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JPC - Journal of Planar Chromatography - Modern TLC
Authors:
Ágnes Móricz
,
Györgyi Horváth
,
Péter Molnár
,
Béla Kocsis
,
Andrea Böszörményi
,
Éva Lemberkovics
, and
Péter Ott

The composition of the essential oil of Thymus vulgaris L. has been determined by GC-FID and GC-MS. Because separation of thymol, carvacrol, and linalool, components of the essential oil, was more efficient by overpressured layer chromatography (OPLC) than by conventional thin-layer chromatography (TLC), the forced flow technique was used before biological detection. All three test compounds had antibacterial effect against the phytopathogenic bacterium Pseudomonas syringae pv. maculicola, in bioautography, although in essential oil thymol was present in sufficient quantity to produce an inhibiting zone in the adsorbent layer. In BioArena investigations, when reduced glutathione as a formaldehyde (HCHO) capturer was dissolved in the cell suspension before bioautographic exposure to the essential oil, the characteristic inhibiting activity of thymol and carvacrol against Bacillus subtilis soil bacteria was reduced, whereas the presence of the HCHO precursors NGmonomethyl-l-arginine or N ɛ-monomethyl-l-lysine enhanced their antibacterial effect. These results suggest that HCHO and its reaction products may be involved in the antibacterial activity of thymol and carvacrol.

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Lipopolysaccharide and b-1,3-glucan binding protein (LGBP) is a pattern recognition receptor that can recognize and bind LPS and b-1,3-glucan. LGBP has crucial roles in innate immune defense against Gram-negative bacteria and fungi. In this study, LGBP functions in Portunus trituberculatus innate immunity were analyzed. First, the mRNA expression of PtLGBP in hemocytes, hepatopancreas, and muscle toward three typical pathogen-associated molecular patterns (PAMPs) stimulations were examined using real-time PCR. Results show that the overall trend of relative expressions of the LGBP gene in three tissues is consistent, showing up-down trend. In each group, the highest expression of the LGBP gene was at 3 and 12 h post-injection. The LGBP gene is also expressed significantly higher in the hemocytes and hepatopancreas than in the muscle. The highest level of LGBP was in the lipopolysaccharides (LPS) and glucan-injected group, whereas the lowest level was in the PGN-injected group. Furthermore, bacterial agglutination assay with polyclonal antibody specifically for PtLGBP proved that the recombinant PtLGBP (designated as rPtLGBP) could exhibit obvious agglutination activity toward Gram-negative bacteria Escherichia coli, Vibrio parahaemolyticus, and V. alginolyticus; Gram-positive bacteria Bacillus subtilis; and fungi Saccharomyces cerevisiae. LGBP in Portunus trituberculatus possibly served as a multi-functional PRR. In addition, LGBP is not only involved in the immune response against Gram-negative and fungi, as manifested in other invertebrates, but also has a significant role in anti-Gram-positive bacteria infection.

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Acta Biologica Hungarica
Authors:
Marijana Kosanić
,
Branislav Ranković
, and
Tatjana Stanojković

In the present investigation the acetone extracts of three macroalgae, Cystoseira amentacea, Cystoseira barbata and Cystoseira compressa, were tested for antioxidant, antimicrobial and cytotoxic potential. As a result of the study, C. amentacea extract had more potent free radical scavenging activity (IC50 = 409.81 μg/mL) than C. barbata and C. compressa extracts. For reducing power, measured values of absorbance varied from 0.0352 to 0.8873. The IC50 values for superoxide anion scavenging activity for different extracts were 521.45–976.62 μg/mL. Total phenol and flavonoid contents in extracts were 39.96–81.28 μg PE/mg and 20.85–64.58 μg RE/mg respectively. Further, all three Cystoseira species exhibited a similar antimicrobial activity. The lowest MIC value (0.312 mg/mL) was shown in the extract obtained from C. compressa against Bacillus subtilis. Finally, extract of C. amentacea expressed the strongest cytotoxic activity toward tested cell lines with IC50 values ranging from 94.72 to 186.55 μg/mL. Based on these results, it can be stated that the tested macroalgae can be used as potential natural antioxidants and antimicrobial and cytotoxic agents.

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Lactic acid bacteria in fermented foods help in the improvement of flavour, preservation of the nutritive values of the raw material, and inhibition of growth or killing of food spoilage and pathogenic bacteria. Beside other metabolites, the produced bacteriocin, which is a ribosomally synthesized antimicrobial peptide, is the major metabolite involved in the killing of food spoilage microorganisms and acts as a biopreservative. In search of a bacteriocin having specific inhibitory activity against food pathogenic bacteria, isolation of bacteriocin producing lactic acid bacteria from various food items was carried out. Based on maximum production of the bacteriocin, strain BS13 was characterized and was further identified as Enterococcus faecium BS13 on the basis of physicochemical properties and 16S rRNA analysis. In MRS medium this isolate presented the maximum production of bacteriocin (27 306 AU ml−1) after 18 h of incubation period. BS13 bacteriocin showed antimicrobial activity against a wide range of bacteria, including Bacillus subtilis, Staphylococcus sp., Pediococcus sp., Listeria monocytogenes and Lactobacillus sp.

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DL-p-chlorophenylalanine (PCPA) and its derivatives were evaluated for genotoxic effects using Escherichia coli and Bacillus subtilis strains lacking various DNA-repair mechanisms in spottest and in suspension test. The mutagenic activity of studied compounds was determined by the Ames test. Reverse mutation test was performed with Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 without S9 mix. 0.02 M nitrosomethylurea (NMU) standard mutagen was used as a positive control. The results showed that the parent nonessential amino acid PCPA had no detectable genotoxic and mutagenic activities in bacteria. The methyl ester of this amino acid and its N-phenylacetyl derivative possessed weak genotoxicity. Meanwhile N-sec-butyloxycarbonyl, N-benzyloxycarbonyl, N-(p-nitrophenylacetyl) and N-(p-nitrophenoxyacetyl) derivatives of DL-p-chlorophenylalanine exhibited appreciable genotoxi- city. Among the seven tested compounds only N-benzyloxycarbonyl and N-(p-nitrophenoxyacetyl) derivatives of DL-p-chlorophenylalanine have been found to be mutagenic. Only parent PCPA possessed antimutagenic properties in respect of nitrosomethylurea. The structural modification, which strongly affects genotoxicity and mutagenicity perhaps may be due to steric hydrance of the substituents, causing interference with enzyme and DNA interactions.

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Acta Phytopathologica et Entomologica Hungarica
Authors:
A. Kamalakannan
,
L. Mohan
,
K. Kavitha
,
S. Harish
,
R. Radjacommare
,
S. Nakkeeran
,
V. K. Parthiban
,
R. Karuppiah
, and
T. Angayarkanni

Five isolates of Trichoderma viride, Pseudomonas fluorescens and four isolates of Bacillus subtilis were evaluated for their ability to control Rhizoctonia solani, the causal agent of stem and stolon rot of peppermint (Mentha piperita Lin.). Of the various isolates of T. viride, P. fluorescens and B. subtilis tested, TVUV10, PFMMP and BSG3 showed the maximum inhibition of mycelial growth of R. solani. Among these isolates, P. fluorescens, PFMMP recorded the highest inhibition zone against R. solani in vitro and was very effective in reducing disease incidence in greenhouse condition. The effective isolates were evaluated for their ability to induce defense related enzymes and chemicals in plants. Increased activity of phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenoloxidase (PPO) and total phenolics were recorded in the biocontrol agents pretreated peppermint plants challenged with R. solani. P. fluorescens isolate PFMMP recorded early and increased synthesis of all defense related enzymes and total phenol. Thus, the present study shows that application of biocontrol agents; induce defense related enzymes involved in phenyl propanoid pathway in addition to direct antagonism which collectively contribute for enhanced resistance against invasion of R. solani in M. piperita.

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Fusarium wilt of tomato is one of the most prevalent and economically important diseases of tomato worldwide especially in tropical regions. The aims of the present study were to isolate and characterize Bacillus bacteria from tomato rhizospheric soil of various regions in Iran and determine the isolates that exhibit high levels of antagonistic efficiency against tomato Fusarium wilt pathogen, Fusarium oxysporum f. sp. lycopersici (Fol) and growth promotion activity. In this study, 303 Bacillus isolates were obtained from tomato rhizospheric soil. Dual culture and volatile metabolite tests were used to screen for antagonism of Bacillus isolates against Fol. Among them, 20 isolates were found to inhibit pathogen growth by 67.77% and 33.33% in dual culture and volatile metabolite tests, respectively. Based on the results of physiological tests and 16S rRNA and gyrA gene sequence analysis of 20 effective isolates, 11, seven and two isolates were identified as B. subtilis, B. velezensis and B. cereus, respectively. The results of greenhouse assessment showed that KR1-2, KR2-7 and A2-9 isolates which were characterized as Bacillus subtilis, reduced the disease index to 16.67% and promoted the plant growth by 80%. These isolates may serve as potential promising biocontrol agents against Fusarium wilt of tomato.

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The efficiency of seven disinfectants (Divomil Forte, Nobactel, Domestos, SU 392, Buraton, Descosal, Cidex) was tested against Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, Candida albicans ATCC 10231, Bacillus subtilis ATCC 6633, Aspergillus niger ATCC 16404. Surface test was used in order to evaluate the efficiency of disinfectants during the everyday sanitizing practice on special industrial surfaces. Test organisms represented pathogenic, spore forming bacteria, yeast and mould. Surface test was started with minimal concentration of agents and was not increased above the maximal concentrations, which were recommended by manufacturers in order not to corrode surfaces or risk the safety of use. Test organisms were inoculated on test areas and after drying inoculated surfaces were treated with disinfectants. Seven disinfectants were tested and four were effective against every test organism. (Buraton: 1%, Descosal: 1%, Cidex: 100%, SU 392: 75%.) Two disinfectants were ineffective against Aspergillus niger (Nobactel: 2%, Domestos: 2%) and 1 against Staphylococcus aureus and Aspergillus niger (Divomil Forte: 2%).

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The objective of this study was to examine antibacterial, antifungal and antiviral properties of selected Phlomis species (Lamiaceae) growing in Turkey. The petroleum ether and methanol extracts of the seven species, namely P. armeniaca Willd., P. bourgaei Boiss., P. leucophracta P.H. Davis & Hub.-Mor., P. lunariifolia Sibth. & Sm., P. lycia D. Don, P. pungens Willd. var. pungens , and P. pungens var. hirta Velen. were tested against Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, Bacillus subtilis , and Enterococcus faecalis for their antibacterial activity using ampicillin and oflaxocin as references. Antifungal activity of the same extracts was determined against Candida albicans using microdilution method with ketocanazole as reference. Both DNA virus Herpes simplex type-1 (HSV-1) and RNA virus Parainfluenza (PI-3) were employed for antiviral assessment of the Phlomis extracts using Madin-Darby Bovine Kidney and Vero cell lines in which acyclovir for HSV-1 and oseltamivir for PI-3 were employed as reference drugs. Although both the petroleum ether and methanol extracts seemed to exert similar antibacterial activity, the methanolic extracts were observed to be more active against S. aureus and E. faecalis . On the other hand, methanolic extract of P. pungens var. pungens possessed notable antiviral activity against both type of viruses.

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A recently introduced microtiter-plate multienzyme-inhibition assay using rabbit liver esterase (RLE), Bacillus subtilis (BS2) esterase, and cutinase from Fusarium solani pizi has been successfully transferred to high-performance thin-layer chromatography. Paraoxon, malaoxon, and carbofuran as esterase inhibitors with high, medium, and low inhibitory activity, respectively, were used to optimize method performance with regard to enzyme concentration, incubation time, and time of immersion in α-naphthyl acetate-fast blue salt B substrate. For paraoxon as strongest inhibitor, limits of detection (LOD) of 1.3, 1.2, and 540 pg per band were determined using RLE, BS2, and cutinase, respectively. Respective LODs were 7.9, 7.4, and 760 ng per band for malaoxon, and 33, 54, and 1420 ng per band for carbofuran. With regard to the LODs of strong, medium, and weak inhibitors, the detectability range is favorably reduced for the low-sensitivity cutinase (0.54–1420 ng per band) whereas it was approximately 3 × 10 4 and 5 × 10 4 for RLE and BS2, respectively.

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