Amarjit Singh

Mechanism of biochemical action of substituted 4-methylbenzopyran-2-ones. Part I: Dioxygenated 4-methyl coumarins as superb antioxidant and radical scavenging agents.

Twenty-three 4-methylcoumarins bearing different functionalities have been examined for the first time for their effect on NADPH-catalysed liver-microsomal lipid peroxidation with a view to establish structure-activity relationship. Dihydroxy- and diacetoxy-4-methylcoumarins produced dramatic inhibition of lipid peroxidation. 7,8-Diacetoxy-4-methylcoumarin and 7,8-dihydroxy-4-methylcoumarin were found to possess superb antioxidant and radical scavenging activities.

Mechanism of biochemical action of substituted 4-methylbenzopyran-2-ones. Part II: Mechanism-based inhibition of rat liver microsome-mediated aflatoxin B1–DNA binding by the candidate antimutagen 7,8-diacetoxy-4-methylcoumarin

7,8-Diacetoxy-4-methylcoumarin (DAMC), with no prerequisite for oxidative biotransformation has been reported to produce suicide inactivation of microsomal cytochrome P-450-catalysed formation of aflatoxin B1-8,9-oxide that binds to DNA. Parenteral administration of DAMC to rats caused significant inhibition of AFB1 binding to hepatic DNA in vivo as well as AFB1-induced micronuclei formation in bone marrow cells. These results highlight the antimutagenic potential of DAMC.

Synthesis, Characterization and In Vitro Anti-invasive Activity Screening of Polyphenolic and Heterocyclic Compounds

Invasion is the hallmark of malignant tumors, and is responsible for the bad prognosis of the untreated cancer patients. The search for anti-invasive treatments led us to screen compounds of different classes for their effect in an assay for invasion. Thirty-nine new compounds synthesized in the present study along with 56 already reported compounds belonging mainly to the classes of lactones, pyrazoles, isoxazoles, coumarins, desoxybenzoins, aromatic ketones, chalcones, chromans, isoflavanones have been tested against organotypic confronting cultures of invasive human MCF-7/6 mammary carcinoma cells with embryonic chick heart fragments in vitro. Three of them (a pyrazole derivative, an isoxazolylcoumarin and a prenylated desoxybenzoin) inhibited invasion at concentrations as low as 1 microM; instead of occupying and replacing the heart tissue within 8 days, the MCF-7/6 cells grew around the heart fragments and left it intact, when treated with these compounds. At the anti-invasive concentration of 1 microM, the three compounds did not affect the growth of the MCF-7/6 cells, as shown in the sulforhodamine B assay. Aggregate formation on agar was not stimulated by any of the three anti-invasive compounds, making an effect on the E-cadherin/catenin complex improbable. This is an invasion suppressor that can be activated in MCF-7/6 cells by a number of other molecules. Our data indicate that some polyphenolic and heterocyclic compounds are anti-invasive without being cytotoxic for the cancer cells.

Mechanism of Biochemical Action of Substituted 4-Methylbenzopyran-2-ones. Part 4: Hyperbolic Activation of Rat Liver Microsomal NADPH-Cytochrome C Reductase by the Novel Acetylator 7,8-Diacetoxy-4-methylcoumarin

The effect of 7,8-diacetoxy-4-methylcoumarin (DAMC) has been studied on hepatic NADPH cytochrome C reductase-- an enzyme participating in the microsomal electron transport. The preincubation of liver microsomes with DAMC resulted in a time-dependent activation of NADPH cytochrome C reductase. The catalytic activity of the enzyme enhanced nearly 600% by 25 microM concentration of DAMC after 10 min of preincubation. The action of DAMC on the reductase resulted in enhanced v(max) while Km remained constant. A plot of 1/v(max) as a function of DAMC concentration resulted in a non-linear, but rectangular hyperbola indicative of hyperbolic activation. DAMC was also proved to be effective in significantly enhancing the activity of NADPH cytochrome C reductase in vivo. 7,8-Dihydroxy-4-methylcoumarin (DHMC), the deacetylated product of DAMC failed to irreversibly activate the enzyme. The activation effect of DAMC upon the enzyme was abolished by p-hydroxymercury benzoate. The role of a transacetylase in transferring the acetyl group of DAMC to the amino acid(s) of the active site of NADPH cytochrome C reductase causing irreversible enzyme activation is enunciated.

PEROXIDASE-CATALYZED CROSSlINKING OF FUNCTIONALIZED POLYASPARTIC ACID POLYMERS

ABSTRACT The formation of biodegradable hydrogels is reported based on enzymatic crosslinking reactions of modified polyaspartic acid. Poly(aspartic acid) polymers functionalized with aromatic groups were crosslinked in aqueous solution via peroxidase with hydrogen peroxide to form hydrogels. The reaction products were characterized based on time to gel, swelling ratio, sol fraction, storage and loss modulus, and entrapped enzyme. Optimum reaction conditions for gel synthesis were determined. Poly(aspartic acid) hydrogels have significant potential for use in biomedical applications such as in drug delivery due to the aqueous, non-toxic synthesis conditions as well as their inherent biodegradability by proteases.

Mechanism of biochemical action of substituted 4-Methylbenzopyran-2-ones. Part 3: A novel mechanism for the inhibition of biological membrane lipid peroxidation by dioxygenated 4-Methylcoumarins mediated by the formation of a stable ADP-Fe-Inhibitor mixed ligand complex

7,8-Dihydroxy-4-methylcoumarin (DHMC) and 7,8-diacetoxy-4-methylcoumarin (DAMC) have been reported to effectively inhibit in-vivo lipid peroxidation in rat tissues induced by CCl4 and paraquat. DHMC was found to readily impart green colour to the lipid peroxidation incubation mixture containing ADP and Fe3+, whereas DAMC formed green complex only upon incubation with liver microsomes, confirming our earlier observation that liver microsomal deacetylase hydrolyses DAMC to DHMC. Sensitive pH metric technique revealed the formation of ADP-Fe-DHMC ternary complex with highest stability, while Fe-DHMC and ADP-DHMC had negligible stabilities concluding that ADP-perferryl ion formation is prevented by DHMC resulting in the production of stable ternary mixed ligand complex (ADP-Fe-DHMC), thereby inhibiting the formation of O2-, and eventually other reactive oxygen species (ROS) responsible for membrane lipid peroxidation.

PEROXIDASE, HEMATIN, AND PEGYLATED-HEMATIN CATALYZED VINYL POLYMERIZATIONS IN WATER

Horseradish peroxidase-, hematin- and pegylated-hematin mediated polymerization of sodium styrene sulfonate and sodium acrylate in water is reported. Molecular weight and yields were influenced by the concentrations of hydrogen peroxide and initiator, 2,4-pentanedione. Hematin and pegylated-hematin were studied in lieu of peroxidase at pH 11.0 and 7.0 in aqueous solution, respectively. Polymer with a high molecular weight (Mn = 223,520) was formed when the pegylated-hematin was used as the catalyst. The results demonstrate vinyl polymerizations in an all aqueous process in high yield and molecular weight catalyzed by peroxidase as well as biomimetic catalysts.

Chemoprevention of carcinogen-DNA binding: the relative role of different oxygenated substituents on 4-methylcoumarins in the inhibition of aflatoxin B1-DNA binding in vitro☆

Eighteen 4-methylcoumarins bearing methoxy/hydroxy/acetoxy functionalities have been reported to effectively inhibit the rat liver microsome-mediated aflatoxin B1-DNA binding in vitro. The contribution of functionality on coumarin nucleus towards the inhibition of AFB1-DNA binding is in the order acetoxy > hydroxy > methoxy. The results illustrate the structure-activity relationship.

Neolignans and a lignan from Piper clarkii

Abstract Four new compounds, a lignan and three neolignans, together with one known neolignan have been isolated from the leaves and stems of Piper clarkii . Their structures were established on the basis of 1 H, 13 C and 1 H 1 H COSY NMR, NOE and mass spectral data and CD curves. The novel lignan was identified as (2 S ,5 S )-diveratryl-(3 R ,4 S )-dimethyltetrahdrofuran. The structures of the new neolignans were established as (7 R ,8 R ,1′ S )- Δ 8′ -1′-methoxy-3,4-methylenedioxy-1′,6′-dihydro-6′-oxo-7.0.4′,8.3′-lignan[2 R ,3 R ,5 S )-2-(1,3-benzodioxol-5-yl)-3,5-dihydro-5-methoxy-3-methyl-5-(2-propenyl)-6(2H)-benzofuranone], (7 S ,8 S ,3′ R )- Δ 8′ -3,3′,4-trimethoxy-3′,6′-dihydro-6′-oxo-7.0.4′,8.3′-lignan[2 S ,3 S ,3 aR )-2-(3,4-dimethoxyphenyl)-3,3a-dihydro-3a-methoxy-3-methyl-5-(2-propenyl)-6(2H)-benzo-furanone] and rel-(7 S ,8 S ,1′ R ,3′ R )- Δ 8′ -5′-methoxy-3,4-methylenedioxy-1′,2′,3′,4′-tetrahydro-2′,4′-dioxo-7.3′,8.1′- lignan[(7 S ,6 S ,5 R ,1 R )-7-(1,3-benzodioxol-5-yl)-3-methoxy-6-methyl-5-(2-propenyl)bicyclo[3.2.1]oct-3-ene-2,8-dione]. The known neolignan has been identified as denudatin B.

Enzyme-Based Vinyl Polymerization

In vitro enzyme-mediated polymerization of vinyl monomers is reviewed. Oxidoreductase enzymes have been used for the polymerization of styrene, derivatives of styrene, acrylates, and acrylamide in water and water-miscible co-solvents. Kinetic and mechanistic studies revealed that a ternary system (horseradish peroxidase, H2O2, initiator, or β-diketone) is required for efficient polymerization and the initiator controls the polymer characteristics.