FR2697133A1 - Synergistic biocidal and biostatic compsns, active against eg bacteria, fungi and viruses - contg a sesquiterpene, eg pinene, and an aromatic cpd, eg carvacrol - Google Patents

Abstract

A biocidal and/or biostatic composition comprises at least one sesquiterpene (I) containing one oxygen atom, of formula C15HxO, where x is 20-26, and at least one aromatic synergistic agent (II). (I) may be a monohydroxy sesquiterpene such as viridiflorol, nerolidol, farnesol, isolongifolol, gaiol, cedrol, glubulol, bisabolol, ledol, cadinol, T-muurolol their isomers, esters, and ethers, or it may be a keto sesquiterpene such as vetivone. The aromatic cpd. (III) may, for example, be isoeugenol, methyl eugenol, safrol, isosafrol, cinnamaldehyde, cinnamyl alcohol, methyl benzoate, benzyl benzoate, thymol, or pref. eugenol or carvacrol. USE - The compsns. are active against Gram positive and negative bacteria, fungi, viruses, plants, molluscs, and insects. They may be used in anti-fouling paints, plant protection compositions, and for disinfection of food, medical and pharmaceutical premises.

Description

BIOCIDAL AND / OR BIOSTIC COMPOSITION AND ITS APPUCATIONS
The field of the present invention is that of the active principles, in particular useful as biocides and / or biostats, derived from vegetable essential oils. The active ingredients more specifically considered are sesquiterpene compounds.

 The present invention relates to biocidal and / or biostatic compositions comprising sesquiterpenic compounds, as well as the applications of this composition as a means of combating bacteria (Gram + or Gram ~), fungi, viruses, plants, molluscs or insects.

 The search for active products against the proliferation of living parasitic organisms, harmful to the health of living beings of the animal or higher plant kingdom, or likely to attack objects or constructions belonging to our environment, has always been one constant concerns of biology.

 In addition to direct chemical synthesis and fermentative biosynthesis, the plant world can be considered as an important source of biostatic compounds, that is to say, limiting the development of undesirable organisms, and / or biocides, that is, eradicating such organisms, because the plants are able to produce themselves, in some cases, the substances useful for their defense against the attacks of the external environment.

 This is how many essential oils or essences have already shown their bactericidal, fungicidal or other power. It has been possible to isolate and identify a plurality of active constituents of these species.

 Among these, we know well the family of sesquiterpenes which are terpenes in Cis and which are contained in the essences of a large number of plants.

 Oxygen derivatives of these aromatic substances have been repeatedly described as having interesting biocidal and / or biostatic properties.

The publication "Studies on Warburganal, Muzigadial and Related
Compounds ", from NAKANEHI et al., Israel Journal of Chemistry, Vol 16, 1977, pp. 28-31, discloses the warburganal and muzigadial which are two new sesquiterpenes isolated from two medicinal plants, respectively Warburgia ugandensis and Warburgia. stuhlmanni, more precisely, two monohydroxylated sesquiterpenes with aldehyde functions, which have proved active against worms, molluscs, bacteria, yeasts or other fungi and parasites.

 The authors of this publication have, in particular, found minimum inhibitory concentrations (MIC) of the order of 2-2.5 micrograms per disc for Candida, Saccharomyces and Trichophyton type yeasts.

 According to these authors, the molluscicidal activity of these compounds results, essentially, from the presence of aldehyde groups.

 Japanese Patent Application JP 74 00 69 describes the extraction and separation, from Picea polita leaves, of monohydroxylated sesquiterpenes, namely T-cadinol, T-muurolol, delta-cadinol, alpha -cadinol and oploganone. This document also teaches that these isolated compounds are useful as insecticides and insect repellents.

 Another patent application JP 61 03 31 29 relates to pharmaceutical formulations comprising a sesquiterpene alcohol and a polar compound, such as glycerin. The sesquiterpene alcohols considered are farnesol, hexahydro-farnesol, nerolidol, santalol, etc. According to this patent application, they would have antibiotic and antifungal properties, among other pharmacological effects.

 In search of a product having a high antibacterial activity specific for cariogenic bacteria, the Applicant of the patent application EP 0 130 027 proposes a monohydroxylated sesquiterpene, called 1-a-cadinol, as an antibiotic effective against anaerobic bacteria.

 1-ex-cadinol is described as having essentially bacteriostatic and, to a lesser extent, bactericidal activity.

 The document "Bacteriostatic activity of some Australian essential oils" by BEYLIER and Al appeared in Perfumer and Flavorist, vol. 4, April-May 1979, discloses the essential oils, including those extracted from Melaleuca quinquenervia containing 80% of monohydroxylated sesquiterpenol formed by Dnolidolidol, those extracted from Melaleuca viridiflora containing methyl cinnamate as the major constituent (75%) and and finally, those extracted from Australian sandalwood, containing 80% of a mixture of two monohydroxylated sesquiterpenols: alpha and beta-santalol and farnesol.

 it appears from this document that the essential oil containing D-nerolidol has bactericidal and fungicidal activities relatively inconsistent. In contrast, the mixture of santalol and farnesol is one of the most active active ingredients against Gram + bacteria and yeasts.

 However, this mixture has the disadvantage of being derived from Australian sandalwood, which is a protected plant and therefore can not be used as raw material for industrial production.

 The teaching that can be drawn from this document is that monohydroxylated sesquiterpenols do not exhibit a spectrum of antibacterial and antifungal extended activity and that their inhibitory performance is relatively average. It should be noted, also, availability problems specific to essential oils containing these active compounds.

 Finally, recent work on this subject shows that sesquiterpenols, in particular viridiflorol and nerolidol exert an antibacterial activity, all in all limited (MIC ranging from 17 to 37 micrograms per milliliter), even if these MICs are at least up to five times lower than the minimum MICs measured for the farnesol / santalol mixtures tested in the document BEYLIER et al mentioned above.

 These recent works contradict results previously described by some authors, as to the synergies existing between the constituents of essential oils mainly containing monohydroxylated sesquiterpenes.

 it thus follows from the foregoing that sesquiterpenols do not constitute, a priori, compounds of choice for the preparation of biocidal and / or biostatic compositions of high efficiency.

 In this state of the art, one of the essential objects of the present invention is to provide a biocidal and / or biostatic composition which offers an important inhibitory efficacy with regard to bacteria, fungi, viruses, molluscs, insects, algae and other plant or animal pests.

 Another object of the invention is that this biocidal and / or biostatic composition has a broad spectrum of activity for each of the above-mentioned species.

 Another object of the invention is that the constituents of such a composition can be obtained easily and economically.

 These objects and others have been achieved by the Applicant, who discovered, quite surprisingly and unexpectedly, that it was appropriate to combine at least one oxygenated sesquiterpene of formula Cl5HxOS x ranging from 20 to 26, and less a compound of aromatic structure, so as to obtain a synergistic effect, both from the point of view of the inhibitory performance, and from the point of view of the variety of living organisms parasites susceptible.

 The present invention therefore relates to a biocidal and / or biostatic composition characterized in that it contains at least one monooxygenated sesquiterpene of general formula ClsExOX x being between 20 and 26, and at least one synergistic agent consisting of a compound of aromatic structure.

 It is to the merit of the Applicant to have attempted such an association, notwithstanding the teaching of the prior art, which tends rather to rule out the use of monohydroxylated sesquiterpenes combined or not with other substances, given the results. previously unsuccessful, with regard to the biocidal and / or biostatic activities of these compounds.

 According to a first embodiment of the invention, the selected sesquiterpene belongs to the family of monohydroxylated sesquiterpenes.

 These compounds have the formula Cl 5 H x OH n x being between 20 and 26.

- trans-nerolidol:

- famesol:

- isolongifolol: - gaiol: - cedrol:

- bisabolol:

- ledol: - globulol: -α-cadinol:

Among the preferred compounds, there may be mentioned, by way of examples, the following products - viridiflorol:

- trans-nerolidol:

- famesol:

- isolongifolol: - gaiol: - cedrol:

- bisabolol:

- ledol: - globulol: - α -cadinol:

- T-muurolol:

- T-muurolol:

 It goes without saying that the invention is not limited to these compounds alone, but also extends to their isomers, such as the trans-trans, trans-cis and cis trans isomers of farnesol, or the isomers a and T a-cadinol, as well as their derivatives including, in particular, esterified or etherified derivatives.

According to a second embodiment of the invention, sesquiterpene is selected from the monocetone sesquiterpenes or their derivatives of the general formula C15HX O, x being between 20 and 26, such as the α-vétivone of formula:

 The other essential component of the composition according to the invention, namely the aromatic synergistic agent, is a hydrocarbon compound comprising at least one C6 ring, of a benzene nature.

dans laquelle R1, Rz, R3 et R4 sont des radicaux hydrogènes, hydroxyles, allyles, alcényles, alkoxyles, araikyles ou aryles, cyclisés ou non.More precisely, it must be considered that it corresponds to the following general formula (V):

in which R1, Rz, R3 and R4 are hydrogen, hydroxyl, allyl, alkenyl, alkoxyl, aryl or aryl radicals, cyclized or not.

 Without wishing to be bound by the theory, it is presumed that the existence of a Cs benzene ring in the aromatic synergistic agent is of some importance as to the synergistic pharmacological effect characteristic of the two types of constituents of the aromatic synergistic agent. composition according to the invention.

eugenol isoeugénol methyl safrol
eugenol

Examples of aromatic synergistic agents which are particularly suitable for the purposes of the invention are given below

eugenol isoeugenol methyl safrol
eugenol

<tb><SEP> fS <SEP> cinnamaldehyde <SEP> cinornyl <SEP> benzoate <SEP> from
<tb><SEP> alcohol <SEP> methyt
<tb> CH3
<tb><SEP> CÔO <SEP> OH <SEP>
<tb><SEP> o <SEP> H
<tb><SEP> H2C <SEP> CH <SEP> CH
<tb> benzoate <SEP> of <SEP> CH3 <SEP> CH3 <SEP> CH3 <SEP> CH3
<tb><SEP> benzyl <SEP> carvacrol <SEP> thymol
<Tb>
It has been demonstrated that eugenol and carvacrol are particularly preferred as constituents of the present invention.

 The biocidal and / or biostatic composition according to the invention may be in any suitable form. In particular, it may be an aqueous emulsion stabilized with an emulsifier, preferably inert and chemically stable, present for example at 10% by weight in the composition. Various emulsifiers can be used. Examples that may be mentioned include the emulsifiers sold under the names MIRJ, TWEEN, SPAN.

 The composition according to the invention is, in practice, a stabilized active ingredient, in liquid or solid form (powder), to be easily used in various more complex formulations.

 Ultimately, the composition comprises a mono-oxygenated sesquiterpene / aromatic synergistic agent combination of at least 5, preferably 10 and more preferably 15% by weight.

 According to an advantageous characteristic, the weight ratio of the (or) sesquiterpene (s) to the aromatic agent (s), expressed in parts by weight, is between 90 and 10: 90, preferably between 30:70 and 70:30, and more preferably between 40:60 and 60:40, the ratio 50:50 being particularly preferred.

 It is perfectly within the abilities of those skilled in the art to choose the sesquiterpene / aromatic synergistic agent weight ratio as a function of the final application aimed at.

 Similarly, it goes without saying that the amount of composition included in the final application formulation will be easily adjusted depending on the desired final dose and on the active combination content of the composition used.

 The composition according to the invention may be used in pure form or diluted in a support, optionally in the presence of an emulsifier, or else as a bactericidal and / or fungicidal and / or virucidal and / or mollusquicidal ingredient and / or phytocide and / or insecticide in elaborate formulations.

 In particular, it can be used as an agent against the deposit of molluscs, algae, fungi or bacteria on support surfaces, such as hulls of boats. The application here concerned is the elimination of the deposit of marine parasites, on the outside of the hulls of boats which, as we know, must be regularly maintained, in order to avoid their early deterioration.

This technique is also known as antifouling. The composition according to the invention can, for example, be perfectly integrated into a formulation of paints and varnishes intended to be applied to the hulls of boats, as antifouling treatment.
Another application of the composition according to the invention can be carried out as a phytosanitary agent for the control of cryptogamic or bacterial diseases of plants, or else as a disinfectant for food, medical or pharmaceutical purposes (hospitals, laboratories, food factories or refectories, ...).

 The mono-oxygenated sesquiterpene and the aromatic synergistic agent typical of the composition of the invention are either products obtained by chemical synthesis, or constituents of the essential oils or essences of certain plants V or their concrete or absolute. In the latter case, they are obtained by extraction according to appropriate methods, which are perfectly known to those skilled in the field under consideration. These are distillation techniques, for example under vacuum or steam, extraction and condensation supplemented by various purifications.

 By way of illustration, it can be stated that the viridiflorol and nerolidol mentioned above are obtained by distillation of a plant called niaouli, in particular of the species Melaleuca vindiflora growing mainly in Australia.

The table below gives the main sources of some of the preferred sesquiterpene compounds of the invention.

<tb><SEP> SESQUITERPENES <SEP> SOURCES
<tb><SEP> MONOOXYGENES
<tb> Farnesol <SEP> Abelmoschus <SEP> moschatus <SEP>
<tb> Trans-tran <SEP> s <SEP> farnesol <SEP> Hibiscus <SEP> abelomoschus
<tb> Trans-ci <SEP> s <SEP><SEP> farnesol <SEP> Synthesis <SEP> Chemical
<tb> Cis-trans <SEP> farnesol <SEP> Petigrain
<tb> Cedrol <SEP> Juniperus <SEP> Virginiana <SEP>
<tb><SEP> Dacrydtum <SEP> elatum <SEP>
<tb><SEP> Cypress
<tb><SEP> Bisabolol <SEP> Synthesis <SEP> Chemical
<tb><SEP> Guaiol <SEP> Wood <SEP> from <SEP> Guaiac
<tb><SEP> Isolongifolol <SEP> Synthesis <SEP> Chemical
<tb><SEP> a-cadinol <SEP> Chamaecyparis <SEP> lawsoniana
<tb><SEP> Junipencs <SEP> communis
<tb><SEP> 8-cadinol <SEP> Juniperus <SEP> communis
<tb><SEP> Pilgerodendron <SEP> Uniforms <SEP>
<tb><SEP> Pinus <SEP> albicaulis <SEP>
<tb><SEP> Pinus <SEP> annantll <SEP>
<tb><SEP> T-cadinol <SEP> Wood <SEP> from <SEP> Taiwania <SEP> cryptomeroids
<tb><SEP> a-vetivone <SEP> Vetiveria <SEP> zizanioides
<Tb>

Insofar as the essential oils of plants, their concrete or absolute, are sources for the various active compounds of the compositions according to the invention, these essential oils, these concretes or these absolutes, or mixtures of them, can directly form galenic forms
usable as is. To that end, they must preferably be purified,
for example by removal of their most volatile non-active compounds (cg
terpenes) and, above all, that they contain at least one mono-oxygenated sesquiterpene
and at least one synergistic agent according to the invention.

It is then advantageous for this (or these) sesquiterpene (s) and this (or these)
synergistic agent (s) are present in appropriate relative proportions and
preferred, as indicated above.

It therefore appears that the compositions according to the invention can be
formed by essential oils, concretes or absolutes, or
compositions of them, directly applicable.

The composition according to the invention, its advantages, as well as its
applications, will be better understood in the light of the example given below as
non-limiting.

EXAMPLE
EVALUATION OF THE BACTICIDE AND FUNGICIDE ACT
OF DIFFERENT COMPOSITIONS FOLLOWING THE INVENTION,
BY MEASURING MIC ON GRAM +, GRAS, YEAST BACTERIA
AND MUSHROOMS.

1- PREPARATION OF SAMPLES:
The mono-oxygenated sesquiterpene compounds used in the present
examples are nerolidol, farnesol, cedrol and globulol.

All these compounds are commercial quality products called
"puriss" and marketed by the company FLUKA, to a degree of purity
greater than 99%.

It is obvious that for a large scale production of compositions
according to the invention, it is advantageous to integrate, in the process of
preparation, extraction of essential oils from plants by distillation, as well as
that the separation and purification of the sesquiterpenes sought and
in these essential oils, by standard chromatography techniques
column and / or crystallization, for example.

 Thus nerolidol can be isolated from the essential oil of Niaouli.

The aromatic synergistic agents tested here are eugenol and carvacrol. he
also commercial products of quality "puriss" marketed
by FLUKA, at a purity level of over 99%.

The same remark as for the sesquiterpenes is to be formulated as regards
concerns the supply of these aromatic substances.

In the inhibitory potency tests, a control
constituted by pinene, an aromatic substance known for its lack of
antimicrobial capacity vis-à-vis the strains tested.

Various mixtures of mono-oxygenated sesquiterpenes and aromatic agents
synergistic were tested. The weight ratio of sesquiterpene / substance
aromatic used is 50:50 parts by weight.

2- METHODOLOGY:
The antimicrobial efficacy of the compositions according to the invention is measured at
using an aromatogram (or antibiogram) by the minimum concentration
Inhibitor or MIC, which is the lowest concentration of products
completely inhibiting the visible growth of microbial cells. She is
in vitro measurement and it is expressed here in milligrams per milliliter of
middle.

The technique used is that of ALLEGRINI and DE
BUOCHBERG (Prod Prob Pharm., (1972), 27, 891-897).

It consists in preparing a stable and homogeneous emulsion of the compositions
according to the invention or of the controls, by using an inert emulsifier and
chemically stable, at 10% by weight relative to the total mixture.

The emulsifier used in this example is the one marketed under the
Tween denomination.

Increasing dilutions in distilled water of this mother emulsion are then
poured into petri dishes, in which the culture medium is poured
employee. Emulsion concentrations vary from one thousandth to one thousandth.

The tested germ is then seeded on the surface. The boxes are incubated at
37 C for bacteria and 300 C for yeasts and fungi, during
18 to 24 hours.

The nutrient medium used for bacteria is "Nutrient Broth" (DIFCO),
while for yeasts and fungi use is made of
Sabouraud. These media are dosed from 10 to 16 g / l.

The strains tested come from INSTITUT PASTEUR's collection.
PRODUCTION (IPP). MICs are determined by visual reading of the
growth or non-growth of strains on Petri dishes.

Alternatively, one could also use the culture method in
liquid suspension with nephelometry reading or the technique of
the antibiogram with disks.

3 - RESULTS AND COMMENTS
The MIC determination results are given in Tables I and II
(Plate 1/1) corresponding, respectively, to the tests with eugenol and
carvacrol as synergistic aromatic agents.

The results presented above show that the combination of a sesquiterpenol
mono-oxygenated and, more precisely, monohydroxylated with a synergistic agent
aromatic having a benzene ring, such as eugenol or carvacrol,
provides antimicrobial efficacy exacerbated compared to that
typical of sesquiterpene used alone or that of the pinene control, and this
Gram +, Gram ~, yeasts and fungi.

It may in some cases be multiplied by a factor of 200
at 600.

Claims (12)

CLAIMS:  1-biocidal and / or biostatic composition, characterized in that it contains at least one mono-oxygenated sesquiterpene, of general formula Cl5ErO, x being between 20 and 26, and at least one synergistic agent consisting of a compound of aromatic structure .  2- Composition according to claim 1, characterized in that sesquiterpene is a sesquiterpene monohydroxyl selected from the following compounds: viridiflorol, nerolidol, famesol, isolongifolol, gaiol, cedrol, globulol, bisabolol, ledol, cadinol, T-muurolol or else an isomer or an ester or ether derivative thereof.  3 - Composition according to claim 1, characterized in that the sesquiterpene is a mono-ketonic sesquiterpene, such as vettivone.  4 - Composition according to any one of claims 1 to 3, characterized in that the aromatic synergistic agent is a hydrocarbon compound comprising at least one benzene C6 ring.  5 - Composition according to claim 4, characterized in that the aromatic synergistic agent corresponds to the following general formula:

 6 - Composition according to claim 5, characterized in that the synergistic aromatic agent is chosen from the following list of products:  eugenol, iso-eugenol, methyleugenol, safrol, isosafrol,  cinnamaldehyde, cinnamylalcohol, methyl benzoate, benzoate  benzyl, carvacrol, thymol, eugenol and carvacrol being particularly preferred.  7 - Composition according to any one of the preceding claims, characterized in that it comprises a combination sesquiterpenes / synergistic agent in a proportion of at least 5, preferably 10 and, more preferably still, 15% by weight.  8 - Composition according to any one of the preceding claims, characterized in that the weight ratio of (or des) sesquiterpene (s) on the (or) aromatic agent (s), expressed in parts by weight, is between 90: 10 and 10: 90, preferably between 30: 70 and 70: 30 and, more preferably still, between 40: 60 and 60: 40, the ratio 50: 50 being particularly preferred.  9 - Composition according to any one of the preceding claims, characterized in that it consists of an essential oil, a concrete or an absolute, or a mixture of essential oils, concretes and / or absolutes.  10 - Application of the composition according to any one of claims 1 to 9 as bactericidal and / or fungicidal and / or virucidal and / or molluscicidal and / or phytocidal and / or insecticidal.  il - Application of the composition according to any one of claims 1 to 9 as an agent against the deposit of molluscs, algae, fungi or bacteria on support surfaces, such as hulls of boats.  12 - Application of the composition according to any one of claims 1 to 9 as a phytosanitary agent for controlling fungal or bacterial plant diseases.  13 - Application of the composition according to any one of claims 1 to 9 as a disinfectant.