IT202000028106A1 - FINISHED PRODUCT COMPRISING NATURAL EXTRACTS AND A PAINT PRODUCT HAVING ANTIBACTERIAL OR BACTERIOSTATIC ACTIVITY ALSO FOR GRAM-NEGATIVE BACTERIA, AND ITS USES - Google Patents

Description

DESCRIPTION of the invention entitled:

?Finished product comprising natural extracts and a paint product having activity? antibacterial or bacteriostatic also for Gram-negative bacteria, and its uses?

The present invention refers to a mixture M comprising or, alternatively, consisting of (a) a usnic acid and/or (b) a salt thereof, preferably said usnic acid and/or its salt being in the racemic or dextrorotatory form D( +). Furthermore, the present invention refers to a semi-finished product PS, preferably in the form of a cream or semi-solid paste, comprising said mixture M and a resin, as well as refers to a finished product PF, preferably in the form of a liquid or a dispersion, comprising said semi-finished product PS and a painting product. Said mixture M, said semi-finished product PS and finished product PF show an activity? anti-bacterial, anti-proliferative, bacteriostatic, microbicidal, anti-mold, anti-yeast, anti-fungal or anti-fungal, preferably a? against Gram-positive and/or Gram-negative bacteria, such as for example those with the scientific name Klebsiella, Enterobacteriaceae, Enterobacter, Pseudonomas and Escherichia. Finally, the present invention relates to a method for making a surface anti-bacterial, anti-proliferative, bacteriostatic, microbicidal, anti-mould, anti-yeast, anti-fungal or anti-mycotic, preferably against Gram- positive and/or Gram-negative, said method provides for the application by spray technique, roller or brush of said mixture M, or said semi-finished product PS or finished product PF, onto said surface.

The present invention also relates to an inclusion compound (ci) comprising or, alternatively, consisting of: (i) D-usnic acid as enantiomer, or a salt thereof, or mixtures thereof, of natural origin and (ii) beta- cyclodextrins. Said inclusion compound (ci) has an activity? antibacterial or bacteriostatic both against Gram-positive and Gram-negative pathogenic bacteria, such as those with the scientific name Klebsiella, Enterobacteriaceae, Enterobacter, Pseudonomas and Escherichia. The present invention also relates to a use of said inclusion compound (ci) as an antibacterial or bacteriostatic agent for Gram-negative and Gram-positive bacteria. The present invention also relates to a liquid composition comprising or, alternatively, consisting of: (a) said inclusion compound (ci); (b) an acrylic resin, a polyurethane resin or an acrylpolyurethane resin, or mixtures thereof; (c) optionally a pigment or opacifying agent; and (d) water. The present invention also relates to a use of said liquid composition as a paint or architectural coating of surfaces and walls, preferably as an antibacterial or bacteriostatic architectural coating, both against Gram-positive and Gram-negative pathogenic bacteria, such as for example those with scientific name Klebsiella, Enterobacteriaceae, Enterobacter, Pseudonomas and Escherichia. Finally, the present invention finally relates to a use of cyclodextrins, preferably of betacyclodextrins such as for example of (2-hydroxypropyl)-?cyclodextrin, as selective complexing agents of D-usnic acid, or a salt thereof, or mixtures thereof, from a mixture racemic form of usnic acid, the latter obtained by means of an extraction process which is also the subject of the present invention, starting from a natural material.

Pathogenic microorganisms, also called pathogens, are biological agents responsible for the onset of the disease condition in the host organism. They are divided into: viruses; prokaryotes: bacteria; eukaryotes: fungi and protozoa. The pathogenicity?, or the ability? generic to determine a morbid state, ? defined by two factors: (i) virulence, which indicates the greater or lesser capacity? to generate disease; (ii) invasiveness?, i.e. the ability? to invade host tissue and multiply inside. The invasiveness, in turn, depends on factors such as: adhesiveness, that is? the capacity? of the pathogen to bind with its external surface structures to the receptor sites of the host cells; production of extracellular enzymes that facilitate the destruction of host tissues; production of antiphagocytic substances or presence of anti-phagocytic capsule, which allow the pathogen to resist the host's defense mechanisms. The increase in the incidence of nosocomial infections (infectious diseases associated with care within a health facility), due to a transmission of multiresistant microorganisms, is? long been the subject of study and research. Numerous scientific studies demonstrate that hospital environmental surfaces play a prominent role in the contamination, persistence and spread of a variety of of microorganisms in the nosocomial environment; these surfaces therefore become a durable reservoir of pathogens within the hospitalization structures. In the hospital environment, but not only, ? felt the need? to limit the nocosomial bacterial load (and not only) allowing the hospitalization of patients in an environment as much as possible? sterile as possible. The same speech can be applied to schools, kindergartens, playgrounds or in public places such as supermarkets and shopping centers where there is? very often a very high bacterial load due to the large number of people. In addition, infecting (pathogenic) microorganisms have evolved into strains capable of resisting most of the antibiotics currently available on the market. Therefore, it could be very useful to have a treatment for surfaces, for example surfaces in fabric, leather, wood, glass, plastic, steel, linoleum or concrete walls and floors, which uses active substances or compounds still unknown to living microorganisms , including drug-resistant ones, in order to make it difficult or reduce as much as possible? the proliferation of microorganisms on the treated surfaces is possible. Examples of surfaces to be treated, without limitation, can be found for example in a doctor's surgery, emergency room, hospital, dentist's office, playground, kindergarten, school or toilets and toilets present, for example, in public or private structures, or for example in supermarkets and shopping centers or playgrounds. In Italy, the probability to contract an infection during a hospital stay? by 6%, with a number of cases ranging from 450,000 to 700,000 each year and with an annual number of deaths estimated at around 7,800. This last statistic gives Italy an unfortunate primacy among European countries. Currently, the environmental contamination of surfaces? countered by synthetic detergent or disinfectant compounds which, in addition to being ineffective or in any case unsuitable to prevent short-term re-contamination (within 30 minutes of disinfection), have a significant environmental impact.

Therefore, ? felt the need? to be able to have a surface treatment, an active compound and an active composition having a reduced environmental impact and, possibly, completely of natural origin. A sanitation carried out only with synthetic agents cannot? therefore guaranteeing a healthy and safe environment in a hospital or kindergarten or school, as it proves unable to keep the environmental surfaces sanitized over time. ? so felt the need? to be able to have a treatment, an active compound and an active composition with a reduced environmental impact able to reduce or counteract the pathogenic bacterial load or effectively counteract the contamination, persistence and transmission of pathogenic bacteria in public environments and spaces and private individuals such as in a hospital or kindergarten or school, preventing pathogenic bacteria from spreading and/or developing resistance.

The Applicant, after a long and intense activity? of research and development, has developed a mixture M, a semi-finished product PS containing said mixture M and a resin, a finished product PF containing said semi-finished product PS and a varnishing product, an inclusion compound and a composition thereof capable of provide an adequate response to the limitations, drawbacks and existing problems. Not only that, but the Applicant has perfected a surface treatment method which allows to make said surfaces treated with a mixture M, a semi-finished product PS containing said mixture M and a resin, a finished product PF containing said semi-finished product PS and a varnish, an inclusion compound and a composition thereof, anti-bacterial, anti-proliferative, bacteriostatic, microbicidal, anti-mould, anti-yeast, anti-fungal or anti-fungal, preferably against Gram-positive bacteria and/ or Gram-negative.

Examples of surfaces where it is possible to apply said mixture M, said semi-finished product PS containing said mixture M and a resin, said finished product PF containing said semi-finished product PS and a painting product, said inclusion compound and a composition thereof, are for example horizontal surfaces or vertical surfaces, for example floors, walls or ceilings made for example in cement, lime or plasterboard, linoleum, or in PVC polyvinyl chloride, PA polyamide, PE polyethylene, PES polyester or PTF polyethylene terephthalate. These types of surfaces, without limitation, can be found for example in a doctor's surgery, emergency room, hospital, dentist's office, playground, kindergarten, school or toilets and toilets present, for example, in public or private structures or, for example, in supermarkets and shopping malls or playgrounds. Or they can be surfaces of a fabric, a non-woven fabric (TNT), natural leather, artificial or synthetic leather, leather, wood, glass, plastic, polymer, aluminum, steel, linoleum.

The object of the present invention is a mixture M comprising a usnic acid and/or a salt thereof, preferably a sodium salt of the usnic acid, said usnic acid and/or a salt thereof being preferably of natural origin in the racemic or dextrorotatory form D( +), having the characteristics as reported in the appended claims.

The object of the present invention is a use of said mixture M as an anti-bacterial, anti-proliferative bacterial, bacteriostatic, microbicide, anti-mould, anti-yeast (for example Candida), anti-fungal or antifungal (for example Saccharomycetes), preferably against of Gram-positive and/or Gram-negative bacteria, such as for example those having the scientific name Klebsiella, Enterobacteriaceae, Enterobacter, Pseudonomas and Escherichia, said use having the characteristics as reported in the attached claims.

The object of the present invention is a method for rendering a surface antibacterial, bacterial antiproliferative, bacteriostatic, microbicidal, anti-mould, anti-yeast, anti-fungal or anti-mycotic, preferably against Gram-positive and/or Gram -negatives, such as for example those having the scientific name Klebsiella, Enterobacteriaceae, Enterobacter, Pseudonomas and Escherichia, said method provides for the application by spray technique, roller or brush of said mixture M on said surface, said method having the characteristics as reported in the combined claims.

Preferably, said surface to be treated can also be subjected to a pre-treatment first to increase adhesion, stability? or the effectiveness of said mixture M on said surface. The pretreatment can for example be of the mechanical type, for example a mechanical abrasion of the surface by emery, or be of the chemical type, for example by applying an impregnating solution or a coating film, for example a polymeric film or a paint or a fixative or gripper.

The object of the present invention is a semi-finished product PS comprising said mixture M, and a resin, having the characteristics as reported in the accompanying claims. By way of example , the resins are for example those known to the expert in the field of varnishes, enamels and paints (water-based or organic solvent; glossy or opaque, or colored transparent ones) for example mono- or bi-component resins. The resins are added to said mixture M by means of the procedures and equipment known to the expert in the sector.

The object of the present invention is a use of a semi-finished product PS as an antibacterial, bacterial antiproliferative, bacteriostatic, microbicide, anti-mould, anti-yeast (for example Candida), anti-fungal or anti-mycotic (for example Saccharomycetes), preferably in comparisons of Gram-positive and/or Gram-negative bacteria, such as for example those having the scientific name Klebsiella, Enterobacteriaceae, Enterobacter, Pseudonomas and Escherichia, said use having the characteristics as reported in the appended claims.

The object of the present invention is a method for rendering a surface antibacterial, bacterial antiproliferative, bacteriostatic, microbicidal, anti-mould, anti-yeast, anti-fungal or anti-mycotic, preferably against Gram-positive and/or Gram -negatives, such as for example those having the scientific name Klebsiella, Enterobacteriaceae, Enterobacter, Pseudonomas and Escherichia, said method provides for the application by spray technique, roller or brush of said semi-finished product PS on said surface, said method having the characteristics as reported in the joined claims.

Preferably, said surface to be treated can also be subjected to a pre-treatment first to increase adhesion, stability? or the effectiveness of said semi-finished product PS on said surface. The pretreatment can for example be of the mechanical type, for example a mechanical abrasion of the surface by emery, or be of the chemical type, for example by applying an impregnating solution or a coating film, for example a polymeric film or a paint or a fixative or gripper.

The object of the present invention is a finished product PF comprising said semi-finished product PS, and a painting product, having the characteristics as reported in the accompanying claims. By way of example, the paint products are for example those known to the expert in the field of varnishes, enamels and paints (water-based or organic solvent; glossy or opaque, or colored transparent ones). The paint products are added to said semi-finished product PS by means of the procedures and equipment known to the expert in the sector.

The object of the present invention is a use of a finished product PF as an antibacterial, bacterial antiproliferative, bacteriostatic, microbicide, anti-mould, anti-yeast (for example Candida), anti-fungal or antifungal (for example Saccharomycetes), preferably in comparisons of Gram-positive and/or Gram-negative bacteria, such as for example those having the scientific name Klebsiella, Enterobacteriaceae, Enterobacter, Pseudonomas and Escherichia, said use having the characteristics as reported in the appended claims.

The object of the present invention is a method for making a surface anti-bacterial, anti-proliferative, bacteriostatic, microbicidal, anti-mould, anti-yeast (for example Candida), anti-fungal or anti-mycotic (for example Saccharomycetes), preferably against Gram-positive and/or Gram-negative bacteria, such as for example those with the scientific name Klebsiella, Enterobacteriaceae, Enterobacter, Pseudonomas and Escherichia, said method provides for the application by spray technique, roller or brush of said finished product PF on said surface , said method having the characteristics as reported in the appended claims.

In one embodiment, said surface to be treated may also be subjected to a pre-treatment first to increase adhesion, stability? or the effectiveness of said finished product PF on said surface. The pretreatment can for example be of the mechanical type, for example a mechanical abrasion of the surface by emery, or be of the chemical type, for example by applying an impregnating solution or a coating film, for example a polymeric film or a paint or a fixative or gripper.

In one embodiment, the finished product PF, preferably in the form of a liquid or dispersion, comprises said semi-finished product PS, and a varnished product, for example a coloured, opacifying or transparent varnishing product. Said paint product pu? for example be a varnish, an enamel or a paint, water-based or organic solvent. The combination or association between said semi-finished product PS, preferably in the form of a semi-solid cream or paste, with a varnish or enamel or paint, preferably in the form of a liquid or dispersion, d? result in a finished product in the form of a coloured, opaque or transparent varnish, or a finished product in the form of a coloured, opaque or transparent glaze, or a finished product in the form of a coloured, opaque or transparent paint. The latter finished products in the form of varnish, enamel or paint can be applied with a spray or roller or brush technique on a surface, possibly pre-treated, so as to give said surface, for example a wooden or steel or glass surface or a concrete wall in a hospital, a property? anti-bacterial, anti-proliferative, bacteriostatic, microbicidal, anti-mould, anti-yeast, anti-fungal or anti-fungal, preferably against Gram-positive and/or Gram-negative bacteria.

In another embodiment, the semi-finished product PS or the finished product PF can be added or added for example to a polymeric material (for example PVC, PE or PTF) of those generally used to prepare a coating film or a colored, opaque or transparent film. The polymeric material in film or foil is then positioned and fixed, for example with a glue or by heat heating, on the surface of a table, or a countertop in a kitchen or a wall for example made of wood or plastic or aluminum or steel.

In another embodiment, the semi-finished product PS or the finished product PF can be added or added for example to a solution or cream of those generally used to treat or polish the natural or synthetic leather of, for example, a chair or armchair .

Furthermore, the object of the present invention is an inclusion compound (ci) comprising or, alternatively, consisting of: (i) D-usnic acid as enantiomer, preferably as pure enantiomer, or a salt thereof, or mixtures thereof, and (ii) betacyclodextrins, having the characteristics as defined in the accompanying claims. Said compound(s) D-usnic acid ? of natural origin why? ? extracted through a process from a natural material.

Another object of the present invention is the use of said inclusion compound (ci) as an antibacterial or bacteriostatic agent for Gram-negative bacteria and for Gram-positive bacteria, having the characteristics as defined in the accompanying claims.

The object of the present invention is also a liquid composition comprising or, alternatively, consisting of: (a) said inclusion compound (ci); (b) an acrylic resin, a polyurethane resin or an acryl-polyurethane resin, or mixtures thereof; (c) optionally a pigment or opacifying agent; and (d) water, having the characteristics as defined in the appended claims.

Furthermore, an object of the present invention is a use of said liquid composition as a paint or architectural coating of surfaces and walls, preferably as an antibacterial or bacteriostatic architectural coating both against Gram-positive and against Gram-negative bacteria, having the characteristics as defined in the appended claims.

Finally, the object of the present invention is the use of cyclodextrins, preferably of beta-cyclodextrins, such as for example of (2-hydroxypropyl)-?-cyclodextrin, as selective complexing agents of D-usnic acid, or a salt thereof, or their mixtures, having the characteristics as defined in the appended claims.

The present invention will be now illustrated with reference to the attached tables, provided purely by way of example, and therefore not limiting, in which:

- Figure 1 exemplifies a flow diagram of a process for the production of usnic acid, according to a possible embodiment;

Figure 2 shows an average distribution of solid particles of D-usnic acid according to a possible embodiment;

- Figures 3 and 4 illustrate activities? bacteria R respectively related to Example 2 and to Example 3; - Figure 5 represents the buffers used in Example 4;

Figure 6 illustrates a reduction in bioburden as discussed in Example 4;

- Figures 7, 8, 9 and 10 show results of the microbiological monitoring discussed in Example 5;

- Figure 11 refers to a microscope image relating to the initial formation of a lattice of crystals (initiation of the cross-linking) of usnic acid and/or a salt thereof, after a finished product PF, according to the present invention, ? been applied to a surface;

- Figure 12 refers to a microscope image relating to the flowering of crystals (continuation of cross-linking) of usnic acid and/or a salt thereof, after a finished product PF, according to the present invention, is ? been applied on a surface and the solvent, contained in said finished product PF, starts to evaporate from the surface;

- Figure 13 refers to a microscope image relating to the complete formation of crystals of usnic acid and/or a salt thereof, after a finished product PF, according to the present invention, is formed. been applied on a surface and the solvent, contained in said finished product PF, ? evaporated from the surface;

- Figure 14 refers to a microscope image relating to the perforation action caused by the crystal of usnic acid and/or its salt damaging the cell wall of the bacterium present on the surface treated with a finished PF product, in accordance with present invention.

Therefore, the object of the present invention is an inclusion compound (ci) comprising or, alternatively, consisting of: (i) a D-usnic acid as enantiomer, preferably as a pure enantiomer, or a salt thereof, or mixtures thereof, and ( ii) cyclodextrins. Said(s) D-usnic acid ? of natural origin why? ? extracted with a process, also object of the present invention, starting from a natural material. Also called (ii) beta-cyclodextrins are of natural origin. Consequently also called inclusion compound (ci) ? of natural origin. Said inclusion compound (ci) advantageously has an activity? bacteriostatic or antibacterial against both Gram-positive and Gram-negative pathogenic bacteria.

Within the present description, with the expression ?inclusion compound? means a chemical structure of the type that of a chemical complex in which a chemical compound (host / host) can? have cavities? (for example one or two or three cavities, preferably one) of certain dimensions, equal or different from each other, in which molecules can be allocated or positioned or established (for example one or two or three molecules, preferably one) having similar dimensions, to those of the respective cavities, of a second chemical compound (host/guest), where the host and the guest are bonded in a non-covalent way, generally through intermolecular forces such as forces of the van der Waals type. Cyclodextrins are natural cyclic oligosaccharides formed by 6, 7 or 8 monomers of D-(+)glucopyranose joined together with a ?,1-4 glucosidic bond and closed in a ring which have cavities (for example one or two cavities?) of certain dimensions, equal or different from each other. The cyclodextrins (ii) of the present invention form a molecular cage which delimits a cavity lipophilic capable of hosting D-usnic acid as an enantiomer, or its salt, or mixtures thereof (i) of the present invention. Preferably, said cyclodextrins (ii) used in the inclusion compound (ci) are selected from the group comprising or alternatively consisting of ?cyclodextrins, ?-cyclodextrins, ?-cyclodextrins and mixtures thereof. Pi? preferably, said cyclodextrins (ii) are beta-cyclodextrins.

In one embodiment, (ii) cyclodextrins are selected from beta-cyclodextrins. The (ii) cyclodextrins comprise or alternatively consist of (2-hydroxypropyl)-?-cyclodextrin (CAS No. 128446-35-5). In fact, (2-hydroxypropyl)-?-cyclodextrin is found to be, together with others, advantageously a selective complexing agent of d-usnic acid, or a salt thereof, or mixtures thereof, from a racemic (or racemic) mixture of usnic acid, obtained by the process of the present invention.

Racemic usnic acid (CAS No. 125-46-2) ? the bioactive secondary metabolite of lichens. The large-scale use of said usnic acid? always been limited due to the low solubility? in water (0.06 mg/cm<3>, at room temperature of 20°C and pressure of 1 atmosphere).

Therefore, the solubility? of usnic acid in water has always represented, and still represents, a major limitation to its use. Furthermore, as regards the effectiveness of usnic acid (or one of its relative salts) in terms of activity? anti-bacterial ? It has been seen by the inventors of the present invention that it depends both on the origin (natural or synthetic) of the usnic acid and on the type of isomer (levorotatory (-) and/or dextrorotatory (+)) used. ? been seen that the dextrorotatory form (+) of natural origin from Usnea ? more stable, effective and active dextrorotatory (+) form of synthetic origin.

The inventors of the present invention have surprisingly found, after an intense and extensive activity, of research, that the solubility? in water of the D enantiomer of usnic acid, or its salt, or mixtures thereof (the) can? be increased by various orders of magnitude (for example up to about 4.2 mg/cm<3>, at the same temperature of 20°C and pressure 1 atmosphere) through the formation of the inclusion compound (ci). In this compound (there), as mentioned above, the cavity? lipophilic of cyclodextrins (host) hosts D-usnic acid, or its salt, or mixtures thereof (i) (guest), through a noncovalent interaction of a reversible nature. Indeed, during the formation of the inclusion compound (ci), no covalent bond is formed, and no covalent bond is broken. What mechanism? at least in the main street? promotes the formation of the inclusion compound ? the release of solvent molecules (preferably water molecules), highly enthalpic exchange reaction, from the cavity? of cyclodextrins (ii). Thanks to this host/guest interaction (reversible electrostatic chemical bond), the inventors of the present invention have surprisingly found that D-usnic acid, or a salt thereof or mixtures thereof (i) is not? trapped / bound or unable to perform its function in the inclusion compound but, on the contrary, is easily released from the cavity? lipophilic of its host, and therefore readily available to carry out its activity. Furthermore, the inclusion compound (ci) renders D-usnic acid (or a salt thereof) compatible in an aqueous solution or an aqueous dispersion or an aqueous suspension. When said aqueous solution or dispersion or suspension, containing said inclusion compound (ci), is applied manually, by spray or mechanically on a surface such as that of a wall or floor of a room of a hospital or asylum or of a school, said acid or a salt thereof contained in said inclusion compound (ci) ? capable of disposing, adhering and coating said surface in a uniform and homogeneous manner as if it were a coating paint.

Said D-usnic acid as enantiomer (i), preferably as pure enantiomer, is of natural origin and could be associated, as chemical structure, with that of the corresponding synthetic compound having CAS No. 7562-61-0, ie the dextrorotatory enantiomer of said acid. D-usnic acid as a pure enantiomer object of the present invention ? soluble in chloroform and in ethyl acetate. While, ? moderately soluble in ethanol, and ? insoluble in water. Said D-usnic acid as a pure enantiomer has a melting point ranging from 192?C to 204?C, a flash point? of 223?C and a boiling point of 605?C. Preferably, said salt of D-usnic acid as pure enantiomer ? a sodium salt. Said inclusion compound (ci) preferably comprises solid particles of D-usnic acid as pure enantiomer, or its salt, or mixtures thereof (i). Pi? preferably, said solid particles have an average particle distribution ranging from 0.01 µm to 50 µm, preferably ranging from 0.1 µm to 30 µm, plus? preferably included from 0.15 ?m to 20 ?m, even more? preferably between 0.2 ?m and 15 ?m. Said mean particle distribution ? been determined and measured with a laser diffraction method, according to the GB/T 19077-2016 standard, for example with a Malvern Mastersizer 3000 instrument. of this patent application. Preferably, said solid particles have an average particle distribution such that D10 = 0.236 ?m, D50 = 1.570 ?m, and D90 = 31.800 ?m. In an embodiment of the present invention, said solid particles have a distribution according to the diagram of Figure 2. Said solid particles are dispersed in the aqueous phase of the liquid composition, to give an aqueous liquid dispersion of D-usnic acid as a pure enantiomer.

D-usnic acid, or its salt or mixtures thereof (i) ? advantageously a natural and non-synthetic D-usnic acid and is preferably extracted from lichens. There are several methods of classifying lichens; one of these methods consists in examining the different forms of growth according to which we have:

- Fruticose lichens. The lichen species that belong to this group are Letharia vulpina, or those belonging to the genus Usnea, otherwise known as Beard of the woods and of the genus Ramalina.

- Leafy lichens. This typology includes lichens of the genus Parmelia, Collema, Physcia, Physconia, Xanthoria.

- Crustose lichens.

- Gelatinous lichens.

- Squamulous lichens. Some lichens that can be found in this category are: Catapyrenium psoromoides, Cladonia coniocraea, Cladonia bimbriata, Cladonia macilenta, Cladonia pyxidata, Normandina pulchella.

The lichens are preferably selected from the group which includes or alternatively consists of Usnea, Cladonia, Hypotrachyna, Lecanora, Ramalina, Evernia, Parmelia, Alectoria and combinations thereof, plus preferably from Usnea, even more? preferably from Usnea Longissima Ach., by means of an extraction process object of the present invention.

The attached figure 1 schematically shows a flow diagram of an embodiment of a process, in accordance with a possible embodiment of the same, for obtaining D-usnic acid of natural origin. In accordance with this process embodiment, the usnic acid in dried form is obtained following the following steps: (a.1) maceration and extraction from a plant material selected from a lichen preferably selected from the group comprising or, alternatively , consists of Usnea, Cladonia, Hypotrachyna, Lecanora, Ramalina, Evernia, Parmelia, Alectoria and their combinations, plus preferably of Usnea, even more? preferably of Usnea Longissima Ach., with an organic solvent, preferably a solvent selected from the group comprising or, alternatively, consisting of benzene, hexane, acetone, chloroform, trichlorethylene, or an alcoholic solvent, still more preferably ethanol, to give an extraction solution, and concentration of said extraction solution to give a concentrated extraction solution and a residual solvent;

(a.2) crystallization and filtration of the concentrated extraction solution, obtained from step (a.1), to give a crystallized and filtered extraction product;

(a.3) dissolving, filtering and concentrating the crystallized and filtered extraction product, obtained from step (a.2), to give a concentrated extract and a residual solvent;

(a.4) crystallization, filtration, and subsequently drying and grinding, of the concentrated extract, obtained from step (a.3), to give a ground dry extract of usnic acid, preferably having a titre ranging from 80% to 99.9 %, more preferably between 90% and 99.5%, even more? preferably ranging from 95% to 98%.

The maceration and extraction of step (a.1) is preferably carried out in an extraction tank, preferably made of stainless steel, equipped with stirring and heating means. Preferably, in the maceration and extraction of step (a.1) a ratio [weight of plant material]:[volume of organic solvent] ranging from 10:1 to 1:50, preferably ranging from 5:1 to 1:40 is used , even more? preferably ranging from 1:1 to 1:35. The maceration and extraction of step (a.1) are preferably carried out at ambient pressure, and at a temperature ranging from 10?C to 80?C, preferably ranging from 20?C to 70?C, even more? preferably between 25?C and 60?C.

The concentration of phase (a.1) is preferably carried out in a concentrator (or evaporator), more? preferably single effect, even more? preferably stainless steel.

In phase (a.1) the thallus (sprout or cutting) of Usnea (Longissima Ach.) is used together with the solvent ethyl acetate, for example in a quantity for example of 350 Kg of vegetable part and 2600 liters of solvent. The maceration is preferably carried out at a temperature of about 25°C and a pressure of 1 atmosphere for a period of time ranging from 2 hours to 10 hours, preferably from 4 hours to 8 hours, for example from 5 hours to 6 hours. The maceration can be carried out in a reactor equipped with stirring, heating and liquid recirculation means. In practice, the maceration is carried out by making a continuous recirculation of the distillate (solvent) on the vegetable part. The extraction, performed as a single phase, is carried out at a temperature of about 25°C and a pressure of 1 atmosphere. The concentration of the extraction solvent used, containing the usnic acid extracted from the vegetable part, is carried out by considering the boiling temperature of the ethyl acetate which is ? of about 77.1°C, possibly also by acting on the extraction pressure. A dense concentrated liquid is obtained and the solvent is almost completely recovered.

In the crystallization and filtration of step (a.2), subsequent to step (a.1), an organic solvent is preferably used to give the crystallized and filtered extraction product selected from the group comprising or, alternatively, consisting of benzene, hexane , acetone, chloroform, trichlorethylene, or an alcoholic solvent, even more? preferably ethanol. In the crystallization and filtration of step (a.2) a volume ratio [concentrated extraction solution]:[organic solvent] ranging from 10:1 to 1:40, preferably ranging from 5:1 to 1:30, is preferably used, even more preferably ranging from 1:1 to 1:20. In the crystallization of step (a.2), the concentrated extraction solution obtained from step (a.1) is preferably cooled to promote crystallization, more? preferably at a temperature between 1?C and 20?C at ambient pressure, even more? preferably ranging from 5°C to 15°C at ambient pressure. At the end of phase (a.2) a material in crystals is obtained with a purity of at least 80%, from 85% to 90% and in a quantity of about 20 kg, if you start from about 350 kg of vegetable part.

In step (a.3), following step (a.2), the crystallized and filtered extraction product obtained from step (a.2) is dissolved, filtered and concentrated to give a concentrated extract and the residual solvent. In step (a.3) an organic solvent is preferably used, more preferably selected from the group which includes or alternatively consists of benzene, hexane, acetone, chloroform, trichlorethylene, or an alcoholic solvent, still more preferably ethanol. In the dissolution of step (a.3) a ratio [weight of crystallized and filtered extraction product]:[volume of organic solvent] ranging from 10:1 to 1:40, preferably ranging from 5:1 to 1:30 is preferably used, even more preferably ranging from 1:1 to 1:20. In step (a.3) 2x20 kg are dissolved in chloroform to give 20 kg with a minimum purity of 98% of usnic acid.

In step (a.4) following step (a.3), the concentrated extract obtained from step (a.3) is crystallized, filtered, and subsequently dried and ground, to give the ground dry extract of usnic acid. In the crystallization of step (a.4) an organic solvent is preferably used, more preferably selected from the group which includes or alternatively consists of benzene, hexane, acetone, chloroform, trichlorethylene, or an alcoholic solvent, still more preferably ethanol. In the crystallization of step (a.4) a ratio [concentrated extract weight] is preferably used:

[organic solvent volume] ranging from 10:1 to 1:40, preferably ranging from 5:1 to 1:30, even more? preferably ranging from 1:1 to 1:20. In the crystallization of step (a.4), the concentrated extract obtained from step (a.3) is preferably cooled to favor crystallization, more preferably at a temperature between 1?C and 20?C at ambient pressure, even more? preferably ranging from 5°C to 15°C at ambient pressure. The drying of phase (a.4) ? preferably carried up to a residual solvent content ranging from 0.5% to 10% by weight, preferably ranging from 1% to 5% by weight, even more? preferably ranging from 1.5% to 3% by weight, with respect to the total weight of the dry extract of usnic acid. Preferably, the grinding of step (a.4) is carried out by means of a mill, more preferably a rotary ball mill. The drying of the filtered and crystallized solid obtained from step (a.4) is completed when a residual solvent content of about 2%-5% by weight remains, with respect to the initial weight. A plate dryer (without pressure vacuum) is used at a temperature of about 95?C-99?C with air circulation. The ground solid has an average particle distribution ranging from 20 mesh to 40 mesh and contains usnic acid at 98% by weight (HPLC with the Sigma Aldrich method. Starting from 2x350 kg of vegetable part at the beginning of the process (starting material) obtains about 3%-4% of material yield (dry solid) at the end of the process, which is equivalent to about 14Kg-28Kg of usnic acid with a content of 98% by weight (13.72 Kg-27.44Kg). The obtained usnic acid is in the form D(+) pure usnic acid at 99.9%, or as a racemate.

After obtaining the ground dry extract of usnic acid (step (a.4)) as a racemic mixture, said dry extract is selectively complexed with cyclodextrins, preferably betacyclodextrins, to give said inclusion compound (ci). Preferably, said selective complexation ? obtained by co-precipitation of D-usnic acid, or its salt, or mixtures thereof (i) and cyclodextrins (ii). The inclusion compound (ci) is preferably obtained by co-precipitation of D-usnic acid, or its salt, or mixtures thereof (i) and cyclodextrins (ii), preferably betacyclodextrins. Pi? precisely, the cyclodextrins (ii) are initially dissolved in water or other suitable aqueous solvent, and subsequently the ground dry extract of usnic acid of step (a.4) is added, while the aqueous solution containing the cyclodextrins (ii) is kept under agitation. In the presence of a sufficiently high concentration of cyclodextrins (ii) in solution, it will start? to have precipitation of the compound of inclusion (there) as you proceed? the complexation reaction of D-usnic acid, or its salt, or mixtures thereof (i) by cyclodextrins (ii). Preferably, the solution containing the inclusion compounds (ci) may need to be cooled to a temperature ranging from 1?C to 18?C, preferably under stirring, so that precipitation begins. The inclusion compounds (ci) can be collected by decantation, centrifugation or filtration. The inclusion compound (ci) ? preferably a water-soluble clathrate or a water-suspendable clathrate, in which said D-usnic acid as a pure enantiomer, or its salt, or mixtures thereof (i) is housed in a cavity? of said clathrate, once said D-usnic acid, or its salt, or mixtures thereof (i) ? brought into contact with said cyclodextrins (ii). D-usnic acid, or its salt, or mixtures thereof (i) and the cyclodextrins (ii), preferably the betacyclodextrins, are present in the inclusion compound (ci) preferably in a ratio by weight ranging from 3:1 to 1 :3, preferably in a weight ratio of 2:1 to 1:2, plus? preferably between 1.5:1 and 1:1.5, even more? preferably being 1:1. In case (2-hydroxypropyl)-?cyclodextrin is used, the weight ratio with D-usnic acid ? 1:1. The inclusion compound (ci) ? preferably used as an antibacterial or bacteriostatic agent for both Gram-negative and Gram-positive pathogenic bacteria, preferably for Gram-negative bacteria, for which said inclusion compound (ci) is? proved to be particularly effective.

Preferably, the Gram-negative bacteria in relation to which the inclusion compound (Cl) exhibits an antibacterial or bacteriostatic function are selected from the group comprising or, alternatively, consisting of: Escherichia Coli, Klebsiella, Acinetobacter baumannii, and combinations thereof. Preferably, the Gram-positive bacteria in relation to which the inclusion compound (Cl) exhibits an antibacterial or bacteriostatic function are selected from the group comprising or, alternatively, consisting of: Staphylococcus aureus, methicillin resistant Staphylococcus aureus (MRSA), Enterococcus, Enterococcus spp. vancomycin resistant (VRE), Actinobacter, Actinobacter spp., Clostridium difficile, and combinations thereof. Preferably, in this use, said inclusion complex (ci) is added in the process of preparing a product in the form of a film or layer made of plastic, resin or thermoplastic polymer, polyethylene PE, polyvinyl chloride PVC, polyethylene terephthalate PET, latex; or said inclusion complex (ci) is spread or positioned on the surface of said article in an amount ranging from 0.1% to 20% by weight, with respect to the weight of the manufactured article.

The object of the present invention is also a liquid composition which comprises or, alternatively, consists of:

(a) said inclusion compound (ci);

(b) an acrylic resin, a polyurethane resin or an acryl-polyurethane resin, or mixtures thereof;

(c) optionally a pigment or opacifying agent; (of water.

Said liquid composition has an?Activity? bacteriostatic or antibacterial against both Gram-positive and Gram-negative pathogenic bacteria, such as those with the scientific name Klebsiella, Enterobacteriaceae, Enterobacter, Pseudonomas and Escherichia. This liquid composition can be in the form of an aqueous solution or an aqueous dispersion or an aqueous suspension or an aqueous emulsion.

The acrylic resin (b), used in conjunction with (a) said inclusion compound (ci) in said composition, preferably comprises monomers selected from the group comprising or, alternatively, consisting of acrylic acid, acrylic acid ester, methacrylic acid, ester of methacrylic acid, styrene, vinyltoluene, vinyl acetate, vinylester of carboxylic acids higher than acetic acid, acrylonitrile, acrylamide, butadiene, ethylene, vinyl chloride, and mixtures thereof. Pi? preferably, the acrylic resin (b) comprises or alternatively consists of a methacrylic acid copolymer? styrene.

The optional pigment or opacifying agent (c), present in said liquid composition together with the inclusion compound (ci) and the acrylic resin (b), is preferably selected from the group consisting of iron oxides, titanium oxides, cobalt-based dyes, phthalates, azo dyes, and mixtures thereof. Preferably, said pigment or opacifying agent comprises or alternatively consists of titanium dioxide.

The water (d), present in said liquid composition together with the inclusion compound (ci), the acrylic resin (b) and the optional pigment or opacifying agent (c), has no particular limitations. Preferably, the water (d) ? tap water, purified water, or deionized water.

Said liquid composition preferably comprises: (a) the inclusion compound (ci) in an amount ranging from 0.1% to 15% by weight, preferably ranging from 0.2% to 10% by weight, even more? preferably ranging from 0.3% to 7% by weight, with respect to the total weight of said liquid composition;

(b) said acrylic resin, said polyurethane resin or said acryl-polyurethane resin in an amount ranging from 1% to 80% by weight, preferably ranging from 2% to 75% by weight, even more? preferably ranging from 5% to 70% by weight, with respect to the total weight of said liquid composition;

(c) optionally said pigment or opacifying agent in an amount ranging from 10% to 40%, preferably ranging from 15% to 35% by weight, even more? preferably ranging from 20% to 30% by weight, with respect to the total weight of said liquid composition;

(d) water in a quantity? ranging from 1% to 40%, preferably ranging from 2% to 30% by weight, even more? preferably ranging from 3% to 22% by weight, with respect to the total weight of said liquid composition.

The object of the present invention is a use of said liquid composition as an architectural paint or coating, preferably as a masonry coating or paint or for floors, for example for linoleum floors, more preferably as an antibacterial or bacteriostatic architectural coating for both Gram-positive and Gram-negative pathogenic bacteria.

Finally, the object of the present invention is the use of cyclodextrins, preferably of beta-cyclodextrins, more preferably of (2-hydroxypropyl)-?cyclodextrin, as selective complexing agent(s) of D-usnic acid, or a salt thereof, or mixtures thereof (i), from a racemic (or racemic) mixture of usnic acid of natural origin .

Examples of surfaces where the mixtures or products of the present invention can be applied are horizontal or vertical surfaces, for example floors, walls or ceilings made for example in cement, lime or plasterboard, linoleum, or in PVC polyvinyl chloride, PA polyamide, PE polyethylene, PES polyester or PTF polyethylene terephthalate. These types of surfaces, without limitation, can be found for example in a doctor's surgery, emergency room, hospital, dentist's office, playground, kindergarten, school or toilets and toilets present, for example, in public or private structures or, for example, in supermarkets and shopping malls or playgrounds.

The object of the present invention is a mixture M comprising or, alternatively, consisting of (a) a usnic acid of natural origin and/or (b) a relative salt thereof.

Said (a) a usnic acid of natural origin, contained in said mixture M, ? a combination or C/A association between a natural dextrorotatory D(+) usnic acid and a natural levorotatory L(-) usnic acid.

With the term ?combination? in the context of the present invention it is meant that for example the natural dextrorotatory usnic acid D(+) and the natural levorotatory usnic acid L(-) are together, simultaneously present in contact with each other, before their use, while with the term ? Association? in the context of the present invention it is understood that for example the natural dextrorotatory usnic acid D(+) and the natural levorotatory usnic acid L(-) are separated from each other, before their use, and can be brought into contact with each other, to time of their use. The present meaning of ?combination? and ?association? between substances? also applicable for example to the salt of usnic acid, as well as to other substances or compounds used in the present invention.

With the term ?natural? or ?of natural origin? or ?usnic acid or salt of natural usnic acid or of natural origin? it means that said usnic acid or salt of? usnic acid ? obtained from a plant, in particular from a plant of the family Usneaceae, genus Usnea.

Preferably, the dextrorotatory form D(+), in said (a) a usnic acid of natural origin, is present in a quantity ranging from 0.1% to 99.9% by weight, with respect to the total weight of the C/A combination or association, while the levorotatory form L(-), in said (a) a usnic acid of natural origin, is present in a quantity ranging from 99.9% to 0.1% by weight, based on the total weight of the C/A combination. For example, usnic acid can? be present as racemic 50% (+) and 50% (-), or for example as 100% dextrorotatory form D(+).

Said (b) a salt of usnic acid, contained in said mixture M, is a salt of an alkali or alkaline earth metal. Preferably, said (b) a salt of usnic acid ? a salt of the dextrorotatory D (+) form of usnic acid and which can? be present in a quantity? by weight ranging from 0.1% to 99.9% by weight, with respect to the total weight of the C/A combination or association, and the L(-) levorotatory form which can? be present in a quantity? ranging from 99.9% to 0.1% by weight, based on the total weight of the C/A combination. For example, the salt of usnic acid, preferably the sodium salt of usnic acid, can be present as racemic 50% (+) and 50% (-), or for example as the sodium salt of dextrorotatory usnic acid D(+).

Said (a) a usnic acid of natural origin and said (b) a relative salt thereof are present, in said mixture M, in a weight ratio ranging from 1:10 to 10:1, preferably from 1:5 to 5: 1, even more preferably from 1:3 to 3:1; for example 3:1, 2.5:1, 2:1, 1.5:1 or 1:1. Said mixture M pu? be in a solid or semi-solid state, in a dispersed or suspended form, in the form of a cream or paste or gel, or in a liquid state; preferably said mixture M pu? be in the form of flakes, granules, powders, pellets, or pu? be an aqueous, or hydroalcoholic solution or, in organic solvents. Said (a) a usnic acid of natural origin and/or said (b) a relative salt thereof are in solid powder form with an average granular size ranging from 1 micron to 100 micron, preferably from 5 micron to 50 micron, even more ? preferably from 10 microns to 20 microns.

The usnic acid can? be represented, for example, as follows: (+)-usnic acid 2,6-Diacetyl-7,9-dihydroxy-8,9-dimethyldibenzo[b,d]furan-1,3(2H,9bH)-dione; (+)-usnic acid from Usnea; CAS: 7562-61-0, EC: 231-456-0. The sodium salt of usnic acid can? be represented, for example, as follows: 2,6-diacetyl-7,9-dihydroxy-8,9b-dimethyldibenzofuran-1,3(2H,9bH)-dione monosodium salt; CAS: 34769-44-3, EC: 252-204-6. The purity of said (a) a usnic acid and/or of said (b) a salt of usnic acid ? between 95% and 99.9%, preferably between 96% and 99.5%, even more? preferably from 97% to 98%, for example 98%.

The object of the present invention is a semi-finished product PS, preferably in the form of a cream or semi-solid paste, comprising said mixture M, and a resin.

Said mixture M comprises or, alternatively, consists of said (a) a usnic acid of natural origin and/or said (b) a relative salt thereof, preferably said mixture M ? present in said semi-finished product PS in a quantity? by weight ranging from 20% to 80%, preferably from 35% to 65%, even more? preferably from 40% to 50%, for example 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48% or 49%, with respect to the total weight of said semi-finished product PS.

In one embodiment said mixture M, contained in the semi-finished product PS, comprises only said (a) a usnic acid. In this case, said (a) a usnic acid ? present in said semi-finished product PS in a quantity? by weight ranging from 20% to 80%, preferably from 35% to 65%, even more? preferably from 40% to 50%, for example 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48% or 49%, with respect to the total weight of said semi-finished product PS.

In another embodiment said mixture M, contained in the semi-finished product PS, comprises both said (a) a usnic acid and said (b) a relative salt thereof, preferably a sodium salt. In this case, said usnic acid ? present in said semi-finished product PS in a quantity? by weight ranging from 10% to 60%, preferably from 20% to 50%, even more? preferably from 30% to 40%, for example 24%, or 32%, with respect to the total weight of said semi-finished product PS. While, said (b) salt of usnic acid ? present in said semi-finished product PS in a quantity? by weight ranging from 5% to 50%, preferably from 10% to 40%, even more? preferably from 15% to 30%, for example 16%, 18%, 20%, 22%, 24%, 26% or 28%, with respect to the total weight of said semi-finished product PS.

Said resin? present, together with the mixture M, in said semi-finished product PS in a quantity? by weight ranging from 20% to 70%, preferably from 30% to 60%, even more? preferably from 35% to 50%, for example 38%, 40%, 42% or 45%, with respect to the total weight of said semi-finished product PS.

PS semi-finished product can? comprise, in addition to the mixture M and the resin, preferably also (i) water in a quantity? by weight ranging from 5% to 30%, preferably from 10% to 20%, for example 15%, with respect to the total weight of the semi-finished product PS; (ii) additives, preservatives and a glycol, such as a propylene glycol or diethylene glycol, in an amount by weight ranging from 0.5% to 5%, preferably from 1% to 1.5%, for example 2%, with respect to the total weight of the semi-finished product PS.

Preferably, the PS semi-finished product can contain, in addition to said mixture M and said resin, also a preservative, for example, as a mixture of two preservatives, 5-chloro-2-methyl-2H-isothiazol-3-one [EC no.

247-500-7] and 2-methyl-2H-isothiazol-3-one [EC no. 220-239-6] in a weight ratio of 3:1, Index Number: 613-167-00-5 and CAS: 55965-84-9.

Preferred embodiments of a PS semi-finished product of the present invention are shown in Table 1.

Table 1

The resins are selected from the group comprising or, alternatively, consisting of polyurethanes, urethanes, polyacrylics, acrylics, polyvinyls, vinyls, polyamides or amides known to the person skilled in the art.

The object of the present invention is a finished product PF comprising said semi-finished product PS, and a varnishing product.

Said semi-finished product PS contained in said finished product PF? present in a quantity by weight ranging from 0.1% to 10%; preferably from 0.5% to 8%; even more preferably from 1% to 6%, for example 1%, 2%, 3%, 4%, or 5%, with respect to the weight of the coating product. In one embodiment, said coating product can be present in said finished product PF, together with said semi-finished product PS, preferably in the liquid state, dispersion or aqueous dispersion.

In one embodiment, said coating product can be preferably selected from the group comprising or, alternatively, consisting of water-soluble varnishes, enamels, paints or paints; preferably said varnishes, enamels or paints, for external or internal surfaces, are preferably selected from those based on water, or based on organic solvents. For example, can be used a water-based paint for exterior or interior surfaces.

In one embodiment, said water-based paint product can be preferably chosen among the one or two-component ones compatible for surfaces, for interiors or exteriors, in masonry, linoleum or wood for application by spray, brush or roller.

In another embodiment, said painting product based on an organic solvent can be preferably chosen among the two-component acrylic and/or methacrylic and/or urethane and/or polyurethane based ones and/or for surfaces, for interiors or exteriors, for example surfaces in glass, aluminium, steel, plastic, polymer, linoleum, fabric, natural leather, synthetic leather, wood, natural fabric, artificial fabric or synthetic fabric for application by spray or roller or brush.

The finished product PF of the present invention can? be viewed as a fluid solution with a polymer matrix and a solute (dextrorotatory usnic acid and/or a salt thereof, such as a sodium salt). For this reason, water-based and solvent-based paints, varnishes and enamels can be considered to fall within painting products. The varnishes to give rise for example to a transparent film. Furthermore, taking into account that paints, varnishes and enamels have multiple applications, the raw materials such as solvents, polymeric matrices (resins), additives and pigments/fillers are listed below. common and used in the paint, varnish and enamel sector for professional use. In the case of paints, for example transparent, you can? talk about solutions; while we speak of dispersions, when the various components are dispersed, not dissolved.

List of solvents pi? commonly used for the preparation of varnishes (clears), enamels (colored) and paints (building products): water, butyl alcohol, isopropyl alcohol, ethyl acetate, n-butyl acetate, iso-butyl acetate, toluene, xylene , naphtha solvent, white spirit, acetone, methyl ethyl ketone, methyl isobutyl ketone, butyl glycol, dibutyl glycol, glycol ethers, methoxypropyl acetate.

List of polymeric matrices pi? common: resins are usually found in solvent solution or in aqueous emulsion. The polymer matrices are dissolved in solvents and in emulsion/dispersion in water where they do not dissolve. For example, synthetic or vegetable oils, vegetable fatty acids, castor, saturated or unsaturated fatty acids.

Until now we have seen the categories of components that make up paints (which deposit a more or less glossy transparent film on the product), for paints (low in resinous polymer) and enamels (rich in resin) pigments and fillers and colorants in specific cases. Then there are the functional components. Fillers characterize paints and primers, pigments enamels. For example, as fillers we can consider: calcium carbonate, mica, talc, barium sulphate, quartz; functional pigments such as: zinc phosphate, iron oxide; anticorrosive pigments: aluminum paste; pigments: titanium dioxide, iron oxides, yellow, orange, red, green, blue, magenta violet organic pigments; effect pigments (optical interference).

In one embodiment, the PF finished product for applications such as natural or synthetic leather or leather can be added to a base (paint product) having for example the following composition: (i) water in a quantity? by weight ranging from 75% to 85%, preferably from 78% to 80%; (ii) SiO2 in an amount? by weight between 1% and 8%, preferably between 2% and 5%; (iii) di-propyl glycol dimethyl ether in an amount by weight ranging from 0.5% to 5%, preferably from 1% to 2%; (iv) siloxanes and silicones in a quantity? by weight ranging from 2% to 5%, preferably from 2.5% to 3.5%; and (v) polymers in an amount? by weight ranging from 10% to 20%, preferably from 16% to 18%. In this case you can also use a crosslinker to carry out a crosslinking to obtain a film with characteristics such as to resist abrasion tests, such as the Taber test, rubbing with alcohol and petrol. As for the fabrics, for example a non-woven fabric (TNT) in polypropylene or polyester, you can? use the following solution (finished product) containing 100 parts by weight of demineralised water, 0.6 parts by weight of racemic usnic acid, or the dextrorotatory form D(+), and 0.9 parts by weight of beta-cyclodextrins.

In another embodiment, the finished product PF for applications for example on walls or ceilings and surfaces (horizontal and vertical) in cement or plasterboard, linoleum or wood can be added to a base (varnishing product ? mono- and bi-component transparent opaque water-based top coat) having for example the following composition: (i) resin (as part of the varnishing product) in a quantity? by weight ranging from 60% to 80%, preferably 70%, for example 72%; (ii) inert additives in a quantity? by weight ranging from 2% to 10%, preferably from 3.5% to 8%, for example 6.5%; (iii) water in a quantity? by weight ranging from 10% to 30%, preferably from 15% to 25%, for example 17%; (iv) dipropylene glycol monomethyl ether in an amount by weight ranging from 0.5% to 5%, preferably from 1% to 3%, for example 2%; (v) diethylene glycol in an amount? by weight ranging from 1% to 4%, preferably from 1.5% to 3%, for example 2.5%. This basis? for applications, for example, for wooden or parquet surfaces, also for exteriors. This base has excellent surface hardness, abrasion resistance, chemical resistance and UV resistance. To further increase its chemical resistance you can, for example, add a quantity by weight ranging from 3% to 15%, preferably 10%, with respect to the total weight of the finished product PF, of a catalyst which comprises for example an amount? by weight ranging from 70% to 90%, preferably 80% of a polyisocyanate resin and an amount by weight ranging from 10% to 30%, preferably 20% of a propylene carbonate, with respect to the total weight of the catalyst. This PF finished product can? be applied by spray, roller or brush technique.

In another embodiment, the finished product PF for applications for example on glass, aluminum or steel can be added to a base (paint product - two-component acrylic glossy transparent varnish) having for example the following composition: (i) acrylic resin in a quantity? by weight ranging from 60% to 85%, preferably from 70% to 80%, for example 75%; (ii) xylene in a quantity? by weight ranging from 10% to 30%, preferably from 15% to 25%, for example 20%; (iii) additives in a quantity? by weight ranging from 0.5% to 4%, preferably from 1% to 3%, for example 2%. This basis? capable of giving a highly resistant and tough coating with high resistance to light and, therefore, suitable for exterior and interior applications. To further increase the chemical resistance of the PF finished product, when applied to glass, it can be for example, add a quantity? by weight ranging from 1% to 10%, preferably from 3% to 5% with respect to the total weight of the finished product PF of a catalyst which comprises an aliphatic polyisocyanate resin in an amount? by weight ranging from 30% to 50%, preferably from 35% to 45%, for example 40%; xylene in a quantity? by weight ranging from 20% to 40%, preferably from 25% to 35%, for example 30%; methyl ethyl ketone in a quantity? by weight ranging from 20% to 40%, preferably from 25% to 35%, for example 30%, with respect to the total weight of the formulation. This PF finished product can? be applied by spray technique.

Preferred embodiments FPn of the present invention are reported below.

FP1. An inclusion compound (ci) comprising or, alternatively, consisting of: (i) a D-usnic acid as enantiomer, or a salt thereof, or mixtures thereof, of natural origin, and (ii) beta-cyclodextrins.

FP2. The inclusion compound (ci) according to FP1, wherein D-usnic acid, or a salt thereof, or mixtures thereof (i) and beta-cyclodextrins (ii), preferably (2-hydroxypropyl)-?- cyclodextrins, are present in said inclusion compound (ci) in a weight ratio ranging from 3:1 to 1:3, preferably ranging from 2:1 to 1:2, plus? preferably between 1.5:1 and 1:1.5, even more? preferably being 1:1.

FP3. The inclusion compound (ci) according to FP1 or FP2, wherein D-usnic acid, or a salt thereof, or mixtures thereof (i) is? extracted from lichens, preferably selected from the group which includes or, alternatively, consists of Usnea, Cladonia, Hypotrachyna, Lecanora, Ramalina, Evernia, Parmelia, Alectoria and combinations thereof, plus? preferably from Usnea, even more? preferably from Usnea Longissima Ach., and wherein said cyclodextrins (ii) comprise or, alternatively, consist of betacyclodextrins, preferably ? the (2-hydroxypropyl)-?cyclodextrin, in a 1:1 ratio.

FP4. The inclusion compound (ci) according to any one of FP1-FP3, wherein said inclusion compound (ci) comprises solid particles of Dusnic acid as a pure enantiomer, or its salt, or mixtures thereof (i), wherein said solid particles have an average particle distribution ranging from 0.01 ?m to 50 ?m, preferably ranging from 0.1 ?m to 30 ?m, plus? preferably included from 0.15 ?m to 20 ?m, even more? preferably between 0.2 ?m and 15 ?m.

FP5. Use of an inclusion compound (Cl) according to any of FP1-FP4 as an antibacterial or bacteriostatic agent for both Gram-negative and Gram-positive bacteria; wherein said bacteria are preferably selected from the group comprising or, alternatively, consisting of: Escherichia Coli, Klebsiella, Acinetobacter baumannii, Staphylococcus aureus, methicillin resistant Staphylococcus aureus (MRSA), Enterococcus, Enterococcus spp. vancomycin resistant (VRE), Actinobacter, Actinobacter spp., Clostridium difficile, and combinations thereof.

FP6. Use according to FP5, wherein said inclusion complex (ci) is added in the process of preparing a product in the form of film or layer in plastic, resin or thermoplastic polymer, polyethylene PE, polyvinyl chloride PVC, polyethylene terephthalate PET, latex; or said inclusion complex (ci) is spread or positioned on the surface of said article in an amount ranging from 0.1% to 20% by weight, with respect to the weight of the manufactured article.

FP7. A liquid composition comprising or alternatively consisting of:

(a) an inclusion compound (c1) according to any one of claims 1-4;

(b) an acrylic resin, a polyurethane resin, an acryl-polyurethane resin, or mixtures thereof;

(c) optionally a pigment or opacifying agent; (of water.

FP8. The liquid composition according to FP7, comprising or, alternatively, consisting of: (a) the inclusion compound (ci) in an amount? ranging from 0.1% to 15% by weight, preferably ranging from 0.2% to 10% by weight, even more? preferably ranging from 0.3% to 7% by weight, with respect to the total weight of said liquid composition;

(b) said acrylic resin, said polyurethane resin, said acryl-polyurethane resin or mixtures thereof, in an amount ranging from 1% to 80% by weight, preferably ranging from 2% to 75% by weight, even more? preferably ranging from 5% to 70% by weight, with respect to the total weight of said liquid composition;

(c) optionally, said pigment or opacifying agent in an amount ranging from 10% to 40%, preferably ranging from 15% to 35% by weight, even more? preferably ranging from 20% to 30% by weight, with respect to the total weight of said liquid composition;

(d) water in a quantity? ranging from 1% to 40%, preferably ranging from 2% to 30% by weight, even more? preferably ranging from 3% to 22% by weight, with respect to the total weight of said liquid composition.

FP9. Use of the liquid composition according to any of FP7-FP8 as an architectural paint or coating, preferably as a masonry or wall and floor coating or paint, for example for linoleum floors, more preferably as an antibacterial or bacteriostatic architectural coating for Gram-negative and Gram-positive bacteria.

FP10. Use of beta-cyclodextrins, preferably of (2-hydroxypropyl)-?-cyclodextrin, as a selective complexing agent of D-usnic acid as a pure enantiomer, or its salt, or mixtures thereof (i), from a racemic (or racemic) mixture of usnic acid of natural origin.

Some examples, provided by way of non-limiting example, of the present invention are given below. Is ISO 22196 being considered to measure the activity? antibacterial of the mixture M, the semi-finished product PS and the finished product PF, all in accordance with Table 1, applied on the plastic surfaces.

Advantageously said mixture M, said semi-finished product PS and said finished product PF meet the requirements of the following standards:

UNI EN ISO 7784:2016 (resistance to abrasion) and UNI EN ISO 18593:2018.

Advantageously said mixture M, said semi-finished product PS and said finished product PF do not require an activator of light or external energy such as for example UV light or light at any wavelength, since once applied on a surface they are able to self-fix.

EXPERIMENTAL PART BY EXAMPLES.

EXAMPLE 1: Efficacy test of the aqueous liquid composition of the present invention in relation to the Gram-positive pathogen S. aureus (MRSA).

The aqueous liquid composition ? tested for efficacy against the Gram-positive pathogen S. aureus, according to the ISO 22196:2007 test method. The aqueous liquid composition comprises the inclusion compound D-usnic acid as a pure enantiomer of natural origin and (2-hydroxypropyl)-?-cyclodextrin.

The type of material tested? state of 6 untreated samples (divided into two groups) and of 6 treated samples (divided into two groups) with the composition according to the present invention so? subdivided:

- References (time 0h): CTRL1, CTRL2, CTRL3;

- References (time 24h): CTRL4, CTRL5, CTRL6;

- Samples treated with the composition according to the present invention: SSC7, SSC8, SSC9;

- Samples treated with the composition according to the present invention subjected to the aging method: SSC10, SSC11, SSC12.

The analyzed sample includes a 50x50 mm square plastic support coated with the material to be tested, treated by painting. ? a polyethylene cover film, square shape, 40x40 mm, thickness 0.1 mm was used. The bacterial strain tested? Methicillin resistant Staphylococcus aureus (MRSA) ATCC 43300 (10<6 >cells/ml), with a bacterial inoculum volume of 0.4ml. The modifications made to the International Standard protocol are a volume of Neutralizer (SCDLP) = 20 mL.

The test ? to be considered valid as it satisfies the following conditions dictated by the ISO 22196:2007 standard:

1) (LOGMAX - LOGMIN)/ LOGMEAN ? 0.2

2) Is the average number of viable bacteria immediately after inoculation of the untreated test (reference) within the range of 6.2 x 10<3 >cells/cm<2 >? 2.5 x 10<4 >cells/cm<2>;

3) The number of viable bacteria in each control sample after the 24-hour incubation does not appear to be less than 6.2 x 10<1 >cells/cm<2>.

The activity bacterial R? calculated according to the following equation:

R = (UT - U0) - (AT - U0) = UT - AT

The activity bacterial R? shown in the attached figure 3.

EXAMPLE 2: Efficacy test of the aqueous liquid composition according to Example 1 in relation to the Gram-negative pathogen E. coli.

Yup ? proceeded as in example 1, but in this case the bacterial strain tested? was a Gram negative pathogenic strain of Escherichia coli ATCC 8739 (6x10<5 >cells/ml).

The activity bacterial R? calculated according to the following equation:

R = (UT - U0) - (AT - U0) = UT - AT

The activity bacterial R? shown in the attached figure 4.

From the previous Example 1 and Example 2 it emerges, after a contact of just 24 hours, a reduction of the vitality? approximately 100% (R% > 99.99 %), a result calculated, as per the guideline, in the logarithmic efficacy index of the antimicrobial material.

This reduction? found indistinctly on both types of microorganisms tested (RE.Coli log = 4.96; RSt.Aureus log= 4.30) with very similar values, advantageously showing an effective capacity? antibacterial against both Gram-positive and Gram-negative pathogenic bacteria.

From this consideration it follows that the composition object of the present invention can be useful for multiple purposes, from everyday life to sectoral environments: business, domestic, clinical-hospital.

EXAMPLE 3: Efficacy test of the aqueous liquid composition according to Example 1 in non-specific terms of the level of bacterial contamination.

The laboratory results obtained from surface swabs to indicate in non-specific terms the level of bacterial contamination of the sampled point are summarized below.

In a first series of tests performed in a hospital, yes? identified a room used as a dental care clinic on whose surfaces, previously,? the aqueous liquid composition of Example 1, object of the present invention, was applied, which made said surfaces resistant to bacterial contamination.

In a second series of tests carried out in a nursery, on the treadable surfaces of the floors of the rooms (common area and rest room) ? the aqueous liquid composition of Example 1, object of the present invention, was applied, which made said surfaces resistant to bacterial contamination.

To be able to have traceability? of the above results? Was the UNI EN ISO 18593:2018 standard ?horizontal method for surface sampling? with contact plate. In particular ? the ?Contact slide 2 Liofilchem? was used. Before applying the aqueous liquid composition of Example 1, object of the present invention, swabs were also carried out on the walls and surfaces to determine the level of bacterial contamination which represents the natural background (initial situation) . After having applied said composition under study, the samplings were programmed.

The activities they took place under normal conditions of use of the dental clinic room (hospital facility), or of the rooms used to look after children (kindergarten). Therefore, the present study ? was conducted in a situation of real presence of the bacterial population, possibly, in some cases, even in the presence of users of the clinic. These conditions confirm the tendency to resistance to re-contamination of the treated surfaces given that point and non-distributed contaminations have been detected on all surfaces.

The materials used for the activities? to verify the bacterial load, in addition to the individual protective devices (overall, gloves and mask), the Liofilchem contact plates item code 525272 for specific use according to the indicated standard, the product for professional use requires the direct use of the surface of the plate contact with the wall of the room or with the floor that can be walked on, the subsequent incubation in a thermostat chamber at 30?C for 24 hours and reading of the results expressed in Units? Colony Formers (CFU)/cm<2>.

For tests in hospital facilities, the agreed protocol provides for the identification of four distinct points for each of the four walls present in the outpatient room with identification of the points in an anti-clockwise direction, from one to four, following a logic of distribution of the surfaces to be sampled as quickly as possible. representative as possible. It must therefore be considered that each sample represents 10 cm<2> in terms of surface area. Therefore, each sampling session analyzed a surface consisting of 16 samples, four against the wall, for a total of 160 cm<2>. The samples were marked by the number from 1 to 4 for the four positions on the wall, therefore starting from the wall on the right from the entrance door of the room, the front wall to the armchair with a small window, the wall with windows and finally the wall to left of the entrance door, as shown by the photos in the attached figure 5.

In the execution of the samplings in hospital yes? followed a distribution logic of the points to be surveyed such as to cover the largest possible surface during the four sampling sessions.

The following Table 2 summarizes the results of the separate samplings in the hospital facility by sampling day:

Table 2

For the tests in the nursery, the agreed protocol provides for the identification of a series of points distributed on the treadable surface of the floors of the rooms used following a logic of distribution of the surfaces to be sampled as quickly as possible. representative as possible. It must therefore be considered that each sample represents 10 cm<2> in terms of surface area.

The test samples in the two areas present in the nursery are summarized in the following Table 3 (common area) and Table 4 (rest room).

Table 3

Table 4

In conclusion, the data collected in the sampling sessions report a resistance to recontamination of the surfaces in overall terms considering the starting values, possibly also the type of colonies that are always punctiform and isolated, considering the initial values, the reduction of the microbial load of the surfaces subjected upon treatment with the composition object of the present invention:

- in tests performed in hospitals, it reaches 90%, as can be seen from the graph shown in figure 6, having gone from just under 3000 CFU/cm<2 > to values close to 300 CFU/cm<2>. Another important fact to note? the numerical constant of the results achieved (468.75 CFU/cm<2>, 468.75 CFU/cm<2>, 405.0 CFU/cm<2>, 312.5 CFU/cm<2>) obtained with a data tending to decrease in the number of microorganisms over time.

- In the tests performed in the nursery, the bacterial load ? residual, between 8% and 12%, as can be seen from Table 3 and Table 4 above.

EXAMPLE 4: Further evidence of efficacy of the aqueous liquid composition according to Example 1 in non-specific terms of the level of bacterial contamination.

The tests were carried out in accordance with ISO 18593, and show excellent repeatability.

The withdrawal conditions were as follows:

Reference sample: Inpatient room / post-sanitization outpatient clinic;

Post-treatment samples: Inpatient / outpatient room in use.

These conditions represent the worst case for verifying the efficacy of the treatment with the composition object of the present invention.

The treatment, after obtaining a series of excellent in vitro results (ISO 22196 and ASTM 2180), is proposed as a candidate for the prevention and control of the bacterial load on nosocomial surfaces.

For the in vivo validation, bacterial load samples were taken using ?Contact slide ISO 18593? before and after said treatment to monitor the trend of the generic charge expressed in CFU/m<2 > (colony forming units per square metre).

Other samples were taken approximately 25 days later in order to monitor the efficacy trend over time.

Laboratory tests using accelerated aging have shown excellent stability? of effectiveness over time (effectiveness guaranteed for 3 years).

Does the number of samples taken guarantee excellent validity? of the statistical data obtained (up to 32 swabs for each room for each step).

This validation considers 5 different hospital environments which can be classified as outpatient clinics and hospital rooms.

Were the tests performed in compliance with the pi? recent guidelines for the microbiological monitoring of hospital environments.

The attached figures 7, 8, 9, 10 show the results, divided by room and by sampling date, and their trend over time. Furthermore, the following Table 5 shows ? figure by figure - the percentages of reduction / abatement of the bacterial load.

Table 5

* sampling data (125 CFU/m2) performed at an anomalous moment, of maximum activity, of the clinic.

Advantageously, the surface treated with the aqueous liquid composition of Example 1, object of the present invention, is constantly sanitized, thanks to the inclusion compound (ci) contained in it. Advantageously, this sanitization efficacy (reduction of the pathogenic charge) ? constant for at least 3 years from application, as verified by tests after accelerated aging according to ISO 22196:2007(E) standard (in the version in force on the priority date of this patent application).

Advantageously, the aqueous liquid composition object of the present invention (such as for example that of Example 1) finds particular use in the hospital environment, since said composition appears suitable for limiting the nocosomial bacterial load (and not only) both against pathogenic bacteria Gram-positive than against Gram-negative ones. This unexpected result advantageously allows patients to stay in an environment that is as close as possible to sterile as possible.

EXPERIMENTAL PART B EXAMPLES.

EXAMPLE 1

1.1 Purpose

Verify the microbicidal effectiveness of devices treated with the antimicrobial agent object of the present invention based on usnic acid and/or a relative salt thereof, of natural origin, preferably the sodium salt, in the racemic or dextrorotatory form D(+). ? a test was performed according to the procedure described, reference standard ASTM E2180-07, using microbial strains considered as indicative.

2.1 Principle of the test method

The ASTM E2180-07 standard describes the test method for quantitatively evaluating the antimicrobial efficacy of agents embedded in or onto polymeric or hydrophobic surfaces. This method involves the inoculation of a fused semi-solid agar (agar slurry) with a standardized culture of microbial cells. A thin layer of inoculated agar slurry ? transferred onto the surfaces to be tested and onto others used as controls. After one or more specified contact times, surviving organisms are recovered by elution of the agar slurry inoculum from the test substrate in a neutralizing agent and extracted by a method that ensures complete removal of the inoculum from the test surface. Serial dilutions are then set up, each seeded by inclusion in a suitable culture medium. After incubation of the plates under the conditions specified for the test organisms used, the number of surviving microbial colonies for each dilution ? contacted and registered. ? then the calculation of the percentage of reduction of the microorganisms was carried out by comparing the surviving microorganisms on samples of surfaces treated with antimicrobials with those recovered on untreated surfaces taken as a reference.

3.1. Reference legislation

The test described in this report refers to the legislation specified below. ASTM E2180-07 ?Standard Test Method for Determining the Activity of Incorporated Antimicrobial Agent(s) in Polymeric or Hydrophobic Materials?.

3.2. Internal references

The tests carried out and described below refer to the following operating procedures and instructions, submitted to the Quality Management System? ISO 9001 and ISO 13485 certified.

- P08 ?Analysis and validation tests? rev. 05 of 01/10/2013;

- P09 ?Infrastructure management? rev. 03 of 01/10/2013;

- P10 ?Instrument management? rev. 03 of 01/10/2013;

- I01 ?Log management? rev. 01 of 10/05/2011;

- I02 ?Management of culture media and reagents? rev. 03 of 02/03/2015.

4. IDENTIFICATION OF THE SAMPLES UNDER TEST

The product under examination consists of devices treated with a mixture M based on usnic acid and/or a relative salt thereof, of natural origin, preferably the sodium salt, in the racemic or dextrorotatory form D(+), to obtain properties antimicrobial; devices of the same material free of antimicrobial agent were used as a reference. The samples of antimicrobial-treated and non-antimicrobial-treated devices used in the study, so? as identified below, they were manufactured according to internal procedures. The samples to be tested appeared as rectangles of dimensions approximately equal to 2 x 8 cm.

Table 6

5. EQUIPMENT AND REAGENTS

The following reagents, materials and laboratory equipment were used for the test:

- diluent for the preparation of microbial suspensions: physiological solution with NaCl 9 g/l Cod. SA279/2015 Expires 10/03/2016;

- culture medium for bacteria: Tryptone Soy Agar (TSA) Cod. SA289/2015 Expired 22/03/2016;

- agar slurry: semi-gelatinous preparation containing agar-agar 3 g/l and NaCl 8.5 g/l Cod. SA292/2015 Expiry 24/12/2015;

- recovery / neutralizing broth: Tryptone Soy Broth solution containing tween 80 30 ml/l, saponin 30 g/l, L-histidine 1 g/l, egg lecithin 3 g/l, sodium thiosulphate 5 g/l Cod SA230/2015 Exp.

01/14/2016;

- thermostatic bath MPM INSTRUMENTS Cod. SA65 controlled at (45 ? 1)?C;

- CHEMICAL OMNIA thermostatic bath Cod. SA15 controlled at (45 ? 1)?C;

- vortex mixer VELP SCIENTIFIC Cod. SA52;

- PID SYSTEM thermostat Cod. SA66 controlled at (36 ? 1)?C;

- VELP SCIENTIFIC refrigerated thermostat Cod. SA82 controlled at (31 ? 1)?C;

- GENESYS 10 spectrophotometer Cod. SA26;

- miscellaneous sterile material (e.g. scissors, pliers, etc.). The preparation of the media and the reagent used ? carried out according to the indications of the manufacturer and/or of the reference method, in compliance with what is reported in the internal operating instructions. Were the soils used in the trials checked for fertility? and sterility?. Equipment management? carried out according to internal procedures; at the time of execution of the tests the devices were in the valid calibration status.

The preparation of the work environment and the management and handling of materials are carried out according to the specifications defined in the relative internal procedures.

6. DESCRIPTION OF THE METHOD

6.1. Experimental conditions

The proof of antimicrobial efficacy ? was performed under the following experimental conditions.

- Microbial strain: Escherichia coli ATCC 10536 (Gram-negative bacteria).

The incubation conditions adopted for the test strain are detailed in the table below.

Table 7 Development conditions of the microbial strain

Contact time: the contact times agreed with the client are specified in the table below. Table 8 Contact times

- Reference: devices not treated with mix M (without usnic acid).

6.2. Test description

The microbial strain? was transplanted on a slant of suitable medium for 24 hours and then diluted in saline until reaching a concentration, estimated by spectrophotometric reading, between 1-5 x 108 cfu/ml. The number of microbial cells in the suspension? was determined through scalar dilutions to base 10 in saline, up to 10-6. Two 1 ml aliquots were taken from this dilution and seeded for inclusion in medium. After incubation and counting of the colonies developed on the plates ? been determined the number of units? colony forming per ml (cfu/ml) in the suspension.

1.0 mL of microbial suspension ? was seeded in 100 ml of agar slurry, kept in fusion at a temperature of 45°C, to obtain a final concentration of cells in each agar slurry between 1-5 x 106 cfu/ml. Were the test and reference devices prepared by inserting n? 5 pieces for contact time defined above. 1.0 mL inoculated agar slurry ? was transferred to each of the test and control samples prepared for the test suspension. The inoculation? was carried out with an angle and a speed? in order to avoid dispersion of the suspension outside the sample. After letting the agar slurry inoculum gel, the samples were placed in the incubator at the temperature suitable for the development of the microbial strain for the defined contact times. To avoid drying of the agar slurry inoculum, inside the thermostat ? was the maintenance of? humidity guaranteed? at a level above 75%, using a tray containing water. At each of the defined contact times, the treated and non-reference device samples were removed from the petri dishes and transferred to a flask containing neutralizing broth in a volume such as to form a 1:10 dilution of the initial inoculum. The flasks were subjected to sonication for 1 minute, followed by a subsequent mechanical mixing using vortexing in order to ensure complete release of the agar slurry from the sample. So the neutralizing broth? was subjected to 1:10 serial dilutions, each seeded by inclusion in suitable medium. The sample ? was seeded by inclusion in molten medium, in order to determine the effectiveness of the release from the treated surface. After incubation, ? The number of colonies developed for each of the prepared dilutions was counted and recorded, calculating the number of surviving microorganisms (cfu/ml) for each contact time.

6.3. Calculation and expression of results

The results were expressed as a percentage reduction in microbial contamination of the treated device sample compared to the untreated one, as defined in the reference standard. ? the geometric mean of the number of microorganisms recovered in the five replicates carried out for the devices treated with antimicrobial and untreated was calculated; so ? the percentage difference between the antilogarithm of the geometric mean of the control sample and the antilogarithm of the geometric mean of the treated sample was calculated.

Geometric mean = (LogR1 LogR2 LogR3 LogR4 LogR5)/5

Where is it:

R1/2/3/4/5 = n? total number of organisms recovered after exposure to the test substance or control and incubation (replications 1/2/3/4/5).

Percentage reduction = (a-b)x100/a

Where is it:

a = antilogarithm of the geometric mean of the reference untreated device

b = antilogarithm of the geometric mean of the treated device

6.4. Validity criteria? of the essay

The wise ? considered valid when the recovery of the initial microorganisms ? equal to or greater than 104 cfu/ml. The ASTM 2180 reference standard requires, in order to declare a device effective under test conditions, a percentage reduction in microbial contamination, evaluated with respect to the untreated reference, equal to or greater than 99%.

7. RESULTS

The results obtained are summarized in the tables below.

Table 9: Strain count (cfu/mL)

Table 10: Mean logarithmic expression of the microorganisms that survived at different times of contact

Table 11: percentage reduction at different contact times

Table 12: logarithmic reduction at different contact times

8. CONCLUSIONS

On the basis of the results obtained, have the validity criteria been met? of the test, you can? conclude that, according to the requirements of the ASTM E-2180-07 standard (greater than 99% reduction):

- devices treated with a usnic acid-based mixture M identified as ?SAMPLE 1? (usnic acid of synthetic origin, not in accordance with the present invention) are effective against the test strain (E.coli) at a contact time of 72 hours;

- devices treated with a usnic acid-based mixture identified as ?SAMPLE 2? (usnic acid of natural origin in accordance with the present invention) are effective against the test strain (E.coli) at a contact time of 24 hours.

EXAMPLE 2

2.1. Purpose

? The microbicidal efficacy of devices treated with a mixture M based on usnic acid and/or a relative salt thereof, of natural origin, preferably the sodium salt, in the racemic or dextrorotatory form D(+) has been verified. ? a test was performed according to the procedure described, reference standard ASTM E2180-07, using microbial strains considered as indicative.

2.2. Principle of the test method

The ASTM E2180-07 standard describes the test method for quantitatively evaluating the antimicrobial efficacy of agents embedded in or onto polymeric or hydrophobic surfaces. This method involves the inoculation of a fused semi-solid agar (agar slurry) with a standardized culture of microbial cells. A thin layer of inoculated agar slurry ? transferred onto surfaces to be tested and onto others used as a control. After one or more specified contact times, surviving organisms are recovered by elution of the agar slurry inoculum from the test substrate in a neutralizing agent and extracted by a method that ensures complete removal of the inoculum from the test surface. Serial dilutions are then set up, each seeded by inclusion in a suitable culture medium. After incubation of the plates under the conditions specified for the test organisms used, the number of surviving microbial colonies for each dilution ? contacted and registered. ? then the calculation of the percentage of reduction of the microorganisms was carried out by comparing the surviving microorganisms on samples of surfaces treated with antimicrobials with those recovered on untreated surfaces taken as a reference.

3.1. Reference legislation

The test described in this report refers to the legislation specified below.

- ASTM E2180-07 ?Standard Test Method for Determining the Activity of Incorporated Antimicrobial Agent(s) in Polymeric or Hydrophobic Materials?.

3.2. Internal references

The tests carried out and described in this report refer to the following procedures and operating instructions, submitted to the Quality Management System? ISO 9001 and ISO 13485 certified.

- P08 ?Analysis and validation tests? rev. 05 of 01/10/2013;

- P09 ?Infrastructure management? rev. 03 of 01/10/2013;

- P10 ?Instrument management? rev. 03 of 01/10/2013;

- I01 ?Log management? rev. 01 of 10/05/2011;

- I02 ?Management of culture media and reagents? rev. 03 of 02/03/2015.

4. IDENTIFICATION OF THE SAMPLES UNDER TEST

The product concerned consists of devices treated with a usnic acid-based mixture M to obtain properties? antimicrobial; devices of the same material free of antimicrobial agent were used as a reference. The samples of antimicrobial-treated and non-antimicrobial-treated devices used in the study, so? as identified below, they were manufactured according to internal procedures.

The samples to be tested appeared as rectangles of dimensions approximately equal to 2 x 8 cm.

Table 13

5. EQUIPMENT AND REAGENTS

The following reagents, materials and laboratory equipment were used for the test:

- diluent for the preparation of microbial suspensions: physiological solution with NaCl 9 g/l Cod. SA 226/2015 Expires 10/01/2016;

- culture medium for bacteria: Tryptone Soy Agar (TSA) Cod. SA 225/2015 Expired 10/01/2016;

- culture medium for yeasts: Agar Sabouraud (SAB) Cod. SA 219/2015 Expired 10/01/2016;

- agar slurry: semi-gelatinous preparation containing agar-agar 3 g/l and NaCl 8.5 g/l SA Code 229/2015 Expiry 10/14/2015;

- recovery / neutralizing broth: Tryptone Soy Broth solution containing tween 80 30 ml/l, saponin 30 g/l, L-histidine 1 g/l, egg lecithin 3 g/l, sodium thiosulphate 5 g/l Cod SA 230/2015 Expires 14/01/2016;

- thermostatic bath MPM INSTRUMENTS Cod. SA 65 controlled at (45 ? 1)?C;

- thermostated bath CHIMICA OMNIA Cod. SA 15 controlled at (45 ? 1)?C;

- vortex mixer VELP SCIENTIFIC Cod. SA 52;

- PID SYSTEM thermostat Cod. SA 66 controlled at (36 ? 1)?C;

- VELP SCIENTIFIC refrigerated thermostat Cod. SA 82 controlled at (31 ? 1)?C;

- GENESYS 10 spectrophotometer Cod. SA 26;

- miscellaneous sterile material (e.g. scissors, pliers, etc.).

The preparation of the media and the reagent used ? carried out according to the indications of the manufacturer and/or of the reference method, in accordance with what is reported in the Studio Ambiente internal operating instructions. Were the soils used in the trials checked for fertility? and sterility?.

6. DESCRIPTION OF THE METHOD

6.1. Experimental conditions

The proof of antimicrobial efficacy ? was performed under the following experimental conditions.

- Microbial strains: Staphylococcus aureus MRSA ATCC 43300 (Gram-positive bacteria), Escherichia coli ATCC 10536 (Gram-negative bacteria), Candida albicans ATCC 10231 (yeasts).

The incubation conditions adopted for the test strains are detailed in the table below.

Table 14

Contact time: contact times are specified in the table below.

Table 15

Reference: devices not treated with said usnic acid-based mixture M.

6.2. Test description

Each microbial strain ? was transplanted on a slant of suitable medium for 24 hours and then diluted in saline until reaching a concentration, estimated by spectrophotometric reading, between 1-5 x 108 cfu/ml. The number of microbial cells in each suspension? was determined through scalar dilutions to base 10 in saline, up to 10-6. Two 1 ml aliquots were taken from this dilution and seeded for inclusion in medium. After incubation and counting of the colonies developed on the plates ? been determined the number of units? colony forming per ml (cfu/ml) in each suspension. 1.0 mL of each microbial suspension ? was seeded in 100 ml of agar slurry, kept in fusion at a temperature of 45°C, to obtain a final concentration of cells in each agar slurry between 1-5 x 106 cfu/ml. Were the test and reference devices prepared by inserting n? 5 pieces for contact time defined above. 1.0 mL inoculated agar slurry ? was transferred to each of the test and control samples prepared for each of the test suspensions. The inoculation? was carried out with an angle and a speed? in order to avoid dispersion of the suspension outside the sample. After leaving the agar slurry inoculum to gel, the samples were placed in the incubator at the temperature suitable for the development of the microbial strains for the defined contact times. To avoid drying of the agar slurry inoculum, inside the thermostat ? was the maintenance of? humidity guaranteed? at a level above 75%, using a tray containing water. At each of the defined contact times, the reference samples of treated and untreated devices were removed from the petri dishes and transferred to a flask containing neutralizing broth in a volume such as to form a 1:10 dilution of the initial inoculum. The flasks were subjected to sonication for 1 minute, followed by a subsequent mechanical mixing using vortexing in order to ensure complete release of the agar slurry from the sample. So, the neutralizing broth? was subjected to 1:10 serial dilutions, each seeded by inclusion in medium suitable for the development of the specific microbial strain. The sample ? was seeded by inclusion in molten medium, in order to determine the effectiveness of the release from the treated surface. After incubation, ? The number of colonies developed for each of the prepared dilutions was counted and recorded, calculating the number of surviving microorganisms (cfu/ml) for each suspension and contact time.

6.3. Calculation and expression of results

The results were expressed as a percentage reduction in microbial contamination of the treated device sample compared to the untreated one, as defined in the reference standard. ? the geometric mean of the number of microorganisms recovered in the five replicates carried out for the devices treated with antimicrobial and untreated was calculated; so ? the percentage difference between the antilogarithm of the geometric mean of the control sample and the antilogarithm of the geometric mean of the treated sample was calculated.

Geometric mean = (LogR1 LogR2 LogR3 LogR4 LogR5)/5

Where is it:

R1/2/3/4/5 = n? total number of organisms recovered after exposure to the test substance or control and incubation (replications 1/2/3/4/5).

Percentage reduction = (a-b)x100/a Where:

a = antilogarithm of the geometric mean of the reference untreated device

b = antilogarithm of the geometric mean of the treated device

6.4. Validity criteria? of the essay

The wise ? considered valid when the recovery of the initial microorganisms ? equal to or greater than 104 cfu/ml. The ASTM 2180 reference standard requires, in order to declare a device effective under test conditions, a percentage reduction in microbial contamination, evaluated with respect to the untreated reference, equal to or greater than 99%.

7. RESULTS

The results obtained are summarized in the tables below.

Table 16: Strain count (cfu/mL)

Table 17: Mean logarithmic expression of the microorganisms survived at different time points

contact

Table 18: percentage reduction at different contact times

Table 19: logarithmic reduction at different contact times

8. CONCLUSIONS

On the basis of the results obtained, have the validity criteria been met? of the test, you can? conclude that the devices treated with usnic acid (subject of the present invention) are effective, according to the requirements of the ASTM E-2180-07 standard (reduction greater than 99%) and in the test conditions at a contact time of 24 hours, against of the representative strain of Gram-positive bacteria (Staphylococcus aureus MRSA).

Claims (8)

1. A finished product PF including a semi-finished product PS, and a paint product; in which: - said semi-finished product PS comprises a mixture M, and a resin; said mixture M comprises or, alternatively, consists of (a) a usnic acid of natural origin and/or (b) a relative salt thereof; - in which said (a) a usnic acid of natural origin ? in racemic form 50% (+) and 50% (-), or in dextrorotatory form D(+), and - in which said (b) a salt of usnic acid of natural origin ? a salt of an alkali or alkaline earth metal; preferably said salt of usnic acid of natural origin ? a sodium salt of usnic acid, and in which - said semi-finished product PS comprises a resin which ? selected from the group comprising or alternatively consisting of polyurethane, urethane, polyacrylic, acrylic, polyvinyl, vinyl, polyamide or amide polymers or resins. 2. The finished product PF according to claim 1, wherein said painted product is? preferably selected from the group comprising or alternatively consisting of varnishes, enamels or paints; preferably said varnishes, enamels or paints are preferably selected from those with a water base or solvent, or with an organic solvent or base. 3. The finished product PF according to claim 1 or 2, wherein said painting product can preferably be in the liquid state, dispersion or aqueous dispersion. 4. The finished product PF according to any one of claims 1-3, wherein said semi-finished product PS is present in said finished product PF in a quantity? by weight ranging from 0.1% to 10%; preferably from 0.5% to 8%; even more preferably from 1% to 6%, for example 1%, 2%, 3%, 4%, or 5%, with respect to the weight of the coating product. 5. A use of a finished product PF according to any one of claims 1-4, wherein said finished product PF is used as an anti-bacterial, anti-proliferative, bacteriostatic, microbicide, anti-mold, anti-yeast (e.g. Candida), anti-fungal or antifungal (e.g. Saccharomycetes), preferably against Gram-positive and/or Gram-positive bacteria - negative, such as for example those with the scientific name Klebsiella, Enterobacteriaceae, Enterobacter, Pseudonomas and Escherichia. 6. A method of rendering a surface anti-bacterial, anti-proliferative, bacteriostatic, microbicidal, anti-mold, anti-yeast (e.g. Candida), anti-fungal or anti-fungal (e.g. Saccharomycetes), preferably against bacteria Gram-positive and/or Gram-negative, such as for example those having the scientific name Klebsiella, Enterobacteriaceae, Enterobacter, Pseudonomas and Escherichia, said method provides for the application by spray technique, roller or brush of said finished product PF on said surface, according to any one of claims 1-4. 7. The method according to claim 6, wherein said finished product PF is applied on/over horizontal or vertical surfaces, for example floors, walls or ceilings made for example in cement, lime or plasterboard, linoleum, or in PVC, polyvinyl chloride, PA polyamide, PE polyethylene, PES polyester or PTF polyethylene terephthalate, preferably said surfaces being present in a doctor's surgery, emergency room, hospital, dental office, playground, kindergarten, school or toilets and toilets present, for example, in public or private structures or at example in supermarkets and shopping centers or playgrounds. 8. The method according to claim 7, wherein said finished product PF is preferably applied on surfaces, for internal or external use, for example made of wood, steel, aluminium, fabric, non-woven fabric, skin, leather or glass; preferably said finished product PF is applied on/over said surfaces by spray, roller or brush technique.