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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.

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Bioautography can be extended to a complex system called BioArena by linking different steps to it, for example dissolving a variety of compounds in the cell suspension to affect biological activity, measuring putative mediators of antibiosis, for example formaldehyde (HCHO) and hydrogen peroxide (H 2 O 2 ) in the inoculated layer, and performing densitometric and ex and in situ spectroscopic examination to follow the changes in the inhibition zones and active compounds (e.g. antibiotics and toxins). Possibilities of the basic elements of BioArena system are illustrated in this paper by results with aflatoxin B1 (AFB1). Target bacterial cells in the logarithmic growth phase were found to be the most sensitive for direct bioautography. Densitometric signals of bioautograms (negative densitometry) of 0.125–1 μg AFB1 spots showed logarithmic correlation with the amount of AFB1. The HCHO capturer L-arginine decreased whereas the HCHO generator-mobilizer Cu(II) ions increased the antibacterial-toxic effect of AFB1. The latter effect was also confirmed by negative densitometry. Besides higher levels of HCHO, a decrease of H 2 O 2 in the toxin spot was found. HCHO could also originate, among other sources, from demethylation of AFB1, which is apparent from the Fourier transform Raman spectra obtained in situ from the AFB1-containing spots. These results support the suggested role of HCHO and its reaction products with H 2 O 2 (e.g. singlet oxygen ( 1 O 2 ), ozone (O 3 )) in the antibacterial-toxic effect of AFB1.

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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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The aim of the present study was the chemical characterization of some medically relevant essential oils (tea tree, clove, cinnamon bark, thyme, and eucalyptus) and the investigation of antibacterial effect of the components of these oils by use of a direct bioautographic method. Thin-layer chromatography (TLC) was combined with biological detection in this process. The chemical composition of the oils was determined by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Eucalyptol (84.2%) was the main component of the essential oil of eucalyptus, eugenol (83.7%) of clove oil, and trans-cinnamic aldehyde (73.2%), thymol (49.9%), and terpinen-4-ol (45.8%) of cinnamon bark, thyme and tea tree oils, respectively. Antibacterial activity of the separated components of these oils as well as of their pure main components (eucalyptol, eugenol, trans-cinnamic aldehyde and thymol) was observed against the Gram-negative luminescence gene-tagged plant pathogenic bacterium Pseudomonas syringae pv. maculicola (Psmlux) and the Gram-negative, naturally luminescent marine bacterium Vibrio fischeri. On the whole, the antibacterial activity of the essential oils could be related to their main components, but the minor constituents may be involved in this process. trans-Cinnamic aldehyde and eugenol were the most active compounds in TLC-bioautography. The sensitivity of TLC-bioautographic method can be improved by using luminescent test bacteria. This method is more cost-effective and provides more reliable results in comparison with conventional microbiological methods, e.g., disc-diffusion technique.

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Néhány potenciálisan mérgező fém frakcióinak meghatározására alkalmazott analitikai módszer értékelése eltérő fizikai talajféleségű mintákon

Evaluation of analytical methods for the determination of different potentially toxic metal fractions in soils with different physical properties

Agrokémia és Talajtan
Authors:
Katalin Kovács
,
Márk Horváth
,
Gábor Halász
,
Anita Takács
,
György Heltai
,
Norbert Boros
,
Péter Sipos
, and
Zoltán Győri

Munkánk során potenciálisan toxikus fémek frakcióinak meghatározására alkalmazott analitikai módszereket kívántunk értékelni, azok talajféleségtől való függése alapján.

A talajféleségtől való függés mértéke döntően megszabja a módszer használhatóságát. Jelen dolgozat célja, hogy a Cu, a Zn, a Fe és a Ni környezeti mobilitása szempontjából értékelje a vizsgálatba vont egy- és többlépéses kioldásos módszereket.

Vizsgálatainkhoz a TIM adatbázisból származó négy, eltérő fizikai talajféleségű mintát választottunk (homok-, homokos vályog-, vályog- és agyag-talaj sorrendjében növekvő agyag- és humusztartalommal és pH-val).

Öt különböző kivonószerrel végzett egylépéses extrakciót és kétféle szekvens, azaz 3+1 lépéses egymás utáni extrakciós vizsgálatot végeztünk (BCR és McGrath módszerekkel).

Elvégeztük a minták Magyar Szabvány (MSZ 21470-50) szerinti, mikrohullámú kezeléssel egybekötött, H2O2+HNO3 eleggyel való roncsolását is. Az így megállapított elemtartalom környezetvédelmi szempontból teljes („összes”) elemtartalomnak tekinthető.

A mérési eredmények alapján megkíséreltük kiválasztani a vizsgálatba vont módszerek közül azokat, amelyek a talajból már biztonsággal meghatározható mennyiségű elemet vonnak ki, de a kivont mennyiség még nem a teljes elemtartalommal arányos.

A mikrohullámú feltárással kapott Zn-, Cu-, Fe- és Ni-tartalmak, a homok-, homokos vályog-, vályog- és agyag-talaj sorrendben, vagyis az agyag- és humusztartalmukkal, valamint pH-jukkal párhuzamosan nő. Egy olyan összetett rendszerben, mint a talaj, az egyes talajtulajdonságok, így az agyag- és humusztartalom, valamint a pH hatása vizsgálataink alapján nem különíthető el, de mint várható volt, a nagyobb agyag- és humusztartalmú és magasabb pH-jú talajok ezekből az elemekből többet halmoztak fel.

Mivel a kelátképzőket tartalmazó kivonószerek az összes réztartalomnak mintegy harmadát kivonták, a kivont mennyiség a réztartalommal volt arányos, vagyis a réztartalom növekedésével párhuzamosan nőtt.

Ezek a kivonószerek tehát Cu esetében kevésbé alkalmasak az egyes talajféleségek toxicitása közti különbségek kimutatására.

A rézzel ellentétben, a másik három elemnél az egyes talajféleségeknél kivont kis elemmennyiségek esetenként nagyságrendileg is különböztek, így az elméleti meggondolásunknak megfelelően feltételezhető, hogy az egyes talajféleségek toxicitása közti különbséget mutatják.

A két szekvens módszernél a vasnál kapott mérési eredményeket azok szórása miatt nem lehetett értékelni.

Megállapítható, hogy a BCR agresszívebb kivonószereket használ, mint a McGrath módszer, ennek megfelelően a BCR módszerrel a talajok átlagában az elemek 36, a McGrath módszerrel pedig csak 9%-át vontuk ki.

A BCR módszernél nincs vízszerű gyenge kivonószer, amiből a különböző talajok összes elemtartalmából felszabaduló ionok mennyiségére lehetne következtetni. Az ecetsav, a leggyengébb kivonószer, de a kicserélhető elemtartalom mellett kivonja a karbonátokhoz kötött, vagyis biztosan nem szabad ionos elemtartalmat is.

Mivel a BCR módszer minden kivonószerével sok elemet vonunk ki, nincs érdemi különbség az elemek egyes talajoknál mért kivonási százaléka között, ami figyelembe véve, hogy a talajok elemtartalma a homoktól az agyagtalajig nő, azt jelenti, hogy a kivont mennyiség az összes elemtartalommal arányos.

Lényegében ugyanezt mondhatjuk a McGrath módszer két agresszívebb kivonószeréről is, annak ellenére, hogy lényegesen kevesebb elemet vonnak ki. Ezzel szemben a módszer leírása szerint, a 0,1 mólos kalcium-kloridos kivonatból a vízoldható és kicserélhető elemtartalomra lehet következtetni, így minden bizonnyal a talajok toxicitását mutatja.

In this study, we aimed to evaluate some analytical methods used to determine the fractions of potentially toxic metals, based on their dependence on soil type.

The degree of dependence on soil type determines the applicability of the method. The aim of the present paper is to evaluate the single- and multi-step extraction methods included in the study in terms of the environmental mobility of Cu, Zn, Fe and Ni.

For the studies, we selected four samples with different physical soil types from the Soil Information and Monitoring System database.

In order to establish trends, soil samples were selected so that their clay and humus content, i.e., their adsorption capacity as well as their pH increased in the order of sand, sandy loam, loam and clay soils.

One-step extractions with five different extractants and two sequential extraction analyses including 3+1 steps were performed (BCR and McGrath methods).

We also performed the digestion of the samples with H2O2 + HNO3 solvent combined with microwave treatment according to the Hungarian Standard (MSZ 21470-50). The element content determined in this way can be considered as the "total" element content from the environmental point of view.

Based on the results of the analyses, we tried to select from the methods included in the study those that already extract a safe amount of elements from the soil, but the extracted amount is not yet proportional to the total element content.

The Zn, Cu, Fe and Ni contents resulting from microwave digestion increased in the order of sand, sandy loam, loam and clay soils, i.e. in parallel with the clay and humus content and pH of the soils.

As the extractants containing chelating agents extracted about one-third of the total copper content, the amount extracted was proportional to the copper content, i.e. it increased in parallel with the increase in copper content. Thus, in the case of copper, these extractants are less suitable for detecting differences in the toxicity of different soil types.

In contrast to copper, in the case of the other three elements, the small amounts extracted from each soil type also differed in order of magnitude, so according to our theoretical consideration, it can be assumed that they show a difference between the toxicity of each soil type.

The results obtained with iron using the two sequential methods could not be evaluated due to their standard deviation.

It can be stated that BCR uses more aggressive extractants than the McGrath method, accordingly, the BCR method extracted 36%, whereas the McGrath method only 9% of the elements on average of the soils.

The BCR method does not have an aqueous weak extractant, which would indicate the amount of ions released from the total element content of the different soils. Acetic acid is the weakest extractant, but in addition to the exchangeable element content, it also extracts the ionic element content bound to carbonates, which is certainly not free.

Because a large amount of elements is extracted with each extractant in the BCR method, there is no significant difference in the percentage of elements extracted for each soil, which, given that soil element content increases from sand to clay soil, means that the extracted amount is proportional to total element content.

Basically, the same can be said for the two more aggressive extractants of the McGrath method, despite the fact that they extract significantly less amount of elements. In contrast, as described in the method, the water-soluble and exchangeable element content can be inferred from the 0.1 M calcium chloride extract, thus it certainly indicates soil toxicity.

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