Donatella Mares

Chemical characterization and antifungal activity of essential oil of capitula from wild Indian Tagetes patula L

Summary.The essential oil extracted by steam distillation from the capitula of Indian Tagetes patula, Asteraceae, was evaluated for its antifungal properties and analyzed by gas chromatography and gas chromatography–mass spectrometry. Thirty compounds were identified, representing 89.1% of the total detected. The main components were piperitone (24.74%), piperitenone (22.93%), terpinolene (7.8%), dihydro tagetone (4.91%), cis-tagetone (4.62%), limonene (4.52%), and allo-ocimene (3.66%). The oil exerted a good antifungal activity against two phytopathogenic fungi, Botrytis cinerea and Penicillium digitatum, providing complete growth inhibition at 10 µl/ml and 1.25 µl/ml, respectively. The contribution of the two main compounds, piperitone and piperitenone, to the antifungal efficacy was also evaluated and ultrastructural modifications in mycelia were observed via electron microscopy, evidencing large alterations in hyphal morphology and a multisite mechanism of action.

A simple colorimetric method for detecting cell viability in cultures of eukaryotic microorganisms

The dye methylene blue can be taken up by dead or severely damaged cells, but not by living cells. Based on this fact, a method was devised which permits quantitative determinations of injured cells in populations of microorganisms such asSaccharomyces cerevisiae, Rhodotorula glutinis, andEuglena gracilis. The percentage of damaged cells was determined by measuring, at 664 nm, the optical density of cell suspensions pretreated with 0.15 mM methylene blue for 6 min, a condition that does not affect cell integrity as determined by oxygen consumption and release of potassium ions. This technique is faster and simpler than the classical dye-exclusion and plate-counting methods.

Chicory extracts from Cichorium intybus L. as potential antifungals

In this work extracts from roots of the common vegetable Cichorium intybus L., highly appreciated for its bitter taste, were studied to investigate their possible biological activity on fungi from a variety of ecological environments: some are parasites on plants (phytopathogens) or of animals and humans (zoophilic and anthropophilic dermatophytes), others live on the soil and only seldom parasitize animals (geophilic dermatophytes). The extracts were ineffective on geophilic species and on tested phytopathogens, with the exception of Pythium ultimum, whereas they inhibited the growth of zoophilic and anthropophilic dermatophytes, in particular Trichophyton tonsurans var. sulfureum, whose treatment caused morphological anomalies, here observed by scanning electron microscopy. This behaviour is discussed on the basis of the presence in the chicory extract of the two main sesquiterpene lactones, 8-deoxylactucin and 11β,13-dihydrolactucin.

Antimicrobial activity of protoanemonin, a lactone from ranunculaceous plants

Protoanemonin, a component of Ranunculus bulbosus, was tested as an antifungal agent on selected strains of dermatophytes and yeasts. The minimum inhibitory concentrations ranged from 2.0 to 7.5×10−4 M and the minimum lethal concentrations from 3.8×10−4 M to >1.0×10−3 M. The most sensitive dermatophyte tested was Epidermophyton floccosum, and the most sensitive yeast Rhodotorula glutinis. The effects of different culture media and of light on the sensitivity of Rhodotorula glutinis to protoanemonin were also tested. Structural analogies between protoanemonin and other cytotoxic unsaturated lactones, and the reversal by the amino acid cysteine of the antifungal action suggest a possible mechanism of action.

Electron microscopy of Microsporum cookei after in vitrö treatment with protoanemonin: a combined SEM and TEM study

The ranunculaceous derivative protoanemonin (PrA) was studied as an antifungal agent on the dermatophyte Microsporum cookei. The ultrastructural changes that PrA brought about in this fungus were observed with both the transmission and scanning electron microscopes. The main anomalies noted were abnormally shaped hyphae and within the cytoplasm, multimembranous bodies which were irregular in shape and size, and tubules of 25 and 60 nm in diameters. Mitochondria, nuclei and vacuoles were also variously affected by PrA. Although multifarious, the observed cellular alterations in M. cookei can be considered the result of a PrA interaction with cytoplasmic microtubules. Since these cell structures contain a great number of ASH groups, our previous hypothesis, that sulphydryl groups are the primary targets of this molecule, appears to be supported.

The photodynamic effect of 5-(4-hydroxy-1-butinyl)-2,2’-bithienyl on dermatophytes

The thiophene 5-(4-hydroxy-1-butinyl)2,2′-bithienyl (BBTOH) strongly inhibited in vitro eight different dermatophytes. Epidermophyton floccosum proved most sensitive to all doses of BBTOH when applied in conjunction with uv-A irradiation. BBTOH also proved quite active against Nannizia cajetani , the only dermatophyte which was also strongly inhibited when treated (50 μg ml −1 ) and kept in the dark. For this reason, N. cajetani was chosen as the test organism for TEM and SEM aimed at determining what treatment-induced ultrastructural and morphological modifications had occurred. TEM revealed that the photoactive mechanism of BBTOH was similar to that of 2,2′:5′,2″-terthienyl (α-T). SEM, on the other hand, showed that early culture aging resulted from treatment.

Mannan changes induced by 3-methyl-5-aminoisoxazole-4-thiocyanate, a new azole derivative, on Epidermophyton floccosum

The antifungal activity of 3-methyl-5-aminoisoxazole-4-thiocyanate, a new azole derivative, was studied on the dermatophyte Epidermophyton floccosum. The compound strongly inhibited the in vitro growth of two different strains of the fungus and even induced profound morphogenetic anomalies. Optical and electron microscopy showed that such treatment targets the endomembrane system, particularly the plasmalemma, causing abnormal extrusion of the wall mannans. This results in improper arrangement of the different parietal materials; the walls are thus weak and subject to subapical rupture which terminates cell growth and elongation of the hypha. The morphological results and the preliminary biochemical data on fungal sterols suggest that this compound employs an action mechanism similar to that of other azoles used in therapy.

A cytochemical approach to the wound repair mechanism inUdotea petiolata (Siphonales)

SummaryWhen injured, the thalli of the coenocytic algaUdotea petiolata undergo a rapid sealing process mainly due to the extrusion of two successive plugs. In the first, external and transitory plug, sulphated polysaccharides are the predominant components. In the second, permanent and internal plug, roundish bodies having a complex polysaccharidic composition are embedded in a fibrillar matrix of still unknown nature. The sulphated sugars were identified and located by means of Alcian Blue staining and X-ray microanalysis. A periodic acid-thiocarbohydrazide-silver proteinate technique proved useful especially in the study of the roundish bodies and in the compositional and structural comparison of the siphon wall with the wound wall. Phosphotungstic acid at low pH was used to evidentiate an extensive plasma membrane activity in the repairing cytoplasm.

Ultraviolet-mediated antimycotic activity of α-terthienyl on Microsporum cookei

Alpha-terthienyl (alpha-T) in the presence of UV-A irradiation reduced the growth rate of Microsporum cookei. In the dark, alpha-T accumulated in small diffuse vacuoles within the hyphae. After UV-A treatment, alpha-T caused damage to the membranes of the nucleus, mitochondria and endoplasmic reticulum. Plasmolytic and autolytic changes occurred resulting in plasma membrane breakage and cell wall aberrations. UV-A activated alpha-T would appear to target membrane proteins.

Antifungal activity of essential oil from fruits of Indian Cuminum cyminum

The essential oil of fruits of Cuminum cyminum L. (Apiaceae), from India, was analyzed by GC and GC-MS, and its antifungal activity was tested on dermatophytes and phytopathogens, fungi, yeasts and some new Aspergilli. The most abundant components were cumin aldehyde, pinenes, and p-cymene, and a fraction of oxygenate compounds such as alcohol and epoxides. Because of the large amount of the highly volatile components in the cumin extract, we used a modified recent technique to evaluate the antifungal activity only of the volatile parts at doses from 5 to 20 µL of pure essential oil. Antifungal testing showed that Cuminum cyminum is active in general on all fungi but in particular on the dermatophytes, where Trichophyton rubrum was the most inhibited fungus also at the lowest dose of 5 µL. Less sensitive to treatment were the phytopathogens.