Study on chemical analysis, antioxidant and in vitro antifungal activities of essential oil from wild Vitex agnus-castus L. seeds growing in area of Argan Tree of Morocco against clinical strains of Candida responsible for nosocomial infections.

OBJECTIVE: To study the composition, the antioxidant activity and the in vitro antifungal action anti-Candida species of essential oils extracted from seeds of Vite xagnus-castus L. MATERIALS AND METHOD: The essential oils were extracted using Clevenger-type apparatus and analyzed by gas chromatography/mass spectrometry (GC/MS). The antioxidant activity was analyzed using the DPPH free radical-scavenging method. Susceptibility tests for Candida albicans (12), C. dubliniensis (1), C. glabrata (3), C. krusei (3), C. parapsilosis (6), C. lusitaniae (1), C. famata (1) and C. tropicalis (3) were expressed as inhibition zone by the disc-diffusion method and as minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) by the broth macrodilution method, compared to amphotricin B and fluconazol as standard drugs. RESULTS: Major components were: 1,8-cineole (19.61%), sabinene (14.57%), α-pinene (9.76%), ß-farnesene (6.04%), ß-caryophyllene oxide (5.83%) and ß-caryophyllene (5.02%). A low antioxidant activity was found (IC50=1.072mg/ml), but it can be exploited. V. agnus-castus seeds essential oils disosed a 35-58mm zone of inhibition (mean: 49mm) against all 30 isolates tested. In broth macrodilution method, all the tested Candida species were susceptible to the essential oils and this activity was concentration-dependent. MIC values varied from 0.13 to 2.13mg/ml V. agnus-castus seeds essential oils. CONCLUSION: Results of this study indicated that the oils of plant origin could be used as potential anti-Candida species causative agents of nosocomial infections. These oils exhibited a noticeable antifungal activity against the selected fungi. The natural antifungal substances are inexpensive and have fewer side effects, they may represent alternative therapies for candidiasis.

Natural extracts stimulate membrane-associated mechanisms of resistance in Gram-negative bacteria.

UNLABELLED: Several mechanisms are involved in the bacterial resistance towards antimicrobial agents. The membrane-associated mechanisms of resistance were studied in Escherichia coli strains after incubation with Thymus maroccanus essential oil, its major components (carvacrol and thymol) or with certain antibiotics. The minimum inhibitory concentration (MIC) and the expression of membrane proteins, porins and efflux pumps were determined in wild type and derivative strains. Derivative strains adapted to different compounds displayed a high level of resistance to all tested antibiotics. The MIC increase is associated with an overexpression of an efflux pump immunorelated to AcrAB-TolC in various variants. Interestingly, the expression of outer membrane proteins slightly decreases in these strains. We demonstrate that the increase in antibiotic resistance correlates with membrane changes observed in the variants. This type of bacterial adaptation to natural compounds can occur in vivo providing the emergence/selection of bacteria less susceptible to clinically used antibiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: Thymus maroccanus essential oil and some major components are able to select variants that modify the expression of transporters involved in the influx (porins) and in the efflux (AcrAB family) of various drugs. Importantly, these membrane proteins are involved in the transport of natural compounds and several antibiotic families. This special 'membrane adaptation' can explain the persistence of less susceptible/tolerant bacteria in the environment where natural compounds are present and the continuous stimulation of efflux systems in these bacteria.

Thymus maroccanus essential oil, a membranotropic compound active on Gram-negative bacteria and resistant isolates.

AIMS: The effects of Thymus maroccanus essential oil (EO) on the integrity of the cell membranes and the permeability of the outer membrane (OM) and inner membrane (IM) of Escherichia coli, Enterobacter aerogenes and Salmonella enterica Typhimurium were investigated. METHODS AND RESULTS: The bacterial release of intracellular proteins, cytoplasmic ß-galactosidase and periplasmic ß-lactamase induced by T. maroccanus EO was compared to the membranotropic activity of polymyxin B (PB) known as an effective permeabilizer of the membrane of Gram-negative bacteria. Results showed that T. maroccanus EO increased the permeability of the OM and IM of studied bacteria and induced the release of intracellular proteins into the external medium. CONCLUSIONS: The effect of T. maroccanus EO on the outer membrane was comparable to that of PB, and both T. maroccanus EO and PB induce similar levels of ß-lactamase release. In addition, it also promoted the release of the cytoplasmic ß-galactosidase. Moreover, the lipopolysaccharide molecules and the overexpression of efflux pumps seem to play a crucial role in the level of susceptibility of studied bacteria to the permeabilizing effect of T. maroccanus EO. SIGNIFICANCE AND IMPACT OF STUDY: These results demonstrate that T. maroccanus EO can restore antibiotic activity by targeting the two bacterial membranes and would be attractive candidates for developing new adjuvants for combating resistant Gram-negative bacteria.

Anticandidal activity of the essential oils of Thymus maroccanus and Thymus broussonetii and their synergism with amphotericin B and fluconazol.

The discovery of antifungal drugs had eradicated some infections that ravaged the humankind. But their indiscriminate use has led to the development of multidrug resistant pathogens. One strategy employed to overcome these resistance mechanisms is the use of combination of the essential oils (EOs) of medicinal plants and conventional drugs. In this study, we investigated a possible synergistic effect of the EOs of two Moroccan endemic thymes (Thymus broussonetii and T. maroccanus) with amphotericin B (Amp B) and fluconazol against Candida albicans. The fractional inhibitory concentration indices (FICI) of T. maroccanus and T. broussonetii EOs combined with Amp B and fluconazol, calculated from the checkerboard titer assay, were 0.49, 0.27, 0.37 and 0.3, respectively. Also, our results indicate that the synergistic effect of EOs with fluconazol was stronger than the combination with Amp B. All these data highlight that the EOs tested potentiate the antifungal action of Amp B and fluconazol, suggesting a possible utilization of these EOs in addition to antifungal drugs for the treatment of some candidiasis due to C. albicans. The use of these combinations is likely to reduce the minimum effective dose of the drugs, thus minimizing their toxic side effects and the treatment cost.

Co-treatment of olive-mill and urban wastewaters by experimental stabilization ponds.

Olive oil mill wastewater (OMW) constitutes a source of environmental problems in Morocco due to its significantly high organic load, its phytotoxic properties and its relatively low biodegradability. An effective option for its disposal is its agricultural use after co-treatment with urban wastewater (UWW). The main objective of this investigation was to evaluate the potential of this co-treatment, using experimental waste stabilization ponds, in removing OMW phytotoxicity. We examined the influence of the organic load, at the entry of the treatment system, on the evolution of some physicochemical (chemical oxygen demand and polyphenols) and microbiological (fecal coliforms and fecal streptococci) parameters. The results showed a removal of the organic, phenolic and microbial load throughout the treatment which differed from one system to another according to the OMW load applied to each system. The results concerning the germination assays of Zea mays and Solanum lycopersicum suggested that the co-treatment of OMW with UWW would decrease the phytotoxicity of this waste.

Application of lime and calcium hypochlorite in the dephenolisation and discolouration of olive mill wastewater.

The application of hypochlorite for the removal of soluble COD, phenolic and polyphenolic like compounds, and other organic compounds responsible for the olive mill wastewater (OOWW) colour has been experimentally studied. After the OOWW filtration on a sand column, the effluent was subjected to a fast liming under optimal conditions. Lime application reduced polyphenols, COD and SS contents to half of their initial values but an important blackening of the treated OOWW was observed, especially when adding high concentrations of lime (10% (W/V) and 15% (W/V)). A second stage of treatment was applied using calcium hypochlorite. In this stage, removal of the studied compounds reached as much as 95% at higher concentrations, and particularly the colouring of OOWW which is generally difficult to eliminate was greatly reduced. The OOWW hypochloration acted through coagulation-flocculation and a rapid oxidation of the organic matter proceeded from the first 5min. The kinetic study of the degradation of the waste polluting compounds from liming showed that Ca(ClO)(2) reacts similarly in the elimination of organic compounds, polyphenols, SS and colouration. The analysis of the organochloride compounds generated by the reaction between hypochlorite and the organic compounds showed that DDD, DDT and the heptachlor contents exceeded the values recommended by the International and European drinking water standards.

[Impact of olive oil wastewater on the physicochemical and biological quality of groundwater in the Haouz plain, south of Marrakesh (Morocco)]. / Impact des effluents des huileries d'olive sur la qualité physico-chimique et biologique des eaux souterraines dans la plaine du Haouz au sud de Marrakech (Maroc).

Olive Oil Waste Water (OOWW) is acidic water known for its antibacterial and phytotoxic effects. In Morocco, these effluents are generally drained into the surrounding environment without any treatment or are collected and retained in large evaporation basins, which can constitute a source of potential pollution of groundwater. The analysis of water from 14 wells, in an agricultural zone, where there are four storage basins of OOWW coming from various types of oil mills, showed that their physicochemical and biological quality is largely influenced by the presence of these basins. Indeed, high values of COD (400 mg O2 L(-1)), polyphenols (180 mg l(-1)) and electrical conductivity (1.89 mS cm-') were recorded in P5 water. This well is located only 5 m from a storage basin of OOWW coming from the H4 oil mill which rejects OOWW charged with organic matter (more than 290 g of O2 L(-1) of COD, on average). However, the settlement of a basin, dug close to a modern oil mill (H3), which produces large OOWW volumes but is loaded with low organic pollutant concentration (100 g of O2L(-1) of COD), reduced notably the effect of the OOWW infiltration on groundwater. This resulted in the improvement of the groundwater physicochemical quality near this basin (P7 and P8); the organic matter contents expressed by COD vary from 26.7 mg l(-1) to 30 mg l(-1) and the polyphenol contents are on average between 41 mg l(-1) and 47.6 mg l(-1). The analysis of the total well water fauna showed a great taxonomic richness with variations in time and space. The biodiversity decreases in groundwater polluted by the oil mill waste and the underground fauna disappears completely if the degree of contamination of groundwater is high.

Chemical composition and antifungal activity of essential oils of seven Moroccan Labiatae against Botrytis cinerea Pers: Fr.

Essential oils of seven Moroccan Labiatae were chemically analysed by GC-MS and evaluated for their in vitro antifungal activity against Botrytis cinerea. Among them, Origanum compactum and Thymus glandulosus greatly inhibited the growth of the mycelium. The inhibition of Botrytis cinerea was 100% for both oils at 100 ppm, while the IC(50)s were 35.1 and 79.2 ppm, respectively. Mentha pulegium exhibited moderate activity at 250 ppm since the inhibition of the mycelial growth was 58.5% and the IC(50) was 233.5 ppm. The main constituents of the studied oils were also examined. Thymol and carvacrol that are the two main constituents of Thymus glandulosus and Origanum compactum exhibited the strongest antifungal activity with 100% of inhibition at 100 ppm, respectively.