Ratul Sarkar

Alteration of Zeta potential and membrane permeability in bacteria: a study with cationic agents

In the present study, we have tried to establish the correlation between changes in Zeta potential with that of cell surface permeability using bacteria (Escherichia coli and Staphylococcus aureus). An effort has been made to establish Zeta potential as a possible marker for the assessment of membrane damage, with a scope for predicting alteration of cell viability. Cationic agents like, cetyl trimethyl ammonium bromide and polymyxin B were used for inducing alteration of Zeta potential, and the changes occurring in the membrane permeability were studied. In addition, assessment of poly-dispersity index (PDI), cell viability along with confocal microscopic analysis were performed. Based on our results, it can be suggested that alteration of Zeta potential may be correlated to the enhancement of membrane permeability and PDI, and it was observed that beyond a critical point, it leads to cell death (both Gram-positive and Gram-negative bacteria). The present findings can not only be used for studying membrane active molecules but also for understanding the surface potential versus permeability relationship.

Anti-biofilm activity of Marula - a study with the standardized bark extract.

ETHNOPHARMACOLOGICAL RELEVANCE Marula (Sclerocarya birrea; family - Anacardiaceae) is an African plant, which enjoys wide socio-economic importance particularly in southern part of Africa. The fruits are consumed as food and also as alcoholic beverage (cream liquor). In different parts of Africa, the decoction of the bark is traditionally used for the treatment of dysentery, diarrhoea, and various other infectious conditions. The aim of the study was to investigate the anti-biofilm properties of the methanol extract of Marula bark (stem bark of Sclerocarya birrea), with a view towards combating the emergence of antimicrobial resistance often associated with bacterial biofilms. MATERIALS AND METHODS The standardized methanol extract was initially tested for its antimicrobial property. The crystal violet assay was used for evaluating anti-biofilm (biofilm formation by Pseudomonas aeuginosa) activity. Further in order to study the mechanism of anti-biofilm activity, the same was evaluated for understanding its role on various quorums sensing mediated phenomenon (swarming motility assay, protease and pyoverdin assay) that are known to be associated with the formation of biofilms and pathogenicity. RESULTS The methanol extract showed no inhibition of bacterial growth up to a concentration of 200 µg/ml. Interestingly, the sample produced anti-biofilm activity (around 75% decrease; 100 µg/ml) at sub-lethal concentration. Further it also significantly reduced the QS mediated swarming motility. The release of various virulent factors (protease and pyoverdin) was found to be lowered when pre-treated with the extract. CONCLUSION The present study illustrates the anti-biofilm property Sclerocarya birrea. The standardized extract significantly disrupted the quorum sensing mediated production of biofilm formation and also inhibited swarming ability of the cells. The extract displayed a regulatory role on the secretion of protease and pyoverdin, two QS dependent pathogenic factors found in Pseudomonas aeruginosa. This study also validates the ethnobotanical use of Marula.

Pharmacological studies on Buchanania lanzan Spreng.-A focus on wound healing with particular reference to anti-biofilm properties

OBJECTIVE To evaluate the wound healing activity of the methanolic root extract of Buchanania lanzan Spreng. (B. lanzan), with a focus on antimicrobial and anti-biofilm properties. METHODS The extract was evaluated for its wound healing properties (excision and incision models) as evident from the analysis of tensile strength and wound contraction. The extract was also screened for antibacterial properties against different Gram positive and Gram negative bacterial strains. B. lanzan was also studied for its effect on biofilm formation and disruption of preformed biofilms. The synergistic effect of B. lanzan was determined in combination with gentamicin. RESULTS Topical application of B. lanzan (10% w/w ointment) significantly increased (40.84%) the tensile strength in the incision wound model. B. lanzan also showed significant wound healing activity in excision model and such significant activity was observed from the 9th day. Whereas Soframycin displayed significant wound healing activity from the 6th day. It was found that root extracts of B. lanzan revealed significant inhibition against all tested pathogens. B. lanzan displayed antimicrobial activity against Gram positive (MIC 0.625 mg/mL) and Gram negative (MIC 0.625-1.25 mg/mL). B. lanzan was able to reduce biofilm formation and also caused disruption of preformed biofilms in a manner similar to ciprofloxacin. However, gentamicin was found to be ineffective against biofilms formed by Gram negative organism. According to the fractional inhibitory concentration index, B. lanzan displayed synergistic activity when it was combined with gentamicin. CONCLUSIONS From this study it may be concluded that the root extract of B. lanzan revealed significant wound healing potential, which was supported and well correlated with pronounced antibacterial activity of the tested plant parts.

Antimicrobial properties of Kalanchoe blossfeldiana: a focus on drug resistance with particular reference to quorum sensing-mediated bacterial biofilm formation

This study attempts to investigate the antimicrobial properties of Kalanchoe blossfeldiana with a particular reference to quorum sensing (QS)‐mediated biofilm formation.

Synthesis and characterization of 5-amino-2-((3-hydroxy-4-((3-hydroxyphenyl) phenyl) diazenyl) phenol and its Cu(II) complex – a strategy toward developing azo complexes for reduction of cytotoxicity

A major drawback of azo compounds is their associated toxicity, often carcinogenic, which is related to the reduction of the azo bond. This study intends to re-investigate this behavior by studying 5-amino-2-((3-hydroxy-4-((3-hydroxyphenyl) phenyl) diazenyl) phenol (AHPD), a compound containing two azo bonds. Interaction of AHPD and its dimeric Cu(II) complex with bacterial strains Escherichia coli and Staphylococcus aureus revealed the complex was less toxic. Reductive cleavage of the azo bond in AHPD and the complex followed using cytochrome c reductase (a model azo-reductase) as well as azo-reductase enzymes obtained from bacterial cell extracts. Degradation of the azo bond was less in the complex allowing us to correlate the observed cytotoxicity. Cyclic voltammetry on AHPD and the complex support observations of enzyme assay experiments. These were particularly useful in realizing the formation of amines as an outcome of the reductive cleavage of azo bonds in AHPD that could not be identified through an enzyme assay. Results suggest that complex formation of azo compounds could be a means to control the formation of amines responsible for cytotoxicity. Studies carried out on bacterial cells for mere simplicity bear significance for multicellular organisms and could be important for human beings involved with the preparation and utilization of azo dyes.

Inhibition of arachidonic acid metabolism and pro-inflammatory cytokine production by Bruguiera gymnorrhiza leaf

Bruguiera gymnorrhiza is a mangrove plant of the family Rhizophoraceae. The roots and leaves of the plant have traditionally been used for treating burns and mild inflammatory lesions. In the present study an attempt was made to evaluate the anti-inflammatory activity of the Bruguiera gymnorrhiza leaves. The methanolic fraction of Bruguiera gymnorrhiza leaves (BRG) was evaluated using different in vitro and in vivo models to evaluate the anti-inflammatory properties and also to obtain an insight on the probable mechanism of such activity. The leaf extract produced significant inhibition of both carrageenan induced rat paw oedema and acetic Acid induced peritoneal capillary permeability. The above observations were further supported by our findings from in vitro experimental models, where the plant extract produced significant inhibition of both cyclooxygenase (COX) and lipoxygenase (LOX) enzymes. Moreover, BRG inhibited the production of proinflammatory cytokines (TNF- α, IL-6 and IL-1β) in lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) in a dose dependent manner. It was also found to possess significant free radical (DPPH, superoxide and oxygen radical) scavenging activity. From the results obtained from the various in vitro and in vivo test models, it may be concluded that the methanolic extract of Bruguiera gymnorrhiza leaves display significant anti-inflammatory properties, probably mediated through blockade of both COX – LOX pathway, coupled with scavenging effect on free radicals.

5,7-dihydroxy-2-(3-hydroxy-4, 5-dimethoxy-phenyl)-chromen-4-one-a flavone from Bruguiera gymnorrhiza displaying anti-inflammatory properties

Objective: Bruguiera gymnorrhiza (BRG) (L.) Lamk (Rhizophoraceae), a mangrove species, is widely distributed in the Pacific region, eastern Africa, Indian subcontinent, and subtropical Australia. The leaves of this plant are traditionally used for treating burns and inflammatory lesions. This study isolates the bioactive compound from the methanol extract of BRG leaves and evaluates the possible mechanisms of anti-inflammatory activity involved. Materials and Methods: Bioassay-guided fractionation of BRG was performed to identify the bioactive fraction (displaying inhibition of cyclooxygenase 2 [COX2] - 5-lipoxygenase (5-LOX) activities and tumor necrosis factor-alpha (TNF-α) production at the tested concentrations of 100 and 10 μg/ml). The fractionation was performed by solvent extraction and preparative high-performance liquid chromatography. The bioactive compound was characterized by ultraviolet–visible, liquid chromatography–mass spectrometry and nuclear magnetic resonance spectroscopy. The antioxidant potential was evaluated by electron spin resonance spectrum of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical at 250 μM. The effect of the compound was also studied on TNF-α converting enzyme and nuclear factor kappa B (NF-κB) activities at the concentrations 100, 10 and 1 μg/ml. Results: Bioassay-guided purification of BRG revealed the presence of a flavone (5,7-dihydroxy-2- [3-hydroxy-4,5-dimethoxy-phenyl]-chromen-4-one) of molecular weight 330Da. It demonstrated more than 80% inhibition against COX2, 5-LOX activities and TNF-α production at 100 μg/ml. It also displayed 40% inhibition against DPPH radical at the tested concentration along with 23.1% inhibition of NF-κB activity at 100 μg/ml. Conclusions: The isolated methoxy-flavone may play a predominant role in the anti-inflammatory properties displayed by BRG leaves. Such activity may involve multiple mechanisms, namely (a) modulation of oxidative stress (b) inhibition of arachidonic acid metabolism and (c) downregulation of pro-inflammatory cytokines probably through NF-κB inhibition.

Anti-biofilm activity and food packaging application of room temperature solution process based polyethylene glycol capped Ag-ZnO-graphene nanocomposite

Present work reports on synthesis and anti-biofilm activity as well as food packaging application of Ag-ZnO-reduce graphene oxide (rGO)-polyethylene glycol (PEG) (AZGP) nanocomposites via adopting room temperature solution process by varying silver nitrate content (up to 0.1 M) with fixed content of graphene oxide and PEG used in the precursors. Presence of Ag and ZnO nanoparticles (NPs) distributed uniformly over rGO nanosheets has been confirmed by X-ray diffraction and transmission electron microscopic analyses whereas FTIR, Raman, UV-Visible and X-ray photoelectron spectral studies have been performed to confirm the existence of chemical interaction/complexation that happened between the available oxygen functionalities of rGO and PEG with the inorganic moieties (Ag-ZnO/Zn2+) of AZGP samples. A formation mechanism of AZGP nanocomposite is proposed based on the experimental results. Anti-biofilm activity has been studied on Staphylococcus aureus and Pseudomonas aeruginosa bacteria to confirm the efficiency of the nanocomposites for killing the bacterial cells. It is found that 0.05 M silver nitrate based AZGP nanocomposite at 31.25 μg/mL sample dosage shows about 95% inhibition activity towards the biofilm formation as well as eradication of preformed biofilm. Also, agar based AZGP film has been fabricated and characterized by X-ray diffraction study for the purpose of food packaging application. Textural analysis of agar based film shows an enhanced film tensile strength. The film also shows an excellent antimicrobial activity even after keeping it for a prolong period of about 90 days. This cost effective simple synthesis strategy can make an avenue for development of Ag incorporated other biocompatible metal oxide based rGO-PEG nanocomposites for potential food packaging application.

Evaluation of Natural Products against Biofilm-Mediated Bacterial Resistance

Since ages, infections have claimed more lives than any great war. The advent of antibiotics was believed to bring about a possible solution to this problem. Irrational use of antibiotics has ultimately lead to a rapid increase in microbial resistance, thus making it a matter of global concern and this problem is further compounded by a drastic slowdown in the pharma pipeline. Therefore, finding novel strategies to combat the growing menace of drug-resistant microbes is the need of the hour. Among various mechanisms of drug resistance, formation of bacterial biofilms (an environment providing protection to the microbes from external stress), is considered to be a predominant one. Apart from synthetic molecules, products of plant origin (particularly secondary metabolites), owing to their wide chemical diversity, are being explored for disruption of extracellular polysaccharide as well as blocking of signaling pathways (quorum sensing (QS)), by interfering with acylhomoserine lactone production and suppression of QS-regulated virulence factor.