Bioautographic Detection Research Articles

Isolation, Identification, and Antimicrobial Evaluation of Secondary Metabolite from Serratia marcescens via an In Vivo Epicutaneous Infection Model.

Microbial secondary metabolites, which play a pivotal role in struggling with infectious diseases, are the new source for controlling bacterial contaminations and possess a strong antimicrobial potential. The present study is designed to evaluate the in vitro and in vivo bactericidal activities of prodigiosin against Staphylococcus aureus. For this purpose, Serratia marcescens was used to produce prodigiosin. Characterization of the prodigiosin was carried out using NMR. In addition, bioautographic detection of prodigiosin was detected by TLC. Antibacterial assays, in vivo epicutaneous infection tests, swap analyses, and histopathological examinations were determined. The results revealed that prodigiosin was detected by NMR and TLC. According to antimicrobial susceptibility tests, prodigiosin is an efficient bactericidal compound that demonstrated strong antibacterial activity toward S. aureus. In vivo, animal studies determined that the strong inhibition of S. aureus-caused epidermal infection occurs by prodigiosin at 48 h. Histopathological results showed that S. aureus + prodigiosin skin sections consist of improved and healthy tissues without any infection area compared with the S. aureus and control groups. The in vivo study verified the antibacterial results with swap analyses, and histopathological findings showed that prodigiosin is a promising microbial metabolite effective against S. aureus infection. This study proved that prodigiosin with excellent bioactivity exhibited antibacterial properties, which might possess massive potential for new therapeutic approaches using micro-organisms.

TLC Bioautographic Detection and Characterization of Antibacterial Compound from the Cyanobacterium Anabaena oryzae

TLC Bioautographic Detection and Characterization of Antibacterial Compound from the Cyanobacterium Anabaena oryzae

A Comparison of Antioxidant, Antibacterial, and Anticancer Activity of the Selected Thyme Species by Means of Hierarchical Clustering and Principal Component Analysis

In this study, six numerical data sets are presented valid for eighteen thyme (Thymus L.) species and characterizing three biological properties of these herbs, i.e., antioxidant, antibacterial, and anticancer activity. Four data sets characterize antioxidant properties, one data set characterizes antibacterial property, and one data set characterizes anticancer activity. Antioxidant properties were measured with two free radical standards (DPPH and ABTS), two free radical scavenger standards (trolox and gallic acid), and three analytical techniques (EPR spectroscopy, ultraviolet–visible [UV–vis] spectrophotometry, and the dot blot test with bioautographic detection). Antibacterial activity was tested upon the Gram-positive Bacillus subtilis (ATCC 6633) strain, and anticancer activity was evaluated upon the human colon adenocarcinoma cells (HCT116). It was found out that the thyme extracts characterize with all three biological activities (yet with anticancer activity not very strongly pronounced) and that in quantitative terms, each activity strongly depends on the thyme species considered. An ultimate goal of this study was to investigate if any quantitatively confirmed correlation exists among these three biological activities, which might point out to a common mechanism of their action. To this effect, six sets of numerical data underwent hierarchical clustering and Principal Component Analysis. Based on the results obtained, no quantitative correlation was established among antioxidant, antibacterial, and anticancer activity of the thyme species, which seems indicative of different molecular mechanisms of these three actions.

Thin-layer chromatography: Direct bioautography as a method of examination of antimicrobial activity of selected Potentilla species

ABSTRACTSimple method for preparing biologically active material from plant extract may consist of four steps: obtaining an extract from selected plant, analytical chromatography with biological detection and transition to preparative scale, preparative isolation of biologically identified bands/groups of bands from chromatogram, rechromatography of isolated fractions. The next step of proceeding may be a quality analysis of separated material by, e.g., thin-layer chromatography (TLC)-MS Interface or biological studies after isolation. Extracts obtained from four Potentilla species: P. erecta, P. collina, P. megalantha, and P. crantzi were separated on silica plates, developed with the nonaqueous eluent. Immersion—direct bioautography was chosen as a method of detection of bands’ activity for their selection for isolated bands. Antibacterial/bacteriostatic properties of marked areas was the preliminary criterion of choice the samples to the further examinations. Preparative thin layer chromatography allowed isolation of various number of fractions of each extracts. Obtained fractions were examined in various TLC systems (silica/nonaqueous mixture of solvents) with the various modes of development to obtain the best separation effect. The next step was a once more bioautographic detection of separated fractions on the TLC plates. The found chromatographic systems will be used to mass spectrometric determination of biologically active compounds contained in examined extracts. Bioautographic indication will be the method of choice for subsequently isolated compounds or groups of compounds.

Bioautographic Screening of Antibacterial Properties of Selected Juices, Herbal Infusions, and Cosmetic Ingredients

Polyphenols are the products of plant immune system and they characterize with well pronounced antioxidant, antibacterial and antifungal properties. Owing to a wide spectrum of native polyphenols, herbal infusions, fruit juices, wines, and cosmetic substrates are among the most popular health-care products and for this reason, they undergo intense scientific investigations. Antibacterial properties of botanical material can prove particularly helpful in efforts aiming to lower the amounts of preservatives in alimentary, pharmaceutical, and cosmetic products. In this study, a comparison is presented of antibacterial properties for (i) nine samples of red wines and the wine extracts, (ii) 13 samples of plant extracts and the extracts thereof used in cosmetic preparations, and (iii) fruit juices and infusions prepared of dried fruits. All these botanical materials are widely used in pharmaceutical, cosmetic, and/or alimentary industry. As a rapid screening technique, a simplified version of thin-layer chromatography with bioautographic detection (TLC-BD) was applied, known as the dot blot test with bioautographic detection. This test makes use of chromatographic plates and all details of the procedure are the same as for TLC-BD, except for the fact that the chromatogram is not developed and an assessment of antibacterial properties is carried out for a whole sample spotted on to chromatographic plate, and not for its separated fractions. As a result, the most potent antibacterial action was observed with the pasteurized pure raspberry juice and an infusion made of dried black elderberry fruit.

Direct bioautographic detection of antibacterial components of clary sage and spearmint essential oils

The antibacterial effect of the components of clary sage (Salvia sclarea L.) and spearmint (Mentha spicata L. var. crispa (Bentham) Danert) was investigated by means of high-performance thin-layer chromatography-direct bioautography against the Gram-positive soil bacterium Bacillus subtilis (Bs) and Gram-negative bacteria such as a pepper pathogen Xanthomonas euvesicatoria (Xe), a luminescence gene-tagged Arabidopsis pathogen Pseudomonas syringae pv. maculicola (Psm) and a luminescent marine Aliivibrio fischeri (Af). Sclareol, linalool, and linalyl acetate were identified as active components of clary sage and carvone as the antibacterial substance in spearmint. Sclareol inhibited all tested bacteria, linalool and carvone showed antibacterial effect against all Gram-negative strains tested, while linalyl acetate only against Xe and Af. Some minor components of the clary sage essential oil also gave a zone of inhibition when tested on Gram-negative bacterium strains.

Detection of acetylcholinesterase-inhibiting natural products from Illicium angustisepalum by bioautographic screening

Alzheimer's disease (AD) is the most common type of dementia and a leading neurodegenerative disorder. According to the cholinergic hypothesis, memory impairment in AD patients results from the dysfunctions in the central neurotransmission of acetylcholine. Inhibitors of acetylcholinesterase (AChE) currently form the basis of pharmacotherapy of AD. In the course of a search for natural AChE inhibitors, Illicium angustisepalum A.C. Smith (Schisandraceae) was found to be active by using a bioautographic assay. Since former studies have identified the Illicium genus to be a potential source of AChE inhibitors, this under-studied member of Illicium was further investigated. In the present study, an essential oil and four extracts (i.e., ethyl acetate, butanol, petroleum ether and water extracts) of I. angustisepalum were tested for AchE-inhibiting activity, and positive results were observed in the essential oil and the ethyl acetate fraction. Isolation work is underway and this poster will report the most recent findings in the attempt to identify active principles based on bioautographic detection.

Separation and Identification of Antibacterial Chamomile Components Using OPLC, Bioautography and GC-MS

Components of 50% aqueous ethanol chamomile (Matricaria recutica L.) flower extract, previously found antibacterial in a TLC-bioautographic study, were separated and isolated by the use of on-line overpressured layer chromatography (OPLC). This system consisted of an OPLC 50 BS system, an on-line coupled flow-through UV detector, and a manual fraction collector. The collected fractions were investigated by GC-MS analysis and by TLC re-chromatography with subsequent visualization, performed after use of the vanillin-sulphuric acid reagent, or under UV illumination, or applying bioautographic detection. The main compounds of the collected 11 fractions were identified by GC-MS. The results showed that the antibacterial effect of 50% aqueous ethanol extract of chamomile is ascribable to cis-, trans-spiroethers, and the coumarins like herniarin and umbelliferone.

Thin‐Layer Chromatography‐Direct Bioautography of Flumequine Residues in Milk

Fluoroquinolone antibiotics are a relatively new group of synthetic antibiotics derived from 3‐quinolone carboxylic acid. They are widely used in both human and veterinary medicine. Flumequine is frequently used in fish, poultry, and cattle husbandry for the treatment and prevention of diseases. It is also used to enhance weight gain of livestock as a feed or water additive. Hence, the antibiotic residues may be present in edible products, e.g., in meat, eggs, or milk. Thin‐layer chromatography–direct bioautography combines TLC with microbiological detection. In the present paper, conditions for bioautographic detection of flumequine were determined for the antibiotic standards. The established method was applied for semi‐quantitative determination of flumequine residues in milk. Matrix solid‐phase dispersion was used as a pre‐separation method.

Characterization and TLC-bioautographic detection of essential oils from someThymustaxa. Determination of the activity of the oils and their components against plant pathogenic bacteria

The chemical composition of the oils of four thyme (Lamiaceae) chemotypes ( Thymus vulgaris L., Thymus serpyllum L., Thymus x citriodorus (Pers.) Schreb., and Thymus x citriodorus “Archer’s Gold”) has been determined by gas chromatography (GC). Thymol was the main component of the oils of Thymus vulgaris and Thymus serpyllum , geraniol was the main component of the oil of Thymus x citriodorus , and carvacrol was the main component of the oil of Thymus x citriodorus “Archer’s Gold”. The bioactivity of the volatile oil of Thymus vulgaris and of the three main components of the oils against Gram negative plant pathogenic bacteria was examined by direct bioautography. They had an inhibitory effect on all of the test microorganisms. Two bacterial strains ( Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. phaseolicola ) were most sensitive in the bioautographic system and use of these bacteria would considerably shorten the process of bioautographic detection. The two antibiotics gentamycin a...

Semiquantitative Estimation of Enrofloxacin and Ciprofloxacin by Thin‐Layer Chromatography–Direct Bioautography

Fluoroquinolones, relatively new chemotherapeutics, are widely used in the treatment of both human and veterinary diseases because of their broad spectrum of antibacterial activity and good pharmacokinetic properties. Enrofloxacin is used in veterinary medicine, while its main metabolite, ciprofloxacin, is one of the most frequently used human antibiotics in the world. Hence, there is a need for assays capable of determining and distinguishing between these drugs. Thin‐layer chromatography–bioautography (TLC–B) combines TLC with microbiological detection. In the present paper, semiquantitative determination of enrofloxacin and ciprofloxacin standards by TLC–B using Chrom Biodip® Antibiotics Test Kit is presented. The optimal conditions for bioautographic detection were established. The exponential relations were proposed for approximation of dependencies between areas of zone inhibitions and logarithm of the amounts applied. The detection limit for standards of both enrofloxacin and ciprofloxacin was 0.01 ppm, lower than maximum residue limits (MRLs) established for various food products.

Separation and detection of aflatoxins using overpressured-layer chromatography and bioautography

It has been established, by use of bioautographic detection with the phytopathogenic bacteria Pseudomonas savastanoi pv. phaseolicola , that the common aflatoxins have antimicrobial activity after OPLC separation. Our preliminary results suggest this antimicrobial activity originates from the activity of formaldehyde formed by the bacterial cells and/or from the methoxy groups of aflatoxin molecules.

Tissue-Dependent Degradation of Monensin Residues in Chicken Tissues with Prolonged Freezer Storage

Monensin, a monocarboxylic acid ionophore, is widely used in the poultry, cattle, and swine industries? Concern over its toxicity has risen in recent years with several reports of poisoning outbreaks.’ Monensin is known to be readily absorbed through the gastrointestinal tract of most animals. Residue concentrations, however, do not reach high levels, even in severely intoxicated animals. 4 This produces a diagnostic problem, especially if a history of clinical signs that could implicate monensin is not presented with the case upon postmortem examination. Because of the likelihood of low residue concentrations and the possibility of residue degradation while in freezer storage, it is desirable to know which tissue to select for monensin testing. The purpose of this study was to determine a suitable marker organ by testing poultry tissues using a sensitive thin-layer chromatography/ bioautographic detection method for monensin. A marker organ as defined for this study is that tissue in which monensin can be detected in greatest quantities after a specified period of freezer storage. The animals in this study were cared for in accordance with the standards set by the Canadian Council on Animal Care. 2 Four 7-week-old male broilers (Hubbards) a were given an oral dose (1,000 mg/kg body weight) of monensin- sodium salt b via direct intubation to the crop. Samples of cardiac muscle, skeletal muscle, liver, and kidney were taken immediately after death. The tissues of the 4 birds were pooled and homogenized. c The samples were then placed in air-tight plastic d bags and stored in a freezer at -20 C. At

Bioautographic detection of T-2 and HT-2 toxins

Bioautographic detection of T-2 and HT-2 toxins

Chromatographic methods for the analysis of bleomycin complex

Three thin-layer chromatographic methods were developed for the separation of the components of bleomycin which allowed direct bioautographic detection. The change in the composition of heat degraded samples of bleomyein was monitored by these methods and by high-performance liquid chromatography. It was found that the microbiological potency estimates of the heated material did not accurately reflect the changes which had occurred during degradation.

Bioautographic detection of mycotoxins on thin-layer chromatograms

A method for the bioautographic detection of mycotoxins on thin-layer chromatograms bu using Artemia salina larvae is described. The method was tested on standard samples of mycotoxins (aflatoxin B 1, kojic acid and sterigmatocystin) and on the extracts from toxicogenic fungi isolated from different sources.

Bioautography in paper and thin-layer chromatography and its scope in the antibiotic field

A critical review of the application of bioautography as a special detection method in paper and thin-layer chromatographic studies on antibiotics is presented. Examples are given to show various aspects of the bioautographic detection of antibacterial, antifungal, antiprotozoal, antiphage, phage-inducing, antiviral and cytotoxic substances. Documentation possibilities and the use of bioautography in quantitative analysis are also discussed.

Augmented inhibition by kinetin and related compounds of growth of pteridine-inhibited Lactobacillus arabinosus

Application of a previously published microbiological assay procedure for thymidine determination has been made to the detection of kinetin, certain 6-(substituted)aminopurines, and certain other related compounds, by means of the augmented inhibitory effects exerted by these compounds upon growth of pteridine-inhibited L. arabinosus in the absence of thymidine. The method can be used for bioautographic detection of such inhibitory activities on paper chromatograms.