Multidisciplinary Approach to Determine the Optimal Time and Period for Extracting the Essential Oil from Mentha suaveolens Ehrh.

A comprehensive study on essential oils (EOs) extracted from some Mentha suaveolens L. samples, collected in the countryside of Tarquinia, is reported. In this study, the procedure for essential oil preparation, in terms of harvesting and extraction time, was analyzed in detail for the first time. The GC/MS analysis, carried out on 18 samples, revealed that piperitenone oxide (PO), the main essential oils' chemical constituent, is primarily responsible for the related antifungal activity. Nevertheless, EOs with lower PO content indicate that other chemicals, such as para-cymenene, may participate in exerting the EOs' antifungal effect. Furthermore, the bacterial reverse mutation assay highlighted lack of mutagenic effect in all tested samples. Analysis of the results indicated that for higher activity, the essential oils should be produced with 3 h maximum hydrodistillation, regardless of the harvesting time. Differently, the maximum essential oil yield can be obtained in August and the highest piperitenone oxide percentage is obtainable in July.

Beneficial effect of Mentha suaveolens essential oil in the treatment of vaginal candidiasis assessed by real-time monitoring of infection.

BACKGROUND: Vaginal candidiasis is a frequent and common distressing disease affecting up to 75% of the women of fertile age; most of these women have recurrent episodes. Essential oils from aromatic plants have been shown to have antimicrobial and antifungal activities. This study was aimed at assessing the anti-fungal activity of essential oil from Mentha suaveolens (EOMS) in an experimental infection of vaginal candidiasis. METHODS: The in vitro and in vivo activity of EOMS was assessed. The in vitro activity was evaluated under standard CLSI methods, and the in vivo analysis was carried out by exploiting a novel, non-invasive model of vaginal candidiasis in mice based on an in vivo imaging technique. Differences between essential oil treated and saline treated mice were evaluated by the non-parametric Mann-Whitney U-test. Viable count data from a time kill assay and yeast and hyphae survival test were compared using the Student's t-test (two-tailed). RESULTS: Our main findings were: i) EOMS shows potent candidastatic and candidacidal activity in an in vitro experimental system; ii) EOMS gives a degree of protection against vaginal candidiasis in an in vivo experimental system. CONCLUSIONS: This study shows for the first time that the essential oil of a Moroccan plant Mentha suaveolens is candidastatic and candidacidal in vitro, and has a degree of anticandidal activity in a model of vaginal infection, as demonstrated in an in vivo monitoring imaging system. We conclude that our findings lay the ground for further, more extensive investigations to identify the active EOMS component(s), promising in the therapeutically problematic setting of chronic vaginal candidiasis in humans.

Cell wall composition and biofilm formation of azoles-susceptible and -resistant Candida glabrata strains.

In the present study, three strains of Candida glabrata have been investigated to shed light on the mechanisms involved in azole resistance during adherence and biofilm formation. In particular, a clinical isolate, susceptible to azole-based drugs, DSY562 and two different resistant mutagenic strains deriving from DSY562, SFY114 and SFY115, have been analysed with different approaches for their cell wall composition and properties. A proteomic analysis revealed that the expression of six cell wall-related proteins and biofilm formation varied between the strains. The SFY114 and SFY115 strains resulted to be less hydrophobic than the susceptible parental counterpart DSY562, on the other hand they showed a higher amount in total cell wall polysaccharides fraction in the total cell wall. Accordingly to the results obtained from the hydrophobicity and adherence assays, in the resistant strain SFY115 the biofilm formation decreased compared to the parental strain DSY562. Finally, the total glucose amount in resistant SFY115 was about halved in comparison to other strains. Taken together all these data suggest that azole drugs may affect the cell wall composition of C. glabrata, in relation to the different pathogenic behaviours.

Antibacterial activity of essential oils mixture against PSA.

Pseudomonas syringae pv. actinidiae (PSA) is the causal agent of bacterial canker of kiwifruit. It is very difficult to treat pandemic disease. The prolonged treatment with antibiotics, has resulted in failure and resistance and alternatives to conventional antimicrobial therapy are needed. The aim of our study was to analyse the phenotypic characteristics of PSA, identify new substances from natural source i.e. essential oils (EOs) able to contain the kiwifruit canker and investigate their potential use when utilised in combination. Specially, we investigated the morphological differences of PSA isolates by scanning electron microscope, and the synergic action of different EOs by time-kill and checkerboard methods. Our results demonstrated that PSA was able to produce extracellular polysaccharides when it was isolated from trunk, and, for the first time, that it was possible to kill PSA with a mixture of EOs after 1 h of exposition. We hypothesise on its potential use in agriculture.

In vitro inhibition of herpes simplex virus type 1 replication by Mentha suaveolens essential oil and its main component piperitenone oxide.

Several essential oils exert in vitro activity against bacteria and viruses and, among these latter, herpes simplex virus type 1 (HSV-1) is known to develop resistance to commonly used antiviral agents. Thus, the effects of the essential oil derived from Mentha suaveolens (EOMS) and its active principle piperitenone oxide (PEO) were tested in in vitro experimental model of infection with HSV-1. The 50% inhibitory concentration (IC50) was determined at 5.1µg/ml and 1.4µg/ml for EOMS and PEO, respectively. Australian tea tree oil (TTO) was used as control, revealing an IC50 of 13.2µg/ml. Moreover, a synergistic action against HSV-1 was observed when each oil was added in combination with acyclovir. In order to find out the mechanism of action, EOMS, PEO and TTO were added to the cells at different times during the virus life-cycle. Results obtained by yield reduction assay indicated that the antiviral activity of both compounds was principally due to an effect after viral adsorption. Indeed, no reduction of virus yield was observed when cells were treated during viral adsorption or pre-treated before viral infection. In particular, PEO exerted a strong inhibitory effect by interfering with a late step of HSV-1 life-cycle. HSV-1 infection is known to induce a pro-oxidative state with depletion of the main intracellular antioxidant glutathione and this redox change in the cell is important for viral replication. Interestingly, the treatment with PEO corrected this deficit, thus suggesting that the compound could interfere with some redox-sensitive cellular pathways exploited for viral replication. Overall our data suggest that both EOMS and PEO could be considered good candidates for novel anti-HSV-1 strategies, and need further exploration to better characterize the targets underlying their inhibition.

Effects of Mentha suaveolens Essential Oil Alone or in Combination with Other Drugs in Candida albicans.

Candidosis is the most important cause of fungal infections in humans. The yeast Candida albicans can form biofilms, and it is known that microbial biofilms play an important role in human diseases and are very difficult to treat. The prolonged treatment with drugs has often resulted in failure and resistance. Due to the emergence of multidrug resistance, alternatives to conventional antimicrobial therapy are needed. This study aims to analyse the effects induced by essential oil of Mentha suaveolens Ehrh (EOMS) on Candida albicans and its potential synergism when used in combination with conventional drugs. Morphological differences between control and EOMS treated yeast cells or biofilms were observed by scanning electron microscopy and transmission electron microscopy (SEM and TEM resp.,). In order to reveal the presence of cell cycle alterations, flow cytometry analysis was carried out as well. The synergic action of EOMS was studied with the checkerboard method, and the cellular damage induced by different treatments was analysed by TEM. The results obtained have demonstrated both the effects of EOMS on C. albicans yeast cells and biofilms and the synergism of EOMS when used in combination with conventional antifungal drugs as fluconazole (FLC) and micafungin (MCFG), and therefore we can hypothesize on its potential use in therapy. Further studies are necessary to know its mechanism of action.

Glutathione metabolism in Candida albicans resistant strains to fluconazole and micafungin.

Currently available therapies for candidiasis are based on antifungal drugs belonging to azole and echinocandin families that interfere with different aspects of fungal metabolism. These drugs, beyond their specific effects, elicit also a cellular stress including an unbalance of redox state that is counteracted not only utilizing antioxidant species but also increasing the outcome export by transporters to detoxify the internal environment. These cellular actions are both based on the cytosolic concentration of reduced glutathione (GSH). In this paper we investigated the effects of two antifungal drugs fluconazole and micafungin on the redox state of the cell in C. albicans to understand if the resistance to these drugs is accompanied by variation of glutathione metabolism. Analyses of resistant strains showed a marked difference in glutathione contents in strains resistant to fluconazole (CO23RFLC) or micafungin (CO23RFK). In CO23RFLC, the total amount of glutathione was more than doubled with respect to CO23RFK thanks to the increased activity of γ-glutamilcysteine synthetase, the key enzyme involved in GSH synthesis. We demonstrated that the GSH increase in CO23RFLC conferred to this strain a clear advantage in counteracting oxidative toxic agents while assignment of other roles, such as a more efficient elimination of the drug from the cell, should be considered more speculative. As far as MCFG resistance is concerned, from our data a role of glutathione metabolism in supporting this condition is not evident. Overall our data indicate that glutathione metabolism is differently affected in the two resistant strains and that glutathione system may play an important role in the global organization of C.albicans cells for resistance to fluconazole. Such scenario may pave the way to hypothesize the use of oxidant drugs or inhibitors able to deplete reduced glutathione level as a novel approach, for counteracting the resistance to this specific antifungal drug.

Characterization of biofilms in drug-sensitive and drug-resistant strains of Candida albicans.

In this study, we investigated the biofilm formation in strains of Candida albicans susceptible (CO23) or resistant to fluconazole (CO23RFLC) or micafungin (CO23RFK). The effect of drug resistance on biofilm formation was investigated through the cell surface hydrophobicity and the mannan content. Moreover, biofilm formation was evaluated after 24, 48 and 72 hours with crystal violet assay, dry weight, as well as scanning electron microscopy. Our results showed an increase in hydrophobicity, polysaccharides content, metabolic activity and dry weight. Observation of sensitive and resistant strains confirmed the differences in cell morphology. Finally, the expression of genes involved in biofilm formation, such as HWP1 and EFG1, evaluated with relative real-time RT-PCR. Resistant strains proved to up- regulate the expression of HWP1. These results demonstrated the existence of important differences between drug-susceptible and drug-resistant strains biofilm of C. albicans.

The cell wall protein Rhd3/Pga29 is over-expressed in Candida albicans upon micafungin treatment.

Candida albicans cell wall constitutes a sensitive boundary that undergoes molecular changes upon environmental injuries. Antimycotics exert an intense action on cell wall eliciting both qualitative and quantitative changes of resident proteins. The emergence of drug resistance is marked by a modulation of cell wall proteomic profile. In this study, we monitored, at the proteome level through a two-dimensional gel electrophoresis-based approach, differences of cell wall proteins in sensitive and resistant strains of C. albicans, and variations occurring upon treatment of these strains with antifungal drugs. We identified Rhd3/Pga29, a glycophosphatidylinositol (GPI)-anchored protein, as the main over-expressed protein in micafungin resistant strain with respect to the sensitive control cells. A further increase of Rhd3/Pga29 took place when these resistant strains were treated with sub-lethal dose of micafungin. These results were also confirmed in other two clinical isolates resistant to caspofungin. Results were validated by Western blot analyses and RT-PCR and immunoelectron microscopy images confirmed the increase of the Rhd3/Pga29 on the cell wall as well as in the cytosolic compartment of the micafungin-treated resistant cells. Rhd3/Pga29 over-expression upon echinocandin treatment could represent a strategy of C. albicans to counteract the toxic action of this drug. A role of this protein has also been claimed in the virulence of the fungus, suggesting an involvement of Rhd3/Pga29 in the relationship between C. albicans and the host.