Lazar Mathew

Free radical scavenging activity from leaves of Acacia nilotica (L.) Wild. ex Delile, an Indian medicinal tree.

The present study compares the two extraction methods and evaluates the free radical scavenging activity of Acacia nilotica. Results indicated that the sequential extraction method was effective in concentrating the active principles in the ethanol extract as compared to the maceration method in DPPH assay. Based on the results, free radical scavenging property of the extracts obtained from sequential extraction method was analyzed in different assays to find out the possible antioxidant mechanism. Our results indicate that ethanol extract rich in phenolic and flavonoid contents had potent antioxidant activity and were significant in comparison with all the positive controls used in this study. The possible antioxidant mechanism of the ethanol extract can be due to its hydrogen or electron donating and direct free radical scavenging properties. Hence, the ethanol extract represents a source of potential antioxidants that could be used in pharmaceutical and food preparations.

Kinetics, equilibrium and thermodynamic studies on biosorption of Ag(I) from aqueous solution by macrofungus Pleurotus platypus

Reports are available on silver binding capacity of some microorganisms. However, reports on the equilibrium studies on biosorption of silver by macrofungi are seldom known. The present study was carried out in a batch system using dead biomass of macrofungus Pleurotus platypus for the sorption of Ag(I). P. platypus exhibited the highest silver uptake of 46.7 mg g(-1) of biomass at pH 6.0 in the presence of 200 mg L(-1) Ag(I) at 20°C. Kinetic studies based on fractional power, zero order, first order, pseudo-first order, Elovich, second order and pseudo-second order rate expressions have been carried out. The results showed a very good compliance with the pseudo-first order model. The experimental data were analyzed using two parameter isotherms (Langmuir, Freundlich, Dubinin-Radushkevich, Temkin and Halsey), three parameter isotherms (Redlich-Peterson, Sips, Khan, Koble-Corrigan, Hill, Toth, Radke-Prausmitz, Jossens, Langmuir-Freundlich), four parameter isotherms (Weber-van Vliet, Fritz-Schlunder, Baudu) and five parameter isotherm (Fritz-Schlunder). Thermodynamic parameters of the biosorption (ΔG, ΔH and ΔS) were also determined. The present study confirmed that macrofungus P. platypus may be used as a cost effective efficient biosorbent for the removal of Ag(I) ions from aqueous solution.

6-Shogaol inhibits monosodium urate crystal-induced inflammation – An in vivo and in vitro study

Gout is a rheumatic disease that is manifestated by an intense inflammation secondary to monosodium urate crystal deposition in joints. In the present study, we assessed the effect of 6-shogaol (isolated active principle from ginger) on monosodium urate crystal-induced inflammation in mice; an experimental model for gouty arthritis and compared it with that of the non-steroidal anti-inflammatory drug, indomethacin. Paw volume and levels/activities of lysosomal enzymes, lipid peroxidation, anti-oxidant status and inflammatory mediator TNF-alpha were determined in control and monosodium urate crystal-induced mice. The levels of beta-glucuronidase and lactate dehydrogenase were also measured in monosodium urate crystal-incubated polymorphonuclear leucocytes (PMNL) in vitro. The levels of lysosomal enzymes, lipid peroxidation, and inflammatory mediator tumour necrosis factor-alpha and paw volume were increased significantly and the activities of anti-oxidant status were in turn decreased in monosodium urate crystal-induced mice, whereas these changes were reverted to near normal levels upon 6-shogaol administration. In vitro, 6-shogaol reduced the level of beta-glucuronidase and lactate dehydrogenase in monosodium urate crystal-incubated polymorphonuclear leucocytes in concentration dependent manner when compared to control cells. The present results clearly indicated that 6-shogaol exerted a strong anti-inflammatory effect and can be regarded as useful tool for the treatment of acute gouty arthritis.

Aqueous extract of Phyllanthus amarus inhibits chromium(VI)-induced toxicity in MDA-MB-435S cells.

Cr(VI) (hexavalent) is a very strong oxidant which causes high cytotoxicity through oxidative stress in tissue systems. Its abundance in groundwater and drinking water in several parts of the world has been noted to cause severe toxicity to both flora and fauna. This study evaluated the effects of aqueous extract of Phyllanthus amarus Schum. & Thon. against Cr(VI)-induced oxidative toxicity in vitro in MDA-MB-435S human breast carcinoma cells, along with an estimation of its antioxidant potential, inhibition of lipid peroxidation and determination of its polyphenolic composition. The extract showed significant (P<0.05) potential in scavenging free radicals (DPPH() and ABTS()(+)) and Fe(+3), and in inhibiting lipid peroxidation. A distinct decline in Cr(VI)-induced cytotoxicity was noticed in MDA-MB-435S cells with an increase in extract dosage. Furthermore, the extract proved to contain a high content of phenolic compounds which were found to have strong and significant (P<0.05) positive correlations to free-radical scavenging potential, lipid peroxidation inhibition capacity and cyto-protective efficiency against Cr(VI)-induced oxidative cellular damage. HPLC analysis identified some of the major phenolic compounds in it. It was concluded that P. amarus aqueous extract has high antioxidant potential (by virtue of its phenolic constituents) which simultaneously inhibits Cr(VI)-induced oxidative toxicity to MDA-MB-435S cells.

Free Radical Scavenging, Cytotoxic and Hemolytic Activities from Leaves of Acacia nilotica (L.) Wild. ex. Delile subsp. indica (Benth.) Brenan

Dietary intake of phytochemicals having antioxidant activity is associated with a lower risk of mortality from many diseases. Therefore, the aim of this study was to determine the free radical scavenging, cytotoxic and hemolytic activities of leaves of Acacia nilotica by using various methods. The results of the present study revealed that ethanol extract was the most effective and IC50 value was found to be 53.6 μg mL−1 for Vero cell lines and 28.9 μg mL−1 for Hela cell lines in cytotoxicity assays. The zone of color retention was 14.2 mm in β-carotene bleaching assay, which was as significant as positive control, butylated hydroxy toluene. None of the tested extracts possessed any hemolytic activity against rat and human erythrocytes revealing their cytotoxic mechanism and non-toxicity. Thus, only the ethanol extract could be considered as a potential source of anticancer and antioxidant compounds. Further phytochemical studies will be performed for specification of the biologically active principles.

Free Radical Scavenging, Cytotoxic, and Hemolytic Activities of an Active Antioxidant Compound Ethyl Gallate from Leaves of Acacia Nilotica (L.) Wild. Ex. Delile Subsp. Indica (Benth.) Brenan

UNLABELLED In the present study, free radical scavenging, cytotoxic, and hemolytic activities of the polyphenolic compound ethyl gallate isolated from ethanol extract of Acacia nilotica Wild. Ex. Del. leaves were determined. The free radical-scavenging activities of the ethyl gallate were demonstrated in several in vitro assays in order to evaluate the possible antioxidant mechanism. The results revealed ethyl gallate as hydrogen donor, metal chelator, and free radical scavenger. Ethyl gallate was effective in scavenging 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radicals and the IC₅₀ value was lower than all the positive controls used in this study. Deoxyribose degradation assay revealed that ethyl gallate had more iron-chelating ability than the direct hydroxyl radical-scavenging ability. The results of the cytotoxic study revealed that the compound was moderately active and IC₅₀ value was found to be >100 μg/mL for Vero cell lines and 72 μg/mL for Hela cell lines. The compound possessed no hemolytic activity against rat and human erythrocytes revealing its cytotoxic mechanism and nontoxicity. The results from this work will provide an important information for the food and pharmacological industries with respect to the use of the compound as an antioxidant and a health-related drug. PRACTICAL APPLICATION Antioxidant from plant sources is safe to use, as compared to synthetic products. It also can be used as a supplement to alleviate most of the diseases because of its free radical-scavenging activity.

Evolution, homology conservation, and identification of unique sequence signatures in GH19 family chitinases.

The discovery of GH (Glycoside Hydrolase) 19 chitinases in Streptomyces sp. raises the possibility of the presence of these proteins in other bacterial species, since they were initially thought to be confined to higher plants. The present study mainly concentrates on the phylogenetic distribution and homology conservation in GH19 family chitinases. Extensive database searches are performed to identify the presence of GH19 family chitinases in the three major super kingdoms of life. Multiple sequence alignment of all the identified GH19 chitinase family members resulted in the identification of globally conserved residues. We further identified conserved sequence motifs across the major sub groups within the family. Estimation of evolutionary distance between the various bacterial and plant chitinases are carried out to better understand the pattern of evolution. Our study also supports the horizontal gene transfer theory, which states that GH19 chitinase genes are transferred from higher plants to bacteria. Further, the present study sheds light on the phylogenetic distribution and identifies unique sequence signatures that define GH19 chitinase family of proteins. The identified motifs could be used as markers to delineate uncharacterized GH19 family chitinases. The estimation of evolutionary distance between chitinase identified in plants and bacteria shows that the flowering plants are more related to chitinase in actinobacteria than that of identified in purple bacteria. We propose a model to elucidate the natural history of GH19 family chitinases.

Antioxidant Activity of Lawsonia inermis Extracts Inhibits Chromium(VI)-Induced Cellular and DNA Toxicity

Hexavalent chromium Cr(VI) is a very strong oxidant which consequently causes high cytotoxicity through oxidative stress. Prevention of Cr(VI)-induced cellular damage has been sought in this study in aqueous and methanolic extracts of Lawsonia inermis Linn. (Lythraceae), commonly known as Henna. The extracts showed significant (P < .05) potential in scavenging free radicals (DPPH• and ABTS•+) and Fe3+, and in inhibiting lipid peroxidation. DNA damage caused by exposure of pBR322 to Cr(VI)-UV is markedly inhibited by both extracts in varying degrees. A distinct decline in Cr(VI)-induced cytotoxicity was noticed in MDA-MB-435S (human breast carcinoma) cells with an increase in dosage of both extracts individually. Furthermore, both extracts proved to contain a high content of phenolic compounds which were found to have a strong and significant (P < .05) positive correlation to the radical scavenging potential, lipid peroxidation inhibition capacity and cyto-protective efficiency against Cr(VI)-induced oxidative cellular damage. HPLC analysis identified some of the major phenolic compounds in both extracts, which might be responsible for the antioxidant potential and the properties of DNA and cyto-protection. This study contributes to the search for natural resources that might yield potent therapeutic drugs against Cr(VI)-induced oxidative cell damage.

Cytochrome P450 Reductase: A Harbinger of Diffusible Reduced Oxygen Species

The bi-enzymatic system of cytochrome P450 (CYP, a hemoprotein) and cytochrome P450 reductase (CPR, a diflavoenzyme) mediate the redox metabolism of diverse indigenous and xenobiotic molecules in various cellular and organ systems, using oxygen and NADPH. Curiously, when a 1∶1 ratio is seen to be optimal for metabolism, the ubiquitous CYP:CPR distribution ratio is 10 to 100∶1 or higher. Further, the NADPH equivalents consumed in these in vitro or in situ assemblies usually far exceeded the amount of substrate metabolized. We aimed to find the rationale to explain for these two oddities. We report here that CPR is capable of activating molecular oxygen on its own merit, generating diffusible reduced oxygen species (DROS). Also, in the first instance for a flavoprotein, CPR is shown to deplete peroxide via diffusible radical mediated process, thereby leading to the formation of water (but without significant evolution of oxygen). We also quantitatively demonstrate that the rate of oxygen activation and peroxide depletion by CPR accounts for the major reactivity in the CYP+CPR mixture. We show unambiguously that CPR is able to regulate the concentration of diffusible reduced oxygen species in the reaction milieu. These findings point out that CPR mediated processes are bound to be energetically ‘wasteful’ and potentially ‘hazardous’ owing to the unavoidable nature of the CPR to generate and deplete DROS. Hence, we can understand that CPR is distributed at low densities in cells. Some of the activities that were primarily attributed to the heme-center of CYP are now established to be a facet of the flavins of CPR. The current approach of modeling drugs to minimize “uncoupling” on the basis of erstwhile hypothesis stands questionable, considering the ideas brought forth in this work.

Explaining the Atypical Reaction Profiles of Heme Enzymes with a Novel Mechanistic Hypothesis and Kinetic Treatment

Many heme enzymes show remarkable versatility and atypical kinetics. The fungal extracellular enzyme chloroperoxidase (CPO) characterizes a variety of one and two electron redox reactions in the presence of hydroperoxides. A structural counterpart, found in mammalian microsomal cytochrome P450 (CYP), uses molecular oxygen plus NADPH for the oxidative metabolism (predominantly hydroxylation) of substrate in conjunction with a redox partner enzyme, cytochrome P450 reductase. In this study, we employ the two above-mentioned heme-thiolate proteins to probe the reaction kinetics and mechanism of heme enzymes. Hitherto, a substrate inhibition model based upon non-productive binding of substrate (two-site model) was used to account for the inhibition of reaction at higher substrate concentrations for the CYP reaction systems. Herein, the observation of substrate inhibition is shown for both peroxide and final substrate in CPO catalyzed peroxidations. Further, analogy is drawn in the “steady state kinetics” of CPO and CYP reaction systems. New experimental observations and analyses indicate that a scheme of competing reactions (involving primary product with enzyme or other reaction components/intermediates) is relevant in such complex reaction mixtures. The presence of non-selective reactive intermediate(s) affords alternate reaction routes at various substrate/product concentrations, thereby leading to a lowered detectable concentration of “the product of interest” in the reaction milieu. Occams razor favors the new hypothesis. With the new hypothesis as foundation, a new biphasic treatment to analyze the kinetics is put forth. We also introduce a key concept of “substrate concentration at maximum observed rate”. The new treatment affords a more acceptable fit for observable experimental kinetic data of heme redox enzymes.