FR3056403A1 - ANTIMICROBIAL ACTIVITY OF A TERPENIC MIXTURE OF VEGETABLE ORIGIN - Google Patents

Abstract

The invention relates to a composition comprising a natural product obtained by a controlled distillation process from essential oil of tea tree. This composition has a bactericidal activity on pathogenic bacteria, sensitive or resistant to antibiotics, growing or persistent. The bacteria do not develop acquired resistance when they are exposed to the composition for a long time. Because of its bacteriostatic, bactericidal or biocidal properties, the composition according to the invention is particularly intended for the manufacture of antiseptic or disinfectant formulations intended for humans or animals. The composition can be used to be incorporated into soaps, shampoos, creams, or oral hygiene products.

Description

ABSTRACT

The present invention relates to the bactericidal activity of a mixture of terpene derivatives hereinafter called TTOD, a derivative of the essential oil of tea tree devoid of the majority of its monoterpenes. TTOD activity can be used as an antibacterial agent in the preventive or curative treatment of infections or contaminations.

State of the art

Since their discovery at the beginning of the twentieth century, numerous molecules used as antibiotics have made it possible to treat various infections, hitherto incurable.

Antibiotic resistance is one of the major public health challenges of the twenty-first century, which is driving up hospital costs and mortality (1). It is accompanied by a severe lack of new effective therapeutic molecules. These new molecules are most often synthetic, similar to existing molecules, and therefore unfortunately generate the appearance of resistances, faster than there are new molecules created. Without new treatments, infections with resistant and multi-resistant bacteria will cause 10 million deaths per year by 2050 (1,2). The use of cocktails of active molecules, acting in complementarity, on several targets and quickly killing bacteria, are today the best strategy against infections that have become incurable, and against the appearance of resistance to antibiotics. And it is by drawing inspiration from nature that the most effective cocktails can be created.

The Tea Tree or "tea tree" (Melaleuca alternifolia) was once used by Australian Aborigines to cure many ailments. The plant was traditionally used in inhalation to treat colds, coughs and was dispersed on wounds to promote healing (3, 4). Then in 1992 its essential oil was first described for its incredible disinfectant properties (5).

In the centuries that followed its discovery, essential oil of tea tree has proven its effectiveness as a broad-spectrum antimicrobial agent, but also for its other properties such as anti-inflammatory, antioxidant, or even immunomodulator (6).

Several patents describe the inhibition of growth of bacteria by tea tree essential oil, by techniques of Minimum Inhibitory Concentration (MIC) or disc diffusion. So, for example:

Patent WO2014203263 describes a combination of essential oils, tested in MIC in microdilution on Actinomyces viscosus, inhibiting bacterial growth for applications in oral hygiene.

Patent CA2665983 describes an essential oil of a plant with antimicrobial activity increased by the addition of essential oil of oregano, cinnamon and organic or inorganic polyionic for applications in veterinary medicine.

The patent US2006 / 0068044 describes an extract of essential oil of tea tree inhibiting the growth of microorganisms by the technique of diffusion zones comprising Gram negative and positive bacteria, as well as molds. Its activity on viruses is also described. In the same patent, another extract of essential oil of tea tree has growth inhibiting properties by the CMI technique on Staphylococcus aureus ATCC 33591, and MRSA.

It is well known to those skilled in the art that tea tree essential oil in its original form, can contain up to 40% of toxic and irritant molecules, such as monoterpenes and products resulting from their degradation. which limits its applications. It is therefore essential to separate these harmful molecules from the beneficial compounds present in essential oil of tea tree. The inventor has developed an extraction process which makes it possible to remove the vast majority of toxic and irritant monoterpenes contained in essential oil of tea tree, without affecting its biological properties.

US Patent 2013/0158127 describes the inhibitory activity of a fraction of tea tree essential oil. 11 is characterized as bacteriostatic, according to the MIC technique in micro-dilution, on a set of Gram negative and positive microorganisms bacteria as well as yeasts and molds. The invention is also described as having an antiviral activity tested in vivo on dengue-infected macaques. By comparison, the HFF-1 cell toxicity of tea tree essential oil is higher than that of the fraction described. All of these patents use techniques such as Minimum Inhibitory Concentration (MIC) or disc diffusion which only demonstrate growth inhibitions, also called bacteriostatic properties, and not bactericidal (lethal) properties.

Description of the invention

The present invention is based on the bactericidal activity of TTOD, derived from essential oil of tea tree, on bacteria with active metabolism (division) and slowed down (persistence). The object of the present invention is the rapid bactericidal activity of TTOD associated with the inability of bacteria to develop acquired resistance there, which is by way of example, but not limited to, of interest for the pharmaceutical industries, veterinary, hygiene and cosmetics. Tea tree essential oil contains about 40% monoterpenes, well known to those skilled in the art for their widely described antimicrobial properties. It also contains sesquiterpenes and terpene alcohols. Sesquiterpenes and terpene alcohols have also been shown to have antimicrobial properties. But the work aimed at demonstrating it focused on growth inhibition using research techniques of minimum growth inhibitory concentration (MIC) or growth inhibition by disc diffusion, and not the demonstration of real power. bactericide.

Thus, those skilled in the art can only conclude that based on the available data, the antimicrobial / antibacterial property of tea tree essential oil is partly due to the monoterpenes it contains.

TTOD is obtained through a controlled distillation process from tea tree essential oil. It is almost devoid of monoterpenes (<5% mass / mass), but very largely contains sesquiterpenes and terpene alcohols (> 80% mass / mass).

Thus, on the basis of the arguments previously exposed, a person skilled in the art expects TTOD to have a lower bactericidal power than that of tea tree essential oil, since it is deprived of the essential monoterpenes.

Surprisingly, the inventor found that the antibacterial activity of TTOD was maintained, while TTOD lacked the vast majority of monoterpenes known to be very good antimicrobials. In addition, TTOD has been shown to be at least as bactericidal as tea tree against pathogenic bacteria. The concentration of certain active molecules targeted in TTOD, which are terpinene-4-ol and Γα-terpineol, associated with the depletion of molecules reputed to be active, but also toxic, irritant, or allergenic, does not affect the biological activities of the TTOD.

TTOD, a broad spectrum biocide, can be used, for example, but not exclusively, in formulations to develop disinfectants or antiseptics for humans or animals.

TTOD can also be incorporated into dressings, tissues, and other derivatives, to disinfect wounds, abrasions or other external wounds.

TTOD can also be used in formulations for human or animal hygiene as a soap, cream or lotion with multiple properties, such as for example, but in a non-exhaustive manner: anti-dandruff, sanitation of the skin, anti-acne, repellant.

It can also be used in formulation for intimate hygiene in prevention or treatment of an imbalance of the flora implying or leading to infections of the urovaginal sphere.

TTOD, effective against dental plaque, can be incorporated into oral hygiene products, such as chewing gum, mouthwash, toothpaste, dental floss.

Examples of embodiments The invention and its interest are well illustrated in the examples below, which however are not limiting in scope.

The examples below present tests carried out in a Mueller Hinton medium repeated two to three times independently.

Example 1: Composition of the TTOD

Table 1 shows the composition of tea tree essential oil and TTOD. The data in percentage indicates the maximum and minimum quantities obtained on 3 independent productions of TTOD, the data of essential oil of tea tree correspond to the directives of standard ISO / FDIS 4730: 2004 (n = 3).

TTOD is thus composed of terpenes at a mass / mass concentration of less than 0.2% for a-pinene, 0.2% for sabinene, 0.5% for Γα-terpinene, 0.2% for limonene, 0.6% for p-cymene, 0.5% for 1-8-cineol, 3% for γ-terpinene, 1% for terpinolene.

TTOD also has a mass / mass concentration greater than 68% for terpinen-4-ol, 4% for a-terpineol.

The composition of TTOD in unwanted molecules (monoterpenes) is much lower than that of tea tree essential oil. Indeed, they only make up 4% of the TTOD in mass / mass, while the beneficial molecules are present in more than 71% and 5% for terpinene-4-ol and Γα-terpineol respectively in mass / mass.

Table 1

EXAMPLE 2 Bactericidal Activity of TTOD

Table 2 represents the results of Minimum Inhibitory Concentration (MIC) and Bactericide (CMB) of TTOD on the bacterial strains Staphylococcus aureus, Staphyloccus aureus resistant to methicillin (MRSA), Escherichia coli, Pseudomonas aeruginosa. The concentrations tested in serial dilutions range from 0.0049% to 2.5% of TTOD. An activity control is carried out in parallel with a reference antibiotic: ampicillin (0.020-10 pg / ml), or amoxicillin or gentamicin (0.049-25 pg / ml) (n = 3).

Table 2

Graph 1 (a-h) represents the growth kinetics by enumeration of Staphylococcus aureus, MRSA, Escherichia coli, Pseudomonas aeruginosa in the presence of increasing concentrations of TTOD ranging from 1.25% to 5%. The tests were carried out in parallel with a reference antibiotic and the solvent for TTOD, dimethyl sulfoxide (DMSO) (n = 2). The bactericidal activity of TTOD is first defined by a CMB value identical to that of the MIC. The kinetics of the strains in the presence of TTOD shows bactericidal activity on Gram positive bacteria in only 1 h at 5%. The kinetics of the strains in the presence of TTOD show a bactericidal activity on Gram Negative bacteria in only 1 h at 1.25%. TTOD is at least as effective on sensitive bacteria as on bacteria resistant and multi-resistant to antibiotics, with effective concentrations equal or lower on resistant and multi-resistant bacteria compared to sensitive bacteria.

Example 3: Absence of Resistance to Treatment with TTOD

Table 3 presents the results of the MICs of the bacteria exposed and not exposed to TTOD and to the reference antibiotics during 30 passages. E. coli is tested on amoxicillin; P. aeruginosa on gentamicin; S. aureus on ampicillin. The breakpoint defines the concentration to be reached to consider the strain as resistant to the antibiotic (EUCAST Clinical Breakpoint Table v. 3.1, valid from 2013-02-11) (n = 2).

TTOD shows the same MIC values after 30 passages of bacteria in the presence of sub-inhibitory concentrations of TTOD. The results testify to the inability of bacteria to develop resistance systems to TTOD. For the strains and antibiotics used in controls, P. aeruginosa develops resistance to gentamicin, S. aureus develops resistance to ampicillin, and E. coli approaches the threshold for resistance to amoxicillin.

Table 3

EXAMPLE 4 Activity of TTOD on Growing and Persistent Bacteria

Table 4 presents the results of CMB obtained in the presence of TTOD and its reference antibiotic amoxicillin on E. coli, in the growth phase in comparison with E. coli in the persistence phase.

Persistent bacteria are as sensitive to the bactericidal activity of TTOD as when they are growing. The same CMB, except for a serial dilution of order 2, was measured. On the other hand, E. coli in the persistence phase is more resistant to amoxicillin with no CMB measured, even at the maximum concentration tested of 50 pg / ml, while it is 12.5 pg / ml in the phase of growth.

TTOD is effective against E. coli in the growth and persistence phase with the same intensity.

Table 4

I

Example 5: Topical gel Example of composition of a topical gel

EXAMPLE 6 Cosmetic Product

Example of the composition of an anti-acne cosmetic cream:

Example 7: Cosmetic product Example of an alcoholic antiseptic solution:

Example 8: Exfoliating cream Example of an antiseptic exfoliating cream:

Example 8: Nourishing Balm Example of Nourishing Cleansing Balm:

LEGENDS

Table 1: Chromatographic profile of essential oil (HE) of tea tree and TTOD.

Table 2: MIC and CMB of TTOD and antibiotics. Ampicillin is the reference antibiotic for Staphylococcus aureus, and MRSA, gentamicin is the reference antibiotic for Pseudomonas aeruginosa, and amoxicillin is the reference antibiotic for Escherichia coli. /: No MIC or CMB at the concentrations tested.

Figure 1: Time-Kill curves of E. coli, P. aeruginosa, S. aureus, and MRSA. a, c, e, g) In the presence of antibiotics (ATB); b, d, f, h) In the presence of TTOD. The experiments were carried out in independent triplicate. Diamonds = Growth control, square = CMI, triangles = 2xCMI, and circles = 4xCMI. The mean of the DMSO curves (three concentrations) represents the control of TTOD, while the antibiotics have been replaced by water for their controls.

Table 3: Results of MICs of bacteria exposed and not exposed to reference antibiotics. E. coli tested with amoxicillin; P. aeruginosa with gentamicin; S. aureus with ampicillin. The breakpoint defines the concentration to reach in order to consider the strain as resistant to the antibiotic (EUCAST Clinical Breakpoint Table v. 3.1, valid from 2013-02-11).

Table 4: Results of CBM on E. coli in the growth or persistence phase in the presence of TTD and amoxicillin. /: No CMB at the concentrations tested.

Material and method The efficacy of TTOD is tested on four bacterial strains: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus (MRSA).

Equipment. Ampicillin, amoxicillin, and gentamicin (Sigma-Aldrich) are made in sterile water. To increase the solubility of TTOD, Dimethyl sulfoxide (DMSO) is used (10% TTOD in DMSO 10%). Staphylococcus aureus CIP 4.83, MRSA CIP 107422, Pseudomonas aeruginosa CIP 82.118, and Escherichia coli CIP 53.126, are cultured in broth / agar medium Mueller Hinton (B / GMH).

Minimum inhibitory and bactericidal concentration (MIC / B). The CM1 are carried out in microdilutions according to the directives of CLSI 2009 (7), for 18h at 37 ° C. The maximum concentration tested in TTOD and DMSO is 2.5%, 10 pg / ml for ampicillin, and 25 pg / ml for amoxicillin and gentamicin. 50μ1 of the wells without visible growth are spread on nutritive agars of soy tripticase (GTS) and incubated at least 24 hours for counting. The CMB is defined by a reduction of 4 Logio of the inoculum or 99.99% decrease. A positive control without product and a negative control without bacteria are carried out for each test, as well as a solvent control with DMSO for each concentration used in TTOD preparations. Each test is performed three times independently. To study persistence, the same protocol is used except that the bacteria are inoculated in PBS. The lack of a nutrient encourages bacteria to slow down their metabolism and stabilize in the stationary phase. To emphasize this fact, the PBS suspension is prepared and incubated 3 hours before the launch of the test. The suspension is counted after 3 hours of pre-test incubation (= inoculum), and after 24 hours post-test (CMB). The reading is only done by CMB by spreading all the wells and counting the positive control to validate that it is close to 1 or 2 Logio near the inoculum used for the test.

Growth kinetics by enumeration. The study of growth kinetics by enumeration is adapted from a published protocol (8). Three concentrations of antibiotic, DMSO and TTOD are tested for each bacteria, corresponding to the MIC, 2xCMI and 4xCMI (except for DMSO whose concentrations are identical to the concentrations of TTOD tested). The antibiotic concentrations tested on S ARM are the same as those tested on S. aureus. The bacterial suspension used for the inoculum is prepared at 5.105-106 CFU / mL in BMH. The test is carried out in a 96-well microplate with a round bottom. Each test is carried out on three wells which are then pooled and counted on TS agars for each measurement. In this way, the experimental conditions are similar to that of CMI and CMB which allows a relevant comparison of the results. Concerning the interpretation of the results, the count is expressed in log10 as a function of the contact time. Each test is performed three times independently. Development of acquired resistance. This technique makes it possible to determine the capacity of microorganisms to adapt to an antimicrobial (decrease in its sensitivity). It is adapted from two published methods (9, 10). The technique uses the same plate scheme as the MIC tests, in 96-well round-bottom microplates. The concentrations tested are prepared according to the MIC previously obtained, varying by 0.5% between two consecutive wells. A minimum of thirty passages are made. A passage classically corresponds to a day or two (except for slow growth of the microorganism). During each passage, the well with the highest concentration of active principle having growth is inoculated on a new concentration range. The concentration range can be redefined with each pass according to an adaptation appearance. The plate is then placed in a closed container saturated with humidity, and incubated at the optimum growth temperature until the next passage. Once the 30 passages have been carried out, the exposed strain is tested in standard MIC in the presence of the active principle and the reference antibiotic, in parallel with the same unexposed strain. Every five passages, a Gram stain (plus oxidase test if necessary) is carried out in parallel with an isolate on rich agar in order to validate the purity of the suspension and observe a possible morphological modification (eg activation of the SOS system).

REFERENCES (1) Antibiotic resistance threats in the United States, 2013. U.S. Department of Health and Human Services, Center for Disease Control and Prévention.

(2) Estimating the economy costs of antimicrobial resistance, 2014, RAND (3) Shemesh, A., and W. L. Mayo. 1991. Australian tea tree oil: a natural antiseptie and fungicidal agent. Aust. J. Pharm. 72: 802-803. (4) Low T. 1990. Bush medicine. Harper Collins Publishers, North Ryde, NSW, Australia. (5) Penfold, A. R., and R. Grant. 1925. The germicidal values of sonie Australian essential oils and their pure constituées, together with those for some essential oil isolâtes, and synthetics. Part III. J. R. Soc. New South Wales 59: 346-349 (6) Carson CF, Hammer KA, Riley TV. 2006. Melaleuca altemifolia (Tea Tree) oil: a review of antimicrobial and other medicinal properties. Clin Microbiol Rev.

2006 Jan; 19 (1): 50-62. (7) Clinical and Laboratoiy Standards Institute guidelines, CLSI, 2008 (8) Ojo SKS, Ejims-Enukwe O, Esumeh FI. In-vitro antibacterial Time-Kill assay of Phyllanthus Amarus and Diodia Scandens crude extracts a Staphylococci isolated from wound and bums patients. International J pharmaceutical science invention, Volume 2 issue 8 august 2013 p09-13. (9) Hammer KA, Carson CF, Riley TV. 2012. Effects of Melaleuca altemifolia (tea tree) essential oil and the major monoterpene component terpinen-4-ol on the development of single- and multistep antibiotic resistance and antimicrobial susceptibility. Antimicrob Agents Chemother.

2012 Feb; 56 (2): 909-15. doi: 10.1128 / AAC.05741-11. Epub2011 Nov 14. (10) Cooper RA, Jenkins L, Henriques AF, Duggan RS, Burton NF. 2010. Absence of bacterial resistance to medical-grade manukahoney. Eur J Clin Microbiol Infect Dis.

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Claims (13)

Claims 1. Composition comprising a natural product obtained by a controlled distillation process from essential oil of tea tree or Melaleuca altemifolia as a bacteriostatic, bactericidal or biocidal agent. 2. Composition according to claim 1, characterized in that the natural product obtained from the essential oil of tea tree has a mass / mass concentration of less than 0.2% for Γα-pinene, 0.2% for the sabinene, 0.5% for Pa-terpinene, 0.2% for limonene, 0.6% for p-cymene, 0.5% for 1-8-cineol, 3% for γ-terpinene, 1 % for terpinolene. 3. Composition according to claim 1, characterized in that the natural product derived from essential oil of tea tree has a mass / mass concentration greater than 68% for terpinèn-4-ol, to 4% for l 'a-terpineol. 4. Composition according to claims 1 to 3 characterized in that it has a bactericidal activity either a reduction of the inoculum of at least 41og or 99.99% on pathogenic bacteria in less than an hour, at a concentration greater than 1.25% on Gram negative bacteria, and 5% on Gram positive. 5. Composition according to claims 1 to 3 is characterized in that it is bactericidal on bacterial strains sensitive or resistant to one or more antibiotics with the same effectiveness. 6. Composition according to one of claims 4 or 5 characterized in that the bacteria do not develop acquired resistance. 7. Composition according to one of claims 4 or 5 characterized in that it is at least as bactericidal on bacteria in persistence as on growing bacteria. 8. Use of the composition according to claims 1 to 3, for the manufacture of formulations for developing disinfectants or antiseptics for humans or animals. 9. Use of the composition according to claim 8 for its incorporation in dressings and other derivatives. 10. Use of the composition according to claims 1 to 3, for the manufacture of formulations for human or animal hygiene. 11. Use of the composition according to claim 10 for its incorporation in soaps for the body, face, hand, intimate hygiene, or in shampoo. 12. Use of the composition according to claim 10 for its incorporation in creams for the body, the hands, the face, the feet. 13. Use of the composition according to claim 10 for its incorporation in oral hygiene products, such as chewing gum, mouthwash, toothpaste, dental floss.