Michele Leonardi

The Role of Diallyl Sulfides and Dipropyl Sulfides in the In Vitro Antimicrobial Activity of the Essential Oil of Garlic, Allium sativum L., and Leek, Allium porrum L.

The in vitro antibacterial activity of essential oils (EOs) obtained from fresh bulbs of garlic, Allium sativum L., and leek, Allium porrum L. ( Alliaceae), was studied. A. sativum (garlic) EO showed a good antimicrobial activity against Staphylococcus aureus (inhibition zone 14.8 mm), Pseudomonas aeruginosa (inhibition zone 21.1 mm), and Escherichia coli (inhibition zone 11.0 mm), whereas the EO of A. porrum (leek) had no antimicrobial activity. The main constituents of the garlic EO were diallyl monosulfide, diallyl disulfide (DADS), diallyl trisulfide, and diallyl tetrasulfide. The EO of A. porrum was characterized by the presence of dipropyl disulfide (DPDS), dipropyl trisulfide, and dipropyl tetrasulfide. The antimicrobial activities of the DADS and DPDS were also studied. The results obtained suggest that the presence of the allyl group is fundamental for the antimicrobial activity of these sulfide derivatives when they are present in Allium or in other species (DADS inhibition zone on S. aureus 15.9 mm, P. aeruginosa 21.9 mm, E. coli 11.4 mm). Copyright

Antibacterial activity of essential oils, their blends and mixtures of their main constituents against some strains supporting livestock mastitis

Ten of the most known and used commercial essential oils (Cinnamomum zeylanicum L., Citrus bergamia Risso, Eucalyptus globulus Labill., Foeniculum vulgare Mill., Origanum majorana L., Origanum vulgare L., Rosmarinus officinalis L., Satureja montana L., Thymus vulgaris L. ct. carvacrol, Thymus vulgaris L. ct. thymol) were tested against six bacteria strains Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus sciuri, Staphylococcus warneri, Staphylococcus xylosus and Escherichia coli, responsible for mastitis in animals. The best results were achieved by S. montana, T. vulgaris ct. thymol and O. vulgare. Two binary mixtures of essential oils (EOs) were prepared of S. montana and T. vulgaris ct. thymol (ST) and of S. montana and O. vulgare (SO). The ST mixture exhibited the best inhibitory activity against all the tested bacterial strains. Two artificial mixtures of carvacrol/thymol (AB) and carvacrol/thymol/p-cymene (CD) were prepared and tested against all of the bacterial strains used. The results exhibited a general reduction of the inhibitory activity of mixture AB, although not reaching the inhibition of the ST and SO mixtures. However the mixture CD presented an apparent strong inhibition against S. aureus and S. sciuri. The EO mixtures and the mixture CD represent promising phytotherapic approaches against bacteria strains responsible for environmental mastitis.

Aroma characterisation and UV elicitation of purple basil from different plant tissue cultures

Exposure to stressful environmental conditions can induce severe metabolic variations in basil (Ocimum basilicum) aroma. The aromatic profiles of Dark Opal and Red Rubim varieties (in vivo plants, in vitro shoots, callus, and suspension cultures) were investigated for the first time. The established calli represented the most interesting miniaturised aromatic plant systems, as they were able to emit many typical basil volatiles with very low amounts of phenylpropanoids (1-2%). The hydrocarbon monoterpenes and oxygenated volatiles emitted from calli of both varieties were greatly and conversely affected by UV-C and UV-B, in comparison with the non-irradiated samples. As calli of both varieties still maintained very low levels of phenylpropanoids even after UV elicitation, they might be regarded not only as efficient in vitro plant models to study volatile compounds under UV stress conditions, but also as safe aromatic biomass in comparison with in vivo basil plants.

Essential oil composition of Helichrysum italicum (Roth) G. Don ssp. italicum from Elba Island (Tuscany, Italy).

The composition of 21 essential‐oil samples isolated from Helichrysum italicum collected in seven locations of Elba Island (Tuscany, Italy), characterized by different soil types, during three different periods (January, May, and October 2010) was determined by GC‐FID and GC/EI‐MS analyses. In total, 115 components were identified, representing 96.8–99.8% of the oil composition. The oils were characterized by a high content of oxygenated monoterpenes (38.6–62.7%), while monoterpene and sesquiterpene hydrocarbons accounted for 2.3–41.9 and 5.1–20.1% of the identified constituents, respectively. The main oxygenated derivatives were nerol (2.8–12.8%) and its ester derivative neryl acetate (5.6–45.9%). To compare the chemical variability of the species within Elba Island and between the island and other localities within the Mediterranean area, studied previously, multivariate statistical analysis was performed. The results obtained showed a difference in the composition of the essential oils of H. italicum from Elba Island, mainly due to the environment where the plant grows, and, in particular, to the soil type. These hypotheses were further confirmed by the comparison of these oils with essential oils obtained from H. italicum collected on other islands of the Tuscan archipelago.

Morphogenetic changes in essential oil composition of Hypericum perforatum during the course of ontogenesis.

Context: In the past few years, an increasing interest in the volatile secondary metabolites of Hypericum perforatum L. (Guttiferae) has been arising. Objective: The present study is a contribution to better understand the relationship between the morphological variations and volatile composition during the phenological cycle. Materials and methods: Leaves at the stages of vegetative, floral budding, flowering and green capsule, buds, full opened flowers and green capsules were assayed for essential oil (EO) components by gas chromatography-flame ionization detector (GC-FID) and GC-mass spectrometry (MS). Results: Significant amounts of sesquiterpenes (oxygenated 26–50% and hydrocarbons 20–40%) and oxygenated hydrocarbons (13–38%) characterized the all analyzed samples showing peculiar fluctuations during the seven phenological stages. Although monoterpenes were present in much lower amounts (monoterpene hydrocarbons 0.4–6%; oxygenated monoterpenes 0.8–6%) they were considered also important discrimination for several stages. The green capsules and the full opened flowers collected at flowering stage were clearly distinguished in terms of EO compositions from the other samples. Discussion: For the first time, the EO composition of Turkish wild Hypericum perforatum was monitored by the hydrodistillation of different plant organs collected at different seven stages in order to point out the modification of target volatiles related to each phenological step. Conclusions: Based on the EO composition monitored during these seven morphological stages by GC-MS, principal component analysis and cluster analysis, significant metabolite modifications were observed during the phenological cycle which involved the levels of specific volatile target compounds belonging to the chemical classes of hydrocarbons, monoterpenes and sesquiterpenes.

Molecular cloning of SoHPPR encoding a hydroxyphenylpyruvate reductase, and its expression in cell suspension cultures of Salvia officinalis

Rosmarinic acid (RA) is a naturally-derived compound present in medicinal plants, herbs and spices, and is well-known for its antioxidant, antibacterial and medicinal properties. It has been recently considered for use as a food preservative, nutraceutical, and for medicinal purposes. Undifferentiated cells (callus and cell suspensions) cultured in vitro are often used for the production of secondary metabolites, and for identifying biosynthetic pathways. In this work Salvia officinalis L. (common sage) cell suspension cultures were established, and a specific cell line was selected for the high antioxidant capacity of its methanolic extract, which was characterized by a high content of RA. Scavenger activity (DPPH test) and total RA content were evaluated during cell growth. Gene coding for Hydroxyphenylpyruvate reductase, the key-enzyme responsible for the RA metabolic biosynthesis, was cloned from common sage (SoHPPR). Its transcript expression level was monitored during cell suspension cultures, and showed a relationship with scavenger activity and RA yield. Our results suggest the potential use of this gene as a marker and target for the modulation of RA production in controlled conditions.

Antimycotic Activity of Some Aromatic Plants Essential Oils Against Canine Isolates of Malassezia pachydermatis: An in vitro Assay

The antifungal activity and the chemical composition of essential oils (EOs) from some Mediterranean autochthonous plants were investigated against Malassezia pachydermatis, a fastidious opportunistic yeast usually involved in canine external otitis. Minimum inhibitory concentrations (MICs) of Anthemis nobilis, Citrus limon, Citrus paradisi, Illicium verum, Lavandula hybrida, Mentha piperita, Ocimum basilicum, Origanum vulgare, Origanum majorana, Rosmarinus officinalis, Salvia sclarea, Thymus serpillum were assessed by microdilution test; minimum fungicidal activity (MFC) was also determined. O. vulgare, T. serpillum and O. basilicum showed the lowest MIC and MFC values (0.8%) followed by C. limon and M. piperita (1%). EOs from the tested plants showed variable degrees of anti-malassezia activity, putatively related to their chemical composition. The effectiveness, manageability and pleasant organoleptic properties of O. vulgare, T. serpillum, O. basilicum, C. limon and M. piperita EOs make them advisable as promising new natural antifungal drugs in the management of M. pachydermatis otitis in dog.

Clinical and mycological evaluation of an herbal antifungal formulation in canine Malassezia dermatitis

OBJECTIVE Malassezia pachydermatis is a common cause of more widespread dermatitis in dogs (CMD). Recurrences are common, and this disorder can be very troubling for both dogs and for the pet owner. MATERIAL AND METHODS The treatment of 20 dogs affected by dermatitis due to M. pachydermatis, with Malacalm(®), a commercially available mixture consisting of essential oils (Citrus aurantium 1%, Lavandula officinalis 1%, Origanum vulgare 0.5%, Origanum majorana 0.5%, Mentha piperita 0.5% and Helichrysum italicum var. italicum 0.5%, in sweet almond oil and coconut oil) is reported. The effectiveness of the whole mixture, of component essential oils and of their more represented compounds against clinical isolates was evaluated by a microdilution test. Twenty animals were topically administered the mixture twice daily for 1 month. Ten animals were treated with a conventional therapy based on ketoconazole 10mg/kg/day and chlorhexidine 2% twice a week for 3 weeks. At the end of both treatments animals significantly improved their clinical status. Adverse effects were never noticed. Follow-up visit performed on day 180th allowed to observe a recurrence of clinical signs in all the subjects treated conventionally, while not significant clinical changes were referred in dogs treated with Malacalm(®). The overall MIC value of Malacalm(®) was 0.3%. O. vulgare showed the lowest minimum inhibitory concentrations (MIC), being active at 0.8%, followed by M. piperita (1%), O. majorana (1.3%), C. aurantium (2%) and L. officinalis (4%) while H. italicum did not yield any antimycotic effect up to 10%. Active major compounds were thymol, carvacrol, p-cymene, 1,8-cineol, limonene and menthol. CONCLUSION The phytotherapic treatment achieved a good clinical outcome, and no recurrence of skin disorders on day 180th was recorded. This herbal remedium appeared to be a safe tool for limiting recurrences of CMD.

A herbal antifungal formulation of Thymus serpillum, Origanum vulgare and Rosmarinus officinalis for treating ovine dermatophytosis due to Trichophyton mentagrophytes

A number of herbal products with anti‐inflammatory, antiseptic and antimycotic properties are available for dermatological usage. The successful treatment of 13 sheep affected by ringworm due to Trichophyton mentagrophytes with a mixture consisting of essential oils (EOs) of Thymus serpillum 2%, Origanum vulgare 5% and Rosmarinus officinalis 5% in sweet almond (Prunus dulcis) oil. The effectiveness of EOs and of the major components of the mixture (thymol, carvacrol, 1,8 cineole, α‐pinene, p‐cymene, γ‐terpinene) against the fungal clinical isolate was evaluated by a microdilution test. Thirteen animals were topically administered with the mixture twice daily for 15 days. The other sheep were administered with a conventional treatment (seven animals) or left untreated (two animals). Minimum inhibitory concentration (MIC) values were 0.1% for T. serpillum, 0.5% for O. vulgare, 2.5% for I. verum and 5% for both R. officinalis and C. limon. Thymol and carvacrol showed MICs of 0.125% and 0.0625%. A clinical and aetiological cure was obtained at the end of each treatment regimen in only the treated animals. Specific antimycotic drugs licenced for food‐producing sheep are not available within the European Community. The mixture tested here appeared to be a versatile tool for limiting fungal growth.

Synthesis, biological assays and QSAR studies of N-(9-benzyl-2-phenyl-8-azapurin-6-yl)-amides as ligands for A1 adenosine receptors.

2-Phenyl-9-benzyl-8-azapurines, bearing at the 6 position an amido group interposed between the 8-azapurine moiety and an alkyl or a substituted phenyl group, have been synthesised and assayed as ligands for adenosine receptors. All the compounds show high affinity for the A(1) adenosine receptor, and many of them also show a good selectivity for A(1) with respect to A(2A) and A(3) adenosine receptors. Based on the quite rich library containing such compounds and relevant biological data, QSAR models, able to rationalise the results and to give a quantitative estimate of the observed trends were also developed. The obtained models can assist in the design of new compounds selectively active on A(1) adenosine receptor.