Characterization and TLC-Bioautographic Detection of Essential Oils from Some Thymus Taxa. Determination of the Activity of the Oils and their Components Against Plant Pathogenic Bacteria

JPC – Journal of Planar Chromatography – Modern TLC - The chemical composition of the oils of four thyme (Lamiaceae) chemotypes (Thymus vulgaris L., Thymus serpyllum L., Thymus x...     

Beta-eudesmol, a new sesquiterpene component in intact and organized root of chamomile (Chamomilla recutita)

Gas chromatography (GC) and GC-mass spectrometry are used to identify a new sesqiterpene, beta-eudesmol, which seems to be a characteristic essential oil component of the intact and in vitro organized root of chamomile [Chamomilla recutita (L.) Rauschert]. It is identified on three types of stationary phases by GC. The confirmation of identity is carried out by comparison of mass spectra with those reported in the literature and measured from a reference compound. The percentage evaluation of the oil component is made by area normalization, on the basis of three parallel measurements. Among the cultivated and wild chamomile species examined, the wild species from the areas of Szeghalom contain the highest quantity of beta-eudesmol (9.25% in the total essential oil).     

GC–MS Studies of Thiophenes in the Supercritical Fluid CO2 and Solvent Extracts of Tagetes patula L.

The intact plant parts and genetically modified hairy root clone #TpA6 of Tagetes patula were extracted with supercritical fluid CO₂ extraction (SFE) and a conventional solvent extraction. SFE optimization included the variation of fluid CO₂ pressure, dynamic time, and the addition of methanol modifier co-solvent. The four characteristic thiophene metabolites, 5-(3-buten-1-ynyl)-2,2′-bithienyl (BBT), 2,2′:5′,2″-terthiophene (α-T), 5-(4-acetoxy-1-butynyl)-2,2′-bithienyl (BBTOAc), and 5-(3,4-diacetoxy-1-butynyl)-2,2′-bithienyl [BBT(OAc)₂], were analysed by GC–MS. The proposed SFE method allowed the selective extraction of thiophenes in 60 min dynamic time with supercritical CO₂ without modifier co-solvent, at 30 MPa and 40 °C. The SFE and the reference solvent extraction yielded similar results. The SFE of intact roots and flowers yielded 717 ± 31.3 and 480 ± 26.6 μg g^?1 α-T, respectively, while the leaves did not contain considerable amounts of thiophenes. Remarkable amounts of BBT, BBTOAc, and BBT(OAc)₂ were characteristic of the SFE of hairy root cultures.     

Analysis of Non-volatile Constituents in Dracocephalum Species by HPLC and GC-MS

In this paper we identify/determine the composition of the extracts of Dracocephalum moldavica L. and D. ruyschiana L. with special emphasis on their flavonoids, aliphatic, aromatic carboxylic acids and sugars. The plant materials were extracted using methanol-acetone 1:1 (v/v) acidified with HCl (0.05%). Composition of extract's most red fractions was identified by HPLC, while the constituents of the entire extract were identified and quantified as their trimethylsilyl-(oxime) ether/ester derivatives by GC-MS. On the basis of HPLC analyses - (performed on Phenomenex Luna 5 µm C18 column, 250× 4.6 mm I.D.; eluent: acetonitrile - trifluoroacetic acid (0.1 %); isocratic/gradient conditions) - delphinidin, cyanidin and pelargonidin (in traces) were identified by spectral analysis and on the basis of authentic standard's addition. GC-MS analyses were carried out immediately with extracts, as well as subsequently to extract's hydrolysis (trifluoroacetic acid: 2M, 2h, 4h). Constituents were identified and quantified as their trimethylsilyl-(oxime) ether/ester derivatives, from a single run, on the basis of their total (TIC) and selective fragment ion (SIM) responses. Calculations were related to the dry matter content of extracts. As main constituents monosaccharides, sugar alcohols di- and trisaccharides, aliphatic (phosphoric, succinic, levulinic, malic, tartaric, fatty acids) and aromatic (quinic, chlorogenic, caffeic, ferulic, 3,4-dihydroxyphenyllactic, rosmarinic) carboxylic acids and their corresponding esters; apigenin, luteolin flavon aglycons and tocopherol, in total 33 constituents were quantitated partly by their TIC, partly by their SIM responses. Identification/quantification proved to be in total of 35–69% (expressed in the dry matter content of extracts).     

GC-MS Identification and GC-FID Quantitation of Terpenoids in Ononidis spinosae Radix

A GC-MS method has been developed for the qualitative analysis of sterol and triterpene (terpenoid) constituents of Ononis spinosa L. (spiny restharrow) root without derivatization. β-Sitosterol, campesterol, stigmasterol, stigmastan-3,5-diene sterol compounds and the triterpene derivatives β-amyrin and α-onocerin were identified. A validated GC-FID quantitative method was developed for measuring β-sitosterol, the main sterol component, in various extracts of this plant, obtained with organic solvents and by supercritical fluid extraction. The extracts were cleaned by saponifying and then the β-sitosterol was quantified in the non-saponifiable fractions by GC-FID with an internal standard. In addition, the relative concentrations of the other terpenoids were also determined. The β-sitosterol content in the non-saponifiable solvent extraction fractions was 0.19–5.5%, that of the supercritical fractions were 4.8–9.2%, depending on the experimental conditions. The hexane and the pilot scale SFE extracts were considered as main sources of terpenoids (71.8%; 93.3%, respectively).     

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 (OPLC) with on-line 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 with various detection methods (UV, vanillin–sulphuric acid reagent, direct bioautography). The antibacterial components were identified with GC–MS as thymol, carvacrol, (−)-linalool, diethyl-phthalate, and α-terpineol. The oil component diethyl-phthalate is an artificial compound, used as a plasticizer or detergent base in the industry. Our results support that exploiting its flexibility and the possible hyphenations, OPLC is especially attractive for isolation of antimicrobial components from various matrixes.

     

GC and GC-MS Studies on the Essential Oil and Thiophenes from Tagetes patula L.

The production and the composition of the compounds (mono-, sesquiterpenes and acetylenic thiophenes) obtained by the steam distillation of Tagetes patula L. have been investigated. The volatile oil was produced by steam distillation. GC was carried out on three types of stationary phase using flame ionization and mass selective detection. Percentage data were calculated by the area normalization method with very good repeatability (RSD below 5%). Oils from flower-heads were rich in β-caryophyllene (53.5%) and the leaves contained terpinolene in high concentration (21.1%). The main volatile component of the hairy roots and intact roots was 5-(3-buten-1-ynyl)-2,2'-bithienyl (BBT) yielding 28.5% and 44.0% in the oils. Three new minor constituents were identified as α-gurjunene, β-caryophyllene and (E)-β-farnesene. A flash chromatographic method was developed for the isolation of thiophenes from a solvent extract of intact roots. The collected fractions were screened by TLC and analyzed by GC-MS. Three thiophene fractions were obtained containing BBT, 5-(4-acetoxy-1-butynyl)-2,2'-bithienyl (BBTOAc) and 5-(4-hydroxy-1-butynyl)-2,2'-bithienyl (BBTOH).     

Role of direct bioautographic method for detection of antistaphylococcal activity of essential oils

The aim of the present study was the chemical characterization of some traditionally used and therapeutically relevant essential oils (thyme, eucalyptus, cinnamon bark, clove, and tea tree) and the optimized microbiological investigation of the effect of these oils on clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA). The chemical composition of the oils was analyzed by TLC, and controlled by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The antibacterial effect was investigated using a TLC-bioautographic method. Antibacterial activity of thyme, clove and cinnamon oils, as well as their main components (thymol, carvacrol, eugenol, and cinnamic aldehyde) was observed against all the bacterial strains used in this study. The essential oils of eucalyptus and tea tree showed weak activity in the bioautographic system. On the whole, the antibacterial activity of the essential oils could be related to their most abundant components, but the effect of the minor components should also be taken into consideration. Direct bioautography is more cost-effective and better in comparison with traditional microbiological laboratory methods (e.g. disc-diffusion, agar-plate technique).     

Investigation of Thyme (Thymus vulgaris L.) Essential Oil by Use of the BioArena System

JPC – Journal of Planar Chromatography – Modern TLC - The composition of the essential oil of Thymus vulgaris L. has been determined by GC-FID and GC-MS. Because separation of thymol,...