Antibacterial Applications of Silver Nanoparticles Synthesized by Aqueous Extract of Azadirachta Indica (Neem) Leaves

Silver nanoparticles are known to have bactericidal effects. A new generation of dressings incorporating antimicrobial agents like silver nanoparticles is being formulated to reduce or prevent infections. The particles can be incorporated in materials and cloth rendering them sterile. Recently, it was found that aqueous silver ions can be reduced by aqueous extract of plant parts to generate extremely stable silver nanoparticles in water. Apart from being environmentally friendly process, use of Neem leaves extract might add synergistic antibacterial effect of Neem leaves to the biosynthesized nanoparticles. With this hypothesis the biosynthetic production of silver nanoparticles by aqueous extract of Neem leaves and its bactericidal effect in cotton cloth against E. Coli were studied in this work. Silver nanoparticles were synthesized by short term (1 day) and long term (21 days) interaction of Neem extract (20% w/v) and 0.01 M AgNO3 solution in 1:4 mixing ratio. The synthesized particles were characterized by UV visible spectroscopy, transmission electron microscopy, and incorporated into cotton disks by (i) centrifuging the disks with liquid broth containing nanoparticles, (ii) in-situ coating process during synthesis, and (iii) coating with dried and purified nanoparticles. The antibacterial property of the nanoparticles coated cotton disks was studied by disk diffusion method. The effect of consecutive washing of the coated disks with distilled water on antibacterial property was also investigated. This work demonstrates the possible use of biologically synthesized silver nanoparticles by its incorporation in cloths leading them to sterilization.

Purification and Characterization of a New Indole Oxygenase from the Leaves of Tecoma stans L.

A new indole oxygenase from the leaves of Tecoma stans was isolated and purified to homogenity. The purified enzyme system catalyzes the conversion of indole to anthranilic acid. It is optimally active at pH 5.2 and 30°C. Two moles of oxygen are consumed and one mole of anthranilic acid is formed for every mole of indole oxidized. Dialysis resulted in complete loss of the activity. The inactive enzyme could be reactivated by the addition of concentrated dialysate. The enzyme is not inhibited by copperspecific chelators, non-heme iron chelators or atebrin. It is not a cuproflavoprotein, unlike the other indole oxygenases and oxidases.

Indolic compounds in the leaves of Tecoma stans

Indole, tryptophan, tryptamine and skatole were isolated from the leaves of Tecoma stans. Anthranilic acid was also identified in its free form, in contrast to its glucoside, in Jasminum grandiflorum. The presence of both indole and anthranilic acid in the leaves of Tecoma stans indicates that they are the true substrate and product of indole oxygenase from the leaves of Tecoma stans.

Indole oxygenase from the leaves of Tecoma stans

A new indole oxygenase from the leaves of Tecoma stans was isolated and purified to near homogeneity. The purified enzyme system catalyses the conversion of indole to anthranilic acid. It is optimally active at pH 5.2 and at 30°C. Oxygen (2 mol) is consumed and anthranilic acid (1 mol) is formed for every mole of indole oxidized. Neither sulfhydryl reagents nor sulfhydryl compounds inhibited the enzyme activity. The oxygenase also attacks, apart from indole, 5-hydroxyindole, 5-bromoindole and 5-methylindole. It is not inhibited by copper specific chelators or non-heme iron specific chelators. Atebrin did not inhibit the enzyme activity suggesting that it is not a flavoprotein, unlike other indole oxygenases and indole oxidases. Dialysis resulted in complete loss of enzyme activity. The inactive enzyme could not be reactivated by addition of various cofactors.

Alterations in the activities of the enzymes of proline metabolism in Ragi (Eleusine coracana) leaves during water stress

Free proline content in Ragi (Eleusine coracana) leaves increased markedly (6 to 85 fold) as the degree of water stress, created by polyethylene gylcol treatment, was prolonged There was also a marginal increase in soluble proteins in the stressed leaves as compared to that in the controls. Water stress stimulated the activities of ornithine aminotransferase and pyrroline-5-carboxylate reductase, the enzymes of proline biosynthesis and markedly inhibited the enzymes involved in proline degradation viz., proline oxidase and pyrroline-5-carboxylate dehydrogenase. These results suggest that increase in free proline content of Ragi leaves could be due to enhanced activities of the enzymes synthesizing proline but more importantly due to severe inhibition of the enzymes degrading proline. These observations establish for the first time, the pathway of proline metabolism in plants by way of detection of the activities of all the enzymes involved and also highlight the role of these enzymes in proline accumulation during water stress.

oxidation of catechol in plants iv. purification and properties of the 3,4,3',4'-tetrahydroxydiphenyl-forming enzyme system from tecoma leaves

Acetone powders prepared from leaf extracts of Tecoma stans L. were found to catalyze the oxidation of catechol to 3,4,3',4'-tetrahydroxydiphenyl. Fractionation of the acetone powders obtained from Tecoma leaves with acetone, negative adsorption of the acetone fraction with tricalcium phosphate gel, and chromatography of the gel supernatant on DEAE-Sephadex yielded a 68-fold purified enzyme with 66% recovery. The enzyme had an optimum pH around 7.2. It showed a temperature optimum of 30° and the Km for catechol was determined as 2 x 10-4 m. The purified enzyme moved as a single band on polyacrylamide gel electrophoresis. Its activity was found to be partially stimulated by Mg2+. The reaction was not inhibited by o-phenanthroline and agr,agr'-dipyridyl. The purified enzyme was highly insensitive to a range of copper-chelating agents. It was not affected appreciably by thiol inhibitors. The reaction was found to be suppressed to a considerable extent by reducing agents like GSH, cysteine, cysteamine, and ascorbic acid. The purified enzyme was remarkably specific for catechol. Catalase affected neither the enzyme activity nor the time course of the reaction. Hydrogen peroxide was not formed as a product of the reaction.

Nitrogenase activity (acetylene reduction) associated with leaves of different cultivars of paddy

An investigation was conducted to study the levels of nitrogen fixation on the leaf or sheath surfaces of four cultivars of paddy plants by using acetylene reduction technique. Varying levels of positive nitrogenase activity were observed on all the leaf surfaces. Sheath of IET 1991 cultivar showed a higher rate of fixation than the leaf surface. All the nitrogen-fixing organisms on the leaf or sheath surfaces belonged to the genus Beijerinckia. There was no correlation between the bacterial density and the level of fixation. Scanning electron microscopic data revealed that the upper surface of IET 1991 leaf was highly silicified and the microflora was either scanty or nil while the lower surface appeared quite different and harboured more micro-organisms. Similarly, the inner surface of sheath was devoid of silicification and showed the presence of micro-organisms.

Nitrogenase activity (acetylene reduction) associated with leaves of different cultivars of paddy (Oryza sativa L.)

An investigation was conducted to study the levels of nitrogen fixation on the leaf or sheath surfaces of four cultivars of paddy plants by using acetylene reduction technique. Varying levels of positive nitrogenase activity were observed on all the leaf surfaces. Sheath of IET 1991 cultivar showed a higher rate of fixation than the leaf surface. All the nitrogen-fixing organisms on the leaf or sheath surfaces belonged to the genus Beijerinckia. There was no correlation between the bacterial density and the level of fixation. Scanning electron microscopic data revealed that the upper surface of IET 1991 leaf was highly silicified and the microflora was either scanty or nil while the lower surface appeared quite different and harboured more micro-organisms. Similarly, the inner surface of sheath was devoid of silicification and showed the presence of micro-organisms.

Variations in the Levels of Chloroplast tRNAs and Aminoacyl-tRNA Synthetases in Senescing Leaves of Phaseolus vulgaris

The relative amounts of chloroplast tRNAs(Leu), tRNA(Glu), tRNA(Phe), tRNAs(Thr), and tRNA(Tyr) and of chloroplastic and cytoplasmic aminoacyl-tRNA synthetases were compared in green leaves, yellowing senescing leaves, and N(6)-benzyladenine-treated senescing leaves from bean (Phaseolus vulgaris, var Contender). Aminoacylation of the tRNAs using Escherichia coli aminoacyl-tRNA synthetases indicated that in senescing leaves the relative amount of chloroplast tRNA(Phe) was significantly lower than in green leaves. Senescing leaves treated with N(6)-benzyladenine contained higher levels of this tRNA than untreated senescing leaves. No significant change in the relative amounts of chloroplast tRNAs(Leu), tRNAs(Thr), and tRNA(Tyr) was detected in green, yellow senescing, or N(6)-benzyladine-treated senescing leaves. Relative levels of chloroplast tRNAs were also estimated by hybridization of tRNAs to DNA blots of gene specific probes. These experiments confirmed the results obtained by aminoacylation and revealed in addition that the relative level of chloroplast tRNA(Glu) is higher in senescing leaves than in green leaves. Transcription run-on assays indicated that these changes in tRNA levels are likely to be due to a differential rate of degradation rather than to a differential rate of transcription of the tRNA genes. Chloroplastic and cytoplasmic leucyl-, phenylalanyl-, and tyrosyl-tRNA synthetase activities were greatly reduced in senescing leaves as compared to green leaves, whereas N(6)-benzyladenine-treated senescing leaves contained higher enzyme activities than untreated senescing leaves. These results suggest that during senescence, as well as during senescence-retardation by cytokinins, changes in enzyme activities, such as aminoacyl-tRNA synthetases, rather than reduced levels of tRNAs, affect the translational capacity of chloroplasts.

Process variables in biomimetic synthesis of silver nanoparticles by aqueous extract of Azadirachta indica (Neem) leaves

Owing to widespread applications, synthesis and characterization of silver nanoparticles is recently attracting considerable attention. Increasing environmental concerns over chemical synthesis routes have resulted in attempts to develop biomimetic approaches. One of them is synthesis using plant parts, which eliminates the elaborate process of maintaining the microbial culture and often found to be kinetically favourable than other bioprocesses. The present study deals with investigating the effect of process variables like reductant concentrations, reaction pH, mixing ratio of the reactants and interaction time on the morphology and size of silver nanoparticles synthesized using aqueous extract of Azadirachta indica (Neem) leaves. The formation of crystalline silver nanoparticles was confirmed using X-ray diffraction analysis. By means of UV spectroscopy, Scanning and Transmission Electron Microscopy techniques, it was observed that the morphology and size of the nanoparticles were strongly dependent on the process parameters. Within 4 h interaction period, nanoparticles below 20-nm-size with nearly spherical shape were produced. On increasing interaction time (ageing) to 66 days, both aggregation and shape anisotropy (ellipsoidal, polyhedral and capsular) of the particles increased. In alkaline pH range, the stability of cluster distribution increased with a declined tendency for aggregation of the particles. It can be inferred from the study that fine tuning the bioprocess parameters will enhance possibilities of desired nano-product tailor made for particular applications.

An indole oxidase isolated from the leaves of Tecoma stans

The presence of an indole oxidase (indole: O2 oxidoreductase) was detected in the leaf extracts of Tecoma stans. The end product of the reaction was identified as anthranil. Formylaminobenzaldehyde, and o- aminobenzaldehyde were detected as intermediates in the overall conversion. Oxygen-uptake studies established that 3 atoms of oxygen were consumed in the formation of anthranil form I molecule of indole. The enzyme showed an absolute requirement for FAD and Cu2+ for maximum activity. FMN was ineffective as a cofactor. The enzyme had an optimum pH of 5.0. Inhibition studies with GSH and p-chloromericuribenzoate showed that a sulfhydrylcupric-ion complex at the active centre is highly essential.

Enzymatic conversion of anthranilic acid to catechol by chloroplast from the leaves of Tecoma stans

An enzyme system which converts anthranilic acid to catechol was detected in the leaves of Tecoma stans, and its properties studied. The system is present exclusively in the chloroplast fraction of the leaves. The optimum pH of the reaction is 5·2 and maximum activity was obtained with citrate-phosphate buffer. There was good stoichiometry between the amounts of anthranilic acid disappeared and the amounts of catechol and ammonia formed. The enzyme system showed an absolute requirement for oxygen and evidence was obtained for the probable participation of NADPH and FAD in the hydroxylation step. The optimum concentration of anthranilic acid was 10−4 M; at higher concentrations the reaction was inhibited to a considerable extent. Cyanide, pyrophosphate, and EDTA also caused inhibition indicating a requirement for metal ions.

Oxidation of catechol in plants : III. Purification and properties of the diphenylene dioxide 2,3-quinone-forming enzyme system from Tecoma leaves

In attempting to determine the nature of the enzyme system mediating the conversion of catechol to diphenylenedioxide 2,3-quinone, in Tecoma leaves, further purification of the enzyme was undertaken. The crude enzyme from Tecoma leaves was processed further by protamine sulfate precipitation, positive adsorption on tricalcium phosphate gel, and elution and chromatography on DEAE-Sephadex. This procedure yielded a 120-fold purified enzyme which stoichiometrically converted catechol to diphenylenedioxide 2,3-quinone. The purity of the enzyme system was assessed by polyacrylamide gel electrophoresis. The approximate molecular weight of the enzyme was assessed as 200,000 by gel filtration on Sephadex G-150. The enzyme functioned optimally at pH 7.1 and at 35 °C. The Km for catechol was determined as 4 × 10−4 Image . The enzyme did not oxidize o-dihydric phenols other than catechol and it did not exhibit any activity toward monohydric and trihydric phenols and flavonoids. Copper-chelating agents did not inhibit the enzyme activity. Copper could not be detected in the purified enzyme preparations. The purified enzyme was not affected by extensive dialysis against copper-complexing agents. It did not show any peroxidase activity and it was not inhibited by catalase. Hydrogen peroxide formation could not be detected during the catalytic reaction. The enzymatic conversion of catechol to diphenylenedioxide 2,3-quinone by the purified Tecoma leaf enzyme was suppressed by such reducing agents as GSH and cysteamine. The purified enzyme was not sensitive to carbon monoxide. It was not inhibited by thiol inhibitors. The Tecoma leaf was found to be localized in the soluble fraction of the cell. Treatment of the purified enzyme with acid, alkali, and urea led to the progressive denaturation of the enzyme.