WO2022018378A1

WO2022018378A1 - Combination of carvacrol with gallic acid and/or curcumin

Info

Publication number: WO2022018378A1
Application number: PCT/FR2021/051358
Authority: WO
Inventors: Maxime GOBIN; Adeline GAND; Damien SEYER; Stéphane Lack; Emmanuel Pauthe; Richard PROUST
Original assignee: Les Laboratoires Brothier; Cy Cergy Paris Université
Priority date: 2020-07-21
Filing date: 2021-07-21
Publication date: 2022-01-27
Also published as: EP4185282A1; FR3112683B1; FR3112683A1

Abstract

The present invention relates to a combination comprising carvacrol with gallic acid and/or curcumin; it being possible for this combination to be formed as a solution, a semi-solid, a powder, a compress, a wick and/or a yarn.

Description

DESCRIPTION

TITLE: COMBINATION OF CARVACROL WITH GALLIC ACID AND/OR

CURCUMIN

Technical field of the invention

The present invention relates to combinations comprising carvacrol with gallic acid and/or curcumin. This combination is advantageously used to inhibit the growth or the formation of bacterial biofilms or to eliminate mature biofilms.

Technical background

In nature, bacteria are found overwhelmingly in the form of biofilm (99% of bacteria). Biofilm is one of the most widespread modes of life on Earth and plays a major role in many elemental cycles. It is present wherever life can exist and is thus capable of colonizing all types of surfaces, whether natural, such as aquatic systems and most organisms such as humans, or artificial such as water pipes, industrial piping and medical devices. ^(1_4)

Formation of biofilms

Biofilm is initiated by the adhesion of planktonic bacteria to a surface. This adhesion becomes irreversible and the bacteria begin to form micro colonies. The micro-colonies develop and organize themselves into three-dimensional clusters which allow the passage of nutrients. After an exponential growth phase and thanks to quorum sensing, which defines the ability of bacteria to detect and react to bacterial molecular signals allowing them to coordinate their collective behavior, the bacteria produce a matrix protective exo-polysaccharide (“slime”) bringing the biofilm to maturation. Finally, the bacteria can disperse alone or in clusters to contaminate other surfaces. ⁽¹ · ² · ⁵⁾ Composition of the biofilm The biofilm consists of a structure of micro-colonies of one or more species of bacteria which adhere to each other and produce a protective matrix. This, which represents nearly 85% of the components of the biofilm, is composed of exopolysaccharides, proteins, glycolipids, extracellular DNA and other compounds that will depend on the species contained in the biofilm. There are thus many elements in this structure and several communication systems that will allow bacteria to form a community while acquiring new properties such as antibiotic resistance. It is recognized that bacteria in biofilms are up to 1000 times more tolerant to antibiotics than planktonic bacteria. Indeed, the biofilm limits the diffusion of antibiotics or antiseptics via interactions with its matrix and the polymers that compose it. The poor growth of bacteria within the mature biofilm makes them unsusceptible to many antibiotics that target bacterial replication mechanisms. The biofilm lifestyle is accompanied by a significant modulation of gene expression that increases tolerance to biocides, the ability to respond to environmental stresses and virulence. 2 6

Complications linked to biofilms Biofilm, if it is not desired and controlled, leads to multiple complications such as the corrosion of industrial equipment, ships, the obstruction of pipes, the contamination of water, the contamination of products, in particular in the food industry, or even infections linked to the presence of biofilms on implantable medical devices or on the surfaces of an organism, in particular wounds. All this leads to increased equipment maintenance efforts in the various industries and considerable additional costs and constitutes a major public health problem. ^{(1 3} · ⁵ · ⁷⁾

Biofilms of Pseudomonas aeruginosa and Staphylococcus aureus The biofilm is composed of a heterogeneity of micro-organisms which will vary according to the environment ⁽² L Among them are found bacterial species such as Pseudomonas aeruginosa and Staphylococcus aureus. P. aeruginosa, which is a Gram-negative bacterium, is found in many different environments such as soil, vegetables, water in general. P. aeruginosa can also grow in hostile conditions such as soap, fuel, antiseptic solution. More generally, the presence of this species is associated with human activity ^{(8 10)} Staphylococcus aureus is a Gram-positive bacterium that is found in various environments and especially in the food industry, on the surface of food and equipment ⁽¹¹ - ¹²⁾ . species are also at the origin of multiple infections in humans and represent, with Escherichia coli, the most common germs found in the case of nosocomial infections ⁽¹³⁾ . acquisition of antibiotic resistance, these bacteria are classified by the WHO in the lists of priority pathogens of critical class for P. aeruginosa and high for S. aureus for the development of new antimicrobial solutions ⁽¹⁴⁾ .

Treatment of biofilms

Many possibilities exist to overcome the installation of the biofilm by playing on the hydrophobicity, the surface roughness. Treatments such as the functionalization of surfaces by metals, polymers, enzymes make it possible to limit the adhesion of bacteria and therefore the formation of biofilms. When the biofilm is already well established, other strategies must be put in place ⁽³ · ⁵ · ⁷⁾ . The use of antibiotics, antiseptics, biocides or even disinfectants has long been unsuccessful. We now know that the biofilm, by its nature, is tolerant or even resistant to these compounds ⁽⁶ - ¹⁵ - ¹⁶⁾ . New avenues are being developed, such as the use of iron chelators, metal particles, bacteriophages, enzymes or even plant extracts to fight against biofilms ⁽³ - ⁵⁾ .

Object of the patent The object of the present invention is to propose a new solution to this major problem through various combinations of active ingredients, derived from plants, comprising carvacrol, gallic acid and curcumin.

The present invention relates to the 3 combinations: carvacrol with gallic acid, carvacrol with curcumin and carvacrol with gallic acid and curcumin.

These combinations can advantageously be prepared in the form of a solution, a semi-solid, a powder, a compress, a wick or a thread.

Detailed description of the invention • Definitions

In this application, the term "antimicrobial" refers to a compound or composition that kills microorganisms or inhibits or arrests their growth, including, but not limited to, bacteria and yeasts in planktonic form.

The term “antibacterial” is an antimicrobial specific to bacteria.

The term "bacterial biomass" refers to the total amount of bacteria in a given space at a given time.

The term "biofilm" refers to communities of one or more species of microorganisms, such as bacteria, stuck in an extracellular matrix composed mainly of polysaccharides and proteins. Bacteria in a biofilm can be up to 1000 times more resistant to antibiotics/antibacterials than their planktonic counterparts. In the context of the present invention, mature biofilm is understood to mean a biofilm which has stability in terms of bacterial biomass, that is to say whose bacterial biomass of which it is composed remains substantially constant.

The term mono-, double- or multi-species biofilm designates a biofilm whose formation results from a single bacterial species, from a mixture of two bacterial species or from a mixture of several bacterial species. The term "antibiofilm" refers to the inhibition of the formation of biofilms or the destabilization or dispersion of preformed or mature biofilms or the elimination of bacteria present in this biofilm.

The term "inhibition" refers to a decrease in bacterial growth and/or biofilm formation.

The term "prevention" refers to the avoidance of biofilm formation.

The term “treatment” refers to an intervention carried out with the aim of eliminating a biofilm that is already present.

The term "solvent" refers to a liquid which makes it possible to solubilize the active principles of the invention.

• Association

The present invention relates to an association comprising carvacrol with gallic acid and/or curcumin; it thus concerns the 3 associations: carvacrol with gallic acid, carvacrol with curcumin and carvacrol with gallic acid and curcumin.

The Applicant has surprisingly observed a synergistic action of the combination of carvacrol and gallic acid, and of the combination of carvacrol and curcumin, as well as of the combination of these three compounds (carvacrol, gallic acid and curcumin) on the reduction of the bacterial biomass present in the biofilms, but also on the biofilms as such, whatever their degree of maturity and whether these biofilms are mono- or multi-species. These advantageous properties thus allow increased destruction of bacteria both in planktonic form and in biofilm form when these active agents are used in combination compared to their isolated actions.

Carvacrol (5-isopropyl-2-methylphenol or cymophenol) is an isomer of thymol and a monoterpenoid. It is found in oregano, thyme, essential oils derived from these plants or even bergamot. This compound, via its structure and its hydrophobic nature, interacts with the bacterial membrane and the permeabilize; it is known in particular to act on bacteria of the Staphylococcus aureus, Staphylococcus epidermis, Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis type. ⁽¹⁷ - ²¹⁾

Gallic acid (3, 4, 5-trihidroxybenzoic acid) is a phenolic acid found in galls, oak bark, tea leaves, tannins or even in persimmon, guava and apple cinnamon . This compound comes from tannic acid. This molecule has an inhibitory action on many bacterial species such as Staphylococcus aureus (both strains sensitive and resistant to antibacterial agents), Escherichia coli, Staphylococcus epidermis or even Pseudomonas aeruginosa. Gallic acid also has great antioxidant power. ^{(22 26)}

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is a phenolic component found in turmeric. Curcumin has properties that inhibit the growth of various bacteria belonging to the genera Streptococcus, Staphylococcus, Lactobacillus, or Helicobacter pylori, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis species. In addition, curcumin also exhibits anti-inflammatory properties. ^(27_30)

By way of example, these active agents are commercially available under the following name: "41086 - Acros Organics" for gallic acid, "W224511 - Sigma Aldrich" for carvacrol and "8.20354.0010 - Merck" for curcumin .

These three active ingredients each inhibit the formation of certain biofilms but have little effect on mature biofilms. ^{(31 33)} Carvacrol has weak activity on mature S. aureus biofilms, and does not eliminate eg P. aeruginosa biofilms. ⁽³⁴⁾

The combinations that are the subject of the present invention can be prepared in different forms or packaging, and in particular in the form of a solution, semi-solid, powder, compress, wick and/or thread for the prevention and/or treatment of biofilm.

In the combination according to the invention, the ratio of carvacrol and gallic acid and that of carvacrol and curcumin is between 0.1 and 10 whether the combination comprises two or three compounds.

Advantageously, the ratio of carvacrol and gallic acid and the ratio of carvacrol and curcumin is between 1.5 and 10, whether the association comprises two or three compounds.

In a first variant, the ratio of carvacrol and gallic acid is between 0.1 and 5, even more preferably between 1.5 and 5, and for example around 5, whether the combination comprises two or three compounds.

In a second variant, the ratio of carvacrol and gallic acid is between 5 and 10, even more preferably between 6 and 10, and for example around 10, whether the combination comprises two or three compounds.

Advantageously, the ratio of carvacrol and curcumin is between 5 and 10, even more preferably between 6 and 10, whether the combination comprises two or three compounds. The ratio of carvacrol and curcumin is, for example, around 10, whether the combination includes two or three compounds.

These ratios can be used regardless of the form of the product comprising the combination according to the invention.

According to a particular embodiment, the combination according to the invention also comprises one or more ingredients chosen from a buffer, a stabilizing agent, a surfactant, a vitamin, a mineral, any element of the extracellular matrix, a pH adjuster , an antibiotic, an antimicrobial and another antibiofilm.

• Different possible forms or packaging of the association

Antibiofilm solution

The present invention also relates to an antibiofilm solution comprising at least one of the combinations according to the invention in an acceptable solvent. Advantageously, the ratio of carvacrol and gallic acid is between 0.1 and 10, preferably between 1.5 and 10, whether the combination comprises two or three compounds.

Advantageously, the ratio of carvacrol and curcumin is between 0.1 and 10, preferably between 5 and 10, even more preferably between 6 and 10, whether the combination comprises two or three compounds. The ratio is for example about 10, whether the association includes two or three compounds. The solution according to the invention may comprise up to 50 mg/mL of carvacrol, up to 10 mg/mL of gallic acid and/or up to 5 mg/mL of curcumin; in particular between 0.1 mg/mL and 50 mg/mL of carvacrol, between 0.5 mg/mL and 5 mg/mL of curcumin and/or between 0.1 mg/mL and 10 mg/mL of gallic acid .

One of the difficulties that the Applicant has solved is to find a common solvent for carvacrol, gallic acid and curcumin.

The acceptable solvent can be water with a stabilizing agent such as a cyclodextrin (in particular hydroxypropyl- -cyclodextrin), ethanol or a mixture of ethanol and water, in which ethanol is between 7.5% and 100% by volume relative to the total volume of the solvent. Preferably, the ethanol is between 30% and 100% by volume relative to the total volume of the solvent, in the mixture of ethanol and water.

A stabilizing agent according to the invention is a compound making it possible to prevent the degradation, precipitation and/or volatilization of one or more active principles (in particular carvacrol). The preparation of the solution according to the invention can be done simply by mixing the active agents in the solvent. By way of example, the antibiofilm solution is heated to approximately 50° C. for a period of 5 to 20 minutes in the presence or absence of stirring.

As demonstrated below (Example 1), the combination according to the invention in solution, makes it possible to obtain a synergistic effect of the antibiofilm activity.

The antibiofilm solution according to the invention can be used as such to inhibit the growth or the formation of biofilms of bacteria or to eliminate mature biofilms.

The antibiofilm solution according to the invention can also advantageously be used for the manufacture and functionalization of materials and products such as semi-solids, powder, compresses, wicks or threads.

The present invention also relates to an antibiofilm treatment product comprising at least one of the combinations according to the invention.

Preferably, the antibiofilm treatment product is chosen from a semi-solid, a powder, a compress, a wick or a thread.

The treatment product according to the invention can be used to inhibit the growth or the formation of bacterial biofilms or to eliminate mature biofilms.

Antibiofilm semi-solid

The present invention also relates to a semi-solid antibiofilm, which can in particular form a haemostatic and/or healing dressing, comprising the combination according to the invention.

A semi-solid can be defined as a gel, cream, ointment, paste, etc. type preparation. consisting of a simple or compound excipient, in which one or more active ingredients are usually dissolved or dispersed. In the semi-solid, the ratio of carvacrol and gallic acid can be between 0.1 and 10, preferably between 1.5 and 10, whether the combination comprises two or three compounds.

Advantageously, in the semi-solid, the ratio of carvacrol and curcumin is between 0.1 and 10, preferably between 5 and 10, even more preferably between 6 and 10, whether the combination comprises two or three compounds . The ratio of carvacrol and curcumin is, for example, around 10, whether the combination includes two or three compounds.

The semi-solid may include up to 800 mg of carvacrol, up to 200 mg of gallic acid and up to 100 mg of curcumin by total weight in 1 g of semi-solid; in particular, between 20 mg and 800 mg of carvacrol, between 5 mg and 200 mg of gallic acid and/or between 2 mg and 100 mg of curcumin, in 1 g of semi-solid. Advantageously, the semi-solid can comprise between 44 mg and 790 mg of carvacrol, between 8 mg and 160 mg of gallic acid and between 8 mg and 80 mg of curcumin, in 1 g of semi-solid.

Advantageously, the semi-solid comprises a matrix based on alginate salt and the combination according to the invention.

As previously defined; the alginate salt in the semi-solid can be selected from calcium alginate, sodium alginate, potassium alginate, lithium alginate, zinc alginate, manganese alginate or ammonium alginate, preferably calcium alginate. The semi-solid preparation is well known to those skilled in the art; By way of example, one method of preparing an antibiofilm semi-solid consists of an emulsion based on an alginate salt with a hydrophilic phase, such as water with a gelling agent and a lipophilic phase, such as refined soybean oil, comprising the active principles according to the combination of the invention.

Antibiofilm powder

The present invention also relates to an antibiofilm powder, which can in particular form a hemostatic and/or healing powder, comprising the combination according to the invention.

In the powder, the ratio of carvacrol and gallic acid may be between 0.1 and 10, preferably between 1.5 and 10, whether the association comprises two or three compounds.

Advantageously, in the powder, the ratio of carvacrol and curcumin is between 0.1 and 10, preferably between 5 and 10, even more preferably between 6 and 10, whether the combination comprises two or three compounds. The ratio is for example about 10, whether the association includes two or three compounds.

The powder may comprise up to 800 mg of carvacrol, up to 200 mg of gallic acid and up to 100 mg of curcumin by total weight in 1 g of powder; in particular between 20 mg and 800 mg of carvacrol, between 5 mg and 200 mg of gallic acid and/or between 2 mg and 100 mg of curcumin, by weight in 1 g of powder (weight of the powder alone without the active ingredients) . Advantageously, the powder may comprise between 44 mg and 790 mg of carvacrol, between 8 mg and 160 mg of gallic acid and between 8 mg and 80 mg of curcumin, by weight in 1 g of powder.

The powder can also be in the form of a plurality of particles of granular form. The particles advantageously have a size of less than 200 miti, preferably a size of the order of 75 μm, which is defined with a sieve or by laser diffraction measurement.

According to one embodiment, the powder is in the form of a plurality of particles of cylindrical shape obtained from fibers and comprising the combination according to the invention.

The particles in cylindrical form can have a length comprised between 20 μm and 2 mm and a diameter comprised between 5 μm and 50 μm.

The particles constituting the powder, whatever their shape, are preferably composed of alginate salt. For example, the alginate salt can be chosen from calcium alginate, sodium alginate, potassium alginate, lithium alginate, zinc alginate, manganese alginate or ammonium alginate, preferably calcium alginate.

In general, the process for manufacturing the powder according to the invention comprises the following steps: preparation of a powder, immersion of this powder in an antibiofilm solution according to the invention containing the active principles to form a fiber, washing and drying fiber by evaporating the solvent from the antibiofilm solution, chopping the fiber to obtain particles in the desired shape (such as granular, cylindrical, etc.) of the powder according to the invention. The powder in the form of a plurality of particles of cylindrical shape according to the invention can be prepared as described in patent EP-B1-2836243 filed by the Applicant.

By way of example and in the particular case of the powder made up of particles in cylindrical form, the active ingredients can alternatively be added after chopping the fibers by immersing the particles in the antibiofilm solution. These particles associated with the active principles are again dried by evaporation of the solvent.

Antibiofilm compress

The present invention also relates to an antibiofilm compress, which can in particular form a haemostatic and/or healing dressing, the compress comprising the combination according to the invention.

In the compress, the ratio of carvacrol and gallic acid may be between 0.1 and 10, preferably between 1.5 and 10, whether the combination comprises two or three compounds.

Advantageously, in the compress, the ratio of carvacrol and curcumin is between 0.1 and 10, preferably between 5 and 10, even more preferably between 6 and 10, whether the combination comprises two or three compounds. The ratio is for example about 10, whether the association includes two or three compounds.

The compress may include up to 800 mg of carvacrol, up to 200 mg of gallic acid and up to 100 mg of curcumin by total weight in 1 g of compress; in particular between 20 mg and 800 mg of carvacrol, between 5 mg and 200 mg of gallic acid and/or between 2 mg and 100 mg of curcumin, in 1 g of compress.

Advantageously, the compress may comprise between 44 mg and 790 mg of carvacrol, between 8 mg and 160 mg of gallic acid and between 8 mg and 80 mg of curcumin, in 1 g of compress.

The content of active principles in the compress can also be expressed in surface density (mg/cm ² of compress).

Thus, the compress can comprise up to 1500 mg of carvacrol, up to 300 mg of gallic acid and/or up to 200 mg of curcumin, in 100 cm ² of compress.

Preferably, the compress may comprise between 50 mg and 1500 mg of carvacrol, between 10 mg and 300 mg of gallic acid and/or between 5 mg and 200 mg of curcumin, per 100 cm ² of compress.

Advantageously, the compress can comprise between 66 mg and 1190 mg of carvacrol, between 26 mg and 240 mg of gallic acid and between 13 mg and 120 mg of curcumin, per 100 cm ² of compress.

The dressing, for example commonly used in the treatment of wounds, can be chosen from hydrofibres, alginates, hydrocellulars, hydrocolloids, hydrogels, petroleum jelly dressings, activated carbon dressings, silver dressings and dressings based on hyaluronic acid.

Advantageously, the compress comprises a matrix based on alginate salt and the combination according to the invention.

As defined previously, the alginate salt in the compress can be chosen from calcium alginate, sodium alginate, potassium alginate, lithium alginate, zinc alginate, alginate manganese or ammonium alginate, preferably calcium alginate. By way of example, a method for preparing an antibiofilm compress consists of impregnating a width with a solution comprising the active principles according to the combination of the invention.

As demonstrated below (Example 2), the combination according to the invention as a compress also makes it possible to obtain a synergistic effect of the antibiotic activity.

Antibiofilm wick

The present invention also relates to an antibiofilm wick, in particular hemostatic and/or healing, comprising the combination according to the invention.

A wick can be defined as a dressing in the form of a band or strand.

In the lock, the ratio of carvacrol and gallic acid can be between 0.1 and 10, preferably between 1.5 and 10, whether the association comprises two or three compounds.

Advantageously, in the lock, the ratio of carvacrol and curcumin is between 0.1 and 10, preferably between 5 and 10, even more preferably between 6 and 10, whether the combination comprises two or three compounds. The ratio is for example about 10, whether the association includes two or three compounds

The wick can comprise up to 800 mg of carvacrol, up to 200 mg of gallic acid and up to 100 mg of curcumin by total weight in 1 g of wick; in particular between 20 mg and 800 mg of carvacrol, between 5 mg and 200 mg of gallic acid and/or between 2 mg and 100 mg of curcumin, in 1 g of lock. Advantageously, the lock can comprise between 44 mg and 790 mg of carvacrol, between 8 mg and 160 mg of gallic acid and between 8 mg and 80 mg of curcumin, in 1 g of lock.

Advantageously, the lock comprises a matrix based on alginate salt and the combination according to the invention.

As previously defined; the alginate salt in the wick can be selected from calcium alginate, sodium alginate, potassium alginate, lithium alginate, zinc alginate, manganese alginate or ammonium alginate, preferably calcium alginate.

By way of example, a method for preparing the antibiofilm wick consists of impregnating a wick with a solution comprising the active principles according to the combination of the invention.

Antibiofilm yarn

The present invention also relates to an antibiofilm yarn comprising the combination according to the invention.

A yarn can be defined as a long, thin strand made up of one or more filaments.

The thread according to the invention can be used for reinforcements, sutures of wounds or as knitting.

In the thread, the ratio of carvacrol and gallic acid can be between 0.1 and 10, preferably between 1.5 and 10, whether the association comprises two or three compounds.

In a second variant, the ratio of carvacrol and gallic acid is between 5 and 10, even more preferably between 6 and 10, and for example approximately 10, whether the combination comprises two or three compounds. Advantageously, in the yarn, the ratio of carvacrol and curcumin is between 0.1 and 10, preferably between 5 and 10, even more preferably between 6 and 10, whether the combination comprises two or three compounds. The ratio is for example about 10, whether the combination comprises two or three compounds.

The yarn may include up to 800 mg of carvacrol, up to 200 mg of gallic acid and up to 100 mg of curcumin by total weight in 1 g of yarn; in particular, between 20 mg and 800 mg of carvacrol, between 5 mg and 200 mg of gallic acid and/or between 2 mg and 100 mg of curcumin, in 1 g of yarn. Advantageously, the thread can comprise between 44 mg and 790 mg of carvacrol, between 8 mg and 160 mg of gallic acid and between 8 mg and 80 mg of curcumin, in 1 g of thread.

Advantageously, the thread comprises a matrix based on alginate salt and the combination according to the invention.

As previously defined; the alginate salt in the yarn can be selected from calcium alginate, sodium alginate, potassium alginate, lithium alginate, zinc alginate, manganese alginate or ammonium alginate, preferably calcium alginate.

By way of example, a method for preparing the antibiofilm yarn consists of impregnating the yarn by passing it through a solution comprising the active principles according to the combination of the invention.

The present invention also relates to the association, the solution, the semi-solid, the powder, the compress, the wick and/or the wire, for their use for the prevention of the formation, the inhibition of the development, the elimination and/or treatment of biofilms, in particular in industrial equipment, pipes, in the food industry, in medical devices or even in any mammalian organism. The bacteria in the biofilm can be, without limitation, Gram-negative bacteria (for example, Pseudomonas aeruginosa, Escherichia coli and/or Acinetobacter baumannii) and/or Gram-positive bacteria (for example, Staphylococcus aureus and/or bacteria of the genus Propionibacterium).

The present invention also relates to a combination comprising carvacrol with gallic acid and/or curcumin or the solution, semi-solid, powder, compress, wick and thread comprising said combination, for use in preventing and/ or treating disorders caused by or associated with biofilms, preferably in a mammalian organism.

Advantageously, the biofilms comprise bacteria which are Gram-negative bacteria and/or Gram-positive bacteria.

For example, the disorders caused by or associated with biofilms can be:

- Cystic fibrosis,

- recurrent urinary tract infections,

- infections associated with chronic wounds,

- oral and otolaryngology (ENT) pathologies; for example oral infections (caries or stomatitis).

In particular, the use may be intended to prevent and/or treat the infection of wounds and/or promote their healing.

By mammal, we mean preferably men and livestock or domestic animals.

The use according to the invention is of marked interest in the veterinary field in that it avoids the use of antibiotics and the phenomena of resistance which may result therefrom.

The present invention also relates to the use of a combination comprising carvacrol with gallic acid and/or curcumin or of a solution comprising said combination as an antibiofilm agent, preferably to prevent the formation and/or limit the development and/or eliminate biofilms in industrial equipment, pipes, in the food industry, in medical devices. Advantageously, the biofilms comprise bacteria which are Gram-negative bacteria and/or Gram-positive bacteria.

Brief description of figures

Other characteristics and advantages of the invention will appear during the reading of the detailed description which will follow for the understanding of which reference will be made to the appended figures in which:

[Fig.1] Figure 1 represents a histogram illustrating the synergistic activity of the combination of carvacrol and gallic acid in solution on single-species biofilms;

[Fig.2] Figure 2 represents a histogram illustrating the antibiofilm activity of the combination of carvacrol and gallic acid in solution on a monospecies biofilm of P. aeruginosa;

[Fig.3] Figure 3 represents a histogram illustrating the antibiofilm activity of the combination of carvacrol and curcumin in solution on a monospecies biofilm of S. aureus;

[Fig.4] Figure 4 represents a histogram illustrating the antibiofilm activity of the combination of carvacrol and gallic acid in solution on bi-species biofilms;

[Fig.5] Figure 5 represents a histogram illustrating the synergistic activity of the combination of carvacrol and gallic acid in a compress on single-species biofilms;

[Fig.6] Figure 6 represents a histogram illustrating the antibiofilm activity of the combination of carvacrol and curcumin in a compress on single-species biofilms;

[Fig.7] Figure 7 represents a histogram illustrating the antibiofilm activity of the association of carvacrol with hydroxypropyl- -cyclodextrin and gallic acid in a compress on a mono-species biofilm of S. aureus. Examples of the Invention

Example 1 - Synergistic antibiofilm effect of the combination in the form of a solution

The antibiofilm activity of the active ingredients (carvacrol referenced K, gallic acid referenced G, curcumin referenced Q), alone or in a mixture, was tested on mature biofilms prepared with isolated strains (mono-species biofilm) or with a mixture of two strains (bi-species biofilm); the species used are Staphylococcus aureus (also referenced by S. aureus) and Pseudomonas aeruginosa (also referenced by P. aeruginosa).

Example 1.1: Test of the association carvacrol + gallic acid on mono-species biofilms Protocol

Different active solutions are prepared by dissolving respectively a mass of 1.22.10 ² g of carvacrol and/or 2.50 g of gallic acid in a volume of 250 mL of a mixture of 75% ethanol and 25% water or water only for gallic acid. For the carvacrol solution, manual stirring is sufficient to obtain the final solution. In the presence of gallic acid, it is necessary to add a step of heating to 50° C. and stirring for 5 min. Carvacrol and/or gallic acid solutions at 50 mg/mL and/or 10 mg/mL respectively are thus obtained.

Solutions of active agents are then diluted to ^1/10 , so as not to alter/bias the effect of the active agents on the biofilms in a highly concentrated solvent.

Preparation of the different mono-'biofilms

to be tested with the solutions

1 mL of a bacterial suspension calibrated at 10 ⁶ CFU/mL made in TS medium

(Tryptycase Soja) or LB (Lysogenie Broth) respectively from a preculture of S. aureus or P. aeruginosa is deposited in wells of 24-well plates containing a 12mm diameter glass slide. The plates are left for 24 h at 37° C. in order to obtain mature mono-species biofilms. The biofilm can then be brought into contact with one or more solutions according to the invention and control solutions (solvent without active ingredient) for 24 hours at 37°C. Depending on the volume of this solution, the concentration of the active ingredients in contact with the biofilm will vary. The concentrations of antibiofilm solution tested are annotated in FIG.

Thus, with reference to FIG. 1, a quantity of 100 μL of solution containing carvacrol is added to a well containing a biofilm and 900 μL of TS or LB medium; a quantity of 205 pL of solution containing gallic acid is added to a well containing a biofilm and 795 pL of TS or LB medium; 205 pL of gallic acid solution and 100 pL of carvacrol solution are added to a well containing a biofilm and 695 pL of TS or LB medium.

A bacterial count is then carried out: after three rinses of the biofilms with a solution of sodium chloride (NaCI) at 9 g/L, the biofilms undergo two 10-minute sonications in the presence of 500 pL of NaCI at 9 g/L both times. . This solution is recovered in Eppendorfs. From these, 7 cascade dilutions at a rate of 10 are made to obtain solutions diluted from 10° to 10 ⁷ . Spreadings on PCA agars (Plat Count Agar) of 100 μl of each of the dilutions are carried out. The agars are incubated for 24 hours at 37°C. After this time, they are photographed and the colonies present on their surface are counted using the Fiji software. Each condition tested is performed in triplicate and therefore the mean, standard deviation and coefficient of variation of the bacterial biomass are determined. The bacterial biomass is expressed in numbers of bacteria/cm ² . Results: effect on mono-species biofilms

The antibiofilm activity of each of the active ingredients alone (carvacrol or gallic acid) or in combination (carvacrol and gallic acid) in solution is tested on single-species biofilms. This makes it possible to control the reduction in the bacterial biomass of the mono-species biofilms obtained for each active ingredient and for the combination of active ingredients. FIG. 1 illustrates the antibiofilm activities of carvacrol alone in solution, of gallic acid alone in solution and of the combination of carvacrol and gallic acid combined in solution in an ethanol solvent.

In Figure 1, carvacrol has the effect of reducing the bacterial biomass of S. aureus and P. aeruginosa. In fact, the bacterial biomass of S. aureus is reduced by -6.2 log (compared to the biofilm without treatment) in the presence of 0.5 mg/mL of carvacrol. The effect observed is less on P. aeruginosa with a decrease in bacterial biomass of -1.5 log in the presence of 0.5 mg/mL of carvacrol.

Thus, carvacrol exhibits greater antibiofilm activity on S. aureus than on P. aeruginosa.

Also in Figure 1, gallic acid does not have the effect of reducing the bacterial biomass of S. aureus and P. aeruginosa. Indeed, a quantity of gallic acid of 2 mg/mL does not reduce the bacterial biomass of S. aureus and P. aeruginosa. Thus, gallic acid alone does not show any antibiofilm activity on S. aureus and on P. aeruginosa.

The antibiofilm activity of the carvacrol and gallic acid combination is also tested on mono-species biofilms.

Remarkably, when the antibiofilm activity of the carvacrol and gallic acid association is tested on single-species biofilms, a synergistic effect of carvacrol and gallic acid is observed on the reduction of the bacterial biomass of S. aureus and P. aeruginosa (Figure 1).

The combination of carvacrol at 0.5 mg/mL and gallic acid at 2 mg/mL completely eliminates the S. aureus biofilm (a reduction in bacterial biomass of - 8.2 log) and greatly reduces the biofilm at P aeruginosa (a reduction in bacterial biomass of approximately -6.3 log). Thus, these results show that the combination of carvacrol and gallic acid has a synergistic antibiofilm effect on S. aureus and P. aeruginosa. Synthesis of the antibiofilm effect of the association carvacrol K and gallic acid G in solution on mature mono-species biofilms (figure 1)

The results demonstrate with the K+G association (ratio 0.25), a biomass reduction of 8.2 log on S. aureus biofilms and 6.3 log on P. aeruginosa biofilms.

This antibiofilm effect of the K+G association in solution is stronger than the antibiofilm effect observed with K and G used alone, demonstrating a synergistic antibiofilm activity of the association.

The antibiofilm effect (in italics) and the synergistic effect (underlined) of the results in Figure 1 are summarized in the following table (Table 1).

[Table 1]

Example 1.2: Test of the carvacrol + gallic acid combination on one of the mono-species biofilms

The test was reproduced on mono-species biofilms of P. aeruginosa, this time using a solution of gallic acid at a concentration of 3.33 mg/mL, a solution of carvacrol at a concentration of 5 mg/mL and a solution of gallic acid and carvacrol at a concentration of 3.33 mg/mL and 5 mg/mL respectively.

Thus, with reference to Figure 2, an amount of 100 pL of solution containing carvacrol is added to a well containing a biofilm and 900 pL of LB medium; an amount of 100 pL of solution containing gallic acid is added to a well containing a biofilm and 900 pL of LB medium; a quantity of 100 μl of gallic acid and carvacrol solution is added to a well containing a biofilm and 900 μl of LB medium. Results

Figure 2 illustrates the antibiofilm activities of carvacrol alone in solution, gallic acid alone in solution and the combination of carvacrol and gallic acid combined in solution.

In Figure 2, carvacrol alone has the effect of reducing the bacterial biomass of P. aeruginosa. Indeed, the bacterial biomass of P. aeruginosa is reduced by -3.2 log (compared to the biofilm without treatment) in the presence of 0.50 mg/mL of carvacrol.

Thus, carvacrol alone exhibits antibiofilm activity against P. aeruginosa. Still in Figure 2, gallic acid (especially at a low amount compared to Figure 1) does not have the effect of reducing the bacterial biomass of P. aeruginosa. Indeed, the bacterial biomass of P. aeruginosa is very slightly reduced (compared to the biofilm without treatment) in the presence of 0.33 mg/mL of gallic acid. Thus, gallic acid alone does not show antibiofilm activity on P. aeruginosa.

The antibiofilm activity of the carvacrol and gallic acid combination (in small quantities) is also tested on single-species biofilms.

Remarkably, a synergistic effect of carvacrol and gallic acid is observed on the reduction of the bacterial biomass of P. aeruginosa (figure 2). The combination of carvacrol 0.50 mg/mL and gallic acid 0.33 mg/mL has the effect of reducing the bacterial biomass of P. aeruginosa (a bacterial biomass reduction of -4.7 log) . Thus, these results show that the combination of carvacrol and gallic acid has a synergistic antibiofilm effect on P. aeruginosa. Synthesis of the antibiofilm effect of the association carvacrol K and gallic acid G (in small quantities) in solution on mature mono-species biofilms (figure 2) The results demonstrate with the association K+G (ratio 1.5) , a biomass reduction of 4.7 log on P. aeruginosa biofilms.

This antibiofilm effect of the K+G association in solution is stronger than the antibiofilm effect observed with K and G used alone, demonstrating a synergistic antibiofilm activity of the association. The antibiofilm effect (in italics) and the synergistic effect (underlined) of the results of FIG. 2 are summarized in the following table (Table 2). [Table 2]

The results of FIG. 2 are repeated in the following table (Table 3) to demonstrate the synergistic effect of the K+G association.

[Table 3]

The antimicrobial effect is calculated by dividing the bacterial biomass measured for the component(s) in solution by the bacterial biomass measured for the solvent.

The synergistic effect of the combination of two components is evaluated according to the Bliss independence method ⁽³⁵⁾ with the formula: Ec=Ea+Eb−Ea×Eb with Ec: activity produced by the combination of components A and B; with Ea: activity of component A at a dose a; with Eb: activity of component B at a dose b. This Bliss independence formula is applied to the results obtained for the association carvacrol and gallic acid on the P. aeruqinosa strain:

Ec = (16.585366) + (99.673171) - [(16.585366) x (99.673171)] = 99.727377 with Ec minimum necessary activity of the carvacrol and gallic acid combination to produce a synergistic effect; with Ea biomass reduction obtained for carvacrol at 0.50 mg/mL; with Eb biomass reduction obtained for curcumin at 0.33 mg/mL.

The results obtained in FIG. 2 for the carvacrol and gallic acid combination show an antimicrobial effect of 99.990098% on the biofilm, greater than the threshold value Ec (99.727377), this therefore confirms a synergistic effect.

Example 1.3: Test of the carvacrol + curcumin combination on one of the mono-species biofilms Protocol

The test was conducted on mono-species biofilms of S. aureus, this time using a solution of curcumin at a concentration of 5 mg/mL, a solution of carvacrol at a concentration of 0.73 mg/mL and a solution of curcumin and carvacrol at a concentration of 5 mg/mL and 0.73 mg/mL respectively.

Thus, with reference to Figure 3, an amount of 175 pL of solution containing curcumin is added to a well containing a biofilm and 825 pL of TS medium; an amount of 175 pL of solution containing carvacrol is added to a well containing a biofilm and 825 pL of TS medium; a quantity of 175 μl of curcumin and carvacrol solution is added to a well containing a biofilm and 825 μl of TS medium. Results

The antibiofilm activity of each of the active ingredients alone (carvacrol or curcumin) or in combination (carvacrol and curcumin) in solution is tested on mono-species biofilms. This makes it possible to control the reduction of the bacterial biomass mono-species biofilms obtained for each active ingredient and for the combination of active ingredients.

Figure 3 illustrates the antibiofilm activities of carvacrol alone in solution, curcumin alone in solution and the combination of carvacrol and curcumin combined in solution.

In FIG. 3, carvacrol alone (in particular in a small quantity) has the effect of reducing the bacterial biomass of S. aureus. Indeed, the bacterial biomass of S. aureus is reduced compared to the biofilm without treatment (biomass activity of −4.6 log) in the presence of 0.13 mg/mL of carvacrol.

Thus, carvacrol alone also exhibits antibiofilm activity on S. aureus, particularly at very low levels.

Still in FIG. 3, curcumin (also in low quantity) has the effect of slightly reducing the bacterial biomass of S. aureus. Indeed, the bacterial biomass of S. aureus is reduced by -2.5 log (compared to the biofilm without treatment) in the presence of 0.88 mg/mL of curcumin. Thus, curcumin alone exhibits weak antibiofilm activity on S. aureus.

The antibiofilm activity of the carvacrol and curcumin combination is also tested on the mono-species biofilm of S. aureus.

Remarkably, when the antibiofilm activity of the carvacrol and curcumin association is tested on the mono-species biofilm of S. aureus, a synergistic effect of carvacrol and curcumin is observed on the reduction of the bacterial biomass of S. aureus (Figure 3).

The combination of carvacrol at 0.13 mg/mL and curcumin at 0.88 mg/mL has the effect of reducing the bacterial biomass of S. aureus (a bacterial biomass reduction of -6.0 log). Thus, these results show that the combination of carvacrol and curcumin has a synergistic antibiofilm effect on S. aureus. Synthesis of the antibiofilm effect of the combination carvacrol K and curcumin Q in solution on one of the mature single-species biofilms (figure 3)

The results demonstrate with the K+Q association (ratio 0.17), a biomass reduction of 6.0 log on S. aureus biofilms. This antibiofilm effect of the K+Q association in solution is stronger than the antibiofilm effect observed with K and Q used alone, demonstrating a synergistic antibiofilm activity of the association.

The antibiofilm effect (in italics) and the synergistic effect (underlined) of the results in Figure 3 are summarized in the following table (Table 4).

[Table 4]

The results in Figure 3 are shown in the following table (Table 5) to demonstrate the synergistic effect of the K+Q combination.

[Table 5]

Bliss independence formula applied to the results obtained for the combination carvacrol and curcumin on the S. aureus strain:

Ec = (99.965098) + (95.664460) - [(99.965098) x (95.664460)] = 99.998487 with Ec minimum necessary activity of the carvacrol and curcumin combination to produce a synergistic effect; with Ea biomass reduction obtained for carvacrol at 0.13 mg/mL; with Eb biomass reduction obtained for curcumin at 0.88 mg/mL. The results obtained in FIG. 3 for the carvacrol and curcumin combination show an antimicrobial effect of 99.998610% on the biofilm, greater than the threshold value Ec (99.998487), this therefore confirms a synergistic effect.

Example 1.4 Test of the Carvacrol+Gallic Acid Association on Bi-Species Biofilms Protocol

Different active solutions are prepared by dissolving respectively a mass of 1.22.10 ^-2 g of carvacrol and/or 2.50 g of gallic acid in a volume of 250 mL of a mixture of 75% ethanol and 25% water or water only for gallic acid. For the carvacrol solution, manual stirring is sufficient to obtain the final solution. In the presence of gallic acid, it is necessary to add a step of heating to 50° C. and stirring for 5 min. Carvacrol and/or gallic acid solutions at 50 mg/mL and/or 10 mg/mL respectively are thus obtained.

A bacterial suspension containing 10 ⁶ CFU/mL of S. aureus and 10 ⁶ CFU/mL of P. aeruginosa is prepared in TS medium from precultures of these two strains. 1 mL of this mixed bacterial suspension is deposited in wells of 24-well plates containing a glass coverslip 12 mm in diameter. The plates are left for 24 h at 37° C. in order to obtain mature bi-species biofilms. The biofilm is brought into contact with one or more solutions of active ingredients for 24 hours at 37°C. Depending on the volume of solutions of actives, the concentration of actives in contact with the biofilm will vary. The different concentrations tested are annotated in Figure 4.

Thus, with reference to FIG. 4, 205 pL or 500 pL of gallic acid solution and 100 pL of carvacrol solution are added to a well containing a biofilm and respectively 695 pL or 400 pL of TS medium. A count as described above in the mono-species biofilm protocol is carried out (washings, dilutions and spreading on agar) with the exception that for each dilution, a spreading is made on mannitol agar and on cetrimide agar to respectively count specifically the bacterial biomass of S. aureus and P. aeruginosa. Result: effect on bi-species biofilms (mixed biofilms)

The antibiofilm activity of the combination of carvacrol and gallic acid in solution is evaluated on bi-species biofilms (figure 4).

Remarkably, an antibiofilm effect of carvacrol and gallic acid is observed on the reduction of the bacterial biomass of the mixed biofilms of S. aureus and P. aeruginosa.

Indeed, the combinations of carvacrol at 0.5 mg/mL and gallic acid at 2 mg/mL or 5 mg/mL strongly reduce the total bacterial biomass of the bi-species biofilm by -3.7 log with specifically a reduction in the bacterial biomass of S. aureus by -6.2 log and that of P. aeruginosa by -3.2 log (for gallic acid at 2 mg/mL) and by -5.4 log with specifically a reduction the bacterial biomass of S. aureus by -6.2 log and that of P. aeruginosa by -4.9 log (for gallic acid at 5 mg/mL).

By definition in the present application, a strong antibiofilm activity is established when the reduction in the bacterial biomass is greater than −4.5 log. When the reduction in bacterial biomass is less than -4.5 log, the antibiofilm activity is qualified as moderate.

Thus, the combination of carvacrol at 0.5 mg/mL and gallic acid at 2 mg/mL allows a preferential antibiofilm action on S. aureus and a weaker antibiofilm action on P. aeruginosa in the bi-species biofilm. The combination of carvacrol and gallic acid in an amount of 0.5/5 mg/mL provides strong antibiofilm action on both S. aureus and P. aeruginosa in bispecies biofilm. Synthesis of the antibiofilm effect of the association carvacrol K and gallic acid G in solution on mature bi-species biofilms (figure 4)

The results show the reduction in biomass of biofilms composed of S. aureus and P. aeruginosa with the K+G combination used in solution at two ratios (0.25 and 0.1):

K at 0.5 mg/mL and G at 2 mg/mL (ratio 0.25): an antibiofilm action preferentially on S. aureus and a weaker antibiofilm action on P. aeruginosa K at 0.5 mg/mL and G at 5 mg/mL (ratio 0.1): strong antibiofilm action on both S. aureus and P. aeruginosa (antibiofilm effect = in italics). The antibiofilm effect (in italics) of the results of FIG. 4 is summarized in the following table (Table 6). [Table 6]

Example 2 - Synergistic antibiofilm effect of the combination in the form of a compress

The antibiofilm activity of the combination according to the invention formulated in a compress based on alginate salt is tested on mono-species biofilms produced with the species S. aureus or P. aeruginosa.

Preparation of the compress A strip of alginate salt is impregnated with a mixture comprising carvacrol and/or gallic acid and/or curcumin using a suitable machine.

For this, a mass of 1.22×10 ² g of carvacrol and/or a mass of 2.50 g of gallic acid and/or a mass of 1.55 g of curcumin are weighed and placed in a container. A volume of 250 ml of a mixture of 75% ethanol and 25% water is added to the container. The whole is heated to 50° C. and with stirring until the active agents have completely dissolved.

Then, a width of a predetermined length is impregnated with this solution; it is then dried and sterilized.

The amounts of the active ingredient(s) combined according to the antibiofilm associations of the invention are shown in the following table (Table 7).

[Table 7]

Example 2.1: Test of the carvacrol + gallic acid combination in the form of a compress on mono-species biofilms Protocol

1 mL of bacterial suspension calibrated at 10 ⁶ CFU/mL from a preculture of S. aureus or of P. aeruginosa is deposited in wells of 24-well plates containing a glass cover slip 12 mm in diameter. The plates are left for 24 h at 37° C. in order to obtain mature mono-species biofilms. The biofilm can then be brought into contact with one or more compresses enriched with carvacrol or with gallic acid or with carvacrol and with gallic acid according to the invention, for 24 hours at 37°C.

A bacterial count is then carried out: after three rinses with a 9 g/L NaCl solution, the biofilms undergo two 10-minute sonications in the presence of 500 μl of 9 g/L NaCl both times. This solution is then recovered in Eppendorfs. From these, 7 cascade dilutions at a rate of 10 are made to obtain solutions diluted from 10° to 10 ⁷ . Spreadings on PCA agars of 100 μl of each of the dilutions are carried out. The agars are incubated for 24 hours at 37°C. After this time, they are photographed and the colonies present on their surface are counted using the Fiji software. Each condition tested is performed in triplicate and therefore the mean, standard deviation and coefficient of variation of the bacterial biomass are determined. The bacterial biomass is expressed in numbers of bacteria/cm ² . Results: effect of the activity on mono-species biofilms The antibiofilm activity of a compress based on alginate salt without active principles is tested. An absence of antibiofilm activity of the compress alone is observed. This is illustrated in Figures 5 and 6.

The antibiofilm activities of the compress based on alginate salt comprising each of the active ingredients alone or in combination, are tested on mono-species biofilms, in order to control the reduction in the bacterial biomass obtained for each active ingredient and for the combination. .

In figure 3, it is shown that a compress with a quantity of gallic acid of 17.7 mg/g reduces the bacterial biomass of S. aureus by -5.52 log and that of P. aeruginosa by -1, 78 log.

A compress with an amount of carvacrol of 88.4 mg/g reduces the bacterial biomass of S. aureus by -7.83 log compared to the control compress. The bacterial biomass of P. aeruginosa is also reduced with a reduction of -2.18 log which is less important than for the bacterial biomass of S. aureus (see figure 5). Thus, compresses based on alginate salt enriched with active ingredients alone exhibit greater antibiofilm activity on S. aureus biofilms than on P. aeruginosa biofilms.

Remarkably, a strong antibiofilm effect and a synergistic antibiofilm effect of the combination of two of the active ingredients in a compress is observed on the reduction of the bacterial biomass of S. aureus and P. aeruginosa respectively (figure 5).

As defined above, strong antibiofilm activity is established when the reduction in bacterial biomass is greater than -4.5 log.

A compress with a combination of carvacrol at 88.4 mg/g and gallic acid at 17.7 mg/g, reduces the bacterial biomass of S. aureus by -7.53 log and that of P. aeruginosa by -4 .51 log.

Thus, alginate salt-based compresses enriched with the combination of carvacrol and gallic acid exhibit strong antibiofilm activity on S. aureus biofilm and a synergistic antibiofilm effect on P. aeruginosa biofilm. Synthesis of the antibiofilm effect of the combination carvacrol K and gallic acid G in a compress on mature mono-species biofilms (figure 5)

The results demonstrate that the K+G combination (ratio 5) in a compress induces a strong reduction in the biomass of S. aureus biofilms (-7.5 log) and P. aeruginosa biofilms (-4.5 log).

This antibiofilm effect of the K+G association in a compress is stronger than the antibiofilm effect of K and G used alone on biofilms of P. aeruginosa, demonstrating a synergistic antibiofilm activity of the association.

The antibiofilm effect (in italics) and the synergistic effect (underlined) of the results in Figure 5 are summarized in the following table (Table 8).

[Table 8]

Example 2.2: Test of the carvacrol + curcumin combination in the form of a compress on mono-species biofilms Protocol

1 mL of bacterial suspension calibrated at 10 ⁶ CFU/mL from a preculture of S. aureus or of P. aeruginosa is deposited in wells of 24-well plates containing a glass cover slip 12 mm in diameter. The plates are left for 24 h at 37° C. in order to obtain mature mono-species biofilms. The biofilm can then be brought into contact with one or more compresses enriched with carvacrol or curcumin or carvacrol and curcumin according to the invention, for 24 hours at 37°C. A bacterial count is then carried out: after three rinses with a 9 g/L NaCl solution, the biofilms undergo two 10-minute sonications in the presence of 500 μL of 9 g/L NaCl both times. This solution is then recovered in Eppendorfs. From these, 7 cascade dilutions at a rate of 10 are made to obtain solutions diluted from 10° to 10 ⁷ . Spreadings on PCA agars of 100 μl of each of the dilutions are carried out. The agars are incubated for 24 hours at 37°C. After this time, they are photographed and the colonies present on their surface are counted using the Fiji software. Each condition tested is performed in triplicate and therefore the mean, standard deviation and coefficient of variation of the bacterial biomass are determined. The bacterial biomass is expressed in numbers of bacteria/cm ² . Results: effect of activity on mono-species biofilms

The antibiofilm activities of the compress based on alginate salt comprising each of the active ingredients alone or in combination, are tested on mono-species biofilms, in order to control the reduction in the bacterial biomass obtained for each active ingredient and for the combination. . In Figure 6, it is shown that a compress with an amount of carvacrol of 88.4 mg/g reduces the bacterial biomass of S. aureus by -7.83 log compared to the control compress. The bacterial biomass of P. aeruginosa is also reduced with a reduction of -2.18 log which is less important than for the bacterial biomass of S. aureus.

A compress with an amount of curcumin of 8.8 mg/g reduces the bacterial biomass of S. aureus by −4.18 log and that of P. aeruginosa by −1.06 log (see FIG. 4).

Thus, alginate salt-based compresses enriched with active ingredients alone have greater antibiofilm activities on S. aureus biofilms than on P. aeruginosa biofilms.

Remarkably, a strong antibiofilm effect and a synergistic antibiofilm effect of the combination of two of the active ingredients in a compress is observed on the reduction of the bacterial biomass of S. aureus and P. aeruginosa respectively (figure 6).

A compress with a combination of carvacrol at 88.4 mg/g and curcumin at 8.8 mg/g, reduces the bacterial biomass of S. aureus by -7.35 log and that of P. aeruginosa by -4.55 log.

Thus, alginate salt-based compresses enriched with the combination of carvacrol and curcumin exhibit strong antibiofilm activity on S. aureus biofilm and a synergistic antibiofilm effect on P. aeruginosa biofilm. Synthesis of the antibiofilm effect of the carvacrol K and curcumin Q combination in a compress on mature mono-species biofilms (figure 6)

The results demonstrate that the K+Q association (ratio 10) in a compress induces a strong reduction in the biomass of S. aureus biofilms (-7.4 log) and P. aeruginosa biofilms (-4.6 log ). This antibiofilm effect of the K+Q association in a compress is stronger than the antibiofilm effect of K and Q used alone on biofilms of P. aeruginosa, demonstrating a synergistic antibiofilm activity of the association.

The antibiofilm effect (in italics) and the synergistic effect (underlined) of the results of FIG. 5 are summarized in the following table (Table 9). [Table 9]

Example 2.3: Test of the association carvacrol with hvdroxvpropvl-b-cvclodextrin + gallic acid in the form of compresses on mono-species biofilms

The test was reproduced on mono-species biofilms of S. aureus, this time using compresses comprising the combination of carvacrol and gallic acid obtained from an aqueous antibiofilm solution. Carvacrol was complexed in the solution with a stabilizing agent: hydroxypropyl- -cyclodextrin to form the K-HPBCD complex. Protocol

The K-HPBCD complex is obtained following a complexation method by sonication adapted according to the literature ⁽ ^36-37) of a solution of carvacrol and hydropropyl- -cyclodextrin, both at 16 mM. The solution is then filtered and lyophilized to obtain K-HPBCD powder. By spectrophotometric assay, the equivalent quantity of carvacrol in the K-HPBCD complexes is determined. This powder is then dissolved with gallic acid in water in order to obtain an antibiofilm solution. This antibiofilm solution was used to enrich the compresses which are then brought into contact with the mature biofilm of S. aureus for 24 hours at 37°C. Results

The antibiofilm activity of compresses containing a combination of carvacrol complexed with hydroxypropyl- -cyclodextrin and gallic acid is tested on a mono-species biofilm. Figure 7 illustrates the antibiofilm activities of these compresses containing a combination of carvacrol, complexed with hydroxypropyl- -cyclodextrin, and gallic acid. The solution of carvacrol complexed with hydroxypropyl- -cyclodextrin has a quantity of carvacrol of approximately 88.4 mg/g.

In figure 7, it is shown that a compress with an amount of carvacrol of 88.4 mg/g and gallic acid of 17.7 mg/g reduces the bacterial biomass of S. aureus by -7.6 log per compared to the control compress.

Thus, compresses based on alginate salt enriched with the combination of carvacrol, complexed with hydroxypropyl- -cyclodextrin, and gallic acid exhibit strong antibiofilm activity on the mature biofilm of S. aureus.

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Claims

1. Combination comprising carvacrol with gallic acid and/or curcumin, characterized in that the ratio of carvacrol and gallic acid is between 0.1 and 10 and the ratio of carvacrol and curcumin is comprised between 5 and 10, whether the association comprises two or three compounds.

2. Combination according to claim 1, characterized in that the ratio of carvacrol and gallic acid is between 1.5 and 10, advantageously between 1.5 and 5, more preferably about 5.

3. Combination according to claim 1, characterized in that the ratio of carvacrol and curcumin is between 5 and 10, more preferably about 10.

4. Combination according to claim 1, characterized in that it comprises carvacrol, gallic acid and curcumin.

5. Combination according to any one of claims 1 to 4, characterized in that it further comprises one or more ingredients chosen from a buffer, a stabilizing agent, a surfactant, a vitamin, a mineral, any element of the matrix extracellular, a pH adjuster, an antibiotic, an antimicrobial and another antibiofilm.

6. Combination according to any one of claims 1 to 5, characterized in that it is formulated in the form of a solution, semi-solid, powder, compress, wick or yarn.

7. Antibiofilm solution comprising the combination according to any one of claims 1 to 5 in an acceptable solvent.

8. Solution according to claim 7, characterized in that the acceptable solvent is water with a stabilizing agent, ethanol or a mixture of ethanol and water, in which the ethanol is between 7, 5 and 100% by volume relative to the total volume of the solvent.

9. Solution according to claim 7 or claim 8, characterized in that it comprises up to 50 mg/mL of carvacrol, up to 10 mg/mL of gallic acid and/or up to 5 mg/mL of curcumin.

10. Antibiofilm treatment product characterized in that it comprises the combination according to any one of claims 1 to 5.

11. Product according to claim 10, characterized in that it is chosen from a semi-solid, a powder, a compress, a wick or a thread.

12. Product according to claim 11, characterized in that the semi-solid, the powder, the compress, the wick or the thread each comprise up to 800 mg of carvacrol, up to 200 mg of gallic acid and up to to 100 mg of curcumin in 1 g of total weight.

13. Product according to any one of claims 10 to 12, characterized in that it further comprises a matrix based on alginate salt.

14. Product according to claim 13, characterized in that the alginate salt is chosen from calcium alginate, sodium alginate, potassium alginate, lithium alginate, zinc alginate, manganese alginate or ammonium alginate, preferably calcium alginate.

15. Combination according to any one of claims 1 to 6, for use in the prevention and/or treatment of disorders caused by biofilms.