Functional and ultrastructural changes in Pseudomonas aeruginosa and Staphylococcus aureus cells induced by Cinnamomum verum essential oil.

AIMS: To study cellular damage induced by Cinnamomum verum essential oil in Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213. METHODS AND RESULTS: The effect of cinnamon bark essential oil on these two strains was evaluated by plate counts, potassium leakage, flow cytometry and transmission electron microscopy (TEM). Exposure to this oil induced alterations in the bacterial membrane of Ps. aeruginosa, which led to the collapse of membrane potential, as demonstrated by bis-oxonol staining, and loss of membrane-selective permeability, as indicated by efflux of K(+) and propidium iodide accumulation. Thus, respiratory activity was inhibited, leading to cell death. In Staph. aureus, cells treated with the oil entered a viable but noncultivable (VNC) state. The oil initially caused a considerable decrease in the metabolic activity and in the replication capacity of these bacterial cells. The loss of membrane integrity appeared later, as indicated by bis-oxonol and Propidium iodide (PI) staining. Data provided by TEM showed various structural effects in response to cinnamon essential oil. In Ps. aeruginosa cells, coagulated cytoplasmic material was observed, and intracellular material was seen in the surrounding environment, while oil-treated Staph. aureus showed fibres extending from the cell surface. CONCLUSIONS: Cinnamon essential oil damages the cellular membrane of Ps. aeruginosa, which leads to cell death. There is evidence of VNC Staph. aureus after exposure to the oil. SIGNIFICANCE AND IMPACT OF THE STUDY: Cinnamon essential oil shows effective antimicrobial activity and health benefits and is therefore considered a potential food additive. To use this oil as a natural food preservative, especially in combination with other preservation methods, a thorough understanding of the mechanism through which this oil exerts its antibacterial action is required.

Evaluation of three essential oils as potential sources of botanical fungicides.

In previous study, thirty essential oils were evaluated in vitro against two citrus pathogens namely Penicillium italicum Wehmer and Penicillium digitatum Sacc. Essential oils of Cinnamomum zeylanicum, Cinnamomum verum and Eugenia caryophyllus were selected because of their high inhibitory activities against both pathogens. The present study was undertaken to evaluate the in vivo activity of these essential oils. Fresh orange fruits were wounded and treated with different concentrations of essential oil (0.5, 1, and 5%) before being infected at the wound site with conidia suspensions of the tested pathogens. When applied at 5%, essential oils tested controlled totally the infections. Among the three essential oils tested, C. zeylanicum seems particularly interesting because of its high protection activity at 1% compare to the others. It reduced the disease incidence from 40 to 70% and the disease severity from 65 to 82%. Moreover no visible damage burn induced on the orange cuticle or skin was observed up to 5% of essential oil. These results strengthen the potential use of essential oils in postharvest disease management of citrus fruit as alternative to chemical fungicides.

Investigation of functional and morphological changes in Pseudomonas aeruginosa and Staphylococcus aureus cells induced by Origanum compactum essential oil.

AIMS: Evaluation of the cellular effects of Origanum compactum essential oil on Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213. METHODS AND RESULTS: The damage induced by O. compactum essential oil on these two strains has been studied using different techniques: plate count, potassium leakage, flow cytometry (FC) and transmission electron microscopy (TEM). The results showed that oil treatment led to reduction of cells viability and dissipated potassium ion gradients. Flow cytometric analysis showed that oil treatment promoted the accumulation of bis-oxonol and the membrane-impermeable nucleic acid stain propidium iodide (PI), indicating the loss of membrane potential and permeability. The ability to reduce 5-cyano-2,3-ditolyl tetrazolium chloride was inhibited. Unlike in Ps. aeruginosa, membrane potential and membrane permeability in Staph. aureus cells were affected by oil concentration and contact time. Finally, TEM showed various structural effects. Mesosome-like structures were seen in oil-treated Staph. aureus cells whereas in Ps. aeruginosa, coagulated cytoplasmic material and liberation of membrane vesicles were observed, and intracellular material was seen in the surrounding environment. Both FC and TEM revealed that the effects in Ps. aeruginosa were greater than in Staph. aureus. CONCLUSIONS: Oregano essential oil induces membrane damage showed by the leakage of potassium and uptake of PI and bis-oxonol. Ultrastructural alterations and the loss of cell viability were observed. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the mode of antibacterial effect of the oil studied is of a great interest in it further application as natural preservative in food or pharmaceutical industries.

Comparison of bacteriostatic and bactericidal activity of 13 essential oils against strains with varying sensitivity to antibiotics.

AIMS: To compare the bacteriostatic and bactericidal activity of 13 chemotyped essential oils (EO) on 65 bacteria with varying sensitivity to antibiotics. METHODS AND RESULTS: Fifty-five bacterial strains were tested with two methods used for evaluation of antimicrobial activity (CLSI recommendations): the agar dilution method and the time-killing curve method. EO containing aldehydes (Cinnamomum verum bark and Cymbopogon citratus), phenols (Origanum compactum, Trachyspermum ammi, Thymus satureioides, Eugenia caryophyllus and Cinnamomum verum leaf) showed the highest antimicrobial activity with minimum inhibitory concentration (MIC) <2% (v/v) against all strains except Pseudomonas aeruginosa. Alcohol-based EO (Melaleuca alternifolia, Cymbopogon martinii and Lavandula angustifolia) exhibited varying degrees of activity depending on Gram status. EO containing 1.8-cineole and hydrocarbons (Eucalyptus globulus, Melaleuca cajeputii and Citrus sinensis) had MIC(90%) > or = 10% (v/v). Against P. aeruginosa, only C. verum bark and O. compactum presented MIC < or =2% (v/v). Cinnamomum verum bark, O. compactum, T. satureioides, C. verum leaf and M. alternifolia were bactericidal against Staphylococcus aureus and Escherichia coli at concentrations ranging from to 0.31% to 10% (v/v) after 1 h of contact. Cinnamomum verum bark and O. compactum were bactericidal against P. aeruginosa within 5 min at concentrations <2% (v/v). CONCLUSIONS: Cinnamomum verum bark had the highest antimicrobial activity, particularly against resistant strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriostatic and bactericidal activity of EO on nosocomial antibiotic-resistant strains.

Investigation of the mutagenic and antimutagenic effects of Origanum compactum essential oil and some of its constituents.

In the present study, the chemical composition of Origanum compactum essential oil was determined by gas chromatography and mass spectrometry, and its mutagenic and antimutagenic activities were investigated by the somatic mutation and recombination test (SMART) in Drosophila melanogaster. No significant increase in the number of somatic mutations was observed with the essential oil tested using both the standard (ST) and high bio-activation (HB) cross. In order to investigate the antimutagenic effect of the essential oil, we have tested the effect on the indirect-acting mutagen urethane (URE), as well as the direct-acting mutagen methyl methanesulfonate (MMS). O. compactum essential oil showed a strong inhibitory effect against URE-induced mutagenicity, especially with the HB cross. However, only a weak inhibitory effect on the mutagenicity induced by MMS was observed. These results suggest that the detected antimutagenicity could be mediated by an inhibitory effect on metabolic activation. The essential oil was fractionated to identify the components responsible of the suppressing effect detected. Seven fractions were obtained: two of them showed the most potent inhibitory effect against URE-induced mutagenicity and were further fractionated. The sub-fractions obtained from the second chromatographic fractionation were tested for their antimutagenic activity, together with carvacrol and thymol. The highest antimutagenic effect obtained with the sub-fractions was similar to the effect of the crude essential oil, as well as to the effect of carvacrol alone. These results suggest the absence of a synergic antimutagenic effect between the components of O. compactum essential oil and indicate that carvacrol was the most active oil component.

Antigenotoxic effects of three essential oils in diploid yeast (Saccharomyces cerevisiae) after treatments with UVC radiation, 8-MOP plus UVA and MMS.

Essential oils (EOs) extracted from medicinal plants such as Origanum compactum, Artemisia herba alba and Cinnamomum camphora are known for their beneficial effects in humans. The present study was undertaken to investigate their possible antigenotoxic effects in an eukaryotic cell system, the yeast Saccharomyces cerevisiae. The EOs alone showed some cytotoxicity and cytoplasmic petite mutations, i.e. mitochondrial damage, but they were unable to induce nuclear genetic events. In combination with exposures to nuclear mutagens such as 254-nm UVC radiation, 8-methoxypsoralen (8-MOP) plus UVA radiation and methylmethane sulfonate (MMS), treatments with these EOs produced a striking increase in the amount of cytoplasmic petite mutations but caused a significant reduction in revertants and mitotic gene convertants induced among survivors of the diploid tester strain D7. In a corresponding rho0 strain, the level of nuclear genetic events induced by the nuclear mutagens UVC and 8-MOP plus UVA resulted in the same reduced level as the combined treatments with the EOs. This clearly suggests a close relationship between the enhancement of cytoplasmic petites (mitochondrial damage) in the presence of the EOs and the reduction of nuclear genetic events induced by UVC or 8-MOP plus UVA. After MMS plus EO treatment, induction of these latter events was comparable at least per surviving fraction in wildtype and rho0 cells, and apparently less dependent on cytoplasmic petite induction. Combined treatments with MMS and EOs clearly triggered switching towards late apoptosis/necrosis indicating an involvement of this phenomenon in EO-induced cell killing and concomitant decreases in nuclear genetic events. After UVC and 8-MOP plus UVA plus EO treatments, little apoptosis and necrosis were observed. The antigenotoxic effects of the EOs appeared to be predominantly linked to the induction of mitochondrial dysfunction.

Cytotoxicity and gene induction by some essential oils in the yeast Saccharomyces cerevisiae.

In order to get an insight into the possible genotoxicity of essential oils (EOs) used in traditional pharmacological applications we tested five different oils extracted from the medicinal plants Origanum compactum, Coriandrum sativum, Artemisia herba alba, Cinnamomum camphora (Ravintsara aromatica) and Helichrysum italicum (Calendula officinalis) for genotoxic effects using the yeast Saccharomyces cerevisiae. Clear cytotoxic effects were observed in the diploid yeast strain D7, with the cells being more sensitive to EOs in exponential than in stationary growth phase. The cytotoxicity decreased in the following order: Origanum compactum>Coriandrum sativum>Artemisia herba alba>Cinnamomum camphora>Helichrysum italicum. In the same order, all EOs, except that derived from Helichrysum italicum, clearly induced cytoplasmic petite mutations indicating damage to mitochondrial DNA. However, no nuclear genetic events such as point mutations or mitotic intragenic or intergenic recombination were induced. The capacity of EOs to induce nuclear DNA damage-responsive genes was tested using suitable Lac-Z fusion strains for RNR3 and RAD51, which are genes involved in DNA metabolism and DNA repair, respectively. At equitoxic doses, all EOs demonstrated significant gene induction, approximately the same as that caused by hydrogen peroxide, but much lower than that caused by methyl methanesulfonate (MMS). EOs affect mitochondrial structure and function and can stimulate the transcriptional expression of DNA damage-responsive genes. The induction of mitochondrial damage by EOs appears to be closely linked to overall cellular cytotoxicity and appears to mask the occurrence of nuclear genetic events. EO-induced cytotoxicity involves oxidative stress, as is evident from the protection observed in the presence of ROS inhibitors such as glutathione, catalase or the iron-chelating agent deferoxamine.

Genotoxicity and antigenotoxicity of some essential oils evaluated by wing spot test of Drosophila melanogaster.

Essential oils extracted from the three medicinal plants; Helichrysum italicum, Ledum groenlandicum and Ravensara aromatica, together with their mixture were tested for their genotoxic and antigenotoxic activities against urethane, a well-known promutagen. We have adopted the somatic mutations and recombination test (SMART) in the wings of Drosophila melanogaster. Three days old larvae, trans-heterozygous for two genetic markers mwh and flr, were treated by essential oil and/or urethane. A negative control corresponding to solvent was also used. Our results do not show any significant effect of the oils tested but they reduce the mutation ratio resulting from urethane. The mixture of the three oils at equal volume seems to be the most effective. The antimutagenic effect of these oils could be explained by the interaction of their constituents with cytochrome P-450 activation system leading to a reduction of the formation of the active metabolite. The effect could also be attributed to certain molecules that are involved in these oils.

Tissue cultures of Taxus baccata as a source of 10-deacetylbaccatin III, a precursor for the hemisynthesis of taxol.

Callus cultures of Taxus baccata L. cv. stricta were induced from hypocotyl and leaf explants on Woody Plant medium (hormone-free or additionated with phytohormones). Under continuous dark condition, adventitious roots were regenerated from hypocotyl- and leaf-derived callus cultures. Antibodies raised in rabbits against 10-succinyl-10-deacetylbaccatin III were used for the detection and the semi-quantitative determination of 10-deacetylbaccatin III in Taxus cultures. The presence of 10-deacetylbaccatin III in callus extracts was confirmed by TLC, HPLC using a photodiode array detector and mass spectrometry (CI-MS). The highest equivalent content of the taxoid derivatives (7.83 mg/100 g dry wt.) was detected in an extract from leaf-derived callus.

Immunological detection and quantitation of 10-deacetylbaccatin III in Taxus sp. plant and tissue cultures.

A high-sensitive ELISA method was developed for the detection and semi-quantitative determination of 10-deacetylbaccatin III and its structurally related compounds in crude extract of Taxus sp. plants and tissue cultures. The antibodies were raised in rabbits using 7- or 10-succinyl-10-deacetylbaccatin III-BSA conjugate as immunogen. The working range of the assay was from 0.003 to 1.000 ng (0.09 to 31.33 nM) of 10-deacetylbaccatin III per assay. The cross-reacting material in crude plant extract was examined by chromatographic (silica gel CC, HPLC) and immunoassay methods. Study on the evaluation of cross-reacting material in crude Taxus plant extracts showed that at least 80% of the immunosignal correspond to 10-deacetylbaccatin III in the extract. The ELISA method was applied to investigate the 10-deacetylbaccatin III equivalent content in crude extracts of 19 plants species including Taxaceae, Taxodiaceae and Pinaceae species. The 10-deacetylbaccatin III-like structure was only detected in Taxus and Torreya sp. The results indicate that this immunoassay is a useful tool for the rapid screening of species, varieties or individual plants out of a wide population. The distribution of 10-deacetylbaccatin III equivalent content in 9-month old Taxus plantlets cultivated in vitro as well as in callus culture was investigated.

Influence of food intake on the pharmacokinetics of a sustained release formulation of sodium valproate.

The effect of food intake on the pharmacokinetics of DEPAKINE CHRONO 500 mg (Sanofi, France), a sustained release formulation containing 333 mg sodium valproate and 145 mg valproic acid, was studied in 12 young healthy female volunteers. Relative to fasting conditions (F), when the tablet was given at the midpoint of the breakfast (NF), the maximum concentration (F: 34.6 +/- 8.9 micrograms ml-1 and NF: 40.9 +/- 7.3 micrograms ml-1; p = 0.014) and the mean cumulative amount absorbed up to time 6 h (F: 76.3 +/- 11.8% and NF: 90 +/- 10.4%; p = 0.0099) were significantly increased. Nevertheless, the extent of absorption (F: 46.7 +/- 9.9 mg l-1; NF: 48.7 +/- 7 mg l-1) was not significantly affected. There was no change in the area under the curve (1129 micrograms.h ml-1), in the mean residence time (28 h), or in the elimination half-life (16 h). On the basis of this study, the question as to whether DEPAKINE CHRONO should be administered to subjects in the fasting or non-fasting state would not appear to be a major consideration when deciding on the regimen.

Effect of a long-term treatment with lovastatin or fenofibrate on hepatic and cardiac ubiquinone levels in cardiomyopathic hamster.

This study was undertaken to determine if long-term oral administration of lovastatin (50 mg/kg per day) or fenofibrate (200 mg/kg per day) was affecting ubiquinone levels in the heart and the liver of cardiomyopathic hamsters. After 23 weeks of treatment, ubiquinone concentrations (CoQ9 + CoQ10) and ubiquinone ratio (CoQ10/CoQ9) were determined in the heart and in the liver. Our results indicate that lovastatin significantly decreased ubiquinone concentrations in the heart (-33%, P < 0.01) but not in the liver (-23%, NS) when compared to controls, whereas fenofibrate did not alter these parameters. Ubiquinone homologues were not equally decreased during lovastatin treatment: the ratio between CoQ10 and CoQ9 was significantly lowered in the heart (-33%, P < 0.001) and in the liver (-75%, P < 0.001) of lovastatin-treated animals. These results suggest that 3-hydroxymethylglutaryl-coenzyme A reductase inhibition (HMG-CoARI) associated with lovastatin treatment in cardiomyopathic hamsters is more marked in the liver than in the heart, while ubiquinone concentrations are more decreased in cardiac than in hepatic tissues. Our data also showed that fenofibrate had no effect on ubiquinone levels.

Automated determination of disopyramide and N-monodealkyldisopyramide in plasma by reversed-phase liquid chromatography with a column switching system.

A rapid and simple column liquid chromatographic method involving a column switching system for the determination of disopyramide and its N-monodealkyl metabolite (NMD) in plasma is described. The deproteinized plasma is applied to an automated system. Purification and concentration were performed using a precolumn connected to a six-position valve; analytical separation was done on-line using a cyano reversed-phase column with a mobile phase consisting of 10 mmol/l trimethylamine (pH 2.5, adjusted with phosphoric acid)-acetonitrile-tetrahydrofuran (78:20:2, v/v/v). Absorbance was measured at 265 nm, with a minimum detectable amount of disopyramide and NMD of 0.1 micrograms/ml. The method can be applied to drug monitoring and pharmacokinetic studies.

Plasma thiamine concentrations after intramuscular and oral multiple dosage regimens in healthy men.

A novel liquid chromatographic method for the determination of thiamine in plasma has been developed and has been used to study plasma thiamine concentrations after multiple dosage regimens for 11 days. The method involves purification, concentration and analytical separation of thiochrome on-line, using a switching column system. Ten healthy men were given 500 mg thiamine i.m. once a day (Group 1) and ten were given 250 mg p.o. every 12 h (Group 2). The times to reach steady state (7 and 5.6 days for Groups 1 and 2, respectively) were not different (P greater than 0.05). The mean elimination half-life was 1.8 days. The mean minimum steady-state concentration after the oral regimen (23 micrograms.l-1) was 78% of that after the intramuscular regime (29 micrograms.l-1).

Effect of plasma exchange on flumequine pharmacokinetics: comparison with control kinetics.

The effect of plasma exchange (PE) on the pharmacokinetics of flumequine (Apurone) was studied in eight patients receiving a single oral dose of 800 mg. The maximum concentration (38 micrograms/ml) and time to maximum concentration (2.6 h) values were not significantly altered by PE beginning 3 h after administration of flumequine. There was no change in the terminal elimination half-lives (6.6 h), in the steady-state volume of distribution (29 L), or in the apparent plasma clearance (2.5 L/h). By contrast, PE decreased the mean residence time by 30% (14.3 +/- 4.1 h without PE; 9.88 +/- 1.36 h with PE; p less than 0.05). The amount of flumequine extracted by PE (72 mg) was proportional to the plasma concentration at the beginning of the exchange. The elimination half-life during PE (3.15 +/- 1.23 h) decreased by 40%. Renal clearance (0.3 L/h) was not affected. PE only partially modifies the pharmacokinetics of flumequine administered in a single oral dose before PE.

Enzyme-linked immunosorbent assay for the detection and the semi-quantitative determination of taxane diterpenoids related to taxol in Taxus sp. and tissue cultures.

An ELISA-assay for the detection and the semi-quantitative determination of taxane diterpenoids structurally related to taxol found in Taxus sp. has been developed. The antiserum was raised in rabbits using a 2'-succinyltaxol-bovine serum albumin conjugate as immunogen. The working range of the assay was from 1 to 100 ng of taxol. In order to improve the production of taxol, preliminary experiments have been performed on crude extracts of several Taxus sp.; the results indicate that the present immunoassay is an useful tool for the rapid screening of species, varieties or individual plants out of a large population as well as for tissue cultures analysis.

[6 beta-hydroxycortisol: a non-invasive reflection of enzymes of drug metabolism]. / Le 6-beta-hydroxycortisol: un reflet non invasif des enzymes du métabolisme des médicaments.

6-beta-hydroxycortisol (6-beta-OHF) is the main unconjugated metabolite of cortisol in human urine. 6-beta-OHF could be assayed in urine by several methods : chromatography followed by colour reaction, high performance liquid chromatography, radioimmunoassay and enzyme immunoassay. Urinary 6-beta-OHF is increased in some physiological conditions (such as pregnancy) and pathological states (such as hypercortisolemia and liver disease). The measurement of 6-beta-OHF in human urine may provide a useful index of enzyme induction, its excretion being enhanced by many inducers of the mixed function oxygenase.

A new enzyme immunoassay of microsomal rat liver epoxide hydrolase.

Antiserum against purified rat liver microsomal epoxide hydrolase was produced in the rabbit. We developed an enzyme-linked immunosorbent assay which is reliable with regard to its analytical criteria. The concentration of epoxide hydrolase was measured in liver microsomes of control rats and animals treated with F 1379 (250 mg/kg/day) for 5, 7, 14, and 21 days. This hypolipidemic drug was able to induce strong epoxide hydrolase activity and enhance protein concentration. The gradual increase in epoxide hydrolase concentration paralleled the increase of epoxide hydrolase activity, with stabilization occurring after the 14th until the 21st day of treatment.

6 beta-hydroxycortisol in serum and urine as determined by enzyme immunoassay on microtitre plates.

We modified our previous enzyme immunoassay (Clin Chim Acta 1986;157:267-76) for estimating 6 beta-hydroxycortisol (6 beta-OHF) to provide a more routine procedure for measuring it in urine and to increase sensitivity for its measurement in serum. Precision, analytical recovery, specificity, and detection limit are reported. 6 beta-OHF and 17-hydroxycorticosteroids in 24-h and 4-h (08:00-12:00 h) urine collections from healthy volunteers showed no significant day-to-day differences in excretion rate for either collection period. Mean values for 6 beta-OHF in serum of men, women, and women taking oral contraceptives showed no significant differences among these groups.

ELISA of 6-beta-hydroxycortisol in human urine: diurnal variations and effects of antiepileptic therapy.

An enzyme-linked immunosorbent assay for urinary 6-beta-hydroxycortisol (6-beta-OHF) was developed. A highly specific antiserum against 6-beta-OHF-21-hemisuccinate bovine serum albumin (6-beta-OHF-21-HS-BSA) raised in rabbit was used. The enzyme-labelled hapten was prepared by the mixed anhydride method using horse radish peroxidase (HRP). The limit of detection is very low (10 pg), the intra-assay variation ranges from 5.2-6.6%, the inter-assay variation is 8.2-8.3%. Cross reactivities of various steroids are very low. The diurnal variations in urinary 6-beta-OHF and it's ratio to 17-hydroxycorticosteroids (17-OHCS) were studied in six healthy volunteers. No significant difference was found within the 24 h for the ratio of 6-beta-OHF to 17-OHCS. A large increase of 8-12 h urinary 6-beta-OHF was observed in patients receiving chronic treatment with antiepileptics.