Meenakshi Balhara

Analysis Toward Innovative Herbal Antibacterial & Antifungal Drugs

The antimicrobial activities of four medicinal plants Argemona mexicana, Achyranthes aspera, Catharanthus roseus, and Syzygium cumini were evaluated against Escherichia coli, Vibrio cholerae, Klebsiella pneumoniae, Proteus vulgaris, Bacillus subtilis, Salmonella typhi and three Aspergillus species. Extracts from Achyranthes aspera and Catharanthus roseus showed the highest antimicrobial potential (MIC 0.375-0.750 mg/ml) while extract from Argemona mexicana and Syzygium cumini, showed less activity. In disc diffusion assay, only eight out of twenty extracts showed antimicrobial activity at a concentration of 25.0 μg/ disc. The GCMS investigation reveals the existence of 2-bornanone; 1, 2-benzenedicarboxylic acid, bis (2-methylpropyl) ester; hexadecanoic acid, methyl ester and hexatriacontane in water extract fraction of C. roseus. The present research article provides a review of some medicinal plants incorporating antimicrobial drugs, together with recent advances in emerging therapeutics in clinical development and related patents for exploitation of herbal medicine.

Genomics of Chronic Obstructive Pulmonary Disease (COPD); Exploring the SNPs of Protease-Antiprotease Pathway

The COPD has been an important respiratory condition that affects people worldwide and its incidence has been alarming. The increasing incidence of this disorder has been attributed to global industrialization and environmental pollution. Although the exposures to environmental pollutants and smoking have been important triggers, the genetic component of individuals has been shown to be important for development and progression of COPD. Recent literature reported that protease-antiprotease imbalance to be important in etiopathogenesis of COPD. The enzymes namely neutrophil elastase and matrix metalloprotienases are considered to be foremost proteolytic molecules released by neutrophils and macrophages during inflammatory events in COPD. Normally, the lungs remain protected from the destructive effect of these two antiproteases by α1-antitrypsin (α1AT) and tissue inhibitors of metalloproteinases (TIMPs) respectively. In this review, we are trying to highlight the work by various research groups in exploring the SNPs of various genes of inflammatory pathways and the protease-antiprotease pathway, which may have some degree of association with COPD.

Siderophores; iron scavengers: the novel & promising targets for pathogen specific antifungal therapy

ABSTRACT Introduction: The recent emergence of resistance, toxicity paradigm and limited efficacy of conventional antifungal drugs necessitate the identification of de novo targets in fungal metabolism. One of the most critical physiological processes during in vivo pathogenesis is maintenance of iron homeostasis. The most life threatening opportunistic human fungal pathogens like Aspergillus, Candida and Cryptococcus exploit the siderophore mediated iron uptake mechanism either for survival, virulence, propagation or resistance to oxidative stress envisaged in vivo during infection. Areas covered: In this review, we will highlight the metabolic pathways; specifically siderophore biosynthesis, uptake and utilisation, triggered in the fungal pathogens in iron starving conditions and the various putative targets viable in these pathways to be recruited as novel therapeutic antidotes either via biosynthetic enzymes catalytic site inhibitors or as drug conjugates through trojan horse approach and further role in the development of fungal specific reliable diagnostic markers. Expert opinion: Siderophores are the weapons released by a pathogen to conquer the battle for iron acquisition. Hence, the fungal siderophore biosynthetic pathways along with their uptake and utilisation mechanisms represent an ideal target for pathogen specific, host friendly therapeutic strategy which would block the proliferation of parasite without causing any harm to the mammalian host.

Advancement in Infection Control of Opportunistic Pathogen (Aspergillus spp.): Adjunctive Agents

There is continuous emergence of resistant strains which leads to urgent need to discover new antifungal agents. The investigation of adjunctive agents for antifungal activity might help to optimize the therapy for Invasive Aspergillosis (IA). The chelating agents Ethylenediamine Tetraacetic Acid (EDTA) & Disodium salt of EDTA (DiEDTA) as adjunct to antifungal drugs have been investigated against 8 pathogenic isolates of Aspergillus spp. The MIC (Minimum Inhibitory Concentration) found by DDA (Disc Diffusion Assay) is 7.50-15.0 μg/disc; by MDA (Microbroth Dilution Assay) is 30.0-49.13 μg/ml & SGIA (Spore germination Inhibition Assay) is 30.0-49.13 μg/ml. Moreover, these agents did not show any toxicity up to a concentration of 312.5 μg/ml. The antifungal activity is also confirmed by another method i.e time kill curve analysis. While these agents were ten times less active than gold standard drug (Amphotericin B; AmpB) but eight times less toxic than AmpB. This leads to preliminary investigation of in vitro combination of chelating agents with antifungal drugs (Polyenes & Azoles) by DDA. These combinations showed a significant increase in zone of inhibition in contrast to single drug used. This preliminary work with chelating agents suggest that EDTA as an enhancing agent with antifungal properties in combination with antifungal drugs can be used in pharmaceutical preparations. Further investigation is in progress.

An anti-Aspergillus protein from Escherichia coli DH5α: Putative inhibitor of siderophore biosynthesis in Aspergillus fumigatus

An antifungal protein designated as anti‐Aspergillus protein (AAP), produced by Escherichia coli DH5α, was purified and characterised. It exhibited a molecular weight of 60 kDa on Sodium dodecyl sulphate–polyacrylamide gel electrophoresis analysis and depicted 99% purity on ultra performance liquid chromatography. The purified protein manifested antimycotic potential against pathogenic isolates of Aspergillus spp., depicting a minimum inhibitory concentration in the range 15.62–31.25 μg ml−1 and 5.0–10.0 μg per disc, using microbroth dilution, spore germination inhibition and disc diffusion assays respectively. In vitro toxicity tests demonstrated that it showed no toxicity against human erythrocytes at doses up to 1000 μg ml−1. Matrix‐assisted laser desorption ionisation–Time‐of‐flight analysis of trypsin‐digested peptides of purified protein and subsequent Mascot search revealed that several peptides of AAP have identity with bacterial siderophore biosynthetic protein, i.e. non‐ribosomal peptide synthetase enzyme, involved in critical step of fungal siderophore biosynthesis. Siderophore‐based inhibition was further corroborated by Chrome azurol S assay. Hence, the antagonistic effect might be the result of impediment in siderophore‐mediated iron uptake and transport process which may cause critical consequences on Aspergillus growth and virulence.

In vitro evaluation of combination of polyenes with EDTA against Aspergillus spp. by different methods (FICI and CI Model)

We investigated in vitro activity of polyene drugs (amphotericin B, Amp B; and nystatin, NYS;) in combination with ethylene diamine tetra acetic acid (EDTA) against 8 pathogenic isolates of Aspergillus spp.

Safety and Efficacy of Green Foliage in the Treatment of Fungal Infections and Associated Clinical Conditions

Forty five crude extracts of nine selected medicinal plants, based on their use in respiratory and other disorders in traditional systems of medicine were analyzed for their potential activity against three pathogenic species of Aspergillus. The presence of phenols, tannins, flavanoids, terpenoid, steroids, alkaloids and saponins in the different extracts was established. The crude extracts were examined for antifungal activity in concentration ranging from 5000.0 to 19.53 µg/ml using microbroth dilution assay in which twenty two extracts exhibited the anti-Aspergilli activity. The petroleum ether extract of Justicia adhatoda and water extract of Commelina bengalensis exhibited the maximum activity against Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger. Their in vitro minimum inhibitory concentrations (MICs) were found to be 156.0-312.0 µg/ml by microbroth dilution and spore germination inhibition assays. In disc diffusion assay, at concentration of 10 µg/disc of some crude extracts showed significant activity against Aspergilli. The toxicity (in vitro and in vivo) of bio-active fractions was evaluated, in which the extracts isolated from J. adhatoda were found to be non-toxic. Gas chromatography-mass spectrometry (GCMS) studies were performed for various extracts (petroleum ether, chloroform and acetone) of J. adhatoda which resulted in the identification of several bioactive compounds. The antifungal activity along with acute toxicity, cyto-toxicity as well as genotoxicity of extract fractions from J. adhatoda justifies the use of such screening in the expedition for new drugs.

Invasive aspergillosis: adjunctive combination therapy

Invasive aspergillosis remains a serious opportunistic fungal infection particularly in patients with a reduced immune defense such as those with hematological malignancies or transplant recipients. The mortality of invasive infections due to Aspergillus spp. is still high. The main reasons for this are the difficulty in diagnosing of these infections and the limited efficacy of antifungal agents. There is no optimal therapy for invasive aspergillosis, and therefore many clinicians have attempted to utilize a combination approach to improve outcomes. The current antifungal classes of drugs targeting the cell wall and cell membrane may need adjunctive agents focused on separate cellular pathways that can be used in combination therapy to maximize the efficacy, a valuable alternative to the monotherapy. The endeavor of this article is to review the literature on combination therapy by using adjunctive agents against Aspergillus spp and assess its eventual usability in the treatment of invasive aspergillosis.

Inhibition of conidiophore development in Aspergillus fumigatus by an Escherichia coli DH5α strain, a promising antifungal candidate against aspergillosis

The opportunistic human pathogen Aspergillus fumigatus produces a massive number of asexual spores (conidia) as the primary means of dispersal, survival, genome protection and infection of hosts. In this report, we investigated secretory and cytosolic proteins of non-pathogenic bacterial species (mostly belonging to human microbiome) for antifungal potential against A. fumigatus, A. flavus and A. niger. Our preliminary results revealed that cytosolic proteins of E. coli DH5α were most active and the less toxic against various pathogenic isolates of A. fumigatus (the major pathogenic species), depicting a minimum inhibitory concentration (MIC) of 62.50 μg/mL, 62.50 μg/mL and 12.50 μg/disc using microbroth dilution assay (MDA), percentage spore germination inhibition assay (PSGI) and disc diffusion assay (DDA), respectively. E. coli protein was non-toxic against human erythrocytes at doses up to 1000 μg/mL as compared to standard drug, amphotericin B which lysed 100% of erythrocytes at a concentration of 37.50 μg/mL. Time kill analysis proved it to be fungicidal in a concentration and time-dependent manner. Scanning electron microscopic studies (SEM) were carried out to prevail what kind of damage it causes to A. fumigatus. SEM results reported that conidiophore (structures forming conidia) development was halted as a major consequence, reducing the number of conidiophores to insignificant values as well as alteration in their morphological attributes. This feature may contribute to the development of new prevention strategies against Aspergillus infections. Hyphal atrophy was also observed, evidenced by shrinking and flattening of hyphal walls and reduced, abrupt hyphal branching. Such actions may effectively reduce the invasive ability of Aspergillus as well as it can sterilize the fungal burden by obstructing the conidiation pathway of A. fumigatus. Hence, E. coli DH5α, being a commensal species, can lead to the development of antifungal molecule with novel targets in fungal metabolism, which will help in combating the antifungal resistance and toxicity associated with current therapy.

Escherichia coli as a Potential Candidate Against Food Borne Bacterial Infections

The expanding familiarity with the dangers emanating from food-related pathogens as well as from the counterfeit synthetic additives used to control them has prompted restored enthusiasm for supposed ‘green advances’ including novel approaches for a minimal processing and exploitation of commensal bacterial strains as a source of novel antibacterial agents or in an alternative as a probiotic. The present article demonstrates detailed analysis of extracellularly released and intracellular proteins of non-pathogenic bacterial strains residing in human gut against pathogens involved in food-borne illness and toxication. Among the human microbiota, the cytosolic proteins of Escherichia coli DH5α (being a prominent and primitive bacterial species in human gut) have been found to be most active against Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, Proteus vulgaris, Salmonella typhi and Vibrio cholerae (the major food-borne pathogens) showing minimum inhibitory concentration (MIC) in the range of 62.50–250.0 μg/ml using resazurin-based microtitre dilution method. In disc diffusion assay, the MIC was found to be in the range of 6.25–25 μg/disc. Hence, Escherichia coli DH5α emerges as a source of novel antimicrobial proteins.