Maria José Gonçalves

Essential oil of Daucus carota subsp. halophilus: Composition, antifungal activity and cytotoxicity

ETHNOPHARMACOLOGICAL RELEVANCE Essential oils are known to possess antimicrobial activity against a wide spectrum of bacteria and fungi. Daucus carota L. is used since olden times in traditional medicine, due to recognized therapeutic properties, namely the antimicrobial activity of their essential oils. AIM OF THE STUDY In the present study the composition and the antifungal activity of the oils of Daucus carota L. subsp. halophilus (Brot.) A. Pujadas (Apiaceae), an endemic plant from Portugal, were evaluated. Moreover, their cytotoxicity in mouse skin dendritic cells at concentration showing significant antifungal activity was also evaluated. MATERIAL AND METHODS The oils were investigated by GC and GC-MS and the antifungal activity (MIC and MLC) were evaluated against yeasts, dermatophyte and Aspergillus strains. Assessment of cell viability was made by the MTT assay. RESULTS The results showed large variations in the compositions during ontogenesis, particularly in the amounts of elemicin that increased significantly in the ripe umbels (5.9% vs. 31.0%). The results also demonstrated that the oil with high amounts of elemicin, which have stronger antifungal activity, showed no cytotoxic effect, at concentrations ranging from 0.16 to 0.64 microl/ml, for as long as 24h. CONCLUSION It is possible to find appropriate doses of Daucus carota oil showing both antifungal activity and very low detrimental effect on mammalian cells.

Chemical composition and antifungal activity of the essential oils of Lavandula viridis L'Her.

In the present work we report for what we believe to be the first time the antifungal activity and mechanism of action of the essential oils of Lavandula viridis from Portugal. The essential oils were isolated by hydrodistillation and analysed by GC and GC/MS. The MIC and the minimal lethal concentration (MLC) of the essential oil and its major compounds were determined against several pathogenic fungi. The influence of subinhibitory concentrations of the essential oil on the dimorphic transition in Candida albicans was also studied, as well as propidium iodide and FUN-1 staining of Candida albicans cells by flow cytometry following short treatments with the essential oil. The oils were characterized by a high content of oxygen-containing monoterpenes, with 1,8-cineole being the main constituent. Monoterpene hydrocarbons were present at lower concentrations. According to the determined MIC and MLC values, the dermatophytes and Cryptococcus neoformans were the most sensitive fungi (MIC and MLC values ranging from 0.32 to 0.64 µl ml⁻¹), followed by Candida species (at 0.64-2.5 µl ml⁻¹). For most of these strains, MICs were equivalent to MLCs, indicating a fungicidal effect of the essential oil. The oil was further shown to completely inhibit filamentation in Candida albicans at concentrations well below the respective MICs (as low as MIC/16). Flow cytometry results suggested a mechanism of action ultimately leading to cytoplasmic membrane disruption and cell death. Our results show that L. viridis essential oils may be useful in the clinical treatment of fungal diseases, particularly dermatophytosis and candidosis, although clinical trials are required to evaluate the practical relevance of our in vitro research.

Chemical characterization and biological activity of essential oils from Daucus carota L. subsp. carota growing wild on the Mediterranean coast and on the Atlantic coast

The essential oils and supercritical CO(2) extracts of wild Daucus carota L. subsp. carota growing spontaneously in Sardinia and in Portugal were investigated. The main components in the Sardinian essential oil of flowering and mature umbels with seeds are beta-bisabolene (17.6-51.0%) and 11-alpha-(H)-himachal-4-en-1-beta-ol (9.0-21.6%); instead, the oils from Portuguese samples are predominantly composed of geranyl acetate (5.2-65.0%) and alpha-pinene (3.5-37.9%). Supercritical extracts contain lower amounts of monoterpenes and higher amounts of sesquiterpene hydrocarbons. Antifungal activities of the Sardinian oils were the highest, particularly for dermatophytes and Cryptococcus neoformans, with MIC values of 0.16-0.64 microL mL(-1).

Chemical composition and antifungal activity of the essential oils of Lavandula pedunculata (Miller) Cav.

The chemical composition and antifungal activity of the essential oils of Lavandula pedunculata (Miller) Cav., harvested in North and Central Portugal, were investigated. The essential oils were isolated by hydrodistillation and analyzed by GC and GC/MS. The minimal‐inhibitory concentration (MIC) and the minimal‐lethal concentration (MLC) of the essential oils and of their major constituents were used to evaluate the antifungal activity against different strains of fungi involved in candidosis, dematophytosis, and aspergillosis. The oils were characterized by a high percentage of oxygenated monoterpenes, the main compounds being 1,8‐cineole (2.4–55.5%), fenchone (1.3–59.7%), and camphor (3.6–48.0%). Statistical analysis differentiated the essential oils into two main types, one characterized by the predominance of fenchone and the other one by the predominance of 1,8‐cineole. Within the 1,8‐cineole chemotype, two subgroups were well‐defined taking into account the percentages of camphor. A significant antifungal activity of the oils was found against dermatophyte strains. The essential oil with the highest content of camphor was the most active with MIC and MLC values ranging from 0.32–0.64 μl/ml.

Essential oils from Distichoselinum tenuifolium: Chemical composition, cytotoxicity, antifungal and anti-inflammatory properties

ETHNOPHARMACOLOGICAL RELEVANCE Several aromatic plants and their essential oils are known to possess antimicrobial and anti-inflammatory properties. Distichoselinum tenuifolium (Lag.) Garcia Martin & Silvestre, an Iberian endemism, is traditionally used in the treatment of contact dermatitis and skin infections. However, the cellular mechanisms through which this plant exerts their beneficial effects are not known. AIM OF THE STUDY The aims of this study were to examine the chemical composition of the essential oil isolated from Distichoselinum tenuifolium, and to test the efficacy of the essential oil as an antifungal and anti-inflammatory potential. MATERIALS AND METHODS The oils were investigated by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) and the antifungal activity (minimal inhibitory concentrations: MIC and minimal lethal concentrations: MLC) were evaluated against yeasts, dermatophyte and Aspergillus strains. Assessment of cell viability was made by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the in vitro anti-inflammatory potential of Distichoselinum tenuifolium oil was evaluated by measuring nitric oxide (NO) production induced by LPS, in the absence or in the presence of the oil, in a mouse macrophage cell line. RESULTS The oils are predominantly composed of monoterpene hydrocarbons, being myrcene the main compound (47.7-84.6%). The oils revealed significant antifungal activity against Cryptococcus neoformans and dermatophyte strains and significantly inhibited NO production stimulated by LPS in macrophages, without affecting cell viability at concentrations ranging from 0.64 microL/mL to 1.25 microL/mL. CONCLUSION These findings add significant information to the pharmacological activity of Distichoselinum tenuifolium essential oils, specifically to its antifungal and anti-inflammatory properties, thus justifying and reinforcing the use of this plant on traditional medicine. Therefore, their beneficial effects and use in disease prevention, especially those related to fungal infections and inflammation, should be explored in more depth.

Antifungal Activity of the Essential Oil of Thymus x viciosoi against Candida, Cryptococcus, Aspergillus and Dermatophyte Species

The essential oil (EO) of Thymus x viciosoi (Pau) R. Morales was isolated and analysed by GC and GC-MS. The antifungal activity of the EO and its major components against clinically relevant yeasts and molds was then measured. Their influence on the germ tube formation in Candida albicans and the influence of the EO on the metabolic function and cytoplasmic membrane integrity in the same yeast, analyzed by flow cytometry, were also studied. The EO showed high contents of carvacrol, thymol, and P-cymene. The total EO, as well as its components carvacrol and thymol, displayed very low minimum inhibitory concentrations and minimum fungicidal concentrations against all tested organisms (0.04 to 0.64 microL mL(-1)), while P-cymene showed weaker activity (2.5 to > 20.0 microL mL(-1)). They also inhibited filamentation at sub-inhibitory concentrations in C. albicans, particularly P-cymene, and the EO led to rapid metabolic arrest, disruption of the plasma membrane and consequently cell death. The EO and its main components were found to display a broad fungicidal activity through the disruption of cytoplasmic membrane integrity leading to leakage of vital intracellular compounds. In conclusion, the phenolic oil of T. x viciosoi may have potential for use in the development of clinically useful antifungal preparations.

Composition of a volatile extract of Eryngium duriaei subsp. juresianum (M. Laínz) M. Laínz, signalised by the antifungal activity

The composition of a volatile extract of Eryngium duriaei subsp. juresianum, signalised by the antifungal activity (MIC values=0.16-0.32 μL mL(-1)) against several dermatophyte species (Trichophyton mentagrophytes, T. rubrum, Epidermophyton floccosum; T. verrucosum, T. mentagrophytes var interdigitale, Microsporum canis and M. gypseum) was established following a combined methodology of GC, GC-MS and an exclusive (13)C NMR technique that does not require prior isolation of compounds. Twenty-five components were identified accounting 84.6% of the whole composition. Major compound was found to be α-neocallitropsene (26.0%) although the dominance of caryophyllane derived compounds, the most probable responsible for the antifungal activity, namely isocaryophyllen-14-al (16.2%), 14-hidroxy-β-caryophyllene (13.4%), caryophyllene oxide (7.6%) and E-β-caryophyllene (6.3%).

Myrtus communis L. as source of a bioactive and safe essential oil

In Algeria, Myrtus communis L. is distributed throughout the Tell Atlas and the coastal regions of Algiers and Constantine. The leaves are used in respiratory disorders, diarrhea and hemorrhoids. The aims of this work were to evaluate the antifungal and anti-inflammatory potential of well characterized essential oils (EO). Since EO can be applied by inhalation, dermal application and oral administration, we used several mammalian cell lines to assess safe bioactive doses. The chemical composition of two samples was investigated by GC-FID, GC-MS and (13)C NMR spectroscopy. Monoterpene derivatives are the main compounds: α-pinene (50.8 and 33.6%), 1,8-cineole (21.9 and 13.3%), linalool (2.7 and 14.8%), and linalyl acetate (0.5 and 9.5%). The antifungal evaluation revealed that the oils were more active against Cryptococcus neoformans (yeast) and Epidermophyton floccosum, Microsporum canis, Trichophyton rubrum (dermatophytes). The anti-inflammatory potential was evaluated using an in vitro model of lipopolysaccharide (LPS)-stimulated macrophages. Assessment of cell viability was made through the MTT assay. Both oils were able to significantly inhibit NO production, without affecting cell viability, in concentrations up to 0.64 mg/mL. These promising results, disclose bioactive concentrations of Myrtle essential oils with a safety profile suggesting a potential oral and topical application or use by inhalation.

New compounds, chemical composition, antifungal activity and cytotoxicity of the essential oil from Myrtus nivellei Batt. & Trab., an endemic species of Central Sahara

ETHNOPHARMACOLOGIC RELEVANCE Myrtus nivellei Batt. & Trab. (Myrtaceae) known as Sahara myrtle is appreciated by the Touaregs as medicinal plant. Infusion of leaves is employed against diarrhea and blennorrhea. Crushed leaves added to oil or to butter ointment have been traditionally used for the treatment of dermatosis. Aim of the study is to consider the traditional medicinal uses and the lack of scientific studies on their biological activities, the present study was designed to elucidate the chemical composition, the antifungal activity of its essential oils against fungi responsible for human infections, as well as, its cytotoxicity in the mammalian keratinocytes. MATERIALS AND METHODS Chemical analysis of Myrtus nivellei essential oil isolated by hydrodistillation of aerial parts (leaves and flowers), was carried out using a combination of chromatographic (CC, GC with retention indices) and spectroscopic techniques (MS, (13)C NMR, 2D NMR). The antifungal activity was evaluated by using broth macrodilution methods for yeasts and filamentous fungi. Cytotoxicity was tested in HaCaT keratinocytes through the MTT assay. RESULTS Ten samples coming from two localities of harvest were investigated. The chemical composition was largely dominated by 1,8-cineole (33.6-50.4%) and limonene (17.5-25.0%). The structure of two new compounds bearing the isoamylcyclopentane skeleton has been elucidated. The oil was more active against Cryptococcus neoformans with MIC of 0.16μL/mL followed by dermatophytes, with MICs of 0.64 and 1.25μL/mL. Furthermore, evaluation of cell viability showed no cytotoxicity in HaCaT keratinocytes at concentrations up to 1.25µL/mL. CONCLUSIONS The composition of Myrtus nivellei oil differed from that of Myrtus communis. The structure of two di-nor-sesquiterpenoids has been elucidated. It was possible to find appropriate doses of Myrtus nivellei oil with both antifungal activity and very low detrimental effect on keratinocytes. These findings add significant information to the pharmacological activity of Myrtus nivellei essential oils, specifically to its antifungal properties, thus justifying and reinforcing the use of this plant in traditional medicine.

Effects of essential oils from Eucalyptus globulus leaves on soil organisms involved in leaf degradation.

The replacement of native Portuguese forests by Eucalyptus globulus is often associated with deleterious effects on terrestrial and aquatic communities. Several studies have suggested that such a phenomenon is linked with the leaf essential oils released into the environment during the Eucalyptus leaf degradation process. However, to date, the way these compounds affect leaf degradation in terrestrial systems i.e. by direct toxic effects to soil invertebrates or indirectly by affecting food of soil fauna, is still unknown. In order to explore this question, the effect of essential oils extracted from E. globulus leaves on terrestrial systems was investigated. Fungal growth tests with species known as leaf colonizers (Mucor hiemalis, Alternaria alternata, Penicillium sp., Penicillium glabrum and Fusarium roseum) were performed to evaluate the antifungal effect of essential oils. In addition, a reproduction test with the collembolans Folsomia candida was done using a gradient of eucalyptus essential oils in artificial soil. The influence of essential oils on feeding behaviour of F. candida and the isopods Porcellio dilatatus was also investigated through food avoidance and consumption tests. Eucalyptus essential oils were lethal at concentrations between 2.5–20 µL/mL and inhibited growth of all fungal species between 1.25–5 µL/mL. The collembolan reproduction EC50 value was 35.0 (28.6–41.2) mg/kg and both collembola and isopods preferred leaves without oils. Results suggested that the effect of essential oils in leaf processing is related to direct toxic effects on fungi and soil fauna and to indirect effects on the quality and availability of food to soil invertebrates.