Inhibition of 12/15 lipoxygenase by curcumin and an extract from Curcuma longa L.

Curcumin (diferuloylmethane) is an orange-yellow secondary metabolic compound from the rhizome of turmeric (Curcuma longa L.), a spice often found in curry powder. It is one of the major curcuminoids of turmeric. For centuries, curcumin has been used in some medicinal preparations or as a food colouring agent. A variety of enzymes that are closely associated with inflammation and cancer were found to be modulated by curcumin. This paper summarized the results of the inhibitory effect of curcumin and a Curcuma longa L. ethanolic extract on lipoxygenase from the rat lung cytosolic fraction. The positional specificity determination of arachidonic acid dioxygenation by RP- and SP-HPLC methods showed that in a purified enzyme preparation from the rat lung cytosol the specific form of lipoxygenase (LOX) is present exhibiting 12/15-LOX dual specificity (with predominant 15-LOX activity). The inhibitory activity of curcumin and Curcuma longa extract on LOX from cytosolic fraction of rat lung was expressed in the percentage of inhibition and as IC50. Lineweaver-Burk plot analysis has indicated that curcumin is the competitive inhibitor of 12/15 LOX from the rat lung cytosolic fraction.

Monitoring of PQQ-dependent glucose dehydrogenase substrate specificity for its potential use in biocatalysis and bioanalysis.

Substrate specificity of 2,7,9-tricarboxypyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase was investigated in biosensor arrangement for understanding the suitability and the limitations of its use in bioanalysis and bioproduction of chemicals. The study demonstrated a very broad substrate specificity of biosensor utilising soluble form of PQQ-dependent glucose dehydrogenase. Nineteen saccharides out of 31 were oxidised by the sensor. Investigation confirmed strong importance of hydroxyl configuration in the positions 2 and 5 of oxidised saccharides. The broad specificity suggests that the PQQ-dependent glucose dehydrogenase could be utilised for analysis of other sugars than glucose in food samples for various production processes and for biofuel cells. In addition, the results showed that the substrate specificity of enzymes can be effectively and generally studied by biosensor arrangement for research purposes. This layout utilising immobilised enzyme allowed performing comprehensive study using a small amount of enzymes and thus saving the costs and time.

Monitoring of monosaccharides, oligosaccharides, ethanol and glycerol during wort fermentation by biosensors, HPLC and spectrophotometry.

The aim of the present study was to analyze sugar levels (namely maltose, maltotriose, glucose and fructose) and alcohols (ethanol and glycerol) during the fermentation process in wort samples by amperometric enzymatic biosensors developed by our research group for industrial application, HPLC and spectrophotometry, and to compare the suitability of the presented methods for determination of individual analytes. We can conclude that for the specific monitoring of maltose or maltotriose only the HPLC method was suitable. On the other hand, biosensors and spectrophotometry reflected a decrease in total sugar concentration better and were able to detect both glucose and fructose in the later stages of fermentation, while HPLC was not. This can be attributed to the low detection limits and good sensitivity of the proposed methods. For the ethanol and glycerol analysis all methods proved to be suitable. However, concerning the cost expenses and time analysis, biosensors represented the best option.

Antioxidant action and cytotoxicity on HeLa and NIH-3T3 cells of new quercetin derivatives.

Quercetin is a natural polyphenol with proven health beneficial activities. In this study 15 new quercetin derivatives were prepared with the aim to enhance their bioavailability. Modification of their physicochemical properties could herewith improve the action in cells. The prepared compounds were tested for their antioxidant and cytotoxic activity. The ability to scavenge free radicals as well as ferric reducing antioxidant power of the new derivatives was not better than that of unmodified quercetin. But for acetylated esters a better cytotoxic activity was found on human cervical cancer cells HeLa than for the initial molecule. The best effect revealed chloronaphtoquinone quercetin (IC50=13.2 µM). For this compound comparable cytotoxic action on non-cancer murine fibroblast cells was detected (IC50=16.5 µM). The obtained results indicate that appropriate lipophilization of the quercetin molecule could improve its cytotoxic action in cells, probably due to its enhanced bioavailability.

Amperometric glucose biosensor utilizing FAD-dependent glucose dehydrogenase immobilized on nanocomposite electrode.

Amperometric glucose biosensors utilizing commercially available FAD-dependent glucose dehydrogenases from two strains of Aspergillus species are described. Enzymes were immobilized on nanocomposite electrode consisting of multi-walled carbon nanotubes by entrapment between chitosan layers. Unlike the common glucose oxidase based biosensor, the presented biosensors appeared to be O(2)-independent. The optimal amount of enzymes, working potential and pH value of working media of the glucose biosensors were determined. The biosensor utilizing enzyme isolated from Aspergillus sp. showed linearity over the range from 50 to 960 µM and from 70 to 620 µM for enzyme from Aspergillus oryzae. The detection limits were 4.45 µM and 4.15 µM, respectively. The time of response was found to be 60 s. The biosensors showed excellent operational stability - no loss of sensitivity after 100 consecutive measurements and after the storage for 4 weeks at 4 °C in phosphate buffer solution. When biosensors were held in a dessicator at room temperature without use, they kept the same response ability at least after 6 months. Finally, the results obtained from measurements of beverages and wine samples were compared with those obtained with the enzymatic-spectrophotometric and standard HPLC methods, respectively. Good correlation between results in case of analysis of real samples and good analytical performance of presented glucose biosensor allows to use presented concept for mass production and commercial use.

Polyphenol fatty acid esters as serine protease inhibitors: a quantum-chemical QSAR analysis.

We investigated the ability of polyphenol fatty acid esters to inhibit the activity of serine proteases trypsin, thrombin, elastase and urokinase. Potent protease inhibition in micromolar range was displayed by rutin and rutin derivatives esterified with medium and long chain, mono- and polyunsaturated fatty acids (1e-m), followed by phloridzin and esculin esters with medium and long fatty acid chain length (2a-d, 3a-d), while unmodified compounds showed only little or no effect. QSAR study of the compounds tested provided the most significant parameters for individual inhibition activities, i.e. number of hydrogen bond donors for urokinase, molecular volume for thrombin, and solvation energy for elastase. According to the statistical analysis, the action of elastase inhibitors is opposed to those of urokinase and thrombin. Cluster analysis showed two groups of compounds: original polyphenols together with rutin esters with short fatty acid chain length and rutin esters with long fatty acid chain length.

Application of electrochemical biosensors in clinical diagnosis.

Analyses in the clinical area need quick and reliable analytical methods and devices. For this purpose, biosensors can be a suitable option, whereas they are constructed to be simple for use, specific for the target analyte, capable of continuous monitoring and giving quick results, potentially low-costing and portable. In this article, we describe electrochemical biosensors developed for clinical diagnosis, namely for glucose, lactate, cholesterol, urea, creatinine, DNA, antigens, antibodies, and cancer markers assays. Chosen biosensors showed desirable sensitivity, selectivity, and potential for application on real samples. They are often designed to avoid interference with undesired components present in the monitored systems.

Multienzymatic amperometric biosensor based on gold and nanocomposite planar electrodes for glycerol determination in wine.

Amperometric biosensors based on gold planar or nanocomposite electrode containing multiwalled carbon nanotubes for determination of glycerol were developed. The biosensors were constructed by immobilization of a novel multienzyme cascade consisting of glycerol kinase/creatine kinase/creatinase/sarcosine oxidase/peroxidase between a chitosan "sandwich." A measuring buffer contained adenosine 5'-triphosphate (ATP), creatine phosphate, and an artificial electrochemical mediator ferrocyanide. The currents proportional to glycerol concentration were measured at working potential of -50 mV against Ag/AgCl reference electrode. The biosensors showed linearity over the ranges of 5-640 µM and 5-566 µM with detection limits of 1.96 and 2.24 µM and sensitivities of 0.80 and 0.81 nA µM(-1), respectively. Both types of biosensors had a response time of 70s. The biosensors demonstrated satisfactory operational stability (no loss of sensitivity after 90 consecutive measurements) and excellent storage stability (90% of the initial sensitivity after 15 months of storage at room temperature). The results obtained from measurements of wines correlated well with those obtained with an enzymatic-spectrophotometric assay. The presented multienzyme cascade can be used also for determination of triglycerides or various kinase substrates when glycerol kinase is replaced by other kinases.

Modulating effect of flavonoids and their derivatives on sarcoplasmic reticulum Ca2+-ATPase oxidized by hypochloric acid and peroxynitrite.

OBJECTIVES: Effect of rutin and its lipophilic derivatives on Ca2+-ATPase of sarcoplasmic reticulum (SERCA) oxidized by hypochloric acid and peroxynitrite was investigated to examine the role of flavonoids in SERCA activity modulation. METHODS: Ca2+-ATPase activity was measured spectrophotometrically at 37 degrees C using NADH-coupled enzyme pyruvate kinase/lactate dehydrogenase assay. SERCA was oxidized by HOCl (3 min) or ONOO- (30 s) after previous treatment with flavonoids (2 min) at 37 degrees C. Lipophilic rutin derivatives were prepared by lipase-catalyzed esterification of flavonoids with fatty acids. RESULTS: Both hypochloric acid (HOCl) and peroxynitrite (ONOO-) decreased ATPase activity concentration-dependently with IC50 of 50+/-10 micromol/l and 150+/-15 micromol/l, respectively. Rutin was found to have a protective effect on SERCA activity in both oxidation systems in the concentration range 5 - 250 micromol/l. Lipophilic rutin derivatives (rutin oleate, rutin linoleate, rutin linolenate) exerted inhibitory effect on ATPase activity both in the presence and absence of oxidants. CONCLUSION: The results suggest that selective lipophilization of the flavonoid skeleton may represent a useful tool for SERCA activity modulation.

Oxidation of liposomal membrane suppressed by flavonoids: quantitative structure-activity relationship.

Antioxidant activity of the set of 12 flavonoids in heterogeneous membrane system of dioleoyl phosphatidylcholine liposomes stressed by peroxidative damage induced by 2,2'-azobis-(2-amidinopropane)hydrochloride azoiniciator and evaluated by the thiocyanate method was assessed. Antioxidant activity (pIC(50)) was correlated with 19 molecule parameters calculated for the minimum energy conformers of the compounds tested. The linear regression analysis revealed that the parameter of hydration energy E(HYDR) (R = -0.747) was the best predictor of antioxidant activity in a liposomal system.

Structural aspects of flavonoids as trypsin inhibitors.

In the search for new proteinase inhibitors we have focused on the screening and Computer Assisted Drug Design (CADD) studies of polyphenolic compounds. In this paper we report CADD of flavonoles and flavones as trypsin inhibitors concomitant by the screening results. 5,7-Dihydroxy flavonoid have been found to be a perspective trypsin/trypsin-like-enzyme inhibitor. Flavanones and isoflavones are less effective trypsin inhibitors due to a lost of the optimal geometry leading to hydrogen bond interactions. Four different interaction modes were observed, flavonoids are stabilised in S(1) region of beta-trypsin by formation of two (apigenin) or at least one hydrogen bond and other significant electrostatic interactions. Quercetin, myricetin and morin have shown to be the best trypsin inhibitors tested. In general, flavonoids with suitably located hydroxy groups and planar conformation are the building blocks able to replace guanidinobenzoyl part of successful inhibitors. Physiological nature of flavonoids reveals biotechnological source of new trypsin inhibitors as antipancreatitis, anticancer and anti-inflammation drugs.

Improved selectivity of microbial biosensor using membrane coating. Application to the analysis of ethanol during fermentation.

A ferricyanide mediated microbial biosensor for ethanol detection was prepared by surface modification of a glassy carbon electrode. The selectivity of the whole Gluconobacter oxydans cell biosensor for ethanol determination was greatly enhanced by the size exclusion effect of a cellulose acetate (CA) membrane. The use of a CA membrane increased the ethanol to glucose sensitivity ratio by a factor of 58.2 and even the ethanol to glycerol sensitivity ratio by a factor of 7.5 compared with the use of a dialysis membrane. The biosensor provides rapid and sensitive detection of ethanol with a limit of detection of 0.85 microM (S/N=3). The selectivity of the biosensor toward alcohols was better compared to previously published enzyme biosensors based on alcohol oxidase or alcohol dehydrogenases. The biosensor was successfully used in an off-line monitoring of ethanol during batch fermentation by immobilized Saccharomyces cerevisiae cells with an initial glucose concentration of 200 g l(-1).

Properties of hydrogel materials used for entrapment of microbial cells in production of fermented beverages.

Approaches using immobilized biological materials are very promising for application in different branches of the food industry, especially in the production of fermented beverages. Materials tested by our team for the process of entrapment belong to the family of charged polysaccharides able to form beaded hydrogels by ionotropic gelation (e.g. alginate, pectate, kappa-carrageenan) and synthetic polymers (e.g. polyvinyl alcohol) forming bead- and lens-shaped hydrogels by thermal sol/gel transition. Concentration of a gel, conditions and instrumentation of gelation process, bead and size distribution, porosity, diffusion properties, mechanical, storage and operational stability, and many other parameters were followed and optimized. Our work has been oriented especially to practical applications of immobilized cells. Brewing yeast cells were successfully immobilized by entrapment materials and used in a process of batch and continual production of beer, including primary and secondary fermentation of wort. Other applications include continual production of ethanol by fermentation of different saccharide substrates (molasses, glucose syrup, wheat hydrolysate), mead and non-alcoholic beverages production.

Indirect evidence of direct electron communication between the active site of galactose oxidase and a graphite electrode.

Bi-enzymatic biosensor based on galactose oxidase (GalOD) and horseradish peroxidase (HRP) using ferrocene as an efficient mediator was constructed. When a dependence of a working potential on the sensor performance was examined, an unusual behaviour was observed. With increasing of an applied working potential a lower concentration of substrate to attain full linear range was needed. A fully linear dependence from the first substrate addition was observed at and above the working potential of 150 mV. This activation of the biosensor response by an applied working potential very well corresponds with a formal potential of GalOD (156 mV). When a membrane prevented GalOD access to the electrode surface was applied, no activation effect of a working potential on the sensor performance was observed. Thus, it can be assumed that direct electron communication between GalOD and the electrode occurred.

Amperometric urea biosensor based on urease and electropolymerized toluidine blue dye as a pH-sensitive redox probe.

The electropolymerized toluidine blue film deposited on the glassy carbon electrode show amperometrically detectable pH sensitivity. This feature of polytoluidine blue (PTOB) film was used for a construction of an amperometric urea biosensor. We have observed a linear shift of the formal redox potential with increasing pH value between 4 and 8 giving the slope of 81 mV(Delta) pH(-1). Polytoluidine blue film has had a significantly increased stability and higher electrochemical activity compared to the adsorbed monomeric dye. The polytoluidine blue urea biosensor has been operating at a working potential of -200 mV vs. SCE. The sensitivity of the biosensor was 980 nA mM(-1) cm(-2). The biosensor showed linearity in concentration range up to 0.8 mM with the detection limit of 0.02 mM (S/N=3).

Monitoring of ethanol during fermentation using a microbial biosensor with enhanced selectivity.

The present study is concerning the construction of ferricyanide-mediated Gluconobacter oxydans cell ethanol biosensor. The size exclusion effect of a cellulose acetate membrane was used for elimination of glucose interferences during ethanol assays in real samples. A typical response time of the biosensor was 13 s with a high sensitivity of 3.5 microA mM(-1). The microbial biosensor exhibits a very low detection limit of 0.85 microM and a wide linear range from 2 to 270 microM. The operational stability was excellent. During 8.5 h of repetitive ethanol assays, no decrease in the sensor sensitivity was observed. The biosensor was successfully used in the off-line monitoring of ethanol fermentation with a good agreement with HPLC measurements (R(2)=0.998).

Production of non-alcoholic beer using free and immobilized cells of Saccharomyces cerevisiae deficient in the tricarboxylic acid cycle.

Production of non-alcoholic beer using Saccharomyces cerevisiae has been studied. Non-recombinant mutant strains with a defect in the synthesis of tricarboxylic-acid-cycle enzymes were used and applied in both free and pectate-immobilized form, using both batch and packed-bed continuous systems. After fermentation, basic parameters of the beer produced by five mutant strains were compared with a standard strain of brewing yeast. Results showed that the beer prepared by mutant yeast cells was characterized by lower levels of total alcohols, with ethanol concentrations between 0.07 and 0.31% (w/w). The organic acids produced, especially lactic acid, in concentrations up to 1.38 g x l(-1) had a strong protective effect on the microbial stability of the final product and thus the usual addition of lactic acid could be omitted. Application of the yeast mutants appears to be a good alternative to the classical methods for the production of non-alcoholic beer.

Stabilization of ferrocene leakage by physical retention in a cellulose acetate membrane. The fructose biosensor.

The prevention of ferrocene leakage from an electrode by physical retention of mediator in a matrix of cellulose acetate membrane is reported. Five types of the cellulose acetate membranes were prepared, containing 1.8%, 5.3%, 8.5%, 20.0% of ferrocene and a membrane containing 1.8% of ferrocene and 0.05 % of Nafion in the matrix. Ferrocene embedded membranes were successfully applied in the construction of a fructose biosensor by immobilization of PQQ-dependent fructose dehydrogenase (FDH). The biosensor comprising a cellulose acetate membrane with 1.8% of ferrocene and 0.05% of Nafion had good stability characteristics, retained almost 40% of the initial response after 8 h of continuous use with an initial sensitivity of 226 nA mM(-1) and response time of 75 s.