Phase diagrams of a CTAB/organic solvent/buffer system applied to extraction of enzymes by reverse micelles

A partial pseudo-ternary phase diagram has been studied for the cethyltrimethylammonium bromide/isooctane:hexanol:butanol/potassium phosphate buffer system, where the two-phase diagram consisting of the reverse micelle phase (L-2) in equilibrium with the solvent is indicated. Based on these diagrams two-phase systems of reverse micelles were prepared with different compositions of the compounds and used for extraction and recovery of two enzymes, and the percentage of enzyme recovery yield monitored. The enzymes glucose-6-phosphate dehydrogenase (G6PD) and xylose redutase (XR) obtained from Candida guilliermondii yeast were used in the extraction procedures. The recovery yield data indicate that micelles having different composition give selective extraction of enzymes. The method can thus be used to optimize enzyme extraction processes. (c) 2007 Elsevier B.V. All rights reserved.

Relationship between global structural parameters and Enzyme Commission hierarchy: Implications for function prediction

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Salacia campestris root bark extract: peroxidase inhibition, antioxidant and antiradical profile

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Determination of phenolic compounds and evaluation of antioxidant capacity of Campomanesia adamantium leaves

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Lipid peroxidation, antioxidant enzymes and glutathione levels in human erythrocytes exposed to colloidal iron hydroxide in vitro

The free form of the iron ion is one of the strongest oxidizing agents in the cellular environment. The effect of iron at different concentrations (0, 1, 5, 10, 50, and 100 µM Fe3+) on the normal human red blood cell (RBC) antioxidant system was evaluated in vitro by measuring total (GSH) and oxidized (GSSG) glutathione levels, and superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px) and reductase (GSH-Rd) activities. Membrane lipid peroxidation was assessed by measuring thiobarbituric acid reactive substance (TBARS). The RBC were incubated with colloidal iron hydroxide and phosphate-buffered saline, pH 7.45, at 37oC, for 60 min. For each assay, the results for the control group were: a) GSH = 3.52 ± 0.27 µM/g Hb; b) GSSG = 0.17 ± 0.03 µM/g Hb; c) GSH-Px = 19.60 ± 1.96 IU/g Hb; d) GSH-Rd = 3.13 ± 0.17 IU/g Hb; e) catalase = 394.9 ± 22.8 IU/g Hb; f) SOD = 5981 ± 375 IU/g Hb. The addition of 1 to 100 µM Fe3+ had no effect on the parameters analyzed. No change in TBARS levels was detected at any of the iron concentrations studied. Oxidative stress, measured by GSH kinetics over time, occurs when the RBC are incubated with colloidal iron hydroxide at concentrations higher than 10 µM of Fe3+. Overall, these results show that the intact human RBC is prone to oxidative stress when exposed to Fe3+ and that the RBC has a potent antioxidant system that can minimize the potential damage caused by acute exposure to a colloidal iron hydroxide in vitro.

Lipoperoxidation and cyclooxygenase enzyme inhibitory piperidine alkaloids from Cassia spectabilis green fruits

Phytochernical work in the search for bioactive metabolites from the methanolic extract of Senna spectabilis green fruits led to the isolation of a new piperidine alkaloid, (+)-3-O-feruloylcassine (1), in addition to the known (-)-spectaline (2) and (-)-3-O-acetylspectaline (3). The isolates were submitted to in vitro evaluation of lipoperoxidation (LPO) and cyclooxygenase enzymes (COX-1 and -2) inhibitory properties and showed moderate antioxidant activities (40-70%) at 100 ppm when compared to commercial standards BHT and vitamin E and moderate inhibition of COX-1 (ca. 40%) and marginal inhibition of COX-2 enzymes (< 10%) at 100 ppm when compared to nonsteroidal anti-inflammatory drugs (NSAIDs) aspirin, rofecoxib, and celecoxib, respectively.

Determination of malic acid in real samples by using enzyme immobilized reactors and amperometric detection

The malate dehydrogenase (MDH) and ascorbate oxidase were immobilized independently, onto silanized controlled porous silica and packed in a tygon tube. The reactors were inserted in the flow system, and the malic acid was determined by measurement of NADH produced by enzymatic reaction. The NADH was reoxidized in a wall jet cell that consisted of spectrographic graphite, Ag/AgCl, KCl(sat), and steel needle as work, reference, and counter electrodes, respectively. The current intensities were measured at 390 mV. The malate calibration curve shows a linear range from 5.0 x 10(-6) to 1.0 x 10(-4) molL(-1), the lifetime was 40 analyses, after that a decrease of 20% on the response is observed. Three different citric juices were analyzed and a good correlation between the proposed method and spectrophotometric commercial kit were obtained.

Construction and application of an electrochemical sensor for paracetamol determination based on iron tetrapyridinoporphyrazine as a biomimetic catalyst of P450 enzyme

This work describes the construction and application of a biomimetic sensor for paracetamol determination in different samples. The sensor was prepared by modifying a glassy carbon electrode surface with a Nafion (R) membrane doped with FeTPyPz. The best performance of the sensor in 0.1 mol L-1 acetate buffer was at pH 3.6. Under these conditions, an oxidation potential of paracetamol was observed at 445 mV vs. Ag vertical bar AgCl. The sensor presented a linear response range between 4.0 and 420 mu mol L-1, a sensitivity of 46.015 mA L mol(-1) cm(-2), quantification and detection limits of 4.0 mu mol L-1 and 1.2 mu mol L-1, respectively. A detailed investigation about its electrochemical behavior and selectivity was carried out. The results suggested that FeTPyPz presents catalytic properties similar to P450 enzyme for paracetamol oxidation. Finally, the sensor was applied for paracetamol determination in commercial drugs and for the monitoring of its degradation in an electrochemical batch reactor effluent.

Stimulation of polygalacturonase production in an immobilized system by Aspergillus sp.: effect of pectin and glucose

The synthesis of polygalacturonases (PG) is known to be influenced by Aspergillus growth conditions, namely, environmental factors and pectin content in the cultivation medium containing a mixed carbon source. Optimal conditions were attained at a temperature of 30 A degrees C and an initial pH of 4.5. PG activity (3.29 and 2.48 U/mL) was determined after a two-day culture of Aspergillus sp. HC1 and Aspergillus sp. CC1, respectively, in a basic medium containing 2% citrus pectin as the sole carbon source. The addition of glucose (2% w/v) to the basic medium led to a 2-fold increase in PG production. However, enzyme synthesis was repressed when a higher concentration of glucose was used in the medium containing the mixed carbon source. Spores from the two fungi were immobilized in a 3% Ca-alginate system and the mechanical strength of the gel beads allowed the use of this process system 6-fold longer (288 h) than the free culture. In the Aspergillus sp. CC1 immobilized system, PG production increased nearly 10-fold in the medium with 2% glucose added (5.95 U/mL) in comparison to the medium without sugar (0.55 U/mL). The results demonstrate that a different response in activity was produced by free and entrapped spore systems. PG production remained approximately constant throughout the six 48 h cycles in the medium containing citrus pectin (2% w/v) as the sole carbon source.

Studies on the activation of the pre-kininogenin-kininogenin (pre-kallikrein-kallikrein) system by sulfated polysaccharides and kaolin

The activation of pre-kininogenin to kininogenin (pre-kallikrein to kallikrein) is one of the steps in the series of reactions of a complex system, linked also to fibrinolysis and coagulation, that leads to kinin release in plasma (See Cochrane et al., 1976; Wuepper, 1976; Kaplan et al., 1976; Kaplan et al., 1976). For human plasma, a test using kaolin as activator and measuring kallikrein activity with the chromogenic substrate Chromozym PK (Nα-benzoyl-prolyl-phenylalanyl-arginyl-nitroanilide, Pentapharm, Basle) is routinely employed. The purpose of this paper is to further study the mechanism of this activation, by means of different activators and using as inhibitor hexadimethrine bromide (Polybrene). Besides kaolin, sulfated polysaccharides, such as heparin and cellulose sulfate are able to activate pre-kininogenin to kininogenin. Hexadimethrine as expected, inhibited the activation by heparin and also that by cellulose sulfate. The activation by kaolin however followed a different pattern suggesting, at least partially, a different mode of action of this activator. © 1979.

Functional evidence that the central renin-angiotensin system plays a role in the pressor response induced by central injection of carbachol

We investigated the effects of losartan, an AT 1-receptor blocker, and ramipril, a converting enzyme inhibitor, on the pressor response induced by angiotensin II (ANG II) and carbachol (a cholinergic receptor agonist). Male Holtzman rats (250-300 g) with a stainless steel cannula implanted into the lateral ventricle (LV) were used. The injection of losartan (50 nmol/l μl) into the LV blocked the pressor response induced by ANG II (12 ng/l μl) and carbachol (2 nmol/l μl). After injection of ANG II and carbachol into the LV, mean arterial pressure (MAP) increased to 31 ± 1 and 28 ± 2 mmHg, respectively. Previous injection of losartan abolished the increase in MAP induced by ANG II and carbachol into the LV (2 ± 1 and 5 ± 2 mmHg, respectively). The injection of ramipril (12 ng/l μl) prior to carbachol blocked the pressor effect of carbachol to 7 ± 3 mmHg. These results suggest an interaction between central cholinergic pathways and the angiotensinergic system in the regulation of arterial blood pressure.

Quick preparative separation of natural naphthopyranones with antioxidant activity by high-speed counter-current chromatography

The natural naphthopyranones paepalantine (1), paepalantine-9O-β-D-glucopyranoside (2) and paepalantine-9-O-β-D-allopyranosyl-(1→6)-O-β-D-glucopyranoside (3) were separated in a preparative scale from the ethanolic extract of the capitula of Paepalanthus bromelioides by high-speed counter-current chromatography (HSCCC). The solvent system used was composed of water-ethanol-ethyl acetate-hexane (10:4:10:4, v/v/v/v). This technique led to the separation of the three different naphthopyranone glycosides in pure form in approximately 7 hours. Paepalantine showed a good antioxidant activity when assayed by the DPPH radical spectrophotometric assay.

In vitro propagation of Maytenus ilicifolia (Celastraceae) as potential source for antitumoral and antioxidant quinomethide triterpenes production. A rapid quantitative method for their analysis by reverse-phase high-performance liquid chromatography

Cell culture of Maytenus ilicifolia were established in order to produce and to quantify the antitumoral and antioxidant quinonemethide triterpenes. In vitro calli were induced from leaf explants of native plants and cultured in semi-solid medium under controlled conditions of humidity, temperature and photoperiod. The quinonemethide triterpenes showed maximum accumulation in the logarithmic phase growth of the cell culture. A rapid, sensitive and reliable reverse-phase HPLC method was used for quantitative determination of the antitumoral and antioxidant quinonemethide triterpenes, 22β-hydroxymaytenin and maytenin in callus of Maytenus ilicifolia. Well resolved peaks with good detection response and linearity in the range 1.0 - 100 μg/mL were obtained. This quantitative work was performed by an external standard method.

Synergistic action of olive oil supplementation and dietary restriction on serum lipids and cardiac antioxidant defences

Caloric intake is higher than recommended in many populations. Therefore, enhancing olive oil intake alone may not be the most effective way to prevent cardiovascular diseases. The purpose of the present study was to analyse the association of olive oil and dietary restriction on lipid profile and myocardial antioxidant defences. Male Wistar rats (180-200 g, n = 6) were divided into 4 groups: control ad libitum diet (C), 50% restricted diet (DR), fed ad libitum and supplemented with olive oil (3 mL/(kg-day)) (OO), and 50% restricted diet and supplemented with olive oil (DROO). After 30 days of treatments, OO, DR, and DROO groups had increased total cholesterol and high-density lipoprotein cholesterol concentrations. DR and DROO animals showed decreased low-density lipoprotein cholesterol. DROO had the lowest low-density lipoprotein cholesterol concentration. Total lipids and triacylglycerols were raised by dietary restriction and diminished by olive oil. OO rats had higher myocardial Superoxide dismutase and lower catalase and glutathione peroxidase activities than C rats. DR and DROO showed enhanced cardiac Superoxide dismutase, catalase, and glutathione peroxidase activities from the control. Olive oil supplementation alone improved the lipid profile but was more effective when coupled with dietary restriction. There was a synergistic beneficial action of dietary restriction and olive oil on serum lipids and myocardial antioxidant defences.

Endothelial and neuronal nitric oxide synthase inhibitors influences angiotensin II pressor effect in central nervous system

The present study investigated the central role of angiotensin II and nitric oxide on arterial blood pressure (MAP) in rats. Losartan and PD123349 AT 1 and AT 2 (selective no peptides antagonists angiotensin receptors), as well as FK 409 (a nitric oxide donor), N W-nitro-L-arginine methyl ester (L-NAME) a constituve nitric oxide synthase inhibitor endothelial (eNOSI) and 7-nitroindazol (7NI) a specific neuronal nitric oxide synthase inhibitor (nNOSI) were used. Holtzman strain, (Rattus norvergicus) weighting 200-250 g were anesthetized with zoletil 50 mg kg -1 (tiletamine chloridrate 125 mg and zolazepan chloridrate 125 mg) into quadriceps muscle anda stainless steel cannula was stereotaxically implanted into their Lateral Ventricle (LV). Controls were injected with a 0.5 μl volume of 0.15 M NaCl. Angiotensin II injected into LV increased MAP (19±3 vs. control 3±1 mm Hg), which is potentiated by prior injection of L-NAME in the same site 26±2 mm Hg. 7NI injected prior to ANG II into LV also potentiated the pressor effect of ANG II but with a higher intensity than L-NAME 32±3 mm Hg. FK 409 inhibited the pressor effect of ANG II (6±1 mm Hg). Losartan injected into LV before ANG II influences the pressor effect of ANG II (8±1 mm Hg). The PD 123319 decreased the pressor effects of ANG II (16±1 mm Hg). Losartan injected simultaneously with FK 409 blocked the pressor effect of ANG II (3±1 mm Hg). L-NAME produced an increase in the pressor effect of ANG II, may be due to local vasoconstriction and all at once by neuronal NOS inhibition but the main effect is of the 7-NIT an specific nNOS inhibitor. The AT 1 antagonist receptors improve basal nitric oxide (NO) production and release. These data suggest the involvement of constitutive and neuronal NOS in the control of arterial blood pressure induced by ANG II centrally, evolving AT 1 receptor-mediated vasoconstriction and AT 2 receptor-mediated vasodilatation. These results were confirmed by the experiment using FK 409. © 2006 Asian Network for Scientific Information.