Antibacterial activity of resveratrol structural analogues: A mechanistic evaluation of the structure-activity relationship.

Structure-activity relationship (SAR) studies have led to significant improvement in desirable biological activity in different classes of molecules. A general consensus about the substitutions that improve the activity remains elusive in stilbene class of molecules especially in regard to antibacterial activity. Lack of this knowledge remains a major hurdle in developing stilbene based antibacterial molecules. A panel of gram positive and gram negative bacteria were employed for screening the comparative efficacy of the stilbenes. In addition, the mechanisms that contribute to the antibacterial activity were investigated and correlated to structural changes. Employing the notorious nosocomial agent S. aureus we show how changes in structure alters not only the antibacterial activity but also the underlying mechanisms. Antibacterial activity by CLSI (Clinical & Laboratory Standards Institute) guidelines, oxidative stress and membrane damage by fluorescence based methods, DNA binding by spectroscopy, DNA cleavage by gel electrophoresis, substrate efflux by efflux mutant and cell wall damage by scanning electron microscopy were investigated. Antibacterial activity varied drastically among stilbenes bearing different functional groups. The best stilbenes in terms of activity also scored higher in one or more molecular events that contribute to cell death. Stilbenes superior to resveratrol in antibacterial acitvity were identified and probable causes for better activity were also identified. Our study revealed dimerization, halogenation and hydroxy group in conjunction with methoxy group resulted in the best antibacterial molecules. Design of stilbene based drugs would be benefitted with the outcome and rationale presented in the current investigation.

Involvement of Antibiotic Efflux Machinery in Glutathione-Mediated Decreased Ciprofloxacin Activity in Escherichia coli.

We have analyzed the contribution of different efflux components to glutathione-mediated abrogation of ciprofloxacin's activity in Escherichia coli and the underlying potential mechanism(s) behind this phenomenon. The results indicated that glutathione increased the total active efflux, thereby partially contributing to glutathione-mediated neutralization of ciprofloxacin's antibacterial action in E. coli However, the role of glutathione-mediated increased efflux becomes evident in the absence of a functional TolC-AcrAB efflux pump.

dl-trans-3,4-Dihydroxy-1-selenolane (DHSred) heals indomethacin-mediated gastric ulcer in mice by modulating arginine metabolism.

BACKGROUND: The importance of the arginine metabolism in gastric ulcer-healing is given relatively less attention. Hence the role of controlling this pathway by dl-trans-3,4-dihydroxy-1-selenolane (DHSred) and omeprazole against indomethacin-induced stomach ulceration in mouse was investigated. METHODS: Swiss albino mice were ulcerated with indomethacin followed by treatment with the test samples, and the activities of myeloperoxidase (MPO), total nitric oxide synthase (NOS) and arginase, the expressions of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS), and the pro-/anti-inflammatory cytokine levels were assayed. NOS-inhibitors were also used to establish the biochemical mechanism. RESULTS: Indomethacin induced maximum ulceration in mice on the 3rd day, associated with reduced arginase activity, eNOS expression, along with increased MPO and total NOS activities, nitric oxide (NO) generation, iNOS expression, and pro-/anti-inflammatory (Th1/Th2) cytokine ratio. Treatment with DHSred (2.5mgkg(-1)×3days) restored the cytokine balance to shift the iNOS/NO axis to the arginase/polyamine axis as revealed from the increased arginase activity and eNOS expression, and reduced iNOS expression, total NOS activity and NO level. CONCLUSIONS: The ulcer-healing property of DHSred, but not of omeprazole was due to a favorable pro-/anti-inflammatory cytokine ratio that shifted the arginine metabolism to the polyamine pathway and increased the eNOS/iNOS ratio. The healing action of omeprazole was not significantly associated with these parameters. GENERAL SIGNIFICANCE: The shift in the ariginine-metabolism from the iNOS/NO axis to the arginase/polyamine axis is guided by Th1/Th2 cytokines ratio and plays an important role in gastric ulcer-healing. The favourable effects of the non-toxic and water-soluble compound, DHSred on these pathways and other COX-dependent and antioxidative parameters suggested it to be a promising anti-ulcer formulation for further studies.

Design, synthesis and development of a dual inhibitor of Topoisomerase 1 and poly (ADP-ribose) polymerase 1 for efficient killing of cancer cells.

Combinatorial inhibition of Topoisomerase 1 (TOP1) and Poly (ADP-ribose) polymerase 1 (PARP1) is an attractive therapeutic strategy which is under active investigation to address chemoresistance to TOP1 inhibitors. However, this combinatorial regimen suffers from severe dose limiting toxicities. Dual inhibitors often offer significant advantages over combinatorial therapies involving individual agents by minimizing toxicity and providing conducive pharmacokinetic profiles. In this study, we have designed, synthesized and evaluated a library of 11 candidate conjugated dual inhibitors for PARP1 and TOP1, named as DiPT-1 to DiPT-11. Our extensive screening showed that one of the hits i.e.DiPT-4 has promising cytotoxicity profile against multiple cancers with limited toxicities towards normal cells. DiPT-4 induces extensive DNA double stand breaks (DSBs), cell cycle arrest and apoptosis in cancer cells. Mechanistically, DiPT-4 has the propensity to bind catalytic pockets of TOP1 and PARP1, leading to significant inhibition of both TOP1 and PARP1 at in vitro and cellular level. Interestingly, DiPT-4 causes extensive stabilization of TOP1-DNA covalent complex (TOP1cc), a key lethal intermediate associated with induction of DSBs and cell death. Moreover, DiPT-4 inhibited poly (ADP-ribosylation) i.e. PARylation of TOP1cc, leading to long lived TOP1cc with a slower kinetics of degradation. This is one of the important molecular processes which helps in overcoming resistance in cancer in response to TOP1 inhibitors. Together, our investigation showed DiPT-4 as a promising dual inhibitor of TOP1 and PARP1, which may have the potential to offer significant advantages over combinatorial therapy in clinical settings.

Dimer stilbene, a resveratrol analogue exhibits synergy with antibiotics that target protein synthesis in eradicating Staphylococcus aureus infection.

Antibiotic resistance has become a major hurdle for successful treatment of several infections resulting in increased length of stay in hospitals and mortality. One of the notorious pathogens that wreaks havoc due to antibiotic resistance is Staphylococcus aureus. There is an urgent need to discover and understand the function of newer molecules that could serve in the arsenal to combat these bacteria. Our recent work identified important structural determinants of stilbenes that could aid in better antibacterial activity and identified Dimer stilbene (DS) as a potent inhibitor of S. aureus. Contrasting reports exist in literature about the combination of stilbenes with different antibiotics. In this study we evaluated the ability of DS to synergize with different classes of antibiotics. A screen revealed DS exhibited positive co-operativity with antibiotics that target protein synthesis. DS exhibited synergy with the aminoglycoside kanamycin and additive effect with tetracycline. Resistance generation to DS was null while to that of kanamycin was rapid. Kanamycin resistant S. aureus was equally susceptible to DS compared to wildtype. The efficacy of DS against clinical isolates susceptible and resistant to methicillin were similar. Laboratory generated kanamycin resistant strain and clinical strains were sensitized to kanamycin by pre-treatment with DS. DS cured S. aureus infection in mice as a standalone drug as well as in conjunction with kanamycin. Synergy with kanamycin was also observed in other stilbenes apart from DS. Thus our study reveals stilbenes could be exploited towards combating S. aureus infections either as standalone drugs or in combination with existing antibiotics.

Iron modulatory property of a polysaccharide from Indian medicinal plant Ocimum sanctum.

Despite being an essential element for normal functioning of cells and organisms, iron, in excess, can induce oxidative stress by generating reactive oxygen species. A water-soluble, non-toxic iron chelator can reduce the iron-induced oxidative stress in the body as well as help in extricating excess iron. Herein, we report an Ocimum sanctum-derived antioxidant polysaccharide (OSP) that inhibits the deleterious effect of iron. Ocimum sanctum is a widely acknowledged medicinal plant contributing toward several biological benefits. Besides showing good hydroxyl radical scavenging activity, OSP could bind to ferric and ferrous ions to prevent their participation in redox reactions as revealed from modified 2-deoxyribose assays, carried out under various conditions. It also acted as an iron modulator to prevent site-specific damage and was effective in protecting mouse fibroblast L929 cells against iron induced death.

Membrane damage precedes DNA damage in hydroxychavicol treated E. coli cells and facilitates cooperativity with hydrophobic antibiotics.

Hydroxychavicol (HC), found abundantly in Piper betle leaves is credited with antimicrobial property. Previously we had shown HC induces reactive oxygen species mediated DNA damage in bacterial cells. HC also resulted in membrane compromise revealing its pleiotropic effects on cellular targets. The kinetics and exact sequence of events leading to inhibition of growth and cell death in E. coli after HC treatment remains poorly understood. We show that sub-lethal concentration (125 µg/mL) of HC causes cellular filamentation within 1 h of treatment, while a higher concentration (750 µg/mL) induces cell breakage. HC-treated cells were found to experience oxidative stress as early as 10 min, while evidence of membrane damage was apparent at 30 min. DNA damage repair genes were found to be activated at 60 min. Interestingly, HC-induced cell permeabilization was inhibited and enhanced by external Mg2+ and EDTA, respectively, suggesting that HC damages the outer membrane. Kinetic experiments revealed that HC-treated cells underwent oxidative stress, membrane damage and DNA damage in that order. Because gram negative bacteria such as E. coli are refractory to several antibiotics due to the presence of the outer membrane, we hypothesized that HC pretreatment would sensitize E. coli to hydrophobic antibiotics. Our study reveals for the first time that HC could sensitize bacteria to clinically used antibiotics due to its outer membrane damaging property.

A Reusable Column Method Using Glycopolymer-Functionalized Resins for Capture-Detection of Proteins and Escherichia coli.

The use of glycopolymer-functionalized resins (Resin-Glc), as a solid support, in column mode for bacterial/protein capture and quantification is explored. The Resin-Glc is synthesized from commercially available chloromethylated polystyrene resin and glycopolymer, and is characterized by fourier transform infrared spectroscopy, thermogravimetry, and elemental analysis. The percentage of glycopolymer functionalized on Resin-Glc is accounted to be 5 wt%. The ability of Resin-Glc to selectively capture lectin, Concanavalin A, over Peanut Agglutinin, reversibly, is demonstrated for six cycles of experiments. The bacterial sequestration study using SYBR (Synergy Brands, Inc.) Green I tagged Escherichia coli/Staphylococcus aureus reveals the ability of Resin-Glc to selectively capture E. coli over S. aureus. The quantification of captured cells in the column is carried out by enzymatic colorimetric assay using methylumbelliferyl glucuronide as the substrate. The E. coli capture studies reveal a consistent capture efficiency of 105  CFU (Colony Forming Units) g-1 over six cycles. Studies with spiked tap water samples show satisfactory results for E. coli cell densities ranging from 102 to 107  CFU mL-1 . The method portrayed can serve as a basis for the development of a reusable solid support in capture and detection of proteins and bacteria.

Supramolecular Structures Generated via Self-Assembly of a Cell Penetrating Tetrapeptide Facilitate Intracellular Delivery of a Pro-apoptotic Chemotherapeutic Drug.

Development of drug carriers, which can chaperone xenobiotics directly to their site of action, is an essential step for the advancement of precision medicine. Cationic nanoparticles can be used as a drug delivery platform for various agents including chemotherapeutics, oligonucleotides, and antibodies. Self-assembly of short peptides facilitates the formation of well-defined nanostructures suitable for drug delivery, and varying the polarity of the self-assembly medium changes the nature of noncovalent interactions in such a way as to generate numerous unique nanostructures. Here, we have synthesized an ultrashort cell-penetrating tetrapeptide (sequence Lys-Val-Ala-Val), with Lys as a cationic amino acid, and studied the self-assembly property of the BOC-protected (L1) and -deprotected (L2) analogues. Spherical assemblies obtained from L1/L2 in a 1:1 aqueous ethanol system have the ability to encapsulate small molecules and successfully enter into cells, thus representing them as potential candidates for intracellular drug delivery. To verify the efficacy of these peptides in the facilitation of drug efficacy, we generated encapsulated versions of the chemotherapeutic drug doxorubicin (Dox). L1- and L2-encapsulated Dox (Dox-L1 and Dox-L2), similar to the unencapsulated drug, induced upregulation of regulator of G protein signaling 6 (RGS6) and Gß5, the critical mediators of ATM/p53-dependent apoptosis in Dox-treated cancer cells. Further, Dox-L1/L2 damaged DNA, triggered oxidative stress and mitochondrial dysfunction, compromised cell viability, and induced apoptosis. The ability of Dox-L1 to mediate cell death could be ameliorated via knockdown of either RGS6 or Gß5, comparable to the results obtained with the unencapsulated drug. These data provide an important proof of principle, identifying L1/L2 as drug delivery matrices.

Hydroxychavicol from Piper betle induces apoptosis, cell cycle arrest, and inhibits epithelial-mesenchymal transition in pancreatic cancer cells.

Pancreatic cancer is a major cause of cancer-related mortality around the world. Currently, options for diagnosis and treatment are extremely limited, which culminates in a very high mortality rate. Intensive research spanning more than four decades has met several roadblocks in terms of improvement in overall survival. In this study, we have evaluated the effect of Hydroxychavicol (HC), a naturally occurring and abundantly isolatable allylarene from Piper betle leaves on pancreatic cancer cells. Our investigation reveals that HC inhibits proliferation and epithelial-mesenchymal transition (EMT) in pancreatic cancer cells. HC induces DNA damage, as evidenced by γ-H2AX, 53BP1 induction and comet assay, which further results in mitotic catastrophe and apoptosis. The apoptosis induced by HC is JNK pathway-dependent and caspase-mediated. HC also inhibits migration and invasion of pancreatic cancer cells via a generalized repression of genes involved in EMT. A quantitative real time PCR-based array revealed at least 14 different genes to be differentially expressed upon HC treatment in pancreatic cancer cells. These results show significant potential of HC as an anticancer agent against pancreatic cancer.

In vitro apoptosis-induction, antiproliferative and BSA binding studies of a oxidovanadium(V) complex.

In our ongoing efforts to develop novel trace metal complexes with therapeutically interesting properties, a neutral mono nuclear oxidomethoxidovanadium(V) complex, [VVO(OCH3)(hpdbal-sbdt)] (1) and a µ-O bridged dinuclear oxidovanadium(V) complex, [{VVO(hpdbal-sbdt)}2µ-O] (2) [H2hpdbal-sbdt (I) is a tridentate and dibasic ONS2- donor ligand obtained through the Schiff base reaction of 2-hydroxy-5-(phenyldiazenyl)benzaldehyde (Hhpdbal) and S-benzyldithiocarbazate (Hsbdt)] have been synthesized and characterized by various analytical techniques such as TGA, EDS, ATR-IR, UV-Vis, CV, 1H NMR, 13C NMR and 51V NMR. Single-crystal X-ray diffraction analysis of 1 confirms the coordination of phenolate oxygen, imine nitrogen and thioenolate sulfur of the ligand to the vanadium center with a distorted tetragonal-pyramidal geometry. The compound 2 triggered apoptotic and reproductive death of the cancer cells in vitro with 76% and 62% growth inhibition of human breast adenocarcinoma (MCF-7) and human lung carcinoma cells (A549) respectively. The compound 2 was found to be sufficiently stable over a wide window of physiological pH. The complex 2 was studied further for its interaction with a drug carrier protein BSA with the aid of spectroscopic techniques viz. fluorescence, temperature controlled UV-vis and deconvoluted IR techniques.

Involvement of cytoplasmic membrane damage in the copper (II)-dependent cytotoxicity of a novel naturally occurring tripyrrole.

In the presence of a nonlethal concentration of Cu(II), washed Escherichia coli ATCC8739 cells were killed by a novel tripyrrole 1, isolated as a red pigment from the Serratia sp. Cell killing was accompanied by a depletion in the potassium pools of the cells due to the damage to the cytoplasmic membrane, without any detectable DNA damage as revealed by the transformed plasmid DNA and phage induction assay. This revealed that the bactericidal activity of compound 1 in the presence of Cu(II) results from membrane damage. Induction of endogenous catalase in the E. coli cells increased their resistance against the combination of compound 1 and Cu(II). Although compound 1 alone generated large amount of reactive oxygen species (ROS), it did not show any cell killing against E. coli in the absence of Cu(II). The Cu(II)-dependent bactericidal activity of compound 1 was suppressed by ethylenediaminetetraacetate, bathocuproine, catalase and superoxide disumutase (SOD), but not by dimethyl sulfoxide. These findings suggest that recycling redox reactions between Cu(II) and Cu(I), involving compound 1 and hydrogen peroxide on the cell surface, must be important in the mechanism of the killing. Compound 1 alone showed selective bactericidal activity against the gram positive bacterium, Bacillus cereus ATCC 6630, possibly due to its differential cellular transport.

Spice-derived phenolic, malabaricone B induces mitochondrial damage in lung cancer cells via a p53-independent pathway.

The spice-derived phenolic, malabaricone B (mal B) showed selective toxicity to human lung cancer (A549), malignant melanoma (A375) and T cell leukemia (Jurkat) cell lines, without showing toxicity to human normal intestinal (INT407), human kidney (HEK293) and lung fibroblast (WI-38) cells. Among the chosen cancer cell lines, mal B showed maximum cytotoxicity to the A549 cells (IC50 = 8.1 ± 1.0 µM), which was significantly better than that of curcumin (IC50 = 26.7 ± 3.1 µM). Further morphological studies by phase contrast microscopy and a clonogenic assay of the A549 cells revealed that mal B treatment increased the number of shrinking cells and also abolished the clonal proliferation of the cells. Mal B induced apoptotic cell death was confirmed by DNA laddering and quantified by cytoplasmic oligonucleosome formation and annexin V/PI assays. The mal B-induced apoptosis was mediated by an increase in the intracellular reactive oxygen species (ROS), because the cell-permeable antioxidants, N-acetylcysteine (NAC) and PEG-SOD, strongly inhibited its cytotoxicity to the A549 cells. Mal B increased the BAX level while simultaneously decreasing the BCL-2 and BCL-XL levels in the A549 cells, triggering the mitochondrial apoptotic pathway as revealed from the release of cytochrome c, and the activation of caspase-9 and caspase-3. Pre-treatment of cells with caspase-9, caspase-3 and pan-caspase inhibitors made them more resistant to mal B treatment. This effect of mal B was strongly associated with the concomitant decrease in anti-apoptotic (IAP1, IAP2 and survivin), angiogenic (growth factors) and cancer invasiveness (matrix metalloproteinase-9, COX-2) modulating proteins. Mal B induced cytotoxicity was unaffected by the shRNA-mediated depletion of p53 in A549 cells. Most importantly, mal B sensitized a wide range of human carcinoma cells regardless of their p53 status. Finally, mal B (100 mg kg-1) also inhibited lung tumor (xenograft) growth in SCID mice.

Resveratrol analogue, trans-4,4'-dihydroxystilbene (DHS), inhibits melanoma tumor growth and suppresses its metastatic colonization in lungs.

The prevalence of melanoma and the lack of effective therapy for metastatic melanoma warrant extensive and systematic evaluations of small molecules in cellular and pre-clinical models. We investigated, herein, the antitumor and anti-metastatic effects of trans-4,4'-dihydroxystilbene (DHS), a natural product present in bark of Yucca periculosa, using in vitro and in vivo melanoma murine models. DHS showed potent melanoma cytotoxicity, as determined by MTT and clonogenic assay. Further, DHS induced cytotoxicity was mediated through apoptosis, which was assessed by annexin V-FITC/PI, sub-G1 and caspase activation assays. In addition, DHS inhibited cell proliferation by inducing robust cell cycle arrest in G1-phase. Imperatively, these inhibitory effects led to a significant reduction of melanoma tumor in pre-clinical murine model. DHS also inhibited cell migration and invasion of melanoma cells, which were examined using wound healing and Transwell migration/invasion assays. Mechanistically, DHS modulated the expressions of several key metastasis regulating proteins e.g., MMP-2/9, N-cadherin, E-cadherin and survivin. We also showed the anti-metastatic effect of DHS in a melanoma mediated lung metastasis model in vivo. DHS significantly reduced large melanoma nodule formation in the parenchyma of lungs. Therefore, DHS may represent a promising natural drug in the repertoire of treatment against melanoma tumor growth and metastasis.

Hydroxychavicol, a key ingredient of Piper betle induces bacterial cell death by DNA damage and inhibition of cell division.

Antibiotic resistance is a global problem and there is an urgent need to augment the arsenal against pathogenic bacteria. The emergence of different drug resistant bacteria is threatening human lives to be pushed towards the pre-antibiotic era. Botanical sources remain a vital source of diverse organic molecules that possess antibacterial property as well as augment existing antibacterial molecules. Piper betle, a climber, is widely used in south and south-east Asia whose leaves and nuts are consumed regularly. Hydroxychavicol (HC) isolated from Piper betle has been reported to possess antibacterial activity. It is currently not clear how the antibacterial activity of HC is manifested. In this investigation we show HC generates superoxide in E. coli cells. Antioxidants protected E. coli against HC induced cell death while gshA mutant was more sensitive to HC than wild type. DNA damage repair deficient mutants are hypersensitive to HC and HC induces the expression of DNA damage repair genes that repair oxidative DNA damage. HC treated E. coli cells are inhibited from growth and undergo DNA condensation. In vitro HC binds to DNA and cleaves it in presence of copper. Our data strongly indicates HC mediates bacterial cell death by ROS generation and DNA damage. Damage to iron sulfur proteins in the cells contribute to amplification of oxidative stress initiated by HC. Further HC is active against a number of Gram negative bacteria isolated from patients with a wide range of clinical symptoms and varied antibiotic resistance profiles.

DNA damage is a late event in resveratrol-mediated inhibition of Escherichia coli.

Resveratrol is an important phytoalexin notable for a wide variety of beneficial activities. Resveratrol has been reported to be active against various pathogenic bacteria. However, it is not clear at the molecular level how this important activity is manifested. Resveratrol has been reported to bind to cupric ions and reduce it. In the process, it generates copper-peroxide complex and reactive oxygen species (ROS). Due to this ability, resveratrol has been shown to cleave plasmid DNA in several studies. To this end, we envisaged DNA damage to play a role in resveratrol mediated inhibition in Escherichia coli. We employed DNA damage repair deficient mutants from keio collection to demonstrate the hypersensitive phenotype upon resveratrol treatment. Analysis of integrity and PCR efficiency of plasmid DNA from resveratrol-treated cells revealed significant DNA damage after 6 h or more compared to DNA from vehicle-treated cells. RAPD-PCR was performed to demonstrate the damage in genomic DNA from resveratrol-treated cells. In addition, in situ DNA damage was observed under fluorescence microscopy after resveratrol treatment. Further resveratrol treatment resulted in cell cycle arrest of significant fraction of population revealed by flow cytometry. However, a robust induction was not observed in phage induction assay and induction of DNA damage response genes quantified by promoter fused fluorescent tracker protein. These observations along with our previous observation that resveratrol induces membrane damage in E. coli at early time point reveal, DNA damage is a late event, occurring after a few hours of treatment.

Radioprotective property of the ethanolic extract of Piper betel Leaf.

The radioprotective activity of Piper betel ethanolic extract (PE) has been studied using rat liver mitochondria and pBR 322 plasmid DNA as two model in vitro systems. The extract effectively prevented gamma-ray induced lipid peroxidation as assessed by measuring thiobarbituric acid reactive substrates, lipid hydroperoxide and conjugated diene. Likewise, it prevented radiation-induced DNA strand breaks in a concentration dependent manner. The radioprotective activity of PE could be attributed to its hydroxyl and superoxide radicals scavenging property along with its lymphoproliferative activity. The radical scavenging capacity of PE was primarily due to its constituent phenolics, which were isolated and identified as chevibetol and allyl pyrocatechol.

Antioxidant properties of a Tinospora cordifolia polysaccharide against iron-mediated lipid damage and gamma-ray induced protein damage.

The antioxidant activity of an arabinogalactan polysaccharide (TSP) isolated from Tinospora cordifolia, an Indian medicinal plant, was studied. The polysaccharide showed good protection against iron-mediated lipid peroxidation of rat brain homogenate as revealed by the thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxide (LOOH) assays. TSP also provided significant protection to protein against gamma-ray induced damage. The protective action can possibly be explained by its very high reactivity towards DPPH, superoxide radicals and the most damaging of radicals, the hydroxyl radical.

Radioprotective property of polysaccharide in Tinospora cordifolia.

Radioprotective activity of a polysaccharide preparation from the Indian medicinal plant. Tinospora cordifolia Miers has been established using Saccharomyces cerevisiae X2180 strain as the in vivo test model. The entire activity could be attributed to the radical scavenging capacity of the preparation, as it did not enhance the expression of the protective enzymes, catalase and superoxide dismutase in the yeast cells.

Resveratrol induced inhibition of Escherichia coli proceeds via membrane oxidation and independent of diffusible reactive oxygen species generation.

Resveratrol (5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol), a redox active phytoalexin with a large number of beneficial activities is also known for antibacterial property. However the mechanism of action of resveratrol against bacteria remains unknown. Due to its extensive redox property it was envisaged if reactive oxygen species (ROS) generation by resveratrol could be a reason behind its antibacterial activity. Employing Escherichia coli as a model organism we have evaluated the role of diffusible reactive oxygen species in the events leading to inhibition of this organism by resveratrol. Evidence for the role of ROS in E. coli treated with resveratrol was investigated by direct quantification of ROS by flow cytometry, supplementation with ROS scavengers, depletion of intracellular glutathione, employing mutants devoid of enzymatic antioxidant defences, induction of adaptive response prior to resveratrol challenge and monitoring oxidative stress response elements oxyR, soxS and soxR upon resveratrol treatment. Resveratrol treatment did not result in scavengable ROS generation in E. coli cells. However, evidence towards membrane damage was obtained by potassium leakage (atomic absorption spectrometry) and propidium iodide uptake (flow cytometry and microscopy) as an early event. Based on the comprehensive evidences this study concludes for the first time the antibacterial property of resveratrol against E. coli does not progress via the diffusible ROS but is mediated by site-specific oxidative damage to the cell membrane as the primary event.