A simple differential pulse polarographic method for the determination of thymoquinone in black seed oil

A reliable and simple differential pulse polarographic method is described for the determination of thymoquinone in black seed oil. The polarographic behaviour of thymoquinone was examined in various buffer systems over the pH range 5.0-10.0. Thymoquinone is reduced in a single, reversible peak at the dropping mercury electrode. The differential pulse polarogram showed a distinct peak in S?rensen buffer:methanol (3:7, v/v; pH 8.5) at a peak potential of -0.095 V (vs. silver/silver chloride electrode), and a plot of peak height against concentration was found to be linear over the range 0.2-15.0 microg/mL (R = 0.9998). The limit of detection was calculated to be 0.054 microg/mL. The polarographic method has been applied to determine thymoquinone in two black seed oil preparations available on the Austrian pharmaceutical market.     

Thymol and carvacrol binding to DNA: model for drug-DNA interaction

Thymol and carvacrol can bind to major and minor grooves of B-DNA. The aim of this study was to examine the interaction of calf thymus DNA with thymol and carvacrol in aqueous solution and physiological pH with thymol/DNA and carvacrol/DNA (phosphate) molar ratios of 1/20, 1/10, 1/5, and 1/1. Fourier transform infrared and UV-visible difference spectroscopy were used to determine the thymol and carvacrol binding mode, binding constant, sequence selectivity, DNA secondary structure, and structural variations of thymol/DNA and carvacrol/DNA complexes in aqueous solution. Spectroscopic evidence showed that the thymol and carvacrol interaction occurred mainly through H-bonding of the thymol and carvacrol OH group to the guanine N7, cytosine N3, and backbone phosphate group with overall binding constant of K(thymol-DNA) = 2.43 x 10(3) M(-1), K(carvacrol-DNA) = 1.55 x 10(3) M(-1). In thymol and carvacrol-DNA complexes, DNA remains in the B-family structure.     

Essential oil variability in Thymbra capitata (L.) Cav. growing wild in Southern Apulia (Italy)

Essential oils of Thymbra capitata (Thymus capitatus) collected from Southern Apulia (Italy) were analysed using gas chromatography and gas chromatography–mass spectrometry techniques, to check for chemical variability. The study showed that among the 75 components of the oils the most recurrent ones were thymol and carvacrol, which always constituted more than 50% of the oils, as well as γ-terpinene, borneol and p-cymene. Cluster analysis led to the identification of three chemotypes: thymol, carvacrol and thymol/carvacrol; this was presumably a crossbreed between the other two chemotypes. Principal component analysis showed the direct correlation among myrcene, α-terpinene and γ-terpinene; anti-correlation between thymol and carvacrol, and the inverse correlation between linalool and myrcene. Moreover, lower thymol concentrations were accompanied by an increase in myrcene, α-terpinene and γ-terpinene.     

Susceptibility of methicillin-resistant staphylococci to oregano essential oil, carvacrol and thymol

The aim of this study was to evaluate the susceptibility of methicillin-susceptible and methicillin-resistant staphylococci (MSS, MRS) to oregano essential oil, carvacrol and thymol. The commercial aerial parts of Origanum vulgare L. were hydrodistilled and the essential oil analysed by gas- chromatography/electron impact mass spectrometry. The inhibition efficacy of this essence and its major components was assayed against 26 MSS and 21 MRS, using an agar dilution method. The methicillin resistance was thoroughly typed by Epsilometer test (E-test), polymerase chain reaction for mecA gene detection and PBP2' latex agglutination test. The results clearly demonstrated that the comparison between the susceptibility of MSS and MRS to oregano oil, carvacrol and thymol showed no significant differences (Fisher's exact test, P > 0.05). The best minimum inhibitory concentration values were reported for carvacrol (0.015-0.03%, v/v) followed by thymol (0.03-0.06%, v/v) and oregano oil (0.06-0.125%, v/v).     

Essential oil production in shoot cultures versus field-grown plants of Thymus caespititius

Thymus caespititius Brot. is an important aromatic species, due to synthesis and production of essential oils for the pharmaceutical and food industries. In the present study, levels of essential oils from two chemotypes, including carvacrol/thymol (CT) and sabinene/carvacrol (SC), were evaluated in proliferating shoot cultures (6–12 subcultures following establishment) and compared to those from field-grown plants. The essential oils were isolated by hydrodistillation and analysed by gas chromatography (GC) and GC–mass spectrometry (GC–MS). Cultures grown under in vitro culture conditions, evaluated over six subcultures, were found to maintain stable composition of essential oils. For the CT chemotype, carvacrol (42 %) and thymol (23 %) were the main essential oil components detected in field-grown plants; in proliferating shoot cultures the levels detected attained 17–25 % in the case of carvacrol and 18–23 % in that of thymol, closely followed by carvacryl acetate (15–23 %) and thymyl acetate (11–15 %). For the SC chemotype, carvacrol (13–28 %), sabinene (18–45 %), and thymol (9–12 %) were the main essential oil components detected in both field-grown and proliferating shoot cultures. Our experiments showed that the essential oil composition in proliferating shoot cultures was not only stable, but also qualitatively similar to that of field-grown plants, notwithstanding minor quantitative differences.     

A rapid and validated HPLC method to quantify racecadotril metabolite, thiorphan, in human plasma using solid-phase extraction

A HPLC method with UV detection was developed and validated for the determination of thiorphan in human plasma. Nevirapine was used as the internal standard. Separation was performed by a Waters sunfire C18 reversed-phase column maintained at 35 degrees C. The mobile phase was a mixture of 0.05 M phosphate buffer with the pH adjusted to 2.6 and acetonitrile (74:26, v/v) at a flow rate of 1.0 mL/min. The UV detector was set at 210 nm. An original pre-treatment of plasma samples was developed, based on solid-phase extraction (SPE) with solid-phase extraction cartridges (Oasis HLB 3 mL, 60 mg). The extraction recovery for plasma samples of thiorphan at 0.1, 0.4 and 2.0 microg/mL was 93.5%, 98.2% and 97.8%, respectively. The calibration curve was linear with the correlation coefficient (r) above 0.9998. Linearity was verified over the range of 0.05-4 microg/mL thiorphan in plasma. The limit of quantification (LOQ) is 0.05 microg/mL. The mean accuracy was 92.7-99.6%. The coefficient of variation (precision) in the within- and between-batch was 2.2-8.4% and 4.1-8.1%, respectively. This method is simple, economical and specific, and has been used successfully in a pharmacokinetic study of thiorphan.     

Chemical and genetic diversity of Zataria multiflora Boiss. accessions growing wild in Iran

Zataria multiflora Boiss. is an aromatic shrub belonging to the Lamiaceae family. Its aerial parts are used in the traditional medicine and in the pharmaceutical and food industries. The terpenoid and genetic profiles of 18 accessions of Z. multiflora, collected in different locations in Iran, have been analyzed by GC/FID and GC/MS or by AFLP (amplified fragment length polymorphism) analyses, respectively. Altogether, 56 compounds were identified in the essential oils, with the major constituents being thymol (6.0-54.9%), carvacrol (0.7-50.6%), linalool (1.2-46.8%), and p-cymene (1.6-14.8%). On the basis of the essential-oil composition, the 18 accessions were divided into four groups. The first group was characterized by a high content of carvacrol, thymol, and linalool, the second group was dominated by carvacrol, thymol, and p-cymene, the third group was characterized by a high concentration of thymol and a low content of carvacrol and p-cymene, and the forth group contained linalool and carvacrol as the main components. The AFLP results revealed that the average genetic similarity (GS) between the accessions was 0.61, ranging from 0.40 to 0.77. The UPGMA (unweighted pair-group method with arithmetic mean) cluster analysis divided all accessions into five groups at a similarity level of 0.60. The two clusters generated, the first based on the essential-oil compositions and the second on the AFLP data, showed a different pattern of relationships among the accessions. The knowledge of the Z. multiflora chemotype diversity, illustrated in this study, will allow an improvement of the homogeneity of the plant material for the production of different types of essential oils, depending on the demands of the pharmaceutical and food industries for specific uses.     

Reverse micelle-based chemiluminescence system for quinine sulphate.

A new chemiluminescence (CL) method is proposed for the determination of quinine sulphate, which is based on the dichloromethane solvent extraction of the ion-pair complex of tetrachloroaurate (III) with quinine sulphate, and luminol CL detection in a reversed micellar medium formed by the cation surfactant cetyltrimethylammonium bromide in a dichloromethane-cyclohexane (3:7, v/v)-water (0.35 mol/L Na2CO3 buffer solution, pH 11.5). The ion-pair complex of tetrachloroaurate (III) with quinine sulphate produced an analytical CL signal when it entered the reversed micellar water pool. In optimum conditions, CL intensities are proportional to the concentrations of the studied drug over the range 0.015-10 microg/mL, with a detection limit of 1.5 ng/mL. The relative standard deviation (RSD) is 1.38% for 2.5 microg/mL quinine sulphate (n=11). The method has been applied to the determination of the studied drug in biological fluids, with satisfactory results.     

Larvicidal activity of essential oils extracted from commonly used herbs in Lebanon against the seaside mosquito, Ochlerotatus caspius

This study investigates the potential of essential oils from commonly used medical and culinary herbs in Lebanon as an environmentally safe measure to control the seaside mosquito, Ochlerotatus caspius. The composition of essential oils extracted from parsley seeds and leaves, alpine thyme inflorescences, anis seeds, and coriander fruits were analyzed by GC-MS, and the major components of these oils were found to be thymol, sabinene, carvacrol, anethole, and linalool, respectively. Mosquito larvicidal assays were conducted to evaluate the LC(50) and LC(90) after 24 and 48h of the essential oils and their major constituents. All of the tested oils proved to have strong larvicidal activity (LC(50): 15-156ppm) against Oc. caspius fourth instars, with the most potent oil being thyme inflorescence extract, followed by parsley seed oil, aniseed oil, and then coriander fruit oil. Toxicity of each oil major constituent was also estimated and compared to a reported larvicidal compound, eugenol.     

High-performance liquid chromatographic determination of inactive carboxylic acid metabolite of clopidogrel in human serum: Application to a bioequivalence study

A sensitive and rapid method is described for determination of clopidogrel carboxylic acid (CCA), the inactive metabolite of anti platelet agent, clopidogrel, in human serum. The analytical procedure involves liquid-liquid extraction of the analyte and an internal standard (phenytoin) with ethyl acetate. A mobile phase consisting of 0.05 M phosphate buffer containing triethylamine (0.5 mL/L; pH 5.7) and acetonitrile (56:44 v/v) was used and chromatographic separation was achieved using C18 analytical column at detector wavelength of 220 nm. The calibration curves were linear over a concentration range of 0.05-10 microg/mL of CCA in human serum. The total run time of analysis was 5.5 min and the lower limits of detection (LOD) and quantification (LOQ) were 0.02 and 0.05 microg/mL, respectively. The method validation was carried out in terms of specificity, sensitivity, linearity, precision, accuracy and stability. The validated method was applied in a randomized cross-over bioequivalence study of two different clopidogrel preparations in 24 healthy volunteers.     

Determination of clarithromycin in human serum by high-performance liquid chromatography after pre-column derivatization with 9-fluorenylmethyl chloroformate: application to a bioequivalence study

A sensitive liquid chromatographic method for the analysis of clarithromycin, a macrolide antibiotic, in human serum using pre-column derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) is described. The method involved liquid-liquid extraction of the drug and an internal standard (amantadine) followed by pre-column derivatization of the analytes with FMOC-Cl. A mixture of 0.05 M phosphate buffer containing triethylamine (2 mL L(-1); pH 3.8) and methanol (17:83, v/v) was used as mobile phase and chromatographic separation was achieved on a Shimpack CLC-ODS column. The eluate was monitored by a fluorescence detector with respective excitation and emission wavelengths of 265 and 315 nm. The analytical method was linear over the concentration range of 0.025-10 microg mL(-1) of clarithromycin in human serum with a limit of quantification of 0.025 microg mL(-1). The assay is sensitive enough to measure drug levels obtained in human single dose studies. In the present method, sensitivity and run time of analysis have been improved, and successfully applied in a bioequivalence study of three different clarithromycin preparations in 12 healthy volunteers.     

Susceptibility of food-borne bacteria to binary combinations of antimicrobials at selected a(w) and pH

AIM: To evaluate the antibacterial susceptibilities of food-borne bacteria to individual and binary mixtures of a synthetic antimicrobial agent with a natural phenolic compound. METHODS AND RESULTS: Antibacterial susceptibilities of Escherichia coli, Listeria innocua, Salmonella Typhimurium and Staphylococcus aureus to individual and binary mixtures of potassium sorbate with a phenolic compound (thymol, carvacrol, or eugenol) were evaluated, at selected water activity (a(w); 0.99 or 0.97) and pH (5.5 or 4.5). The bacteria studied were susceptible to the action of the antimicrobials individually with minimal inhibitory concentrations that varied from 800-ppm potassium sorbate for Staph. aureus at a(w) 0.99, and pH 5.5 to 100-ppm thymol or carvacrol for the four studied bacteria at a(w) 0.97 and pH 4.5. Several binary mixtures of potassium sorbate with thymol, carvacrol or eugenol inhibited bacterial growth. Antimicrobial agent inhibitory concentrations in the mixture varied among bacteria, additionally depending on the a(w) and the pH tested. CONCLUSIONS: Synergistic binary mixtures with fractional inhibitory concentration index <0.6 include 100- or 200-ppm potassium sorbate with 50- or 100-ppm thymol, carvacrol or eugenol. SIGNIFICANCE AND IMPACT OF THE STUDY: The synergistic combinations could be useful in reducing the amounts of antimicrobials needed to inhibit growth, thus diminishing consumer concerns regarding chemical preservatives.     

Simultaneous liquid chromatographic determination of lamotrigine, oxcarbazepine monohydroxy derivative and felbamate in plasma of patients with epilepsy

A very simple and fast method has been developed and validated for simultaneous determination of the new generation antiepileptic drugs (AEDs) lamotrigine (LTG), oxcarbazepine's (OXC) main active metabolite monohydroxycarbamazepine and felbamate in plasma of patients with epilepsy using high-performance liquid chromatography (HPLC) with spectrophotometric detection. Plasma sample (500 microL) pre-treatment was based on simple deproteinization by acetonitrile. Liquid chromatographic analysis was carried out on a Synergi 4 microm Hydro-RP, 150 mm x 4 mm I.D. column, using a mixture of potassium dihydrogen phosphate buffer (50mM, pH 4.5) and acetonitrile/methanol (3/1) (65:35, v/v) as the mobile phase, at a flow rate of 1.0 mL/min. The UV detector was set at 210 nm. Calibration curves were linear (mean correlation coefficient >0.999 for all the three analytes) over a range of 1-20 mg/mL for lamotrigine, 2-40 microg/mL for monohydroxycarbamazepine and 10-120 microg/mL for felbamate. Both intra and interassay precision and accuracy were lower than 7.5% for all three analytes. Absolute recoveries ranged between 100 and 104%. The present procedure describes for the first time the simultaneous determination of these three new antiepileptic drugs. The simple sample pre-treatment, combined with the fast chromatographic run permit rapid processing of a large series of patient samples.     

Composition and infraspecific variability of essential oil from Thymus herba barona Lois

The composition of the essential oils of individual plants of Thymus herba barona Lois. growing wild in Corsica was investigated by GC, GC/MS an carbon-13 NMR. Eight groups of essential oil were distinguished: (i) thymol, (ii) carvacrol, (iii) linalool, (iv) geraniol, (v) -terpenyl acetate, (vi) terpinen-4-ol, (vii) carvone and cis-dihydrocarvone. Three chemotypes -thymol, carvacrol and linalool – are common in the genus Thymus, two others – geraniol, -terpenyl acetate – are scarce, while the latest three ones – terpinen-4-ol, carvone and cis-dihydrocarvone are quite original. It is the first time that the cis-dihydrocarvone chemotype is described in the genus Thymus     

Determination of inositol hexanicotinate in rat plasma by high performance liquid chromatography with UV detection

A HPLC method with UV detection at 262nm was developed to analyze inositol hexanicotinate in rat plasma. Plasma samples were extracted with an equal volume of acetonitrile, followed by dilution with mobile phase buffer (5mM phosphate buffer, pH 6.0) to eliminate any solvent effects. Inositol hexanicotinate and the internal standard (mebendazole) were separated isocratically using a mobile phase of acetonitrile/phosphate buffer (35:65, v/v, pH 6.0) at a flow rate of 1.0mL/min and a reverse-phase XTerra MS C(18) column (4.6mmx150mm, 3.5microm). The standard curve was linear over a concentration range of 1.5-100.0microg/mL of inositol hexanicotinate in rat plasma. The HPLC method was validated with intra- and inter-day precisions of 1.55-4.30% and 2.69-21.5%, respectively. The intra- and inter-day biases were -0.75 to 19.8% and 2.58-22.0%, respectively. At plasma concentrations of 1.5-100microg/mL, the mean recovery of inositol hexanicotinate was 99.6%. The results of a stability study indicated that inositol hexanicotinate was unstable in rat plasma samples, but was stable in acetonitrile extracts of rat plasma for up to 24h at 4 degrees C. The assay is simple, rapid, specific, sensitive, and reproducible and has been used successfully to analyze inositol hexanicotinate plasma concentrations in a pharmacokinetic study using the rat as an animal model.     

A novel high sensitivity HPLC assay for topiramate, using 4-chloro-7-nitrobenzofurazan as pre-column fluorescence derivatizing agent

A new, sensitive and simple high-performance liquid chromatographic method for analysis of topiramate, an antiepileptic agent, using 4-chloro-7-nitrobenzofurazan as pre-column derivatization agent is described. Following liquid-liquid extraction of topiramate and an internal standard (amlodipine) from human serum, derivatization of the drugs was performed by the labeling agent in the presence of dichloromethane, methanol, acetonitrile and borate buffer (0.05 M; pH 10.6). A mixture of sodium phosphate buffer (0.05 M; pH 2.4): methanol (35:65 v/v) was eluted as mobile phase and chromatographic separation was achieved using a Shimpack CLC-C18 (150 x 4.6 mm) column. In this method the limit of quantification of 0.01 microg/mL was obtained and the procedure was validated over the concentration range of 0.01 to 12.8 microg/mL. No interferences were found from commonly co-administrated antiepileptic drugs including phenytoin, phenobarbital carbamazepine, lamotrigine, zonisamide, primidone, gabapentin, vigabatrin, and ethosuximide. The analysis performance was carried-out in terms of specificity, sensitivity, linearity, precision, accuracy and stability and the method was shown to be accurate, with intra-day and inter-day accuracy from -3.4 to 10% and precise, with intra-day and inter-day precision from 1.1 to 18%.     

Selective and rapid liquid chromatography/negative-ion electrospray ionization mass spectrometry method for the quantification of valacyclovir and its metabolite in human plasma

A rapid, sensitive and specific method was developed for the quantification of valacyclovir and acyclovir in human plasma. Sample preparation was performed by protein precipitation with acetonitrile followed by filtration. Valacyclovir, acyclovir and ganciclovir (internal standard) were separated isocratically on a reversed-phase porous graphitized carbon analytical column (2.1 mm x 125.0 mm i.d., particle size 5 microm), using a mobile phase of acetonitrile/water with 0.05% (v/v) diethylamine (50:50, v/v) at a flow rate of 0.15 mL min(-1) in 4.0 min. Detection was performed by negative electrospray ionization using the selected ion monitoring mode of the deprotonated molecular ions at m/z 323.0 for valacyclovir, 224.0 for acyclovir and 254.0 for ganciclovir. The assay had linear calibration curves over the range 0.020-0.800 microg mL(-1) for valacyclovir and 0.100-20.00 microg mL(-1) for acyclovir. Accuracy and precision were within the acceptance limit of 15%. The method was successfully applied to the analysis of plasma samples obtained from patients after oral administration of valacyclovir.     

Essential oil composition is related to the natural habitats: Coridothymus capitatus and Satureja thymbra in NATURA 2000 sites of Crete

The study of essential oils obtained from Coridothymus capitatus and Satureja thymbra collected from different natural habitat types of 11 NATURA 2000 sites scattered all over Crete has shown that they are characterized either by a high amount of carvacrol (up to 75.7%) or thymol (up to 65.6%) or by a more or less equal amount of the two phenols. The results of a discriminant analysis with predefined groups the natural habitat types wherefrom the plants were collected have shown that the oils of both species collected from the dry dwarf-shrub formations of the lowland have a high carvacrol content whereas those collected from the more mesic timber or highland formations have a high thymol content. Furthermore, the results of this study introduce the use of natural habitat unit as a tool for the assessment of essential oil variation.     

Bioactivity of carvacrol and thymol against Frankliniella occidentalis and Thrips tabaci

Abstract: Two constituents of essential oils, carvacrol and thymol, were tested for their effects on the feeding activity, oviposition preference and oviposition rate of Frankliniella occidentalis and Thrips tabaci . Given a choice between treated and untreated leaf discs, F. occidentalis avoided ovipositing on leaf discs treated with 1 or 0.1% thymol. In no-choice experiments, 1 and 0.1% thymol and 1% carvacrol resulted in a reduced oviposition rate of F. occidentalis compared with the control. Both constituents caused a slight reduction of the feeding damage of F. occidentalis . 1 and 0.1% carvacrol had a pronounced deterrent effect on the oviposition site selection of T. tabaci . Both constituents served as anti-ovipositants for both thrips species, albeit F. occidentalis was more affected than T. tabaci .