Reactivity of phenolic compounds towards free radicals under in vitro conditions.

The free radical scavenging activity and reducing power of 16 phenolic compounds including four hydroxycinnamic acid derivatives namely ferulic acid, caffeic acid, sinapic acid and p-coumaric acid, benzoic acid and its derivatives namely protocatechuic acid, gallic acid and vanillic acid, benzene derivatives namely vanillin, vanillyl alcohol, veratryl alcohol, veratraldehyde, pyrogallol, guaiacol and two synthetic antioxidants, butylated hydroxy anisole (BHA) and propyl gallate were evaluated using 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH(•)), 2,2'-Azinobis-3- ethylbenzothiazoline-6-sulfonic acid radical (ABTS(+•)), Hydroxyl radical ((•)OH) and Superoxide radical (O2 (•-)) scavenging assays and reduction potential assay. By virtue of their hydrogen donating ability, phenolic compounds with multiple hydroxyl groups such as protocatechuic acid, pyrogallol, caffeic acid, gallic acid and propyl gallate exhibited higher free radical scavenging activity especially against DPPH(•) and O2 (•-). The hydroxylated cinnamates such as ferulic acid and caffeic acid were in general better scavengers than their benzoic acid counter parts such as vanillic acid and protocatechuic acid. All the phenolic compounds tested exhibited more than 85 % scavenging due to the high reactivity of the hydroxyl radical. Phenolic compounds with multiple hydroxyl groups also exhibited high redox potential. Exploring the radical scavenging and reducing properties of antioxidants especially those which are found naturally in plant sources are of great interest due to their protective roles in biological systems.

Lysosomal destabilization and cathepsin B contributes for cytochrome c release and caspase activation in embelin-induced apoptosis.

XIAP is an important antiapoptotic protein capable of conferring resistance to cancer cells. Embelin, the small molecular inhibitor of XIAP, possesses wide spectrum of biological activities with strong inhibition of nuclear factor kappa B and downstream antiapoptotic genes. However, the mechanism of its cell death induction is not known. Our studies using colon cancer cells lacking p53 and Bax suggest that both lysosomes and mitochondria are prominent targets of embelin-induced cell death. Embelin induced cell-cycle arrest in G(1) phase through p21, downstream of p53. In the absence of p21, the cells are sensitized to death in a Bax-dependent manner. The loss of mitochondrial membrane potential induced by embelin was independent of Bax and p53, but lysosomal integrity loss was strongly influenced by the presence of p53 but not by Bax. Lysosomal role was further substantiated by enhanced cathepsin B activity noticed in embelin-treated cells. p53-dependent lysosomal destabilization and cathepsin B activation contribute for increased sensitivity of p21-deficient cells to embelin with enhanced caspase 9 and caspase 3 activation. Cathepsin B inhibitor reduced cell death and cytochrome c release in embelin-treated cells indicating lysosomal pathway as the upstream of mitochondrial death signaling. Deficiency of cell-cycle arrest machinery renders cells more sensitive to embelin with enhanced lysosomal destabilization and caspase processing emphasizing its potential therapeutic importance to address clinical drug resistance.

Nanocomposite based on modified TiO2-BSA for functional applications.

A novel nanocomposite based on TiO(2)-protein for functional applications was prepared and characterized. The composite was made by covalent immobilisation of bovine serum albumin (BSA) on to the TiO(2) particles. BSA was attached to the TiO(2) powder through a three-step process. This involves the functionalization of the TiO(2) particles with silane and the subsequent coupling by glutaraldehyde to free NH(2) groups of the protein. Formation of Schiff's base was confirmed by IR spectroscopy and the optimum loading of the BSA was found to be 73.61%. Surface morphology of the composite was studied by SEM and TEM. Thermal analysis of TiO(2)-BSA composite was carried out by TGA and DSC. Structural variation of the BSA after immobilisation was studied by CD spectra.

Preparation and characterization of cross-linked enzyme aggregates (CLEA) of subtilisin for controlled release applications.

Enzyme stabilization is one of the major challenges in the biocatalytic process optimization. Subtilisin was aggregated using ammonium sulphate and polyethylene glycol with surfactants like triton X-100 and tween 20. The resultant aggregates on cross-linking with glutaraldehyde produced insoluble and catalytically active enzyme. The effect of pH, temperature, kinetic parameter, thermal stability and stability in organic solvents were studied. The cross-linked enzyme aggregates (CLEA) exhibited broad pH optima of 9.0 and higher temperature optima of 70 degrees C. Reusability and surface morphology of the CLEA were also studied. CLEA of subtilisin has good stability in nonpolar organic solvents, such as hexane, and cyclohexane and it has high thermal stability up to 60 degrees C and therefore can be used as a catalyst for the biotransformation of compounds which are not soluble in aqueous medium. The CLEAs were entrapped in the hydrogel composite beads of alginate:guar gum (3:1) which were resistant to low pH conditions in the stomach and thus was found to be useful for the oral drug delivery. This process can be used to deliver the protein and peptide drugs which involve high concentrations at the delivery stage, and which usually degrades in the stomach before reaching the jejunum. Application of these pH-sensitive beads for the controlled release of subtilisin in vitro was studied and found to be a feasible strategy.

Biosorption of anionic textile dyes by nonviable biomass of fungi and yeast.

The nonviable biomass of Aspergillus niger, Aspergillus japonica, Rhizopus nigricans, Rhizopus arrhizus, and Saccharomyces cerevisiae were screened for biosorption of textile dyes. The selected anionic reactive dyes were C.I. Reactive Black 8, C.I. Reactive Brown 9, C.I. Reactive Green 19, C.I. Reactive Blue 38, and C.I. Reactive Blue 3. Experiments were conducted at initial dye concentration of 50, 100, 150 and 200mg/L. The effect of initial dye concentration, dose of biosorbent loading, temperature, and pH on adsorption kinetics was studied. S. cerevisiae and R. nigricans were good biosorbents at initial dye concentration of 50mg/L, 1g% (w/v) biomass loading and 29+/-1 degrees C. R. nigricans adsorbed 90-96% dye in 15min, at 20 degrees C and pH 6.0. The data showed an optimal fit to the Langmuir and Freundlich isotherms. The maximum uptake capacity (Q(o)) for the selected dyes was in the range 112-204mg/g biomass.

Bioconversions of ferulic acid, an hydroxycinnamic acid.

Ferulic acid is the most abundant hydroxycinnamic acid in the plant world and is ester linked to arabinose, in various plant polysaccharides such as arabinoxylans and pectins. It is a precursor to vanillin, one of the most important aromatic flavor compound used in foods, beverages, pharmaceuticals, and perfumes. This article presents an overview of the various biocatalytic routes, focusing on the relevant biotransformations of ferulic acid using plant sources, microorganisms, and enzymes.

Polyionic hydrocolloids for the intestinal delivery of protein drugs: alginate and chitosan--a review.

The protein pharmaceutical market is rapidly growing, since it is gaining support from the recombinant DNA technology. To deliver these drugs via the oral route, the most preferred route, is the toughest challenge. In the design of oral delivery of peptide or protein drugs, pH sensitive hydrogels like alginate and chitosan have attracted increasing attention, since most of the synthetic polymers are immunogenic and the incorporation of proteins in to these polymers require harsh environment which may denature and inactivate the desired protein. Alginate is a water-soluble linear polysaccharide composed of alternating blocks of 1-4 linked alpha-L-guluronic and beta-D-mannuronic acid residues where as chitosan is a co polymer of D-glucosamine and N-acetyl glucosamine. The incorporation of protein into these two matrices can be done under relatively mild environment and hence the chances of protein denaturation are minimal. The limitations of these polymers, like drug leaching during preparation can be overcome by different techniques which increase their encapsulation efficiency. Alginate, being an anionic polymer with carboxyl end groups, is a good mucoadhesive agent. The pore size of alginate gel microbeads has been shown to be between 5 and 200 nm and coated beads and microspheres are found to be better oral delivery vehicles. Cross-linked alginate has more capacity to retain the entrapped drugs and mixing of alginate with other polymers such as neutral gums, pectin, chitosan, and eudragit have been found to solve the problem of drug leaching. Chitosan has only limited ability for controlling the release of encapsulated compound due to its hydrophilic nature and easy solubility in acidic medium. By simple covalent modifications of the polymer, its physicochemical properties can be changed and can be made suitable for the peroral drug delivery purpose. Ionic interactions between positively charged amino groups in chitosan and the negatively charged mucus gel layer make it mucoadhesive. The favourable properties like biocompatibility, biodegradability, pH sensitiveness, mucoadhesiveness, etc. has enabled these polymers to become the choice of the pharmacologists as oral delivery matrices for proteins.

Enzymatic modification of cassava starch by bacterial lipase.

Enzymatic modification of starch using long chain fatty acid makes it thermoplastic suitable for a myriad of industrial applications. An industrial lipase preparation produced by Burkholderia cepacia (lipase PS) was used for modification of cassava starch with two acyl donors, lauric acid and palmitic acid. Reactions performed with palmitic acid by liquid-state and microwave esterification gave a degree of substitution (DS) of 62.08% (DS 1.45) and 42.06% (DS 0.98), respectively. Thermogravimetric analysis showed that onset of decomposition is at a higher temperature (above 600 degrees Celsius) for modified starch than the unmodified starch (280 degrees Celsius). Modified starch showed reduction in alpha-amylase digestibility compared to native starch (76.5-18%). Swelling power lowered for modified starch as esterification renders starch more hydrophobic, making it suitable for biomedical applications as materials for bone fixation and replacements, carriers for controlled release of drugs and bioactive agents. Thus enzymatic esterification is ecofriendly.

Enzymatic esterification of starch using recovered coconut oil.

Modification of maize and cassava starches was done using recovered coconut oil and microbial lipase. Microwave esterification was advantageous as it gave a DS 1.55 and 1.1 for maize starch and cassava starch, respectively. Solution state esterification of cassava starch for 36 h at 60 degrees C gave a DS of 0.08 and semi-solid state esterification gave a DS of 0.43. TGA and DSC studies showed that the higher DS attributed to the thermostability, since onset of decomposition is at a higher temperature (492 degrees C) than the unmodified (330 degrees C) and was stable above 600 degrees C. alpha-Amylase digestibility and viscosity reduced for modified starch.

Kinetics and thermal stability of two peroxidase isozymes from Eupatorium odoratum.

The Eupatorium odoratum leaf peroxidase exists as at least seven distinct isozymes (three cationic, three anionic, and one neutral). These isozymes were identified and separated by preparative iso-electric focusing. Thermal stability, including the activation enthalpy (deltaH*), free energy of inactivation (deltaG*) and activation entropy (deltaS*), and kinetic studies of two isozymes, one having a pI of 5.0 (E5) and another one having a pI of 7.0 (E7) with mol mass of 43 and 50 kD, respectively, were studied in detail. Of the molecular weight of E5 and E7, 25 and 32% correspond to the carbohydrate content of the isozymes. Optimal pH was in the acidic range of 3.6-3.8 for E5 and 3.8 for E7 with the oxidation of ABTS. E7 and E5 showed activation energy for inactivation, 194.8 and 145.4 kJ/mol, respectively. Both the isozymes showed distinct substrate specificity. The catalytic specificity constant for E5 and E7 were 112 x 105 and 124 x 105/s.M, respectively, when 2,2'-azino-bis-(3-ethylbenz-thiazoline-6 sulfonic acid) was used as the substrate. Maximum affinity (i.e., lowest Km value) to H2O2 was shown by E5 and E7 along with Pyrogallol and was 0.02 and 0.05/s.M, respectively.

Microstructural imaging and characterization of the mechanical, chemical, thermal, and swelling properties of starch-chitosan blend films.

Chitosan has wide range of applications as a biomaterial, but barriers still exist to its broader use due to its physical and chemical limitations. The present study evaluated the properties of the polymeric blend films obtained from chitosan and potato starch by the casting/solvent evaporation method. The swelling properties of the different films studied as a function of pH showed that the sorption ability of the blend films increased with the increasing content of starch. Fourier transform infrared (FTIR) analyses confirmed that interactions were present between the hydroxyl groups of starch and the amino groups of chitosan in the blend films while the x-ray diffraction (XRD) studies revealed the films to exhibit an amorphous character. Thermogravimetric analyses showed that in the blend films, the thermal stability increased with the increasing starch content and the stability of starch and chitosan powders reduced when they were converted to film. The differential scanning calorimetry (DSC) studies revealed an endotherm corresponding to water evaporation around 100 degrees C in all the films and an exotherm, corresponding to the decomposition in the chitosan and blend films. Scanning electron microscopy (SEM) observations indicated that the blend films were less homogenous and atomic force microscopy (AFM) studies revealed the chitosan films to be smooth and homogenous, while the starch films revealed characteristic granular pattern. The blend films exhibited an intermediate character with a slight microphase separation. The starch-chitosan blend films exhibited a higher flexibility and incorporation of potato starch into chitosan films improved the percentage elongation.

In vitro antioxidant activity and scavenging effects of Cinnamomum verum leaf extract assayed by different methodologies.

The free radical scavenging capacity and antioxidant activities of the methanolic extract of Cinnamomum verum leaf (CLE) were studied and compared to antioxidant compounds like trolox, butylated hydroxyl anisole, gallic acid and ascorbic acid. The CLE exhibited free radical scavenging activity, especially against DPPH radical and ABTS radical cation. They also exhibited reducing power and metal ion chelating activity, along with hydroxyl radical scavenging activity. The peroxidation inhibiting activity of CLE recorded using the linoleic acid emulsion system, showed very good antioxidant activity.

Continuous adsorption and recovery of Cr(VI) in different types of reactors.

This study reports the results of experiments on continuous adsorption and desorption of Cr(VI) ions by a chemically modified and polysulfone-immobilized biomass of the fungus Rhizopus nigricans. A fixed quantity of polymer-entrapped biomass beads corresponding to 2 g of dry biomass powder was employed in packed bed, fluidized bed, and stirred tank reactor for monitoring the continuous removal and recovery of Cr(VI) ions from aqueous solution and synthetic chrome plating effluent. Parameters such as flow rate (5, 10 and 15 mL/min), inlet concentration of Cr(VI) ions (50, 100, 150 and 250 mg/L) and the depth of biosorbent packing (22.8, 11.2 and 4.9 cm) were evaluated for the packed bed reactor. The breakthrough time and the adsorption rates in the packed bed column were found to decrease with increasing flow rate and higher Cr inlet concentrations and to increase with higher depths of sorbent packing. To have a comparative analysis of Cr adsorption efficiency in different types of reactors, the fluidized bed reactor and stirred tank reactor were operated using the same quantities of biosorbent material. For the fluidized bed reactor, Cr(VI) solution of 100 mg/L was pumped at 5 mL/min and fluidized by compressed air at a flow rate of 0.5 kg/cm.(2) The stirred tank reactor had a working volume of 200 mL capacity and the inlet/outlet flow rate was 5 mL/min. The maximum removal efficiency (mg Cr/g biomass) was obtained for the stirred tank reactor (159.26), followed by the fluidized reactor (153.04) and packed bed reactor (123.33). In comparison to the adsorption rate from pure chromate solution, approximately 16% reduction was monitored for synthetic chrome plating effluent in the packed bed. Continuous desorption of bound Cr ions from the reactors was effective with 0.01 N Na(2)CO(3) and nearly 80-94% recoveries have been obtained for all the reactors.

Biosoftening of coir fiber using selected microorganisms.

Coir fiber belongs to the group of hard structural fibers obtained from coconut husk. As lignin is the main constituent of coir responsible for its stiffness, microbes that selectively remove lignin without loss of appreciable amounts of cellulose are extremely attractive in biosoftening. Five isolated strains were compared with known strains of bacteria and fungi. The raw fiber treated with Pseudomonas putida and Phanerocheate chrysosporium produced better softened fiber at 30+/-2 degrees C and neutral pH. FeSO4 and humic acid were found to be the best inducers for P. chrysosporium and P. putida, respectively, while sucrose and dextrose were the best C-sources for both. Biosoftening of unretted coir fibers was more advantageous than the retted fibers. Unlike the weak chemically softened fiber, microbial treatment produced soft, whiter fibers having better tensile strength and elongation (44.6-44.8%) properties. Scanning electron microscopy photos showed the mycelia penetrating the pores of the fiber, removing the tylose plug and degrading lignin.

Biosensor for the determination of phenols based on cross-linked enzyme crystals (CLEC) of laccase.

Cross-linked enzyme crystals (CLECs) are a versatile form of biocatalyst that can also be used for biosensor application. Laccase from Trametes versicolor (E.C.1.10.3.2) was crystallized, cross-linked and lyophilized with beta-cyclodextrin. The CLEC laccase was found to be highly active towards phenols like 2-amino phenol, guaiacol, catechol, pyrogallol, catechin and ABTS (non-phenolic). The CLEC laccase was embedded in 30% polyvinylpropylidone (PVP) gel and mounted into an electrode to make the sensor. The biosensor was used to detect the phenols in 50-1000 micromol concentration level. Phenols with lower molecular weight such as 2-amino phenol, catechol and pyrogallol gave a short response time where as the higher molecular weight substrates like catechin and ABTS had comparatively a long response time. The optimum pH of the analyte was 5.5-6.0 when catechol was used as substrate. The CLEC laccase retained good activity for over 3 months.

Ferulic acid: an antioxidant found naturally in plant cell walls and feruloyl esterases involved in its release and their applications.

Ferulic acid is the most abundant hydroxycinnamic acid in the plant world and maize bran with 3.1% (w/w) ferulic acid is one of the most promising sources of this antioxidant. The dehydrodimers of ferulic acid are important structural components in the plant cell wall and serve to enhance its rigidity and strength. Feruloyl esterases are a subclass of the carboxylic acid esterases that hydrolyze the ester bond between hydroxycinnamic acids and sugars present in plant cell walls and they have been isolated from a wide range of microorganisms, when grown on complex substrates such as cereal brans, sugar beet pulp, pectin and xylan. These enzymes perform a function similar to alkali in the deesterification of plant cell wall and differ in their specificities towards the methyl esters of cinnamic acids and ferulolylated oligosaccharides. They act synergistically with xylanases and pectinases and facilitate the access of hydrolases to the backbone of cell wall polymers. The applications of ferulic acid and feruloyl esterase enzymes are many and varied. Ferulic acid obtained from agricultural byproducts is a potential precursor for the production of natural vanillin, due to the lower production cost.

Bioremediation of textile azo dyes by aerobic bacterial consortium.

An aerobic bacterial consortium consisting of two isolated strains (BF1, BF2) and a strain of Pseudomonas putida (MTCC1194) was developed for the aerobic degradation of a mixture of textile azodyes and individual azodyes at alkaline pH (9-10.5) and salinity (0.9-3.68 g/l) at ambient temperature (28 +/- 2 degrees C). The degradation efficiency of the strains in different media (mineral media and in the Simulated textile effluent (STE)) and at different dye concentrations were studied. The presence of a H2O2 independent oxidase-laccase (26.5 IU/ml) was found in the culture filtrate of the organism BF2. The analysis of the degraded products by TLC and HPLC, after the microbial treatment of the dyes showed the absence of amines and the presence of low molecular weight oxidative degradation products. The enzymes present in the crude supernatant was found to be reusable for the dye degradation.

Bioremediation of textile azo dyes by an aerobic bacterial consortium using a rotating biological contactor.

The degradation of an azo dye mixture by an aerobic bacterial consortium was studied in a rotating biological reactor. Laterite pebbles of particle size 850 microm to 1.44 mm were fixed on gramophone records using an epoxy resin on which the developed consortium was immobilized. Rate of degradation, BOD, biomass determination, enzymes involved, and fish bioassay were studied. The RBC has a high efficiency for dye degradation even at high dye concentrations (100 microg/mL) and high flow rate (36 L/h) at alkaline pH and salinity conditions normally encountered in the textile effluents. Bioassays (LD-50) using Thilapia fish in treated effluent showed that the percentage mortality was zero over a period of 96 h, whereas the mortality was 100% in untreated dye water within 26 h. Fish bioassay confirms that the effluent from RBC can be discharged safely to the environment.

Studies on chromium(VI) adsorption-desorption using immobilized fungal biomass.

The aim of this study was to investigate the Cr(VI) biosorption potential of immobilized Rhizopus nigricans and to screen a variety of non-toxic desorbing agents, in order to find out possible application in multiple sorption-desorption cycles. The biomass was immobilized by various mechanisms and evaluated for removal of Cr(VI) from aqueous solution, mechanical stability to desorbents, and reuse in successive cycles. The finely powdered biomass, entrapped in five different polymeric matrices viz. calcium alginate, polyvinyl alcohol (PVA), polyacrylamide, polyisoprene, and polysulfone was compared for biosorption efficiency and stability to desorbents. Physical immobilization to polyurethane foam and coir fiber was less efficient than polymer entrapment methods. Of the different combinations (%, w/v) of biomass dose compared for each matrix, 8% (calcium alginate), 6% (polyacrylamide and PVA), 12% (polyisoprene), and 10% (polysulfone) were found to be the optimum. The Cr sorption capacity (mg Cr/g sorbent) of all immobilized biomass was lesser than the native, powdered biomass. The Cr sorption capacity decreased in the order of free biomass (119.2) > polysulfone entrapped (101.5) > polyisoprene immobilized (98.76) > PVA immobilized (96.69) > calcium alginate entrapped (84.29) > polyacrylamide (45.56), at 500 mg/l concentration of Cr(VI). The degree of mechanical stability and chemical resistance of the immobilized systems were in the order of polysulfone > polyisoprene > PVA > polyacrylamide > calcium alginate. The bound Cr(VI) could be eluted successfully using 0.01 N NaOH, NaHCO3, and Na2CO3. The adsorption data for the native and the immobilized biomass was evaluated by the Freundlich isotherm model. The successive sorption-desorption studies employing polysulfone entrapped biomass indicated that the biomass beads could be regenerated and reused in more than 25 cycles and the regeneration efficiency was 75-78%.

Aerobic degradation of a mixture of azo dyes in a packed bed reactor having bacteria-coated laterite pebbles.

A microbial consortium capable of aerobic degradation of a mixture of azo dyes consisting of two isolated strains (RRL,TVM) and one known strain of Pseudomonas putida (MTCC 1194) was immobilized on laterite stones. The amount of bacterial biomass attached to the laterite stones was 8.64 g per 100 g of the stone on a dry weight basis. The packed bed reactor was filled with these stones and had a total capacity of 850 mL and a void volume of 210 mL. The feed consisted of an equal mixture of seven azo dyes both in water as well as in a simulated textile effluent, at a pH of 9.0 and a salinity of 900 mg/L. The dye concentrations of influent were 25, 50, and 100 microg/mL. The residence time was varied between 0.78 and 6.23 h. It was found that at the lowest residence time 23.55, 45.73, and 79.95 microg of dye was degraded per hour at an initial dye concentration of 25, 50, and 100 microg, respectively. The pH was reduced from 9.0 to 7.0. Simulated textile effluent containing 50 microg/mL dye was degraded by 61.7%. Analysis of degradation products by TLC and HPLC showed that the dye mixture was degraded to nontoxic smaller molecules. The bacteria-coated pebbles were stable, there was no washout even after 2 months, and the reactor was found to be suitable for the aerobic degradation of azo dyes.