to the oxidation of 4-tert-butylcatechol (TBC) to its corresponding quinone, it was possible to evaluate the specific rate constant of active PPO formation, k(3), which showed a value of 0.04 s(-1). This proteolytic activation of latent peach PPO permitted us to characterize the monophenolase activity of peach PPO for the first time using p-cresol as substrate, and it showed the characteristic lag period of the kinetic mechanism of monophenols hydroxylation, which depended on the enzyme and substrate concentration, the pH and the presence of catalytic amounts of o-diphenol (4-methylcatechol). The enzyme activation constant, k(act), was 2 microM.

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Salsolinol, a tethrahydroisoquinoline present in banana and biosynthesized from dopamine, was oxidized by banana pulp polyphenol oxidase to its corresponding salsolinol-o-quinone. This oxidation was pH-dependent and showed a maximum at acidic pH values. At physiological pH of 5.0, the values obtained for the kinetic parameter (V(m) and K(m)) were 62.5 microM/min and 1.7 mM, respectively. When dopamine was added to the reaction medium to imitate physiological conditions, salsolinol was co-oxidized by dopamine-quinone. When this phenomenon was studied oxygraphically, an unexpected activation of dopamine oxidation was found in the presence of salsolinol. This activation was related with the enzyme's kinetic mechanism and was named "kinetic synergism", because a bad substrate activated a good one. A possible physiological role is discussed.

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The reversibility of the SDS-mediated activation of latent peach PPO has been studied using cyclodextrins as strip detergent agent. Cyclodextrins produced a combined inhibitory effect on enzymatic activity of latent peach PPO due to the complexation of detergent and the hydrophobic substrate 4-tert-butylcatechol (TBC) molecules. To study the reversibility of the activation process, this combined effect has to be separated. On the one hand, the enzyme was activated by acid-shocking and the activity was measured in the presence of cyclodextrins, using TBC as substrate. The inhibition curves obtained permitted study of the complexation of TBC into cyclodextrins. On the other hand, the enzyme was activated by SDS and the activity in the presence of cyclodextrins was measured using the highly hydrophilic o-diphenol dopamine as substrate. In this case, the inhibition curves obtained indicated the reversibility of the activation process when SDS was trapped by cyclodextrins. In addition, the complexation constant between SDS and 2-hydroxypropyl-beta-cyclodextrins was calculated by measuring conductivity (K(s) = 3500 M(-1)).

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The effect of cyclodextrins (CDs) on o-diphenol oxidation catalyzed by banana polyphenol oxidase (PPO) was studied. The oxidation of dopamine, the natural substrate of banana, in the presence of cyclodextrins was unaffected, because this hydrophilic phenol does not form inclusion complexes with CDs. However, when a hydrophobic phenol such as tert-butylcatechol (TBC) was used, a marked inhibition was observed with beta-, hydroxypropyl-beta-, and maltosyl-beta- CDs. This inhibition was due to the complexation of TBC in the CD core, demonstrating that banana pulp PPO worked only toward free substrate and not toward the complex TBC-CDs. In addition, the effect of some inhibitors in the presence of CDs and dopamine as substrate was studied. Increasing concentrations of CDs, in the presence of two inhibitors (4-iodophenol and cinnamic acid) were able to activate the inhibited enzyme to reach the noninhibited level by complexing the inhibitors in the hydrophobic core of the CDs. This dual effect of CDs as activator and inhibitor was tested in crude banana pulp extracts, with surprising activation effects never before described being observed.

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PURPOSE: The in vitro formation of DES-cyclodextrins inclusion complexes was characterized using lipoxygenase as enzymatic system. METHODS: DES-cyclodextrins complexes were obtained in aqueous solution. RESULTS: The addition of cyclodextrins to the reaction medium had an inhibitory effect on DES oxidation by lipoxygenase due to the drug's complexation into the cyclodextrin cavity. This inhibitory effect depends on the complexation constant between DES and the cyclodextrins type used. In this case, beta-, 2-hydroxypropyl-beta- and gamma-cyclodextrins have similar complexation constants and therefore produce the same inhibitory effect. Moreover, depending on the type of cyclodextrins used, the solubility of DES can be enhanced up to 956 times, while the lipoxygenase activity remains constant. CONCLUSIONS: These results suggest that the system described may be used as a controlled-release delivery system for DES, since it may diminish the local and systemic adverse side effects caused by high concentrations of the drug.

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The oxidation of xenobiotics by the hydroperoxidase activity of lipoxygenase in the presence of cyclodextrins was studied. These produced an inhibitory effect on xenobiotics oxidation, based on their degree of hydrophobicity and the charge (isoproterenol < 4-methyl-catechol (4MC) < 4-tert-butylcatechol (TBC) < 4-tert-octylcatechol (TOC)). This inhibitory effect was due to the complexation of xenobiotics in the hydrophobic cavity of cyclodextrins. The complexation constant Kc was calculated by nonlinear regression of the inhibition curves obtained in the presence of cyclodextrins, and the values obtained were 400, 16,250, and 35,127 M-1 for 4MC, TBC, and TOC, respectively. The validity of these values was checked at different points of the Michaelis-Menten saturation curve, and a sigmoidal inhibition curve was obtained at the saturating concentration of the o-diphenol, TBC, with no change in the Kc value. This demonstrates the validity of the equations used to calculate Kc for the complete range of the Michaelis-Menten equation.

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The oxidation of Trolox C (a vitamin E analog) by the hydroperoxidase activity of lipoxygenase was studied. Trolox C was oxidized to its corresponding phenoxyl radical in the presence of hydrogen peroxide, evolving through a ketodiene intermediate to the Trolox C quinone. The H2O2/Trolox C quinone molar ratio was 1.0. The overall reaction followed an enzymatic-chemical second-order system and involved a substrate regeneration mechanism. From the equations derived from this mechanism, the dismutation constant of the Trolox C radical was evaluated by non-linear regression as 4 x 10(5) M(-1) x s(-1). The accumulation curve of Trolox C quinone was found to be linear, with no lag period, and dependent on enzyme concentration. No phenoxyl radical was detected when the reaction was carried out in the presence of ascorbate. This synergistic reaction between the Trolox C radical and ascorbate was quantitative and depended on the respective concentrations of enzyme, Trolox C and hydrogen peroxide. The results presented in this paper suggest that the diferences observed in the kinetic behaviour of monophenols (one-electron donors) and diphenols (two-electron donors) stem from the fact that the latter evolve directly into ferric form without taking the slow pathway once the steady state is reached, whereas the monophenols are always forced take the slow way, even in the steady state. This peroxidative oxidation of a vitamin E analog by the hydroperoxidase activity of lipoxygenase together with the oxidation produced by dioxygenase activity suggests that lipoxygenase might be a key enzyme in destroying the lipophilic antioxidant barrier against the reactive oxygen species in membranes.

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The oxidation of diethylstilbestrol (DES), a synthetic carcinogenic estrogen, by the hydroperoxidase activity of lipoxygenase was studied. Lipoxygenase catalyzes the oxidation of DES to its corresponding DES quinone to yield free radical species intermediates (DES semiquinone and DES quinone), which are associated with the adverse effects of this synthetic estrogen. The reaction was dependent on enzyme, DES, and hydrogen peroxide concentrations. Due to the low degree of water solubility of DES, the enzyme works in a range of DES concentrations below K(m). The enzyme presents a high affinity for hydrogen peroxide (5.7 microM), and produces substrate inhibition (Ksi = 2.5 mM). This study is the first demonstration that this reaction, which is known to be catalyzed by a variety of enzymes, including peroxidases, is also catalyzed by lipoxygenase.

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