Bioassay-guided isolation and identification of antimicrobial compounds from thyme essential oil by means of overpressured layer chromatography, bioautography and GC-MS

A simple method is described for efficient isolation of compounds having an antibacterial effect. Two thyme (Thymus vulgaris) essential oils, obtained from the market, were chosen as prospective materials likely to feature several bioactive components when examined by thin layer chromatography coupled with direct bioautography as a screening method. The newly developed infusion overpressured layer chromatographic separation method coupled with direct bioautography assured that only the active components were isolated by means of overrun overpressured layer chromatography with online detection and fractionation. Each of the 5 collected fractions represented one of the five antimicrobial essential oil components designated at the screening. The purity and the activity of the fractions were confirmed with chromatography coupled various detection methods (UV, vanillin-sulphuric acid reagent, direct bioautography). The antibacterial components were identified with GC-MS as thymol, carvacrol, linalool, diethylphthalate, and alpha-terpineol. The oil component diethyl-phthalate is an artificial compound, used as plasticizer or detergent bases in the industry. Our results support that exploiting its flexibility and the possible hyphenations, overpressured layer chromatography is especially attractive for isolation of antimicrobial components from various matrixes

Detection of antibacterial activity of essential oil components by TLC-bioautography using luminescent bacteria

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 their pure main components (eucalyptol, eugenol, trans-cinnamic aldehyde and thymol) was observed against the Gram-negative luminescence 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 with 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

New terpenoids in cultivated and wild chamomile (in vivo and in vitro)

The effect of Chamomilla recutita (L.) Rauschert is made up by several groups of active substances, among which terpenoids in the inflorescences are of greatest importance. Among cultivated species, the Hungarian BK-2 contains more chamazulene in its essential oil than the German Degumil type, which is mainly cultivated for its (-)-alpha-bisabolol. Both components have important antiinflammatory activities. Among wild chamomile populations in Hungary, a population was found in the area of Szabadkigyos containing significant amounts-on average 48%-of (-)-alpha-bisabolol in its inflorescence oil. In vitro cultures were made from this population to obtain propagation material containing a high number of active substances. The intact roots contained no (-)-alpha-bisabolol but the sesquiterpene alcohol beta-eudesmol as new compound was identified by our group. Sterile plantlets, cultured in vitro, were multiplied for phytochernical investigations. Pharmacologically important compounds of the essential oils were followed in great detail. The amount of in vitro cultured terpenoids and polyin compounds was compared with that of in vivo plants. These volatile compounds were identified by comparing their retention times with those of authentic standards, essential oils of known composition and peak enrichment. The confirmation of identity was done by comparison of their mass spectra with those reported in the literature and reference compounds. The percentage evaluation of each component was made by area normalisation. Gas chromatography (GC) and mass spectrometry (MS) showed that sterile chamomile cultures generated the most important terpenoid and polyin compounds characteristic of the parent plant. We identified germacrene-D, berkheyaradulene, 4-(2', 4, 4'-trimethyl-bicyclo[4.1.0]hept-2'-en-3'-yl)-3-buten-2-one, geranyl-isovalerate and cedrol as new components in these sterile cultures. (C) 2003 Elsevier B.V. All rights reserved

Terpenoids in genetically transformed cultures of chamomile

By naturally occurring gene-transformation Ri-plasmids of Agrobacterium rhizogenes have been integrated into the plant genome of Chamomillo recutita, thereby inducing the formation of hairy roots. Clones with the best biosynthetic potential were multiplied for phytochemical investigations. The amounts of terpenoid and polyene compounds in the genetically transformed cultures were compared with those in in-vivo plants. In Hungary a wild chamomile population was found which contained significant amount of (-)-alpha-bisabolol in the oil from the inflorescence. We used biotechnological methods to preserve the genome of this wild type. Transformed chamomile root cultures were obtained by infection of sterile organized cultures with Agrobacterium rhizogenes strains #A-4,# 15834, and #R-1601. Hairy roots freed from bacteria were cultivated on solid medium and then in liquid, hormone-free, B5 and MS media. The qualitative and quantitative composition of the essential oil was examined by gas chromatography and mass spectrometry. The volatile compounds were identified by comparing their retention times with those of authentic standards and of essential oils of known composition, and by peak enrichment. Confirmation of identity was achieved by comparison of mass spectra with those reported in the literature and those of reference compounds. The amount of each component as a percentage of the total was determined by area normalization. GC and GC-MS studies showed that genetically transformed chamomile cultures generated the terpenoid and polyene compounds most characteristic of the parent plant. The main components of hairy root cultures were trans-beta-farnesene, alpha-farnesene, geranyl isovalerate, and cedrol. We identified beta-selinene as a new component of the genetically transformed cultures

Effect of magnesium on essential oil formation of genetically transformed and non-transformed chamomile cultures

Objective: The importance of chamomile (Chamomilla recutita) is widely known in classical and folk medicine, with the largest group of its effective substances forming the essential oil (chamazulene, alpha-bisabolol, trans-beta-farnesene, spathulenol, cis/trans-en-in-dicycloethers). The increasing need for plant-derived high quality drugs cannot be provided by their collection in the wilderness. Method A: To preserve the genome of Szabadkigyos wild type having high (-)-alpha-bisabolol content, we used biotechnological methods. Results: The roots of organized culture contained beta-eudesmol, which we have identified in the intact roots. Our gas-chromatographic and mass-spectroscopic studies showed that sterile chamomile cultures generated the most important terpenoid and polyin compounds characteristics of the intact plant. We identified berkheyaradulene, geranyl-isovalerate and cedrol, as new components in these cultures. Magnesium (Mg) (370 and 740 mg/l MgSO4) has a positive effect on the growth of organized cultures and also on the quality and quantity of essential oil production. Method B: Another possible source of variants is available by the genetic transformation of organized cultures by infection with Agrobacterium rhisogenes. With this method, we cultivated chamomile infected by A4-Y clone and investigated the essential oil production by hairy root cultures cultivated on solid and liquid MS B-5 media. The main component of the essential oil of hairy root cultures was trans-beta-farnesene. Results: We identified a-selinene, as a new component in these hairy roots. We studied the growth rate of A4-Y clone on the cited media, containing MgSO4 concentrations: 0; 185; 370 and 740 mg/l. The cultures grew most in medium containing 740 mg/l of MgSO4. Essential oil content was compared from hairy root cultures of different Mg containing media and measured by GC and GC-MS methods. Mg has a similar effect on hairy roots as on organized cultures