Curcumin from Curcuma longa Linn. (Family: Zingiberaceae) attenuates hypochlorous acid-induced cytotoxicity and oxidative damage to human red blood cells.

Hypochlorous acid (HOCl) is a major oxidant produced by activated neutrophils via the myeloperoxidase catalyzed reaction. The production of HOCl eliminates a wide range of pathogens. However, HOCl can also cause significant oxidative damage in cells and tissues where it is generated. The protective effect of curcumin was studied on HOCl-induced oxidative damage to human red blood cells (RBC). Isolated RBC were incubated with HOCl at 37 °C in absence or presence of different concentrations of curcumin. Hemolysates were prepared and assayed for various biochemical parameters. Treatment of RBC with HOCl alone increased hemolysis, protein carbonyls, heme degradation and chloramines as compared to untreated control cells. This was accompanied by reduction in glutathione level, total sulfhydryls and free amino groups. HOCl also lowered the activities of major antioxidant enzymes and diminished the antioxidant power of RBC. Pre-treatment of RBC with different concentrations of curcumin resulted in concentration-dependent attenuation in all these parameters while curcumin alone had no significant effect. Scanning electron microscopy showed that curcumin prevented HOCl-induced morphological changes in RBC and restored their normal biconcave shape. Thus curcumin can be used as a chemoprotective agent to mitigate HOCl-induced oxidative damage to cells. These results also explain the beneficial effects of curcumin against Helicobacter pylori induced stomach ulcers, caused by excessive production of HOCl at the site of bacterial infection.

Structural alteration in hypochlorous acid modified antithrombin indicates generation of neo-epitopes.

Increased tendency of cancer patients to develop venous thromboembolism (VTE) is associated with high rates of mortality. Elevation of procoagulant proteins and down regulation of naturally occurring coagulation inhibitors appears to form the basis of high risk of VTE in malignancy. A reduced level of anticoagulant protein like antithrombin (AT) will influence both coagulation and angiogenesis, as its cleaved and latent conformations show potent antiangiogenic activity. We show a concentration dependent perturbation in the secondary and tertiary structures of AT conformers exposed to hypochlorous acid (HOCl). Modulated under a very narrow concentration range of HOCl, native AT undergoes oligomerization, aggregation and fragmentation based on spectroscopic, SDS and native-PAGE studies. Factor Xa inhibition assay demonstrated a progressive decrease in inhibition activity of AT on modification by HOCl. Bis-ANS result showed that hydrophobic patches were more exposed in the case of HOCl-modified AT when assessed fluorometrically. Dosage of HOCl-modified AT in experimental animals induced high titer antibodies showing more specificity towards modified forms in comparison to unmodified forms. Auto-antibodies isolated from cancer patients also showed enhanced binding with HOCl-modified AT in comparison to native counterpart. Compared to normal AT, structurally and functionally altered conformation of HOCl-modified AT showed increased immunogenic sensitivity. HOCl modified AT can contribute to prothrombotic and angiogenic environment during cancer progression/development.

Acetaldehyde-induced structural and conformational alterations in human immunoglobulin G: A physicochemical and multi-spectroscopic study.

Acetaldehyde is a reactive aldehyde produced as an intermediate of alcohol metabolism and tobacco pyrolysis. It has the potential to interact with different biomolecules in various tissues which results in the formation of stable, unstable and covalent adducts. This causes structural and functional modifications that may lead to severe complications such as cancer. This study has probed the structural modifications in human immunoglobulin G (IgG) as a function of different concentrations of acetaldehyde in the presence of reducing agent, sodium borohydride. Acetaldehyde mediated modifications in IgG have been characterised by various physicochemical techniques. UV-spectrophotometry showed that acetaldehyde modified IgG exhibited marked increase in hyperchromicity. Fluorescence studies revealed a significant quenching of tryptophan fluorescence which resulted in loss of ß-sheet secondary structure that was confirmed by circular dichroic analysis. Gross structural changes in the morphology of IgG were confirmed by increase in mass and hydrodynamic radius of this glycoprotein along with the appearance of fibrillar structures in modified IgG, when compared to the granular structure of the native form of IgG observed by scanning electron microscope. The results indicate that acetaldehyde causes alterations in the secondary and tertiary structure of the protein leading to diminution of normal function of IgG molecule.

Hypochlorous acid induced structural and conformational modifications in human DNA: A multi-spectroscopic study.

Hypochlorous acid (HOCl) is generated by activated phagocytes at the site of inflammation. Exposure of DNA to HOCl results in base and nucleotide modifications causing DNA damage, which is one of the leading causes of various pathological conditions including carcinogenesis. In the present work, various biophysical techniques were used to study HOCl induced structural and conformational changes in human placental DNA. The HOCl modified DNA showed hyperchromicity, reduced fluorescence and decrease in melting temperature. Circular dichorism (CD) and Fourier transform infra-red (FT-IR) studies exhibited conformational changes and shift in band positions of DNA, respectively, suggesting structural changes. Agarose gel electrophoresis and scanning electron microscopy showed strand breakage and decreased aggregation. These results suggest that HOCl causes conformational and structural perturbations in mammalian DNA, which may consequentially lead to DNA mutations resulting in perturbation of epigenetic signals leading to cancer and autoimmune diseases.

Synthesis and in vitro evaluation of 2-(((2-ether)amino)methylene)-dimedone derivatives as potential antimicrobial agents.

The study was designed with an aim to synthesize a series of 2-(((2-ether)amino)methylene)-dimedone derivatives and evaluate the synthesized compounds for antimicrobial activity. Compound library was synthesized by reaction with alkyl, alkenyl, alkynyl and alicyclic bromo-compounds. Characterization of the synthesized compounds was performed by 1H NMR, 13C NMR and mass spectral techniques. The compounds were evaluated for their antibacterial activity against Gram-positive (Staphylococcus aureus, Bacillus subtilis, Clostridium sporogenes) and Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli). The activity of these compounds was also evaluated against fungi (Aspergillus fumigatus, Penicillium chrysogenum, Fusarium oxysporum, Candida albicans) and molds (A. niger and A. oryzae). Broth microdilution method and CLSI guidelines with minor modification were used for the determination of anti-bacterial and antifungal activity, respectively. Although four compounds (4i, 4j, 4k and 4l) showed good antibacterial activity but compound 4k was found to be most active chemotype in the series. Compound 4k was found to be active against S. aureus, B. cereus and B. subtilis bacterial strains at one dilution lower compared to the control ciprofloxacin. Antibacterial activity of compound 4k was comparable to ciprofloxacin against S. pyogenes and M. luteus. The compound 4d, 4e and 4s showed good antifungal and antimold activity compared to other chemotypes. However, in comparison to fluconazole both the compounds showed lower activity. The results merit the antimicrobial promise of the 2-(((2-ether)amino)methylene)-dimedone analogs.