Jorge M. Canhoto

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.

Somatic embryogenesis and plant regeneration from zygotic embryos of Feijoa sellowiana berg

Abstract Two distinct types of somatic embryos were induced from the cotyledonary region of callused zygotic embryos of Feijoa sellowiana cultured in MS medium supplemented with a wide range of 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations (0.5–5.0 mg/l) singly or in combination with kinetin (Kn) (0.1–0.5 mg/l). Although less effective than 2,4-D, 1-napthaleneacetic acid (NAA) and 3-indolebutyric acid (IBA) could also induce somatic embryogenesis, whereas no embryos were observed if Kn alone was added to the culture medium. Only 5 days of treatment with 2,4-D were required to induce somatic embryogenesis. Longer exposures, until 14 days, increased the number of zygotic embryos responding positively to the treatment. The continued presence of 2,4-D in the culture medium did not prevent embryo development until the cotyledonary stage, but did not improve further the results. A great number of the formed somatic embryos carried different kinds of abnormalities which drastically reduced their germination capacity. Normal embryos germinated after being transferred to a modified Murashige and Skoogs (MS) medium containing gibberellic acid (GA 3 ) (0.5 mg/l) and kinetin (0.1 mg/l). Histological studies of embryogenic material showed that somatic embryos may arise directly from single cells. More often, however, quite extensive clumps or layers of meristematic cells are formed, mainly from dividing epidermal cells, from which numerous embryos are differentiated.

Histodifferentiation of somatic embryos in cotyledons of pineapple guava(Feijoa sellowiana Berg).

SummarySomatic embryos of pineapple guava (Feijoa sellowiana Berg, Myrtaceae) were induced particularly well from the adaxial face of the cotyledons of zygotic embryos cultured on MS medium containing 1.0 mg/l 2,4-D and 0.3 M sucrose. Somatic embryos were never obtained from globular and heart-shaped zygotic embryos and embryos at the torpedo stage produced somatic embryos at lower frequencies than mature zygotic embryos. At the time of explantation, cotyledonary cells were rich in storage proteins and lipids but no starch was found. After the first 5 days of culture most of the reserves had been mobilized in cotyledons of germinating embryos, but were still present in large amounts in cotyledons undergoing embryogenie induction. In contrast to cotyledons following the normal pattern of development, cells of embryogenically-induced cotyledons accumulated starch, especially those cells not involved in the embryogenie process. Two patterns of somatic embryo differentiation were observed: (1) from single epidermal cells or (2) from groups of meristematic cells near the adaxial surface. Comparative observations on cotyledons from germinating embryos and those undergoing embryogenesis suggest that the meristematic layer arises as the result of successive divisions of cells that, under normal conditions, would form the palisade parenchyma. These were the only mesophyll cells that showed mitotic divisions during the normal development.

Micropropagation of Eucalyptus nitens maiden (Shining gum)

SummaryEucalyptus nitens Maiden (shining gum) is a frost-tolerant species of Eucalyptus that can be used as an alternative species to Eucalyptus globulus in some regions of Portugal where winter temperatures are too low. Seedlings and 1-yr-old shoot tips and nodes were used for micropropagation of E. nitens. The best multiplication rate (2.25) was obtained when seedling shoots (<15 mm) were cultured on a medium containing the major nutrients (at half-strength) and minor elements of Murashige and Skoog (1962) medium, the organics of De Fossard medium (De Fossard et al., 1974) and a combination of benzyladenine (0.9 μM) and 1-naphthaleneacetic acid (0.05 μM). Seedling cuttings (4-,8-, and 10-wk-old) rooted well on media containing several concentrations of 3-indolebutyric acid (4.9, 9.8, and 14.8 μM) or 3-indoleacetic acid (5.7, 11.4, and 17.1 μM), giving frequencies of root induction above 80%. With this type of explant, root formation was also found on basal medium without growth regulators. Rooting of in vitro-propagated shoots obtained from seedlings (8-wk-old) after four subcultures (every 3 wk) was more difficult, with the best results obtained on a medium containing 14.7 μM 3-indolebutyric acid (60.0% root induction). No root formation was achieved when shoots from 1-yr-old explants were used. After a period of 4 mo., 96.3% of the plants transferred to the greenhouse survived acclimatization.

Somatic embryogenesis and plant regeneration in myrtle (Myrtaceae)

Somatic embryos of myrtle (Myrtus communis L.) were induced from mature zygotic embryos cultured in MS medium supplemented with several concentrations of 2,4-D (2.26 μM – 18.98 μM) or Picloram (2.07 μM – 16.5 μM) combined with 0.087 M or 0.23 M sucrose. For all the concentrations of 2,4-D or Picloram tested, 0.087 M sucrose proved to be more effective than 0.23 M. The best frequencies of induction were obtained in a medium containing 2.26 μM 2,4-D in which 97.3% of the explants produced somatic embryos. Although most embryos were produced from the adaxial side of the cotyledons, some of them differentiated from the hypocotyl. Secondary somatic embryos were often seen arising from the periphery of the former somatic embryos. Somatic embryo development was not synchronous but practically all the embryos germinated well after being transferred to media containing GA3 (0.29, 0.58 and 1.44 μM) alone. When benzyladenine was combined with gibberellic acid, germinating somatic embryos produced adventitious shoot buds which contributed to an increase in plantlet regeneration. Histological observations suggested that somatic embryos arise from the upper surface of the cotyledons probably from peripheral cells. Polyphenol-rich cells were usually seen in association with meristematic-like cells from which somatic embryos originate or with earlier steps of somatic embryo differentiation. Regenerated plants were phenotypically normal, showing a diploid (2n = 22) set of chromosomes.

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.

EFFECT OF PLANT GROWTH REGULATORS AND GENOTYPE ON THE MICROPROPAGATION OF ADULT TREES OF ARBUTUS UNEDO L. (STRAWBERRY TREE)

Arbutus unedo grows spontaneously around the Mediterranean basin. The species is tolerant to drought and has a strong regeneration capacity following fires making it interesting for Mediterranean forestation programs. Considering the sparse information about the potential of this fruit tree to be propagated in vitro, a project to clone selected trees based on their fruit production was initiated a few years ago. The role of several factors on A. unedo propagation was evaluated. The results showed that 8.9 μm kinetin gave the best results although not significantly different from those obtained with benzyladenine or zeatin. The inclusion of thidiazuron or 1-naphthaleneacetic acid promoted callus growth and had deleterious effects on the multiplication rate. The genotype of the donor plants is also a factor interfering with the multiplication. The results also indicated that the conditions used for multiplication influenced the behavior of shoots during the rooting phase.

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.

Lavandula luisieri essential oil as a source of antifungal drugs.

This work reports the antifungal activity of Lavandula luisieri essential oils against yeast, dermatophyte and Aspergillus strains responsible for human infections and food contamination. The oils cytotoxicity and its effect on the yeast-mycelium transition in Candida albicans, an important virulence factor, were also evaluated. Analyses by GC and GC/MS showed a peculiar composition of irregular monoterpenes. Significant differences between the samples occurred in the amounts of 1,8-cineole, fenchone and trans-α-necrodyl acetate. The oil with higher amounts of irregular monoterpenes was the most effective. The influence of the oils on the dimorphic transition in C. albicans was also studied through the germ tube inhibition assay. Filamentation was completely inhibited at concentrations sixteen times lower than the minimal inhibitory concentration. The results support the use of L. luiseiri essential oils in the development of new phytopharmaceuticals and food preservatives and emphasise its antifungal properties at concentrations not cytotoxic or with very low detrimental effects on mammalian cells.