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.

Larvicidal and repellent activity of essential oils from wild and cultivated Ruta chalepensis L. (Rutaceae) against Aedes albopictus Skuse (Diptera: Culicidae), an arbovirus vector.

Rutaceae are widely recognized for their toxic and repellent activity exerted against mosquitoes. In our research, the essential oils extracted from fresh leaves of wild and cultivated plants of Ruta chalepensis L. (Rutaceae) were evaluated for larvicidal and repellent activity against the Asian tiger mosquito, Aedes albopictus Skuse (Diptera: Culicidae), currently the most invasive mosquito worldwide. In this research, gas chromatography and gas chromatography-mass spectrometry analyses of the essential oils from wild and cultivated plants showed only quantitative differences, in particular relatively to the amounts of ketone derivatives, while the qualitative profile evidenced a similar chemical composition. Both essential oils from wild and cultivated R. chalepensis plants were able to exert a very good toxic activity against A. albopictus larvae (wild plants, LC(50) = 35.66 ppm; cultivated plants, LC(50) = 33.18 ppm), and mortality was dosage dependent. These data are the first evidence of the toxicity of R. chalepensis against mosquitoes. Furthermore, the R. chalepensis essential oil from wild plants was an effective repellent against A. albopictus, also at lower dosages: RD(50) was 0.000215 µL/cm(2) of skin, while RD(90) was 0.007613 µL/cm(2). Our results clearly evidenced that the larvicidal and repellent activity of R. chalepensis essential oil could be used for the development of new and safer products against the Asian tiger mosquito.

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).

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.

Repellent effect of Salvia dorisiana, S. longifolia, and S. sclarea (Lamiaceae) essential oils against the mosquito Aedes albopictus Skuse (Diptera: Culicidae).

Aedes albopictus (Diptera: Culicidae) has been one of the fastest spreading insects over the past 20 years. Its medical importance is due to the aggressive daytime human-biting behavior and the ability to vector many viruses, including dengue, LaCrosse, Eastern Equine encephalitis and West Nile viruses. In this research, the essential oils (EOs) extracted from fresh air dried leaves of Salvia dorisiana, S. longifolia, and S. sclarea (Lamiaceae) were evaluated for their repellent activity against A. albopictus by using the human-bait technique. The EOs chemical composition was also investigated, and EOs were divided in three different profiles on the basis of their chemical composition: EO with large amount of monoterpenes from S. sclarea, EO rich in oxygenated sesquiterpenes from S. dorisiana, and S. longifolia EO characterized by similar percentages of monoterpenes and sesquiterpenes. The efficacy protection from S. dorisiana, S. longifolia, and S. sclarea EOs, at dosages ranging from 0.004 to 0.4 µL cm(-2) of skin, was evaluated during 120 min of observation. Results indicated that S. dorisiana, S. longifolia, and S. sclarea EOs had a significant repellent activity (RD(50) =0.00035, 0.00049, and 0.00101 µL cm(-2), respectively), with differences in repellency rates, as a function of oil, dosage, and observation time. S. dorisiana was the most effective oil: at the two higher dosages, it gave almost complete protection (with a protective efficacy of 90.99% and 95.62%, respectively) for 90 min. The best protection time was achieved with S. dorisiana essential oil. It ranged from 9.2 to 92.4 min. Protection times of S. longifolia and S. sclarea oils ranged from 3.2 to 60 min, and from 3.6 to 64.2 min, respectively. Our findings clearly reveal that these EOs have a good repellent activity against A. albopictus, therefore they can be proposed to improve the efficacy of repellent formulations against the Asian tiger mosquito.

Essential oil composition of Salvia rosmarinus Spenn. wild samples collected from six sites and different seasonal periods in Elba Island (Tuscan Archipelago, Italy).

The hydrodistilled essential oils from eighteen samples of Salvia rosmarinus Spenn. collected in six localities of Elba Island (Tuscany, Italy) during three different seasonal periods were analyzed by GC-MS for the first time. Fifty-five components were identified, representing 96.8-99.6% of the total chemical composition. All the tested essential oils were characterized by a high content of monoterpenes (oxygenated 49.2-80.3%; hydrocarbons 18.7-48.3%). Among them, 1,8-cineole (26.4-49.1%), α-pinene (4.5-34.8%), camphor (1.1-18.8%) and borneol (1.7-16.2%) were the main constituents. The high amount of 1,8-cineole/α-pinene/camphor/borneol may suggest the presence of an intermediate rosemary chemotype. Statistical analysis was also performed on the essential oil (EO) composition evidenced an expected difference depending on the collection seasons, to the geographical areas and soil composition. A comparison with the essential oil composition from S. rosmarinus collected in all the other islands of Tuscan Archipelago (Italy) was also reported, together with a Multivariate Statistical Analysis.

Pollen aroma fingerprint of two sunflower (Helianthus annuus L.) genotypes characterized by different pollen colors.

Samples of fresh pollen grains, collected from capitula in full bloom from two genotypes of sunflower (Helianthus annuus L.) and characterized by a different color, i.e., white-cream (WC) and orange (O), were analyzed by the HS-SPME (headspacesolid phase microextraction)/GC/MS technique. This study defined for the first time the fingerprint of the sunflower pollen, separated from the disc flowers, to define its contribution to the inflorescence aroma. In the GC/MS fingerprints of the WC and O genotypes, 61 and 62 volatile compounds were identified, respectively. Monoterpene hydrocarbons (34% in O vs. 28% in WC) and sesquiterpene hydrocarbons (37% in O vs. 31% in WC) were ubiquitous in all samples analyzed and represented the main chemical classes. α-Pinene (21% in O vs. 20% in WC) and sabinene (11% in O vs. 6% in WC) were the dominant volatiles, but also a full range of aliphatic hydrocarbons and their oxygenated derivatives gave a decisive contribution to the aroma composition (10% in O vs. 12% in WC). In addition, dendrolasin (3% in O vs. 4% in WC) and some minor constituents such as (E)-hex-2-en-1-ol (0.4% in O vs. 0.1% in WC) were pointed out not only for their contribution to the pollen scent, but also for their well-known role in the plant ecological relationships. Having evaluated two pollen morphs with different carotenoid-based colors, the study sought to highlight also the presence of some volatile precursors or derivatives of these pigments in the aroma. However, the pollen aroma of the two selected genotypes made a specific chemical contribution to the sunflower inflorescence scent without any influence on carotenoid derivatives.

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.

The Skin Interactome: A Holistic "Genome-Microbiome-Exposome" Approach to Understand and Modulate Skin Health and Aging.

Higher demands on skin care cosmetic products for strong performance drive intense research to understand the mechanisms of skin aging and design strategies to improve overall skin health. Today we know that our needs and influencers of skin health and skin aging change throughout our life journey due to both extrinsic factors, such as environmental factors and lifestyle factors, as well as our intrinsic factors. Furthermore, we need to consider our microflora, a collection of micro-organisms such as bacteria, viruses, and fungi, which is a living ecosystem in our gut and on our skin, that can have a major impact on our health. Here, we are viewing a holistic approach to understand the collective effect of the key influencers of skin health and skin aging both reviewing how each of them impact the skin, but more importantly to identify molecular conjunction pathways of these different factors in order to get a better understanding of the integrated "genome-microbiome-exposome" effect. For this purpose and in order to translate molecularly the impact of the key influencers of skin health and skin aging, we built a digital model based on system biology using different bioinformatics tools. This model is considering both the positive and negative impact of our genome (genes, age/gender), exposome: external (sun, pollution, climate) and lifestyle factors (sleep, stress, exercise, nutrition, skin care routine), as well as the role of our skin microbiome, and allowed us in a first application to evaluate the effect of the genome in the synthesis of collagen in the skin and the determination of a suitable target for boosting pro-collagen synthesis. In conclusion, we have, through our digital holistic approach, defined the skin interactome concept, as an advanced tool to better understand the molecular genesis of skin aging and further develop a strategy to balance the influence of the exposome and microbiome to protect, prevent, and delay the appearance of skin aging signs and preserve good skin health condition. In addition, this model will aid in identifying and optimizing skin treatment options based on external triggers, as well as helping to design optimal treatments modulating the intrinsic pathways.

Immune Profiling of Deficient Mismatch Repair Colorectal Cancer Tumor Microenvironment Reveals Different Levels of Immune System Activation.

To understand the immune landscape of deficient mismatch repair colorectal cancer (dMMR CRC) tumor microenvironment, gene expression profiling was performed by the nCounter PanCancer Immune Profiling Panel. This study was conducted retrospectively on 89 dMMR-CRC samples. The expression of CD3, CD8, programmed death-1, and programmed death ligand-1 protein was evaluated on a subset of samples by immunohistochemistry, and lymphocyte density was calculated. A subset of deregulated genes was identified. Functional clustering analysis performed on these genes generated four main factors: antigen processing and presentation, with its major histocompatibility complex-II-related genes; genes correlated with the cytotoxic activity of immune system; T-cell chemotaxis/cell adhesion genes; and T-CD4+ regulator cell-related genes. A deregulation score (DS) was calculated for each sample. On the basis of their DS, tumors were then classified as COLD (DS ≤ -3) to select the samples with a strong down-regulation of the immune system and NOT COLD (DS ≥ -2). The COLD group of patients showed a worse prognosis in terms of survival considering all patients (P = 0.0172) and patients with metastatic disease (P = 0.0031). These results confirm that dMMR-CRCs do not constitute a homogeneous group as concerns the immune system activity of tumor microenvironment. In particular, the distinction between COLD and NOT COLD tumors may improve the management of these two subsets of patients.

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.

N6-1,3-diphenylurea derivatives of 2-phenyl-9-benzyladenines and 8-azaadenines: synthesis and biological evaluation as allosteric modulators of A2A adenosine receptors.

Some 1-[4-(9-benzyl-2-phenyl-9H-purin-6-ylamino)-phenyl]-3-phenyl-urea derivatives and some 1-[4-(9-benzyl-2-phenyl-9H-8-azapurin-6-ylamino)-phenyl]-3-phenyl-urea derivatives were synthesised and evaluated for their interaction with adenosine receptors. It was found that some of these compounds can act as positive enhancers of agonist and antagonist radioligands for the A(2A) adenosine receptors. This evidence was also strengthened by functional data. Other compounds can act as negative modulators. Furthermore these compounds show inhibitory properties for A(1) and A(3) adenosine receptors.

QSAR study on a novel series of 8-azaadenine analogues proposed as A1 adenosine receptor antagonists.

8-Azaadenines have been recently proposed as a novel promising class of adenosine A1 receptor antagonists. A QSAR study on 45 derivatives, synthesized in our laboratory as antagonists for A1 receptor, is described here. The use of the CODESSA program allowed obtaining a quite simple equation capable of correlating the structural features of these ligands to their activity toward A1 receptor. The model was investigated for reliability and stability by using statistical analysis criteria stricter than usual. Particular care was put in defining the chemical space where the model gave reliable predictions. The model allowed the identification of relevant structural features required for the interaction with the A1 receptor, enabling the prediction of activity of molecules belonging to focused virtual libraries.

Synthesis, biological activity and molecular modelling of new trisubstituted 8-azaadenines with high affinity for A1 adenosine receptors.

We describe here the synthesis and biological activity of new 8-azaadenines bearing both a phenyl group on C(2) and a 9-benzyl group substituted in the ortho position with a Cl or a F atom or a CF(3) group, to verify the synergistic effect of a combination of these substitution patterns on binding with the A(1) adenosine receptors. In position N(6) aliphatic and cycloaliphatic substituents were chosen which had been shown to bind well with the A(1) receptors. Because of the high lipophilicity of these kinds of molecules, we also introduced a hydroxyalkyl substituent in the same position. The compounds obtained generally showed a very good affinity and selectivity for A(1) receptors. Some of the compounds showed K(i) in the nanomolar range, one even in the subnanomolar range (0.6 M). Molecular docking calculations were performed in order to evaluate the interaction energies between the bovine A(1) receptor model and the selected ligands, and then to correlate these energies with biological activities of the ligands as obtained from the experiments. Molecular docking analysis suggests different binding modes towards A(1) receptors that are plausible for these ligands.

Structural modifications of benzanilide derivatives, effective potassium channel openers. X.

Large-conductance calcium-activated potassium (BK) channels are involved in many fundamental cell functions. Consistently, the ability to activate BK channels by exogenous compounds is considered as a promising pharmacodynamic pattern for the potential treatment of several pathologies. In this perspective, the development of new and selective BK-openers can be considered as an actual field of research. This paper reports the synthesis and pharmacological evaluation of new benzanilides, useful for deepening the comprehension of the structure-activity relationships, emerged in previous studies on this class of BK-activators. From a structural point of view, these benzanilides belong to a general class of BK-activators, showing a common pharmacophoric model, consisting of two aryl groups linked through an appropriate "spacer" and the almost obligatory presence of a phenolic hydroxyl. In particular, a new series of benzanilides, in which the phenyl rings have been widely changed both on the acidic portion and the basic one of the amide spacer, were synthesised. Their vasorelaxing effects, induced through the activation of BK channels, were also evaluated. Although many compounds exhibited effects which could not be attributed to the activation of BK channels, two derivatives showed a clear profile of BK-activators with vasodilator activity comparable to or slightly lower than that recorded for the reference benzimidazolone NS1619. A further molecular modelling approach allowed us to obtain a molecular electrostatic potential feature which suggests a suitable interaction with the receptor site of the BK channel, from a tri-dimensional point of view. This approach seems to represent a further contribution for the development of new BK-activators, designed on the basis of the pharmacophoric model above-mentioned.

Heterocyclic analogs of benzanilide derivatives as potassium channel activators. IX.

On the basis of our previous works, addressed to synthesise new activators of BK potassium channels, and of many suggestions from the international literature, a simple pharmacophoric model, consisting of two suitably substituted phenyl rings bound to various kinds of linkers, was hypothesised. In particular, the effectiveness of the amidic linker was demonstrated, since several benzanilide derivatives showed interesting BK-opener properties. As a development of these benzanilides, in this work we introduced heterocyclic substituents, replacing the aryl ring on the acid side or on the basic one of the amide linker of the above pharmacophore. The pharmacological results indicated some relevant remarks about the structural requirements, needed for a satisfactory BK-opener activity. In particular, the presence of a phenolic function, with a possible H-bond donor role, has been confirmed. Furthermore, the presence of nitrogen heterocycles on the acid side of the amide linker seems to be a negative requirement, while furan and thiophene were well tolerated. On the contrary, the introduction of insaturated heterocyclic rings (pyridine and thiazole) on the basic side of the amide linker, led to satisfactory biological activity, while the presence of aliphatic heterocycles lowered the pharmacological effect.

New N6- or N(9)-hydroxyalkyl substituted 8-azaadenines or adenines as effective A1 adenosine receptor ligands.

In this paper we describe synthesis and biological assays of some A(1) ligands more water-soluble than the effective, but very lipophilic, 8-azaadenines and adenines discovered in the past and obtained introducing on N(6) or N(9) substituent a hydroxy group. Five of the new N(6)-hydroxyalkyl- and N(6)-hydroxycycloalkyl-2-phenyl-9-benzyl-8-azaadenines showed very high affinity (Ki<40 nM) and selectivity for A(1) adenosine receptors. Among the 2-phenyl-9-(2-hydroxy-3-alkyl)-8-azaadenines or adenines prepared, the compounds with the higher A(1) affinity and selectivity resulted 2-phenyl-9-(2-hydroxy-3-propyl)-N(6)-cyclopentyl- and cyclohexyl-8-azaadenine with Ki 2.2+/-0.2 nM and 2.8+/-0.3 nM respectively. From the point of view of water-solubility, 2-phenyl-9-(2-hydroxy-3-propyl)-8-azaadenine was the most interesting compound, having a CLogP of 1.066991 and a water-solubility of 1.2 mg mL(-1).

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.

N-[9-(ortho-fluorobenzyl)-2-phenyl-8-azapurin-6-yl]-amides as potent and selective ligands for A1 adenosine receptors.

A series of N-[9-(ortho-fluorobenzyl)-2-phenyl-8-azapurin-6-yl]-amides were synthesized and tested for their affinity toward A1, A2A , and A3 adenosine receptor subtypes. Biological results demonstrated that the introduction of a fluorine atom at the ortho position of the 9-benzyl group generally enhanced affinity toward A1 subtype and did not significantly affect A2A and A3 affinity. Very interesting is the compound bearing a meta-fluorophenyl substituent on the carbonyl carbon of the amide group, which shows significantly high A1/A2A-A3 selectivity. Compounds of this new series, together with the previously published analogs without the fluorine atom on the 9-benzyl group, constituted the starting dataset for the development of QSAR models. The models obtained were able to rationally describe the affinity trends resulting from biological testing and to enable investigation of the role of different substituents on the 8-azapurine scaffold, as well as the influence of the newly introduced fluorine atom on the benzyl moiety. The said QSAR models can also assist in the design of new compounds selectively active on A1 adenosine receptors. Furthermore, a molecular docking study was carried out to assess hypothetical binding mode of N-[9-(ortho-fluorobenzyl)-2-phenyl-8-azapurin-6-yl]-amides to A1 adenosine receptors.

In vitro production of M. × piperita not containing pulegone and menthofuran.

The essential oils (EOs) and static headspaces (HSs) of in vitro plantlets and callus of Mentha x piperita were characterized by GC-MS analysis. Leaves were used as explants to induce in vitro plant material. The EO yields of the in vitro biomass were much lower (0.1% v/w) than those of the parent plants (2% v/w). Many typical mint volatiles were emitted by the in vitro production, but the callus and in vitro plantelet EOs were characterized by the lack of both pulegone and menthofuran. This was an important difference between in vitro and in vivo plant material as huge amounts of pulegone and menthofuran may jeopardise the safety of mint essential oil. Regarding the other characteristic volatiles, menthone was present in reduced amounts (2%) in the in vitro plantlets and was not detected in the callus, even if it represented the main constituent of the stem and leaf EOs obtained from the cultivated mint (26% leaves; 33% stems). The M. piperita callus was characterized by menthol (9%) and menthone (2%), while the in vitro plantlet EO showed lower amounts of both these compounds in favour of piperitenone oxide (45%). Therefore, the established callus and in vitro plantlets showed peculiar aromatic profiles characterized by the lack of pulegone and menthofuran which have to be monitored in the mint oil for their toxicity.