EP2531203A2

EP2531203A2 - Mentha suaveolens essential oil and therapeutic activities thereof

Info

Publication number: EP2531203A2
Application number: EP11709470A
Authority: EP
Inventors: Letizia Angiolella; Rino Ragno
Original assignee: Universita degli Studi di Roma La Sapienza
Current assignee: Universita degli Studi di Roma La Sapienza
Priority date: 2010-02-01
Filing date: 2011-01-28
Publication date: 2012-12-12
Also published as: ITRM20100035A1; IT1398185B1; WO2011092655A2; WO2011092655A3

Abstract

The present description refers to an essential oil of Mentha suaveolens; in particular, described herein is an oil containing at least 60% of piperitenone oxide, at least 0.5% of al pha-cubebene and at least 0.5% of octanol, its use as medicament, compositions and products containing it.

Description

MENTHA SUAVEOLENS ESSENTIAL OIL AND THERAPEUTIC ACTIVITIES

THEREOF

DESCRIPTION

The present description refers to an essential oil of Mentha suaveolens; in particular, described herein is an oil containing at least 60% of piperitenone oxide, at least 0.5% of alpha-cubebene and at least 0.5% of octanol, its use as medicament, compositions and products containing it.

STATE OF THE PRIOR ART

The last years are witnessing an increasing interest for the study of plant- extracted natural substances, to solve problems of different nature. Among substances of particular interest there are plant-extracted essential oils, which, though having been known and used for centuries for their beneficial effects and therapeutic properties, exhibit potentials and effects not yet known or made clear. Since the Middle Ages, essential oils have been widely utilized as bactericides, virucides, fungicides, pesticides, insecticides and for medicinal and cosmetic applications, being also used as analgesics, sedatives, anti-inflammatory agents, spasmolytics and as local anesthetics. I n nature, essential oils play an important role in plant protection as antibacterials, antivirals, antifungals, insecticides.

Essential oils are extracted from various aromatic plants, generally located in temperate-hot Countries, such as Mediterranean ones, and tropical Countries where they represent a relevant part of traditional Pharmacopeia.

Essential oils are very complex natural mixtures which may contain about 20-60 components with very different concentrations according to the plant type from which they are extracted, but also according to the time period in which the plant material used for the extraction is harvested.

In the literature, essential oils extracted from different plant genera or species are reported, having various therapeutic properties. I n particular, as to essential oils for antimicrobial use, among the most known and effective ones the essential oil extracted from Melaleuca alternifolia, also known as Tea Tree Oil (TTO) needs mentioning (C.F. Carson et al. Melaleuca alternifolia (Tea Tree) Oil: a Review of Antimicrobial and Other Medicinal Properties Clin Microbiol Rev.2006 Jan; 19(1 ):50-62).

Melaleuca alternifolia is an Australian myrtacea from whose leaves the oily essence is obtained. The leaves, subjected to steam current extraction, yield the essential oil, also referred to as TEA TREE OI L, having an activity reported as germicidal, bactericidal, fungicidal; said oil is used in aromatherapy as anti- infective, cicatrizing, antiseptic agent; it is used for the treatment of chronic cystites of colibacillar origin, vaginal infections from Candida, skin infections, it is useful for the treatment of infections from C. albicans, E. coli, S. aureus, P. vulgaris, Enterococci, Enterobacteria, Streptococci, rhinopharyngeal affections, aphthous ulcers, stomatites, pyorrhea, gingivitises, nail and hallux mycosis. Said oil, of proven effectiveness, has a non-negligible drawback in that it is foul-smelling. It is evident that a patient need ing to make use of such a substance having an unpleasant and persistent odor has objective difficulties to accept the treatment, as it will cause him/her embarrassing conditions due to the foul odor.

It is evident that a natural treatment is often preferred over the use of synthetic drugs; however, it is also evident that substances emitting pungent and unpleasant odors are not willingly used by patients, who therefore tend to prefer odorless drugs to the detriment of the use of foul-smelling natural substances.

Other widely used oils are those of plants of genus Mentha (family Lamiaceae).

The constituents of essential oils obtained from species of genus Mentha are very heterogeneous and can vary within the range of the same species, giving origin to essences different by chemistry, pharmacology and toxicology. In particular, the most studied species are Mentha spicata (green mint) and Mentha piperita (peppermint) which, though belonging to th e sa me gen u s, produce essential oils of different composition, each with peculiar therapeutic properties (Lupien et al, 1999; McConkey et al, 2000). Moreover, in the literature it is also reported an essential oil extracted from species Mentha suaveolens, having antibacterial and antifungal properties. This oil owes its properties to the peculiar composition. As reported in a study conducted by Oumzil H . et al, among the different compounds of Mentha suaveolens essential oil, such as pulegone, piperitenone oxide (PEO) and piperitone oxide (PO), pulegone is the component indicated as responsible for the inhibition activity that such oil exerts on the growth of the various microorganisms, and therefore for the antibacterial and antifungal activity observed (Oumzil H . et all., 2002). In particular, as reported in Table 3 of the above-mentioned study, the other components have only a marginal role in such activities, without inducing a statistically significant inhibition of the growth of the various microorganisms tested and therefore, in other words, do not concretely contribute to the therapeutic properties attributed to Mentha suaveolens essential oil.

In the literature it is i n d icated th at PEO possesses antispastic and insecticidal activity.

SUMMARY OF THE INVENTION Th e state of th e known art, as described above, reports various oils obtained from aromatic plants having therapeutic properties. These properties are closely correlated to the specific composition of the oil tested.

As indicated above, essential oils capable of providing extremely effective antifungal and/or antibacterial effects, such as TTO, are known in the literature.

As also indicated above, substances of natural origin sometimes have, in association to undoubtedly effective medical effects, other effects or features that may limit their use; in the case of TTO, the negative effect of greater relevance is the extremely unpleasant, pungent and persistent odor of said substance, inhibiting a topical use thereof by the patient, who should spread such a foul-smelling substance on various parts of his/her body. Thus, it is desirable to find natural products free from the negative features of TTO, which may be used alternatively, with a similar or even greater effectiveness as antifungals and/or antibacterials.

The state of the art indicates that for the oil obtained from the species Mentha suaveolens, the component responsible for the antibacterial and antifungal activity, as already mentioned in the foregoing, is pulegone.

In this description, for the first time an essential oil of Mentha suaveolens is described whose composition is characterized by the presence of piperitenone oxide as major component, havi ng extremely effective a ntifu ngal and/or antibacterial activities, comparable to if not better than those of TTO in the same applications.

This oil, having a negligible percentage of pulegone, surprisingly exhibits therapeutic activities, such as activities of inhibition of the growth of various microorganisms at concentrations lower than those defined for the other essential oils known in the literature.

The present description discloses an essential oil of Mentha suaveolens, in particular containing at least 60% of piperitenone oxide, at least 0.5% of alpha- cubebene an d at least 0.5% of octa nol, having effective antifungal and/or antibacterial and/or antiviral activities, its use as medicament, compositions and products containing it.

Therefore, object of the present invention are:

- an essential oil of Mentha suaveolens containing at least 60% of piperitenone oxide, at least 0.5% of alpha-cubebene and at least 0.5% of octanol;

- an essential oil of Mentha suaveolens containing at least 60% of piperitenone oxide, at least 0.5% of alpha-cubebene and at least 0.5% of octanol as medicament;

- pharmaceutical compositions comprising at least 1 .5% of oil of Mentha suaveolens containing at least 60% of piperitenone oxide, at least 0.5% of alpha- cubebene and at least 0.5% of octanol, and one or more pharmaceutically acceptable vehicles and/or excipients;

- products comprising at least 1 .5% of oil of Mentha suaveolens containing at least 60% of piperitenone oxide, at least 0.5% of alpha-cubebene and at least 0.5% of octanol;

- a method for the extraction of Mentha suaveolens essential oil containing at least 60% of piperitenone oxide, at least 0.5% of alpha-cubebene and at least 0.5% of octanol, comprising the following steps:

a) using plant material of Mentha suaveolens harvested in a period comprised between early June and late September;

b) extracting said oil from said plant material;

d) carrying out the anhydrification of said oil on anhydrous sodium sulphate e) obtaining pure oil by removal of solvent used.

DETAILED DESCRIPTION OF THE FIGURES

Table 1 reports the percentages of components, identified with a mass spectrometer, contained in the Mentha essential oil of the present invention. The relevant feature is represented by the presence of piperitenone oxide in the percentage of 92%, along with other components with very low percentages.

Table 2 reports values of MIC (minimum inhibitory concentration) and MFC (minimum fungicidal concentration) assayed on a number of Candida albicans strains of different provenance; i.e., some are clinical isolates, others are laboratory isolates. The relevant feature is represented by the same MIC value in all strains used, but above all by the fact that there is no great difference between MIC and MFC values, thereby demonstrating a fundamentally fungicidal and not fungistatic effect of the essential oil.

Figure 1 depicts in vivo activity of the essential oil. In Figure 1 A, mouse females infected with a luminescent Candida strain and treated with jasmine oil are photographed; in Figure 1 B, mouse females infected with a luminescent Candida strain and treated with mint oil are photographed; in Figure 1 C, mouse females infected with a luminescent Candida strain a n d treated with sa l i n e a re photographed. All mouse females thus infected and treated were observed after several days to see how the infection proceeded. White spots seen near the tail of the animals correspond to the infection; after 21 days it was possible to observe that animals treated with mint oil exhibited a remarkable decrease of the infection.

In Figure 1 D reference is made to two graphs in which the numbers of live cells found in the vaginal fluids of the 3 groups of treated mice are reported. After 21 days the number of cells in the group of animals treated with mint oil was lower than those found in the other 2 groups. In a direct comparison between jasmine oil and the mint one, it may be observed how the latter performs its antifungal action in the first days of treatment.

Figure 2: in this image there are reported the graphs, in particular 4, related to the cytotoxicity found on two cell lines, Monomac and L929, treated for 2 hours and 24 hours with different concentrations of mint oil, jasmine oil and TTO used as positive control. Values reported on the Y-axis represent the percentage of live cells after treatment with essential oils. Mint essential oil, though being slightly more toxic than TTO, maintains a good therapeutic index.

DETAILED DESCRIPTION OF THE INVENTION

The present description reports for the first time an essential oil of Mentha suaveolens containing at least 60% of piperitenone oxide, at least 0.5% of alpha- cubebene and at least 0.5% of octanol, useful as antifungal and/or antibacterial and/or antiviral.

The essential oil extracted from plant material of Mentha suaveolens, as reported below, has a peculiar composition, with a negligible concentration of pulegone, which from what is known in the literature did not forecast the effective antifungal and/or antibacterial and/or antiviral abilities thereof.

The inventors analyzed the composition of said oil by gas chromatography, highlighting in piperitenone oxide the component present in higher percentage. The essential oil of Mentha suaveolens according to the present description contains at least 60% of piperitenone oxide. In particular, the amount of piperitenone oxide is at least 60%, 65%, 70%, 75%, 80%, 85%, 87% 88%, 89%, 90%, 91 %, 92%. According to the invention, Mentha suaveolens essential oil also contains at least 0.5% of alpha-cubebene, a component normally absent from Mentha suaveolens essential oil, at least 1 %, at least 1 .5%, up to even 2% and over. The above- mentioned oil also contains octanol, it also normally absent or present only in traces in the compositions reported in the literature. Octanol amount will be of at least 0.5%, or of at least 0.7%, at least 1 %, or even >1 .1 %. By way of example, yet without limitative pu rposes, the oil of th e present i nvention could have a composition as that reported in Table 1 .

The oil of the present invention could also be a Mentha suaveolens oil, optionally artificially enriched in piperitenone oxide.

The authors of the present description have observed that an essential oil of Mentha suaveolens with a percentage of piperitenone oxide of at least 60%, of at least 0.5% of alpha-cubebene and of at least 0.5% of octanol acts as an effective antifungal and/or antibacterial a n d/or a ntivi ra l , both u si n g it as medicament, and as aid for the disinfection of materials or of environments, exhibiting in many cases an effectiveness comparable or even superior to those observed to date with essential oils used for the same purpose, such as Tea Tree Oil (TTO).

Given the biological activity demonstrated by the inventors toward various microorganisms, Mentha suaveolens oil with a percentage of piperitenone oxide of at least 60% may be used as medicament.

Therefore, object of the description is a Mentha suaveolens oil having a percentage of piperitenone oxide of at least 60%, of at least 0.5% of alpha- cubebene and of at least 0.5% of octanol as medicament.

The oil described herein exhibits the ability to inhibit and/or slow down the growth of various microorganisms such as, e.g . , bacteria and fungi. From conducted studies, the oil disclosed in the present description has demonstrated an antifungal activity such as to suggest it use in mycoses of different origin. Moreover, the antibacterial activity of the oil described herein has also been demonstrated toward Gram-positive and Gram-negative bacteria. The above-listed features make this oil useful as anti-infective, antiseptic, bactericidal, fungicidal.

Mentha suaveolens oil having a percentage of piperitenone oxide of at least 60%, of at least 0.5% of alpha-cubebene and of at least 0.5% of octanol may be used as medicament with antibacterial and/or antifungal and/or antiseptic and/or anti-infective and/or antiviral action.

In an embodiment, the oil disclosed herein may be used as antibacterial under conditions in which an action of inhibiting or slowing down the growth of bacterial microorganisms is desired. Said antibacterial action could be exerted against bacteria belonging to the families Staphylococcus spp, Streptococcus spp and Escherichia coli spp, like e.g. bacteria selected in the group comprising S. aureus, S. piogens, S. agalactiae.

Moreover, the oil d isclosed herei n may be used as antifungal under conditions in which an action of inhibiting and/or slowing down the growth of fungi is desired. I n a particular embodiment, said antifungal action could be exerted against at least one of the fungi selected in the group: Candida albicans, Cryptococcus neoformans, Tricophyton mentagrophyte, Microsporum canis. The oil of the present invention is particularly effective as antifungal in Candida albicans infections, which often require treatments in specific body regions and where the presence of unpleasant odors may prove disagreeable and/or unbearable for the patient. Surprisingly, as reported in the example below, such effects are exerted at a min imu m inh ibitory concentration (MIC) and at a minimum fungicidal concentration (MFC) lower than that of TTO in comparative experiments (see Table 2). Such antifungal activity, moreover, is observed also toward strains resistant to drugs commonly used against infections from fungi. By way of example, said drugs may be azolic drugs. Therefore, said oil may be used in the treatment of fungal infections not responding to drugs commonly used against said infections. I n a particular embodiment such drugs can be the azolic drugs, like e.g. fluconazole.

Moreover, all of the assays carried out by the inventors have demonstrated that the oil according to the present description does not induce development of resistance in the microorganisms treated.

Surprisingly, the authors of the present invention have also discovered an antiviral effect by the oil as described above; object of the present invention is therefore an essential oil of Mentha suaveolens containing at least 60% of piperitenone oxide, at least 0.5% of alpha-cubebene and at least 0.5% of octanol as described in detail in the present application as antiviral.

In particular, the authors of the present invention have detected how the above-mentioned oil exerts an antiviral activity against herpes simplex Virus type-1 and type-2 (HSV-1 and HSV-2) which is of particular interest for the applications of the oil of the invention indicated herein.

Object of the present invention are also pharmaceutical compositions comprising Mentha suaveolens oil as defined in the present description.

The pharmaceutical compositions of the invention are formulated, metered and administered in ways, i.e., amounts, concentrations, programs, ways, vehicles and administration routes, consistent with a good medical practice. The factors to be considered in this context include the specific mammal to be treated, the clinical condition of the individual patient, the cause of infection, the site of administration of the composition, the administration method, the administration program, and other factors known to the medical staff. The therapeutically effective amount of the compound to be administered will be driven by these considerations, and will be the minimum amount required to prevent, improve or treat the disorder.

The compound of the present invention is typically formulated in forms of pharmaceutical dosage, to give an easily controllable drug dosage and allow patient compliance with the prescribed regimen.

The composition according to the present description is preferably sterile. In particular, the formulations to be used for an in vivo administration should be sterile. Such sterilization is easily carried out, e.g., by filtering through suitable sterile membranes. The oil could be used in such compositions as sole active principle, or even in combination with one or more biologically active substances. In particular, such compositions will comprise at least 1 .5% of sai d oi l , optionally mixed with pharmaceutically acceptable diluents, vehicles, exci pients or stabi l izers (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.).

The specific vehicle, diluent or excipient used will depend on the means and purposes for which the compound of the present invention has been applied. Solvents are generally selected on the basis of solvents reckoned by a technician in the field as safe to be administered to a mammal. In general, safe solvents are non-toxic aqueous solvents such as water and other non-toxic solvents soluble or miscible in water. Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycols (e.g., PEG 400, PEG 300), etc., and mixtures thereof.

Oil suspensions can be formulated by suspending the active ingredient into a vegetable oil (such as peanut oil, olive oil, sesame oil or coconut oil) or a mineral oil (such as liquid paraffin). The oil suspensions may also contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol.

Acceptable for preparing the composition of the present invention are those diluents, vehicles, excipients and stabilizers that are non-toxic for the recipient at the dosages and concentrations used, and include buffers such as phosphate, citrate and other organ ic acids, antioxidants, including ascorbic acid and methionine, preservatives (such as octadecylmethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzetonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low-molecular weight polypeptides (less than about 10 residues) polypeptides; proteins, such as serum albumin, gels, or immunoglobulins; hydrophilic polymers, s u c h a s polyvinylpyrrolidone; amino acids, such as glycine, g l uta mine, asparagine, histidine, arginine, or lys i n e; monosaccharides, disaccharides and other hydrocarbons, including glucose, mannose or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; against salt- forming ions, such as sodium; metal complexes (e.g., aZn-protein complexes); and/or nonionic surfactants such as TWEEN™, PLU RON ICS™ or polyethylene glycol (PEG). The formulations may also include one or more stabilizing agents, surfactants, moistening agents, lubrifying agents, emulsifiers, suspending agents, preservatives, antioxidants.

The pharmaceutical compositions could be suitable for rectal , topical, vaginal administration, for administration by inhalation or nasal administration to a mammal such as a human being.

The compositions could be in any form deemed suitable by a technician in the field, such as solid, semisolid, liquid forms, suspensions, emulsions, jellies, ointments, creams, oils, suppositories, vaginal tampons, vaginal ovules, washings, foams, mouthwashes, sprays for inhalation or for aerosol.

Suppository formulations may be prepared by mixing the active ingredient with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal and/or vaginal temperature, and therefore will dissolve in the rectum or vagina to release the drug. Suitable excipients include, e.g., cocoa butter and polyethylene glycols. Formulations suitable for vaginal administration could be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing, beside the active ingredient, those vehicles known as suitable in the state of the art.

Topical formulations, such as creams, ointments, gels, and aqueous or oily solutions or suspensions, can generally be obtained by formulating an active ingredient with a conventional vehicle or diluent acceptable for topical use, using conventional procedures well known in the state of the art.

Compositions for transdermal administration could be in the form of those transdermal plasters for skin that are well known to a technician in the field.

Formulations suitable for intrapulmonary or nasal administration have a particle size in the range of 0.1 to 500 microns (including particle sizes in the range between 0.1 and 500 microns, in micron increases such as 0.5, 1 , 30 microns, 35 microns, etc.) which is administered by rapid inhalation through the nasal passage or by inhalation through the mouth, so as to reach the alveolar sacs. Suitable formulations include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol or dry powder administration can be prepared according to conventional methods and distributed with other therapeutic agents, such as compounds previously used in disorder treatment or prophylaxis as described below.

The invention also provides veterinary compositions comprising the oil of the present invention pure, also comprising a veterinary vehicle (carrier). Veterinary vehicles are materials useful in order to administer the composition, and they may be solid, liquid or gaseous materials that are either inert or acceptable in the state of the veterinary art and are compatible with the active ingredient. These veterinary compositions may be administered by topical, rectal, vaginal route, or by any other desired route.

The exact dosage and the administration frequency will depend on the specific condition to be treated, the severity of the condition to be treated, as well as on other drugs the patient might be taking, as is well known to those skilled in the art. Moreover, it is evident that said effective amount could be lowered or increased according to the responses of the patient treated and/or according to the assessment of the physician prescribing the compounds of the present invention. The effective dosages indicated herein are therefore merely indicative.

I n the indicative dose of at least 10-20 drops of the oil for application according to the present description in form, said oil carries out its action toward infective diseases in general. At greater doses, of at least 30 drops of pure oil, it carries out its antibacterial and/or antifungal action in case of vaginites and alike inflammations.

For respiratory system infections said oil has a valid use at a dose of at least 10-15 drops of pure oil by inhalatory route, twice per day.

Therefore, the doses in the above-indicated pharmaceutical compositions will easily be inferrable by knowing an effective dosage of the active principle as it is.

Moreover, said oil finds possibility of use and effects obtainable thanks to massage and external use. For instance, in the dose of at least 10 drops of pure oil, diluted with another oil like, e.g., olive oil, it may be useful in case of respiratory tract infections.

Frictions performed with the d iluted essence (lotions at the indicative concentration of 15-20%) are useful for oily and acneic skin, and anyhow in all cases where it is necessary to use a natural disinfectant, like: exanthems, irritations caused by napkins, warts, insect bites and stings.

Diluted in water and oil, it is used by gargles in infections of the lip and of the buccal mucosa, like aphthous ulcers or stomatites. With the same mode, vaginal washes or wraps can be made in case of Candida or other vaginal infections.

In general, the pharmaceutical compositions containing the essential oil of Mentha suaveolens as defined in the present description will be for antibacterial and/or antifungal and/or antiseptic and/or anti-infective and/or antiviral purpose.

Other uses may include the cosmetic industry and not just that; in particular, the oil according to the description might be included in herbal products like soaps, mouthwashes, foams, gels, shampoo, aftershaves, deodorants, disinfectant detergents, gargle liquids, providing products that could also prevent infections by acting daily on healthy individuals and preventing bacterial and/or fungal settlement. Owing to the features demonstrated by the inventors, the MSEO (Mentha suaveolens Essential Oil) besides applications i n th e med ica l, hygiene and cosmetic fields can also have other indications, such as use in environment disinfection, air and inanimate matter purification.

Object of the present description are also products comprising the oil as defined in the foregoing. In a particular embodiment, said products are products useful to body care and hygiene, such as, by way of example, bubble bath, soap, foam, shampoo, balsam, detergent, deodorant, aftershave, etc. Such products will contain in their formulation at least 5% of oil according to the present description, and optionally one or more surfactants and/or solvents and/or fragrances and/or dyes and/or preservatives. The type of surfactant, fragrance, dye and preservative, as well as the relative concentration, will be defined by the technician in the field according to the type of formulation selected for the embodiment. By way of example, useful surfactants are: water, sodium lauryl sulphate, cocamidopropyl betaine, sod ium C14-16 olefin sulfonate, cocamides, cyclopentasiloxane, citric acid, etc. In a further embodiment, said products consist in oral hygiene products such as, e.g., toothpastes, mouthwashes, gargle liquids, etc., containing at least 1 .5% of oil according to the present description. Components optionally additional of such products may be preservatives and/or emulsifiers and/or dyes and/or sweeteners and/or aromas. By way of example, useful preservatives are: sodium sorbate, potassium sorbate, calcium sorbate, sodium benzoate, calcium benzoate, potassium benzoate, propylparaben, sodium propionate, etc. Emulsifiers useful for the manufacturing of said products are, e.g.: lecithin, sodium citrate, sodium phosphate, calcium phosphate, polyethylene, etc. Instead, sweeteners useful for the manufacturing of said products are, by way of example: sorbitol, mannitol, aspartame, saccharin, xylitol. Merely by way of exa m ple, useful dyes are: tartrazine, chlorophyll, carotene, etc. Objects of the present description are also disinfectants for environments and/or inanimate material useful to limit or prevent microorganism growth where desired. Said disinfectants may be made in the form of solutions, emulsions, sprays and will contain at least 5% of the oil as defined in the foregoing. Diffusion in environments such as, e.g., operating rooms, hospital rooms, classrooms, waiting rooms, etc., could occur by heaters, air conditioning filters, diffusers, sterilizing systems.

The essential oil according to the present description is obtained by extraction from plants belonging to the genus Mentha, species suaveolens (Mentha suaveolens), common name: Apple mint. Among its various synonyms, also that of Mentha rotundifolia has been reported (Nagell and Hefendehl, 1974). Mentha suaveolens is a herbaceous perennial plant up to 1 m tall, with a strong pungent odor. It is a plant spontaneously growing in Italy, characteristic of Mediterranean Countries; in particular, it is common in peninsular and insular territories. It may be found spontaneous in high Latium territories, with a balsamic time, defined as the period of the year in which said plant exhibits the highest concentration of active principles, of about 3 months. The balsamic time for plants belonging to the species Mentha suaveolens is between June and late September. Therefore, in order to reach the extraction of the essential oil of Mentha suaveolens according to the present description, plant material of Mentha suaveolens will be used, harvested between early June and late September, period in which there is a maximum production of the essential oil. The starting plant material for obtaining the oil of the present description may be comprised of any portion of the plant potentially containing the oil of interest. In particular, it is known that essences like Mentha suaveolens oil are contained in leaves, flowers and petals, and more specifically in the peculiar glandular hair usually abundant on the surface of said leaves and said petals.

For this reason, the whole plant, and/or leaves and/or flowers and/or petals and/or glandular hair will preferably be selected by the technician in the field as starting material for the extraction of Mentha suaveolens oil. Essential for extraction of the oil described herein is the use of fresh plant material, preferably processed right after harvesting. Therefore, the starting material to the ends of the present invention is represented by the fresh whole plant and/or fresh leaves and/or fresh flowers and/or fresh petals and/or fresh glandular hair.

The extraction method for obtaining the oil of interest could be any method that the person skilled in the art will deem useful for this purpose. In a particular embodiment, the extraction of said oil from said plant material could be carried out by distillation. In particular, two procedures could be adopted:

1 ) Simple hydro-distillation, with direct immersion of the plant material in the vessel heated and brought to a boil; 2) distillation by saturated steam; in this case the material is in direct contact with the steam crossing it. All details related to such extraction techniques are, as is known to the techn ician in the field , amply described in all their steps on different laboratory texts. Merely by way of a non- limiting example, it is therefore possible to place the Mentha suaveolens plant, preferably comminuted, in a balloon containing a suitable volume of water and carry out a steam current extraction for about 3 hours.

Th e essenti a l oi l th u s obta i n ed will be subjected to subsequent anhydrification on a suitable anhydrifying agent, like e.g. anhydrous sodium sulphate. Pure essential oil could be obtained in its pure form by filtering off the anhydrifying agent.

Examples are reported herebelow which have the pu rpose of better illustrating the invention described in the present description; these examples are in no way to be construed as a li mitation of the preceding description and the subsequent claims.

EXAMPLES

Example 1

Plant harvesting and identification, essential oil extraction.

Samples of Mentha suaveolens leaves and flowers were harvested at

Roccaccia-Tarquinia in July 2007, in a suitable time of the day and in its balsamic time. The plant was subjected to microscopy analysis for final identification by experts of the Department of Plant Biology of the "La Sapienza" University of Rome, who actually identified the plant as belonging to genus Mentha, species suaveolens Ehrl. of the family Labiate, subfamily asteroideae. For essential oil extraction a Clevenger-type apparatus (Clevenger J.F. J. Amer. Pharm. Assoc., 1928 17 346) was used. For this purpose, about 100 g of thoroughly comminuted plant were placed in a 500 ml balloon containing 200 ml water, and extracted under steam current for about 3 h. After this period, no separation of further essential oil is observed. Obtained essential oil is anhydrified on anhydrous sodium sulphate. After filtration, pure essential oil is obtained with an overall yield ranging from 0.4 to 0.5%.

Example 2

In vitro biological assays. CLSI 2008 Method. MIC and MFC.

To assay the antifungal activity of Mentha suaveolens essential oil, tests for determination of minimum inhibitory (MIC) and fungicidal (MFC) concentrations were performed. The essential oil was diluted in a stepwise manner in RPMI 1640 medium additioned with L-glutamine in the absence of sodium bicarbonate, in a pH 7.0 buffer containing MOPS (0,165 M). Essential oil final concentration ranged from 0.008 to 4% v/v. A conical-bottom 96-well Costar plate was prepared with 50 μΙ aliquots of each dilution. The test was performed in triplicate. Tween 80 at the final concentration of 0,001 % v/v was added to the solution to facilitate oil solubility. At this concentration, no inhibitory effect due to the presence of the detergent was observed on fungal growth. The fungal inoculum was of about 2.5 x 10³cells/ml. Plates were incubated at 30°C for 48h. For MFC determination, from each well in which no fungal growth was observed, 10 μΙ medium were collected and inoculated on Petri plates containing agarized Sabouraud's medium (SDA) and incubated at 30° C for 48h. Thereafter, colony-forming units (CFUs) were counted to assess MFC, defined as the minimum concentration at which 99.9% mortality is observed. (Pages 6-10 of Clinical and Laboratory Standards I nstitute. Methods for broth dilution antifungal susceptibility testing of yeast. Approved Standard- Third Edition: M27-A3. 2008; 28 (14). Wayne, PA, USA). In parallel, for comparative purposes, the same test was performed using commercial TTO; results are reported in Table 2.

Example 3

Vapour phase.

Vapour phase assay. The effect of the volatile fraction of the essential oil was studied by inverted Petri plate method. Buffers of about 5 mm diameter were imbibed in the diluted solutions of essential oil (prepared as for MIC and MFC determination) at the concentrations corresponding to MIC and those immediately higher and lower. The buffers were applied on the lid of Petri capsules containing 10 ml of agarized Sabouraud's medium inoculated with about 1 x10⁶ cells/ml of fungi, so as not to directly contact the surface of the medium itself. Plates were sealed with Parafilm and incubated at 30° C for 48-72h. Thereafter, lack of fungal growth was observed. (Page 1546, lines 15-26 of the first column of Tullio, V., et al., Antifungal activity of essential oils against filamentous fungi determined by broth microdilution and vapour contact methods. J Appl Microbiol, 2007. 102(6): p. 1544-50)

Example 4

In vivo biological assays

To assess MSEO activity in vivo, an experimental model of murine vaginal infection was used. Three groups, each consisting of four 4-week old female mice, were treated for 5 days with subcutaneous injections of 0.2 mg estradiol valerate diluted in 1 00 μΙ of sesame oil to induce pseudo-estrus conditions. This was repeated each week until study completion. Animals were anesthesized with 2.5 to 3.0% (vol/vol) of isofluorane gas and infected twice at a distance of 24 h with 10 μΙ of a suspension containing 1x10⁹/ml fungal cells, by inserting a pipette in the vaginal lumen up to the uterine cervix. To foster fungal cell contact and absorption, animals were kept with the head down for one minute after infection. Every day after the infection, 10 μΙ coelenterazine was added in the vaginal lumen to enable visualization of the infection. One group of animals was treated with MSEO, one group with physiological solution and finally one group with jasmine essential oil used as negative control. After 1 , 24 and 48h from infection a group of animals was treated either with 50 μΙ MSEO at the concentration of 15 mg/ml, or with 50 μΙ of physiological solution, or with 50 μΙ of jasmine essential oil. On days 5, 7, 9 ,15 and 21 they were observed by fluorescence and vaginal fluids were collected. To determine fungal charge, vaginal fluids were diluted and deposited on Petri plates containing agarized SDA medium. Plates were incubated for 48 h and CFUs counted, (page 4850; lines 5-21 of the first column of Enjalbert B, Rachini A., Vediyappan G., Pietrella D, Spaccapelo R., Vecchiarelli A., Brown A. J. P., d'Enfert C.A Multifunctional, Synthetic Gaussia princeps Luciferase Reporter for Live Imaging of Candida albicans Infections. Infection and Immunity. 2009, 77:4847- 4858; fourth page, lines 6-15 of the first column of Mondello F., De Bernardis F., Girolamo A.,Cassone A., Salvatore G. In vivo activity of terpinen-4-ol, the main bioactive component of Melaleuca alternifolia Cheel (tea tree) oil against azole- susceptible and -resistant human pathogenic Candida species. BMC Infectious Diseases 2006, 6:158).

Example 5

Toxicity assays.

MSEO toxicity was assessed on monocyte and macrophage MonoMac and L929 cell lines. 2.5 x 10⁵ cells were treated with 0.1 , 1 , 10, 100, 1000, 10000 g ml MSEO, for 2 h or 24 h at 37°C. Cell viability was assessed by use of an ATP- bioluminescent kit. 1 00 μΙ of each sample were added into a 96-well plate containing 50 μΙ of lysis reagent and, after 10 min incubation, 1 00 μΙ of ATP- containing reagent (AMRPIus) were added. After 2 min incubation, luminescence was measured by luminometer. (Page 4360, lines 12- 20 of the second column of Pietrella D., Lupo P., Rachini A., Sandini S., Ciervo A., Perito S., Bistoni F., Vecchiarelli A.. A Candida albicans Mannoprotein Deprived of Its Mannan Moiety Is Efficiently Taken Up and Processed by Human Dendritic Cells and Induces T-Cell Activation without Stimulating Proinflammatory Cytokine Production. Infection and Immunity, 2008, 76, 4359-4367).

Example 6

Antiviral activity of Mentha Suaveolens essential oil.

Preliminary studies conducted with non-cytotoxic concentrations of essential oil (MSEO) of monkey kidney cells (Vero) infected with the herpes simplex virus type-1 and -2 (HSV-1 , HSV-2) have highlighted an antiviral activity detected by the reduction of the cytopathic effect (CPE) and determined by colorimetric method as described in Schnitzler P et al 2007, with respect to cells not treated with MSEO and infected with the virus. REFERENCE

1 . Carson C. F. et al . Melaleuca alternifolia (Tea Tree) Oil: Review of Antimicrobial and Other Medicinal Properties Clin Microbiol Rev.2006 ; 19:50-62.

2. Clevenger JF. Apparatus for determination of volatile oil. J.Amer Pharm Assoc. 1928; 17: 346.

3. Enjalbert B et al Multifunctional, Synthetic Gaussia princeps Luciferase Reporter for Live Imaging of Candida albicans Infections. Infection and Immunity. 2009, 77:4847-4858.

4. Lupien S, Karp F, Wildung M, Croteua RB. 1999. Regiospecific cytochrome P450 hydroxylases from mint (Mentha) species: cDNA isolation, characterization, and functional expression of (-)-4S-limonene-3-hydroxylase and (- )-4S-limonene-6-hydroxylase. Arch Biochem Biophys 368: 181 -192.

5. McCon key M E, Gershenzon J , Croteau RB . 2000. Developmental regulation of monoterpene biosynthesis in the glandular trichomes of peppermint. Plant Physiol 122: 215-223.

6. Mondello F et al. In vivo activity of terpinen-4-ol, the main bioactive component of Melaleuca alternifolia Cheel (tea tree) oil against azole-susceptible and -resistant human pathogenic Candida species. BMC Infectious Diseases 2006, 6:158.

7. Nagell and Hefendehl, 1974

8. Oumzil H., Ghoulami S., Rhajaoui M., Ilidrissi A., Fkih-Tetouani S., Faid M., Benjouad A., Antibacterial and antifungal activity of essential oils of Mentha suaveolens . Phytother. Res 2002;16:727-731

9. Pietrella D et al. A Candida albicans Mannoprotein Deprived of Its Mannan Moiety Is Efficiently Taken Up and Processed by Human Dendritic Cells a nd I n d uces T-Cell Activation without Stimulating Proinflammatory Cytokine Production. Infection and Immunity, 2008, 76, 4359-4367

10. Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, Arthur, 1905-, Remington, Joseph P. 1847-1918

1 1 . Clinical and Laboratory Standards Institute. Methods for broth dilution antifungal susceptibility testing of yeast. Approved Standard- Third Edition: M27-

A3. 2008; 28 (14). pages: 6-10

12. Susceptibility of Drug-resistance clinical Herpes simplex virus type 1 , strains to essential oils of ginger, thyme, hyssop and sandalwood. Schnitzler P, Koch C, Reichling J . Antimicrobial Agents and Chemotherapy, 51 ; 1859-1862

2007.

Claims

1 . Essential oil of Mentha suaveolens containi ng at least 60% of piperitenone oxide, at least 0.5% of alpha-cubebene and at least 0.5% of octanol.

2. Oil according to claim 1 , wherein the amount of piperitenone oxide is at least 60%, 65%, 70%, 75%, 80%, 85%, 87% 88%, 89%, 90%, 91 %, 92%.

3. Oil according to claim 1 or 2, as medicament.

4. Oil according to claim 3, wherein said medicament has an antibacterial and/or antifungal and/or antiseptic and/or anti-infective activity.

5. Oil according to claim 4, wherein said antibacterial activity is exerted against bacteria belonging to Staphylococcus spp, Streptococcus spp and Escherichia coli spp.

6. Oil according to claim 5, wherein said bacteria are selected in the group comprising: S. aureus, S. piogens, S. agalactiae.

7. Oil according to claim 4, wherein said antifungal action is exerted against at least one of the fungi selected in the group comprising: Candida albicans, Cryptococcus neoformans, Tricophyton mentagrophyte, Microsporum canis.

8. Oil according to any one of the claims 1 to 7, wherein said oil exerts an antiviral activity against herpes simplex virus of type 1 (HSV-1 ) and 2 (HSV-2).

9. Pharmaceutical compositions comprising at least 1 .5% of th e o i l according to claim 1 or 2, and one or more pharmaceutically acceptable vehicles and/or excipients.

10. Composition according to claim 9, for antibacterial and/or antifungal and/or antiseptic and/or anti-infective use.

1 1 . Products comprising at least 1 .5% of the oil according to claim 1 or 2, selected in the group consisting of: soaps, mouthwashes, foams, gels, shampoo, aftershaves, deodorants, disinfectant detergents, gargle liquids, gargling solutions.

12. A method for the extraction of Mentha suaveolens essential oil containing at least 60% of piperitenone oxide at least 0.5% of alpha-cubebene and at least 0.5% of octanol comprising the following steps:

a) using fresh plant material of Mentha suaveolens harvested in a period comprised between early June and late September;

b) extracting said oil from said plant material

c) carrying out the anhydrification of said oil on anhydrous sodium sulphate.

13. Method according to claim 12, wherein said plant material of Mentha suaveolens is whole plant and/or leaves and/or flowers and/or petals and/or glandular hair.