Antifungal activity of Coriandrum sativum essential oil, its mode of action against Candida species and potential synergism with amphotericin B

The increasing incidence of drug-resistant pathogens and toxicity of existing antifungal compounds has drawn attention towards the antimicrobial activity of natural products. The aim of the present study was to evaluate the antifungal activity of coriander essential oil according to classical bacteriological techniques, as well as with flow cytometry. The effect of the essential oil upon germ tube formation, seen as an important virulence factor, and potential synergism with amphotericin B were also studied. Coriander essential oil has a fungicidal activity against the Candida strains tested with MLC values equal to the MIC value and ranging from 0.05 to 0.4% (v/v). Flow cytometric evaluation of BOX, PI and DRAQ5 staining indicates that the fungicidal effect is a result of cytoplasmic membrane damage and subsequent leakage of intracellular components such as DNA. Also, concentrations bellow the MIC value caused a marked reduction in the percentage of germ tube formation for C. albicans strains. A synergetic effect between coriander oil and amphotericin B was also obtained for C. albicans strains, while for C. tropicalis strain only an additive effect was observed. This study describes the antifungal activity of coriander essential oil on Candida spp., which could be useful in designing new formulations for candidosis treatment.     

Antifungal effects of the volatile oils from Allium plants against Trichophyton species and synergism of the oils with ketoconazole

In an attempt to develop stable and safe antifungal agents from natural products (daily foodstuffs in particular), the activities of essential oils from Allium sativum for. pekinense, A. cepa, and A. fistulosum against three Trichophyton species responsible for severe mycoses in humans were investigated and compared with activity of allicin in this study. The fungistatic activities of Allium oils were evaluated by the broth dilution method and disk diffusion assay. The combined effects of Allium oils with ketoconazole were tested by the checkerboard titer test. Among the tested oils, A. sativum for. pekinense oil exhibited the strongest inhibition of growth of T. rubrum, T. erinacei, and T. soudanense with MICs (minimum inhibiting concentrations) of 64microg/ml, while the activities of A. cepa and A. fistulosum were relatively mild. The inhibiting activities of the oils on Sabouraud agar plates were dose dependent against Trichophyton species. Additionally, these oils showed significant synergistic antifungal activity when combined with ketoconazole in the checkerboard titer test and disk diffusion test.     

Discrepancies between MIC and MLC values of amphotericin B against isolates ofAspergillus species

There is little information addressing the phenomena of discrepancy between minimal inhibitory concentrations (MIC) and minimal lethal concentrations (MLC) values of amphotericin B (AMB) to clinical isolates of fungi. This study assessed in vitro activity of AMB against 70 clinical isolates of aspergilli: 30 strains ofAspergillus fumigatus, 20 strains ofAspergillus flavus and 20 strains ofAspergillus niger. Susceptibility tests were accomplished using a macro broth dilution procedure, with special emphasis on ascertainment of MLCs. AMB exhibited low MIC values against all clinical isolates. While we did not identify any AMB resistant isolates among 70Aspergillus spp. studied as judged by MIC levels, analysis of the data demonstrated a clear discrepancy between the MIC and MLC levels of AMB obtained against clinical isolates ofAspergillus spp. The MLC values of AMB were significantly higher than the MIC values with MIC 50 and MIC 90 of 0.29 and 0.5 µg/ml, respectively, at the second reading time, and MLC 50 and MLC 90 of 2.31 and 9.24 µg/ml, respectively (p<0.001). Additionally, minimal lethal concentrations in 36/70 (51%) of aspergillal isolates studied produced drug concentrations above those which can usually be sustained in patient plasma or tissue.     

In vitro inhibition of Candida albicans growth by plant extracts and essential oils

The opportunistic Candida albicans yeast strain ATCC 10261 grows at 37 °C and gives germ tubes after 3 h on corn meal agar and blood plasma. It produces chlamydospores and assimilates sucrose, dextrose, galactose, maltose, trehalose and xylose among the tested carbon sources. Other growth characters were also investigated. The agar diffusion cut plug technique revealed that 200 l of Foeniculum vulgare Miller (fennel), Mentha piperita L. (peppermint) and Citrus limon (lemon) essential oils showed inhibitory actions. Fumigation test of the three essential oils had complete inhibition on the growth. The essential oil of Eucalyptus occidentalis End1 (eucalyptus, eucalypt) had no influence on C. albicans growth by the two tests. Meanwhile, methanol extracts of both leaves and male cones of the conifer Thuja orientalis (eastern thuya) had an inhibitory influence on the growth by two tests (cut plug and filter paper disc assay). In comparative tests the antibiotics mycostatin and dermatin had an inhibitory activity on C. albicans at the concentrations of 50–80 g per hole. Crude methanol extract of leaves of eastern thuya (10–80 g/ml) reduced C. albicans growth and intercellular protein nitrogen on liquid media.     

The inhibition of Candida albicans by selected essential oils and their major components

Many volatile oils are known to possess antifungal properties and are potentially applicable as antimycotic agents. By studying the efficacy of essential oils against different pathogenic mycetes, we have evaluated the in-vitro inhibiting activity of some essential oils and their main constituents against a strain of Candida albicans. Sixteen commercial essential oils and forty-two pure constituents (alcohols, aldehydes, ketons, phenols and hydrocarbons), were tested by using a semisolid agar antifungal susceptibility (SAAS) method. Gas chromatography/mass spectroscopy analyses of the oils tested were performed. The essential oils of Origanum vulgare, Satureja montana, Mentha piperita, Cinnamomum verum, Cymbopogon flexuosus showed maximum inhibitory activity (MIC = 500 ppm) after 7 days. According to the results of the examination of pure constituents, -phellandrene proved to be the most interesting component among cyclic monoterpenic hydrocarbons as it showed a strong activity (MIC = 50 ppm). The most active of phenols was carvacrol (MIC 100 ppm). The open-chain alcohol 1-decanol was the most active of alcohols at 50 ppm. Finally, among aldehydes, a strong activity was shown by trans-cynnamaldehyde (MIC 50 ppm).     

Determination of amphotericin B, liposomal amphotericin B, and amphotericin B colloidal dispersion in plasma by high-performance liquid chromatography

Amphotericin B is a potent polyene antifungal drug for intravenous treatment of severe infections. It is used as amphotericin B-deoxycholate and in order to reduce amphotericin B toxicity as lipid-formulated complex (liposomal or colloidal dispersion). A sensitive and specific analytical method is presented for the separation of lipid-complexed and plasma protein-bound amphotericin B in human heparinized plasma. This separation, which is required for pharmacokinetic studies, is achieved by solid-phase extraction (SPE) via Bond Elut C₁₈. The protein-bound amphotericin B has a higher affinity to the SPE material and is therefore retained, whereas the lipid-complexed amphotericin B is eluted in the first step. The recovery of the SPE was >75% for high concentrations and >95% for low concentrations. Quantification was performed by reversed-phase HPLC using a LiChrosorb-RP-8 column, UV detection (λ=405 nm) and a mixture of acetonitrile–methanol–0.010 M NaH₂PO₄ buffer (41:10:49, v/v) as mobile phase. The retention time for amphotericin B under the given conditions was 6.7 min. The calibration curves were found to be linear (r≥0.999) in two different ranges (5.0–0.50 μg/ml and 0.50–0.005 μg/ml). Intra- and inter-day precision and accuracy fulfilled the international requirements. No interference from other drugs (typical broad medication for intensive-care patients) or common plasma components was detected in >400 samples analyzed.     

Potential of plant oils as inhibitors of Candida albicans growth

Minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) were determined for 38 oils of plant origin against Candida albicans. Four strains including one standard strain were used in this study. The antifungal agents, Fluconazole and Amphotericin B were used as positive controls. The standard strain (ATCC10231) used in this study was found to be highly resistant to Fluconazole: 3000 microg ml(-1) of Fluconazole was required to inhibit the growth of this strain partially, and complete inhibition could not be achieved. Other Candida strains were sensitive to 5 microg ml(-1) of Fluconazole. All the strains used were sensitive to Amphotericin B. Of the 38 oils tested, 23 were found effective and fifteen were ineffective. Based on their MFCs, effective oils were categorized into three categories. Seven oils, which exerted fungicidal effect at less than 0.15% concentration of oils, were grouped into the most effective class. The oils exhibiting MFCs in the range of 0.16-1.5% concentration were considered moderately effective. Nine oils, which required more than 1.5% concentration, were regarded as less effective. The Fluconazole-resistant strain (MTCC 227) was sensitive to at least 23 of the plant oils. Results of this study indicate that oils of plant origin may find use as potential anti-Candida agents.     

Development of amphotericin B liposomes bearing antibody specific to Candida albicans

Summary Liposomes expressing external antibody specific for Candida albicans and encapsulating amphotericin B were developed and characterized in this study. Antibody was first modified by the covalent attachment of palmitic acid residues. Liposomes were produced by reverse-phase evaporation and modified antibody was incorporated into these liposomes via the hydrophobic interaction between the palmitic acid and the phospholipids composing the liposomes. The liposomes were characterized as to the amount of amphotericin B by spectroscopy and for the presence of antibody by protein analysis and secondary immunolabeling by fluorescent and electron microscopic methods. Immunogold labeling showed that the antibody was being expressed externally on the liposomes in the electron microscopic studies and the specificity of these liposomes for C. albicans was observed by secondary immunofluorescence.     

Effect of amphotericin B on the lipids of five different strains ofCryptococcus neoformans

Cells of five strains ofCryptococcus neoformans were obtained for partial analysis of lipid composition. Quantitative analysis of lipids and sterols were completed, as well as qualitative analysis of sterols by thin-layer chromatography and by the ultraviolet spectra. Such determinations were made on cells cultured in the absence and presence of amphotericin B at sub-MIC (minimal inhibitory concentration) levels. Marked alterations of the lipid and sterol contents were observed in the amphotericin B — treated cells. Moreover, ergosterol disappeared in these antibiotic-exposed cells. It is concluded that amphotericin B altered the lipid profiles, especially sterols ofC. neoformans.     

Antifungal properties of essential oils and their main components uponCryptococcus neoformans

Cryptococcus neoformans opportunistic fungus met in the last phasis of AIDS is inhibited in vitro by several essential oils on natural volatile compounds. The minimal inhibitory concentration may reach 100 µl/l and minimal fungicidal concentration 200 µl/l with Palmarosa or Cinnamon oils. Among phenolic compounds, thymol and carvacrol are most fungitoxic. Terpenoids, citral, geraniol, and citronellol show best activities.     

Comparative efficacy of amphotericin B,clotrimazole and itraconazole againstAspergillus spp.

The susceptibilities of two isolates ofAspergillus flavus, one from a human case of recalcitrant mycotic keratitis, and an environmental isolate ofA. fumigatus, to itraconazole, clotrimazole and amphotericin B were measured. Observations of macroscopic growth and microscopic evaluations of conidia germination both indicated that the two isolates ofA. flavus were markedly more resistant to amphotericin B than to itraconazole and clotrimazole. Itraconazole was more effective than clotrimazole for all isolates. Ourin vitro susceptibility results suggest the use of itraconazole should be a primary consideration in the treatment ofAspergillus keratitis.     

Interaction of amphotericin B with membrane lipids as viewed by H-NMR

The effects of amphotericin B upon the organization and dynamics of multibilayer membranes of dimyristoylphosphatidylcholine (DMPC) were investigated by means of ²H-NMR. At high amphotericin B concentrations (30 mol% with respect to the lipid) and at temperatures above 25°C, DMPC experiences two different environments which are in slow exchange on the ²H-NMR time scale. In one of these, the lipid is immobilized by the antibiotic, in a molar ratio of approximately 1:1, whereas the lipid unsequestered by amphotericin B is more ordered than in its pure state. This ordering effect is perceived at relatively low antibiotic doses (4%). The local lipid order, and the relative percentage, of sequestered DMPC, are temperature-independent (up to 65°C), whereas the ordering of the unsequestered lipid domain is not. The perturbation induced by amphotericin B is manifest similarly at the edges as well as in the center of the bilayer. Antibiotic addition leads to large decreases in the transverse relaxation time, T₂, of the labelled lipid, but not in the spin-lattice relaxation time, T₁. This indicates an increased density of slow motional modes and little change in rapid motions.     

Cholesterol markedly reduces ion permeability induced by membrane-bound amphotericin B

It is widely accepted that amphotericin B (AmB) together with sterol makes a mixed molecular assemblage in phospholipid membrane. By adding AmB to lipids prior to preparation of large unilamellar vesicles (LUV), we directly measured the effect of cholesterol on assemblage formation by AmB without a step of drug's binding to phospholipid bilayers. Potassium ion flux assays based on ³¹P-nuclear magnetic resonance (NMR) clearly demonstrated that cholesterol markedly inhibits ion permeability induced by membrane-bound AmB. This could be accounted for by a membrane-thickening effect of cholesterol since AmB actions are known to be markedly affected by the thickness of membrane. Upon addition of AmB to an LUV suspension, the ion flux gradually increased with increasing molar ratios of cholesterol up to 20 mol%. These biphasic effects of cholesterol could be accounted for, at least in part, by the ordering effect of cholesterol.     

Hairy root cultures of Anethum graveolens (dill): establishment, growth, time-course study of their essential oil and its comparison with parent plant oils

Transformed root cultures of Anethum graveolens were induced by inoculation of aseptically grown seedlings with Agrobacterium rhizogenes carrying plasmid pRi 1855. The main component of the essential oils from the fruits and from the roots of the parent plant was carvone, whereas -phellandrene and apiole were dominant in the oil from, respectively, the aerial parts and the hairy roots. The essential oils from the fruits, aerial parts and roots of the parent plant were at 2%, 0.3% and 0.06% (v/w), respectively, but only 0.02% (v/w) in the hairy root cultures. Growth of the hairy root cultures reached 600 mg dry wt/50 ml medium after 50 days. The essential oil composition did not change significantly during their growth.     

In vitro and in vivo antifungal activities of liposomal amphotericin B,and amphotericin B lipid complex

The in vitro and in vivo antifungal activities of liposomal amphotericin B (L-AMPH) and amphotericin B lipid complex (ABLC), which is composed of amphotericin B and the phospholipids dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol, were compared with those of conventional amphotericin B (Fungizone®, AMPH). The acute intravenous toxicity was markedly lower in BALB/c mice; 50% lethal doses (LD_50s) were 2.75 mg/kg in AMPH, 32.9 mg/kg in L-AMPH and >75 mg/kg in ABLC. In vitro antifungal activities againstCandida albicans, C. parapsilosis, C. tropicalis, C. glabrata, andC. krusei were evaluated by the agar plate dilution method. The activities were unchanged againstC. albicans, but MICs increased more than four fold in 18 of the 20 strains other thanC. albicans in L-AMPH and in 9 of the 20 in ABLC. L-AMPH and ABLC were as efficacious as AMPH in the treatment of mice infected withC. albicans, and at a dose of 0.5 and 1.0 mg/kg of body weight, ABLC was more efficacious on survival. A ten-times larger dose (10 mg/kg) of L-AMPH and ABLC was administered to mice with 100% survival, suggesting improved tolerability as compared to amphotericin B.     

Synergic effect of grape seed extract with amphotericin B against disseminated candidiasis due to Candida albicans

Amphotericin B (Amp B) is considered as a drug of choice for treatment of fungal infections, but it causes severe side effects such as renal damage. To lessen the severity, it is often combined with the azole, but data reporting resistance of Candida albicans to the azole have been recently increasing. Thus, finding a new product that can reduce Amp B dose by combination seems to be important. In the present study, we investigated a synergic effect of grape seed extract (GSE) combined with Amp B against the fungus. Our results showed that the GSE alone inhibited growth of C. albicans yeast cells, and that in a murine model of disseminated candidiasis mice groups given GSE before intravenous inoculation with the yeast cells survived longer than diluent-received (control) mice groups (P<0.05). This GSE antifungal effect was dose-dependent. Upon combination of GSE plus Amp B, the combination therapy strikingly retarded the yeast growth as determined by the broth susceptibility method. Against the disseminated disease, mice given diluent (negative control), Amp B (0.5mg/kg of body weight), or GSE (2mg/kg of body weight) had mean survival times (MSTs) of approximately 11.4, 14.4, and 17.6 days, respectively. However, mice treated with the combination of the doses of Amp B and GSE had a MST value of 38.4 days, surviving an average of 24 days longer than Amp B alone-treated mice groups. This MST value from the combination-received mice group was greater than the MST value from the mice group given four times the Amp B dose (2mg/kg of body weight). All these data indicate that the combination therapy can reduce more than 75% of Amp B dose, implying that GSE has a synergic effect with Amp B.     

Antibacterial effect of some essential oils administered alone or in combination with Norfloxacin

The objective of the present study was that of verifying a possible synergistic antibacterial effect between Pelargonium graveolens [Lis-Balchin, M., Deans, S.G., Hart, S., 1996. Bioactive Geranium oils from different commercial sources. J. Essential Oil Res. 8, 281-290.] essential oil (and its main components) and Norfloxacin antibiotic. As a first step growth inhibition by some types of essential oils was assessed in five microbial species. The antimicrobial effects of P. graveolens oil, as well as those of its components, were evaluated by means of the agar dilution method (ADM) against Bacillus cereus ATCC 11778, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 35218, Staphylococcus aureus ATCC 6538 and S. aureus ATCC 29213. The results obtained highlighted the occurrence of a pronounced synergism between P. graveolens essential oil and Norfloxacin against three of the five bacterial species under study with a FIC index in the 0.37-0.50 range. Such antibacterial effects were also shown to increase, although to a lesser extent, when Norfloxacin was given with the main components of P. graveolens essential oil. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of Norfloxacin with either P. graveolens essential oil, or with some of the main components of this latter, in the treatment of infections caused by some bacterial species is likely to reduce the minimum effective dose of Norfloxacin thus minimizing the side effects of the antibiotic.     

Effect of nystatin,amphotericin B and amphotericin B methyl ester on Saccharomyces cerevisiae with different lipid composition

Saccharomyces cerevisiae was cultured under anaerobiosis in semi-complete medium to which either palmitoleic or oleic acid was added. Cells were grown at 20 °C or 30 °C. The levels of total lipids, total sterols, and phospholipids were higher in cells grown at 20 °C than at 30 °C. The effects of nystatin (NYS), amphotericin B (AMB), and amphotericin B methyl ester (AME) were evaluated by determining cell viability and liberation of intracellular compounds. The loss of cell viability is higher in the first 30 minutes of incubation with the drugs and is the same regardless of the type of cells obtained. Low molecular weight compounds and ions such as K⁺ are liberated a few minutes after incubation with the drugs whereas proteins and substances absorbing at 260 nm are liberated later. Phosphate liberation comes after K⁺ and before compounds of higher molecular weights.     

Interference of melanin in the susceptibility profile of Sporothrix species to amphotericin B

BackgroundThe presence of melanin in the fungal cell is a major virulence factor of the genus Sporothrix since it protects the fungal cells against the defense systems.AimsThe present study aimed to investigate the interference of melanin in the susceptibility of Sporothrix brasiliensis and Sporothrix schenckii sensu stricto to amphotericin B and itraconazole, drugs recommended as therapy for disseminated and subcutaneous sporotrichosis, respectively.MethodsYeast cells were cultivated in minimal medium with or without l-DOPA in order to induce the production of melanin. Microdilution and killing assay methods were used to determine the antifungal activity against yeast cells with different amounts of melanin.ResultsThe killing assay showed that melanization protected isolates within the S. schenckii complex from amphotericin B, particularly in the lower concentrations tested.ConclusionsStudies combining amphotericin B and inhibitors of melanin are required in order to avoid this effect.     

两性霉素B脂质体、氟胞嘧啶和伊曲康唑联合治疗隐球菌性脑膜炎的临床回顾性研究

目的 观察两性霉素B脂质体、氟胞嘧啶和伊曲康唑联合治疗非AIDS、非器官移植隐球菌性脑膜炎患者的疗效和安全性,探讨非AIDS、非器官移植隐球菌性脑膜炎患者诱导治疗的新方案.方法 采用回顾性病例对照研究,18例非AIDS、非器官移植隐球菌性脑膜炎患者诱导期分别接受两性霉素B脂质体、氟胞嘧啶和伊曲康唑联合(研究组n=7)与两性霉素B、氟胞嘧啶和氟康唑联合治疗(对照组n=11).比较两组治疗方案在临床症状、体征变化、病死率、病原学转阴率、脑脊液改变等方面的差异及血常规、肝肾功能损害等不良反应发生情况.结果 治疗前研究组和对照组脑脊液新型隐球菌培养阳性分别为5例和8例(P =0.676),治疗1周后分别为5例和8例(P =0.676),治疗2周后分别为2例和4例(P =0.572),治疗3周后分别为0例和1例(P=0.611),治疗4周后,无死亡病例,两组脑脊液新型隐球菌培养均为阴性,脑脊液生化两组之间差异无统计学意义,对照组较研究组丙氨酸氨基转移酶(156.82±41.30 vs 97.00±22.02,P=0.003)、尿素氮(8.45±3.18 vs 5.54±1.28,P=0.020)升高更明显.结论 两性霉素B脂质体、氟胞嘧啶及伊曲康唑联合治疗非AIDS、非器官移植隐球菌性脑膜炎患者疗效确切,安全性较好,可以作为诱导期治疗的选择.