Extract of Euodia suaveolens Scheff, repellent compositions and use thereof

Abstract

The invention relates to an extract of Euodia suaveolens Scheff. The invention is characterized in that the extract is obtained from the airborne portions of the plant by means of at least one extraction step using an organic or hydro-alcoholic solvent, and in that it comprises evodone at a concentration of between 0.1 and 35% by weight of the total weight of the extract, and in that it has an insect repellent activity. The invention also relates to a repellent composition, characterized in that it comprises, in a cosmetically acceptable medium, evodone at a concentration of between 0.1 and 25% by weight of the total weight of the composition, and to the use of evodone as an active agent for repelling insects, in particular mosquitoes and more particularly the European/Mediterranean mosquitoes of the Aedes aegypti species.

Claims

1. A method of repelling insects, comprising applying an effective amount of an insect-repellant composition comprising evodone to an area in need of being protected from insects, wherein the composition comprises the evodone at a concentration of between 0.8 and 35% by weight, wherein the composition comprises at least one of linalool, alpha-pinene, evodiamine and rutaecapine.

2. The method according to claim 1, wherein the evodone is synthetic evodone.

3. The method according to claim 2, wherein the evodone is synthesized from 5-methylcyclohexan-1,3-dione in the presence of chloroacetone.

4. The method according to claim 1, wherein the composition comprises evodone at a concentration of between 2% and 20% by weight of the total weight of the composition.

5. The method according to claim 1, wherein the composition comprises evodone at a concentration of between 2% and 10% by weight of the total weight of the composition.

6. The method according to claim 1, wherein the composition comprises evodone at a concentration of between 2% and 5% by weight of the total weight of the composition.

7. The method according to claim 1, wherein the composition comprises evodone at a concentration of about 3% by weight of the total weight of the composition.

8. The method according to claim 1, wherein the insects are mosquitoes.

9. The method according to claim 8, wherein the mosquitoes are the European/Mediterranean mosquitoes of the Aedes aegypti species.

10. The method according to claim 1, wherein the composition comprises at least one of linalool, alpha-pinene, evodiamine and rutaecapine at a concentration of less than 0.1% by weight of the total weight of the composition.

11. The method according to claim 10, wherein the composition does not comprise any of linalool, alpha-pinene, evodiamine and rutaecapine at a concentration of more than 0.1% of the total weight of the composition.

12. The method according to claim 1, wherein the composition comprises evodone at a concentration of between 0.8% and 25% by weight, and wherein the composition further comprises a cosmetically-acceptable vehicle selected from the group consisting of water, ethanol, glycerin, propylene glycol, sorbitol, alcohol, and monopropylene glycol.

13. The method according to claim 12, wherein the composition comprises evodone at a concentration of between 2% and 10% by weight of the total weight of the composition.

14. The method according to claim 13, wherein the composition comprises evodone at a concentration of between 2% and 5% by weight of the total weight of the composition.

15. The method according to claim 14, wherein the composition comprises evodone at a concentration of about 3% by weight of the total weight of the composition.

16. The method according to claim 12, wherein the insects are mosquitoes.

17. The method according to claim 16, wherein the mosquitoes are the European/Mediterranean mosquitoes of the Aedes aegypti species.

18. The method according to claim 1, wherein the composition is applied on at least one of clothing and human skin.

19. The method according to claim 12, wherein the composition is coated by topical application on an area to be protected from insects.

20. The method according to claim 19, wherein the composition is coated on at least one of clothing and human skin.

21. The method according to claim 12, wherein the composition is sprayed by topical application on an area to be protected from insects.

22. The method according to claim 21, wherein the composition is sprayed on at least one of clothing and human skin.

Description

(1) The invention and benefits derived therefrom will be better understood after reading the following non-limiting description and embodiments with reference to the accompanying figures, in which:

(2) FIG. 1 is a 3D chromatogram obtained by high-performance thin layer chromatography (HPTLC) at 280 nm using a scanner, revealing the presence of evodone in different extracts of Euodia suaveolens Scheff;

(3) FIG. 2 is a reading at 254 nm before the development of a HPTLC chromatogram, showing different extracts of Euodia suaveolens Scheff, revealing the absence, or simply the presence of trace amounts, of rutaecarpine and evodiamine in the extracts according to the invention;

(4) FIG. 3 is a chromatographic profile obtained by gas chromatography with flame ionization detection on a polar column, which characterizes at least the partial composition of an extract of Euodia suaveolens Scheff according to the invention;

(5) FIG. 4 is a reading of a HPTLC chromatogram after visible light development, showing different extracts of Euodia suaveolens Scheff and revealing the presence of evodone in the extracts according to the invention; and

(6) FIG. 5 is a reading of a HPTLC chromatogram after visible light development, showing different extracts of Euodia suaveolens Scheff and revealing the presence of evodone in the extracts according to the invention.

(7) In this description, unless otherwise specified, it is understood that when a concentration range is given, it includes the upper and lower bounds of said range, and that the concentration is given by weight relative to the total weight of the composition or extract concerned.

(8) The extract of Euodia suaveolens Scheff according to the invention is isolated from the airborne portions of the plant.

(9) By airborne portions of the plant is meant the stems, leaves and flowers of the plant. These can be used fresh or dried, advantageously dried.

(10) To obtain the extract according to the invention, the airborne portions of Euodia suaveolens Scheff are advantageously harvested, dried and coarsely ground.

(11) At least one extraction step is then performed using organic or hydro-alcoholic solvents. This extraction step is different from an extraction step for the production of an essential oil or essence, which is obtained strictly by steam distillation or expression.

(12) The extraction is advantageously repeated several times, for example 3 times.

(13) It is performed with a plant/extraction solvent ratio, by weight/volume, between about 1/1 and about 1/20, for example 1/5 or 1/10.

(14) According to the invention, the temperature of the extraction solvent is greater than or equal to about 15 C., or room temperature. Advantageously, the maximum extraction temperature is the reflux temperature of the extraction solvent. For example, the extraction is carried out at 75 C. with ethanol.

(15) According to the invention, the extraction is advantageously dynamic and is performed under stirring.

(16) According to the invention, the extraction time is between 30 minutes and 5 hours for each extraction step, for example 3 or 4 hours.

(17) Non-limiting examples of organic solvents of the invention include alkanes, ether-oxides, esters, alcohols, halogenated hydrocarbons, carbon dioxide in the supercritical state.

(18) The organic solvent according to the invention is preferably selected from pentane, hexane, heptane, octane, cyclohexane, tetrahydrofuran, dioxane, diethyl ether, ethyl acetate, isopropyl acetate, methanol, ethanol, propanol, isopropanol, butanol, methyl ethyl ketone, dimethyl ketone, methyl isobutyl ketone, dichloromethane, dimethyl carbonate or carbon dioxide in the supercritical state or a mixture of the above-mentioned solvents, preferably hexane and ethanol.

(19) Advantageously, the organic solvent is selected from organically produced solvents, for example alcohols and carbon dioxide in the supercritical state.

(20) According to the invention, the hydro-alcoholic solvent, for example, is an ethanol/water dilution solvent with a ratio of 80/20 by volume.

(21) In the preparation of an extract according to the invention, a separation of the solid phase from the liquid phase is then carried out. The solid portions of the plant are thus separated from the liquid extraction juice by plate filtration or centrifugation, pressing.

(22) The thus obtained filtrate is then concentrated by evaporation, to yield a more or less concentrated extract, if appropriate until total evaporation of the extraction solvent.

(23) Advantageously, the filtrate can be concentrated by vacuum evaporation.

(24) The dark green-brown concentrated extract can then be bleached or refined using activated carbon, or acidic or clayey earths, or molecular distillation or extraction with carbon dioxide in the supercritical state or codistillation.

(25) The extract according to the invention can then be purified. It can be purified by any technique known to one skilled in the art, such as, for example, liquid phase-liquid phase extraction between 2 immiscible solvents, absorption onto a carrier such as silicate or an ion exchange resin, precipitation or crystallization.

(26) The mass yield of the extract is between about 1% and about 25% of the dry matter involved, for example about 15%.

(27) According to the invention, different types of extractions can be performed, such as maceration (static extraction at room temperature), hot static extraction, decoction, leaching.

(28) According to an advantageous embodiment of the invention, from a quantitative point of view in terms of yield, a Soxhlet extraction or dynamic extraction, maceration, static hot extraction, ultrasonic extraction or microwave extraction with ethanol at a concentration greater than about 80%, for example 80%, 96% or 97%, is carried out.

(29) After purification, the extract according to the invention can be dried by total evaporation of the solvent and then dissolution in a compatible and cosmetically acceptable solvent.

(30) Another feature of the extract is that it does not contain linalool, alpha-pinene, or evodiamine, rutaecarpine, or at least not in an effective amount according to the prior art. Even though these compounds are present in the plant, the extract or oleoresins obtained from this plant contain only a small amount of linalool and alpha-pinene and neither evodiamine nor rutaecarpine.

(31) Furthermore, the extract according the invention has a zero or very small titer of these molecules, for example a content of less than 1%, preferably less than 0.1%, and therefore contains only trace amounts of these molecules. Indeed, linalool and alpha-pinene are removed, in particular, during the organic or hydro-alcoholic extraction of the extract according to the invention. These molecules are driven by the distillation of the solvent and are diluted by concentrating the extract. For evodiamine and rutaecarpine, the solvent used for the extraction according to the invention is not selective for these types of alkaloid structures. The extract according to the invention, when tested on European/Mediterranean mosquitoes of the Aedes aegypti species, therefore no longer contains, or contains only trace amounts of these four molecules. According to the invention, the properties displayed by such an extract are not attributable to the presence of such substances.

(32) The extract according to the invention, which, in itself, is a new product, however contains non-volatile compounds such as flavonoids, for example glycosylated flavanols such as rutine or 3 O-rutinosylquercetol, flavonols such as kaempferol and isorhamnetin or methyl-3-quercetin, a flavone, such as a derivative of apigenine, for example vitexin or vitexin 2O rhamnoside, as well as volatile compounds such as bicyclic lactones, in particular evodone or (6S)3-6 dimethyl 6,7 dihydro 5H benzofuran-4-one, menthalactone or mintfuranone or 3,6-dimethyl-5,6,7,7a-tetrahydro-4H-1-benzofuran-2-one, aromacurcumene, and fatty acids, in particular of the acidic, palmitic or oleic type.

(33) In particular, the extract of the invention contains a titer of evodone, having the following chemical structure:

(34) ##STR00008##

(35) Evodone is an oxygenated monoterpene, that is, a benzofuranone derived from menthofuran. Its description can be found, in particular, in Euodia hortensis (Rutaceae). The plant Calamintha ashei (Lamiaceae) also contains oxygenated monoterpenes such as evodone, caryophyllene, menthofuran, and calaminthone. These compounds have been studied for their allelopathic properties for the inhibition of the seeds of certain species such as Lactuca sativa and Schizachyrium scoparium. A mixture of these terpenes appears to inhibit seed germination, but the individual compounds do not seem to have a significant activity.

(36) The synthesis of evodone can be performed from 5-methylcyclohexan-1,3-dione in the presence of chloroacetone (A. Srikrisma and G. Veera Raghava Sharma, A simple synthesis(+/) evodone, Indian journal of Chemistry, 1989, p. 852). 99% pure evodone (racemic mixture) was thus obtained.

(37) The evodone titer of the extract according to the invention advantageously corresponds to a concentration of between about 0.1 and 35% by weight of the total weight of the extract, preferably between 0.2 and 5%.

(38) For example, a crude, unrefined oleoresin according to the invention may comprise between 0.1 and 35% of evodone, for example 0.8% for an unrefined crude extract, and 20.16% when the codistilled oleoresin is taken up in hexane, and a codistilled refined extract may include between 0.1 and 20% of evodone, for example 2%.

(39) As shown in FIGS. 1, and 3 to 5, evodone is present in an extract of Euodia suaveolens Scheff according to the invention.

(40) In FIG. 1, chromatograms 3 to 14 represent the deposited evodone standard at the same concentration and different volumes, and are characterized by the presence of a peak having a baseline (front ratio) of 0.69 for this mobile phase in this particular analysis case. It is observed that the same evodone-characteristic peak is present in chromatograms 1, 2, 15, 16, 17 pertaining to extracts of Euodia suaveolens Scheff, namely, respectively, in an oleoresin obtained from fresh leaves, an oleoresin obtained from dry leaves, a formulation based on a codistilled extract with an evodone content of 0.3%, another formulation based on a codistilled extract with an evodone content of 2.36%, and codistilled oleoresin taken up in hexane.

(41) Furthermore, analysis of the essential oil of Euodia suaveolens Scheff, by comparing the essential oil with the oleoresin extracted according to the invention, reveals various compounds identified by gas chromatography coupled with mass spectrometry.

(42) The Table below shows some of the compounds mentioned above and described by some authors as being responsible for the repellent activity (including alpha pinene and linalool). This Table shows that, based on the obtained results, alpha pinene and linalool, in particular, are not present in the oleoresin extracted according to the invention. However, the oleoresin extracted according to the invention retains a strong repellent activity against mosquitoes.

(43) TABLE-US-00001 Content of compounds Theoretical content analyzed GC/FID of compounds in the oleoresin content in in the oleoresin extracted according the analyzed extracted according to the invention, Compounds essential oil to the invention in GC/FID alpha pinene 0.12% 0.0086% not detected Limonene 18.77% 1.13% not detected Linalool 0.08% 0.00480% not detected Menthofuran 31.30% 0.28% not detected Limonen 10 ol 6.21% 0.38% 2.28%

(44) For information, the theoretical levels given in the Table have been extrapolated from analyses of the essential oil. Based on this data, it is indeed possible to determine the theoretical contents of these compounds in the plant, and then, in the oleoresin extracted according to the invention.

(45) However, the fact that the oleoresin according to the invention is obtained by alcoholic extraction followed by concentration, should be taken into account. Thus, when the solvent is evaporated, most of these molecules, which are volatile molecules, are primarily driven by azeotropy, which explains why they are not found in the extract according to the invention.

(46) In addition, as shown in FIG. 2, rutaecarpine and evodiamine are not present in the extract of the invention, or are present in trace amounts only.

(47) Specifically, in FIG. 2, different extracts of Euodia suaveolens Scheff have been analyzed: lane 1 relates to a concrete obtained by hexane extraction from fresh Euodia suaveolens Scheff leaves; lane 2 relates to an absolute obtained by alcohol extraction of the concrete; lane 3 relates to a bleached oleoresin according to the invention; lane 4 relates to an extract according to the invention, bleached by molecular distillation; lane 5 relates to an essential oil of Euodia suaveolens Scheff; lane 6 is empty; lane 7 corresponds to the evodiamine standard with a baseline (retardation ratio) of 0.35-0.39, shown to be 0.38 in FIG. 2, under these analytical conditions; lane 8 corresponds to the rutaecarpine standard with a baseline (retardation ratio) of 0.53 under these analytical conditions; lane 9 is empty; and lane 10 corresponds to the evodone standard.

(48) In lanes 3 and 4, the absence of a band with a baseline of 0.53 (retardation ratio) is observed, which is characteristic of the presence of rutaecarpine in these extracts. Thus, the extracts according to the invention deposited in lanes 3 and 4 are free of rutaecarpine. It is also observed in lanes 3 and 4 that there is no significant band having a baseline of 0.38 (retardation ratio), which is characteristic of the presence of evodiamine in these extracts. Only trace amounts are distinguished, which might indicate the presence of traces of evodiamine in the extracts according to the invention.

(49) Furthermore, as shown in FIG. 3, the composition of a bleached ethanol extract of Euodia suaveolens Scheff according to the invention has been characterized at least partially by GC/FID chromatography (Agilent Technologies HP 6890) on a polar column (Varian CP8713 CP WAX 52 CB; 50 m0.25 mm0.2 mm) following a rise in temperature of 70 C. for 5 min, then of 2 C./min up to 240 C. for 50 min, and for an injected volume of 1 l. In this extract, peaks that are characteristic of the presence of benzaldehyde (peak 1), carbitol (peak 2), aroma curcumene (peak 3), benzyl alcohol (peak 4), evodone (peak 5), limonene-10-ol or (p-mentha-1,8(10)-dien-10-ol) (peak 6), levojunenol (peak 7), menthalactone isomer 1 (peak 9), menthalactone isomer 2 (peak 10), phytol (peak 12), and peaks that are characteristic of the presence of fatty acids, can be observed.

(50) As illustrated in FIG. 4, HPTLC analyses of evodone and various extracts of Euodia suaveolens Scheff according to the invention were carried out: lane 1 relates to an extract of Euodia suaveolens Scheff obtained by codistillation on monopropylene glycol in bleached oleoresin; lane 2 relates to an extract obtained by codistillation on monopropylene glycol of a non-bleached oleoresin obtained from fresh Euodia suaveolens Scheff leaves; lanes 3 to 7 correspond to the evodone standard; lane 8 relates to a crude unrefined oleoresin obtained by ethanol extraction from fresh Euodia suaveolens Scheff leaves; lanes 9 and 10 correspond to the evodone standard; lane 11 relates to a bleached oleoresin obtained by ethanol extraction from dry leaves of Euodia suaveolens Scheff; lane 12 relates to a bleached oleoresin obtained by ethanol extraction from fresh leaves of Euodia suaveolens Scheff; lane 13 is empty; lane 14 corresponds to the evodone standard.

(51) Thus, the extracts according to the invention that are deposited in lanes 1, 2, 8, 11 and 12 do contain evodone.

(52) As shown in FIG. 5, HPTLC analyses of evodone and various extracts of Euodia suaveolens Scheff according to the invention were carried out: lane 1 relates to an unrefined crude oleoresin obtained by ethanol extraction from dry leaves of Euodia suaveolens Scheff; lane 2 relates to an unrefined crude oleoresin obtained by ethanol extraction from fresh leaves of Euodia suaveolens Scheff; lanes 3 to 14 correspond to the evodone standard; lanes 15 and 16 relate to formulations obtained from codistilled oleoresin extracts; lane 17 is empty; lane 18 relates to hexane extraction of a codistilled oleoresin.

(53) Thus, the extracts of the invention deposited on lanes 1, 2, 15, 16, 18 do contain evodone.

(54) The extract according to the invention is natural. Advantageously, the extract can be certified as organic, using organic extraction solvents such as alcohols or carbon dioxide in the supercritical state.

(55) The extract is biodegradable and non-toxic. Indeed, a toxicity study of an ethanol extract of Euodia suaveolens Scheff was performed at a concentration of 100% pure, non-bleached, on six female Sprague Dawley rats, aged 8 weeks and weighing between 186 g and 210 g. The study began after an acclimatization period of 5 days. All factors such as brightness, temperature and humidity were controlled. The results are obtained by comparison with a control group of rats that received dimethyl sulfoxide only. Each rat received a single effective dose of 2000 mg/kg by body weight of the pure extract of Euodia suaveolens Scheff, diluted in dimethyl sulfoxide and administered orally, in particular by force-feeding, at a volume of 5 ml/kg of body weight. Based on the results obtained, the extract according to the invention is not toxic. Specifically, it has an LD50 of more than 2000 mg/kg of body weight. According to the OECD 423 directive, the LD50 can be considered to be greater than 5000 mg/kg by bodyweight after oral administration in rats.

(56) As an example of a method for extracting and obtaining an extract of Euodia suaveolens Scheff or oleoresin according to the invention, the steps described below, in particular, are carried out.

(57) The previously dried leaves and flowers of Euodia suaveolens Scheff are first coarsely ground.

(58) 600 g of non-finely ground plant are thus added to a 20 L three-necked flask placed in a water bath fed with steam.

(59) The extraction is performed with 96% ethanol. Three extractions are performed.

(60) The first extraction is performed with a volume of 10 times the weight of the ground plant, or 6 L. The extraction is carried out under reflux with mechanical stirring for 3 hours, at a temperature of 75 C.

(61) The filtrate is then discharged and collected.

(62) A second extraction is then performed with a volume equal to 6 times the mass of the initial plant, or 3.6 L, under reflux, with stirring for 3 hours.

(63) Thereafter, a final extraction is performed with a volume equal to 4 times the initial mass of plant, that is 2.4 L.

(64) The three filtrates are combined and then filtered on a Bchner filter.

(65) A clear, dark green extract is obtained.

(66) The extract according to the invention is then concentrated under vacuum using a rotary evaporator at a temperature of 60 C.

(67) An oleoresin according to the invention, dark green in color, extracted from Euodia suaveolens Scheff, is thus obtained. It has a pasty appearance, is solid at room temperature and viscous-liquid when hot.

(68) The extract according to the invention is then bleached with activated carbon at a content of 10% by weight in an 80/20 ethanol/water mixture. The finally obtained extract according to the invention is reddish/brown, glossy, and soluble in an 80/20 ethanol/water mixture.

(69) The extract according to the invention is moderately fragrant.

(70) Alternatively, the extract according to the present invention is bleached with supercritical CO.sub.2.

(71) Various tests using supercritical CO2 were carried out on the above oleoresin, for example at a pressure of 180 bar and at a temperature of 45 C., or at a pressure of 250 bar and at a temperature of 45 C., with 10% ethanol as co-solvent.

(72) The CO.sub.2 test without a co-solvent results in a lemon yellow, odorous extract, but having a non-uniform appearance.

(73) The CO.sub.2 extract obtained with an ethanol-based co-solvent allows a reddish brown, less odorous extract to be obtained.

(74) According to a preferred embodiment, the oleoresin obtained from fresh plants is codistilled on a cosmetically acceptable carrier such as monopropylene glycol. This codistillation provides a pale yellow extract having a high concentration of evodone, for example about 2%.

(75) This extract can then be taken up in hexane to increase the evodone titer.

(76) According to a second aspect, the invention also pertains to a repellent composition comprising, in a cosmetically acceptable medium, evodone at a concentration between 0.1 and 25% by weight of the total weight of the composition.

(77) Preferably, the repellent composition according to the invention comprises evodone at a concentration of between 0.2 and 10% by weight of the total weight of the composition, for example at a concentration of 3% by weight of the total weight of the composition.

(78) The invention also relates to a composition comprising an effective amount of an extract of Euodia suaveolens Scheff as defined above, as the active repellent agent against insects, in particular against mosquitoes. It is incorporated into the composition with any cosmetically acceptable excipient. Such a composition is substantially free of any effective amount of linalool, alpha-pinene, evodiamine or rutaecarpine, and, in particular, has an evodone titer, at a concentration of between 0.1 and 25% by weight of the total weight of the composition.

(79) Advantageously, the extract content in the above composition is between 1 and about 70% by weight of the total weight of the composition, preferably 10.10%, 26.8%, 32% or 35%.

(80) For example, a composition according to the invention may comprise 35% of an ethanol extract (ethanol/water as dilution solvent: 80/20), 10.10% of an extract obtained by codistillation with monopropylene glycol, or 26.8% of an extract obtained by alcoholic extraction, formulated with castor oil and ethanol.

(81) The compositions according to the invention may be more or less fluid and may be in any pharmaceutical form suitable for use in a topical application to the skin, for example, in the form of a milk, gel, cream, lotion, emulsion, spray and preferably in the form of a spray.

(82) Examples of solvents used in the composition according to the invention include: water, ethanol, glycerin, propylene glycol, sorbitol.

(83) Said composition may optionally comprise another active ingredient selected from agents which have a recognized repellent effect on insects, preferably a naturally occurring repellent agent, for example menthoglycol or citriodiol.

(84) This naturally occurring agent is derived from the essential oil of Eucalyptus citriodora.

(85) By way of illustration, some formulation examples of compositions according to the invention are mentioned below, which contain evodone or the above-mentioned extract of Euodia suaveolens Scheff:

EXAMPLE 1

Spray Against European/Mediterranean Mosquitoes (Aedes aegypti) for Topical Application

(86) 32% of extract of Euodia suaveolens Scheff

(87) 1.3% of citriodiol

(88) 65% of 80%-alcohol

(89) 1.7% of monopropylene glycol

EXAMPLE 2

Spray Against European/Mediterranean Mosquitoes (Aedes aegypti) for Topical Application

(90) 3% of evodone

(91) 63% of 100%-alcohol

(92) 34% of monopropylene glycol

EXAMPLE 3

Spray Against European/Mediterranean Mosquitoes (Aedes aegypti) for Topical Application

(93) 26.8% of extract of Euodia suaveolens Scheff

(94) 33.5% of castor oil

(95) 39.7% of 100%-alcohol

(96) In a third aspect, the present invention relates to the use of evodone as an active agent for repelling insects, particularly mosquitoes and more particularly European/Mediterranean mosquitoes of the Aedes aegypti species.

(97) The invention also relates to the use of an extract of Euodia suaveolens Scheff as an active agent, or of compositions as defined above to repel insects, in particular mosquitoes and more particularly European/Mediterranean mosquitoes of the Aedes aegypti species.

(98) In this respect, the invention is illustrated by the following efficacy tests, carried out in the laboratory, where the formulas are not degraded through the effects of abrasion, exudation, sunlight, temperature or even swimming in the sea. These tests are designed to demonstrate the repellent effect of evodone and an extract of Euodia suaveolens Scheff at different concentrations, purified, whether bleached or not, alone or in combination with another natural active repellent agent such as menthoglycol, on European/Mediterranean mosquitoes of the Aedes aegypti species.

(99) The tests are carried out on female Aedes aegypti mosquitoes aged 6 to 10 days having undergone a prior 24-hour period of fasting (no blood meal) in order to increase their aggressiveness.

(100) The blood meal target is a mouse of the hairless type held in a retaining cage (using eye protection) and coated with the test product (0.1g/60 cm.sup.2) applied with a pipette.

(101) 30 minutes after product application, the mouse is introduced into a cage containing approximately 200 female mosquitoes (prior removal of the males using a food gradient).

(102) The test lasts 5 minutes and during this period the number of mosquitoes having landed on the mouse (landing effect: number of mosquitoes that land on the treated area and leave without biting) and the number of effective bites, are recorded.

(103) The test is stopped after 5 effective bites (stop in the Tables below).

(104) The test is repeated with new batches of mosquitoes, at intervals of one hour until a duration of 8 hours is reached, depending on the persistence limit of the product (between the runs, the mouse was released). Based on the outcome of the first repetition, repetitions are performed at time intervals of 30 minutes in order to provide a more accurate limit for the duration of protection.

(105) During and between the runs, the mice are maintained in a tropical like atmosphere under controlled conditions at 27+/1 C. and 70+/5% relative humidity. The cages are changed upon each unit test and placed 3 meters apart.

(106) 3 repetitions are carried out per formula to be tested and on 3 different mice, that is, 9 unit tests per formula.

(107) Test 1:

(108) 4 extracts (supplier: Charabot S. A.) are tested comparatively, the solvent serving as a reference (it is verified that at least 10 landings occur in 30 seconds to validate the test):

(109) Extract 1: ethanol extract of Euodia suaveolens Scheff according to the invention at a concentration of 35% (ethanol/water as dilution solvent: 80/20) non-bleached;

(110) Extract 2: ethanol extract of Sichuan red pepper (Soxhlet extraction) at a concentration of 19.44% (ethanol as dilution solvent);

(111) Extract 3: ethanol extract at a concentration of 35% of menthoglycol;

(112) Extract 4: Spilanthes extract at a concentration of 18.56% (ethanol as dilution solvent); and

(113) Test without any product (reference): solvent.

(114) The results are summarized in the following Table:

(115) TABLE-US-00002 AEDES T0 + 2 H T0 + 4 H T0 + 6 H T0 + 8 H T0 + 10 H T0 + 12 H AEGYPTI mice rep. P L P L P L P L P L P L Extract 1 1 1 0 0 0 0 0 3 stop stop stop stop stop stop 2 0 0 0 1 0 1 stop stop stop stop stop stop 3 0 0 0 1 0 2 stop stop stop stop stop stop 2 1 0 0 0 0 0 1 stop stop stop stop stop stop 2 0 0 0 0 0 2 stop stop stop stop stop stop 3 0 0 0 0 0 4 stop stop stop stop stop stop 3 1 0 0 0 0 0 1 stop stop stop stop stop stop 2 0 0 0 1 0 2 stop stop stop stop stop stop 3 0 0 0 0 0 0 stop stop stop stop stop stop Extract 2 1 1 0 0 0 1 stop stop stop stop stop stop stop stop 2 0 0 0 2 stop stop stop stop stop stop stop stop 3 0 0 0 0 stop stop stop stop stop stop stop stop 2 1 0 0 0 0 stop stop stop stop stop stop stop stop 2 0 0 0 1 stop stop stop stop stop stop stop stop 3 0 0 0 0 stop stop stop stop stop stop stop stop 3 1 0 1 0 1 stop stop stop stop stop stop stop stop 2 0 0 0 1 stop stop stop stop stop stop stop stop 3 0 0 0 1 stop stop stop stop stop stop stop stop Extract 3 1 1 0 0 0 1 0 3 stop stop stop stop stop stop 2 0 0 0 1 0 2 stop stop stop stop stop stop 3 0 0 0 0 0 2 stop stop stop stop stop stop 2 1 0 0 0 0 0 0 stop stop stop stop stop stop 2 0 0 0 1 0 2 stop stop stop stop stop stop 3 0 0 0 0 0 1 stop stop stop stop stop stop 3 1 0 0 0 0 0 0 stop stop stop stop stop stop 2 0 0 0 0 0 1 stop stop stop stop stop stop 3 0 0 0 0 0 0 stop stop stop stop stop stop Extract 4 1 1 0 0 0 0 stop stop stop stop stop stop stop stop 2 0 0 0 1 stop stop stop stop stop stop stop stop 3 0 0 0 0 stop stop stop stop stop stop stop stop 2 1 0 0 0 1 stop stop stop stop stop stop stop stop 2 0 0 0 3 stop stop stop stop stop stop stop stop 3 0 0 0 1 stop stop stop stop stop stop stop stop 3 1 0 1 0 1 stop stop stop stop stop stop stop stop 2 0 0 0 2 stop stop stop stop stop stop stop stop 3 0 0 0 1 stop stop stop stop stop stop stop stop P: number of bites rep: repetition L: landing: number of mosquitoes landing on the treated area without biting stop = the test is stopped when more than 5 bites have been observed

(116) Under these test conditions, with the samples provided, the insect strains and methods considered, extracts 1 and 3 showed a repellent effect against the mosquito, with a persistence of 6 hours after application.

(117) Extracts 2 and 4, at the concentrations used, showed a repellent effect against the mosquito with a persistence of only 4 hours after application.

(118) Extract 1 of Euodia suaveolens Scheff, at a concentration of 35%, purified, non-bleached, thus shows a true repellent effect with a persistence of 6 hours against the Aedes aegypti mosquito.

(119) Test 2:

(120) 3 extracts (supplier: Charabot S. A.) are tested comparatively, with the solvent serving as a reference (it is verified that at least 10 landings occur in 30 seconds to validate the test):

(121) Extract 1: ethanol extract of Euodia suaveolens Scheff according to the invention at a concentration of 15%, bleached by molecular distillation (ethanol/water as dilution solvent: 80/20);

(122) Extract 2: ethanol extract of Euodia suaveolens Scheff according to the invention, bleached at a concentration of 35% (ethanol/water as dilution solvent: 97%);

(123) Extract 3: menthoglycol (20%)+Euodia suaveolens Scheff (15%) mixture (ethanol/water as dilution solvent: 80/20), bleached; and

(124) Test without any product (reference): solvent.

(125) The results are summarized in the following Table:

(126) TABLE-US-00003 AEDES T0 + 2 H T0 + 4 H T0 + 6 H T0 + 8 H T0 + 10 H T0 + 12 H AEGYPTI mice rep. P L P L P L P L P L P L Extract 1 1 1 0 0 0 0 stop stop stop stop stop stop stop stop 2 0 0 0 2 stop stop stop stop stop stop stop stop 3 0 0 0 0 stop stop stop stop stop stop stop stop 2 1 0 0 0 0 stop stop stop stop stop stop stop stop 2 0 0 0 1 stop stop stop stop stop stop stop stop 3 0 0 0 0 stop Stop stop stop stop stop stop stop 3 1 0 0 0 0 stop Stop stop stop stop stop stop stop 2 0 0 0 2 stop Stop stop stop stop stop stop stop 3 0 0 0 0 stop Stop stop stop stop stop stop stop Extract 2 1 1 0 0 0 0 0 3 stop stop stop stop stop stop 2 0 0 0 0 0 2 stop stop stop stop stop stop 3 0 0 0 0 0 0 stop stop stop stop stop stop 2 1 0 0 0 0 0 0 stop stop stop stop stop stop 2 0 0 0 0 0 1 stop stop stop stop stop stop 3 0 0 0 0 0 0 stop stop stop stop stop stop 3 1 0 0 0 1 0 1 stop stop stop stop stop stop 2 0 0 0 0 0 1 stop stop stop stop stop stop 3 0 0 0 1 0 2 stop stop stop stop stop stop Extract 3 1 1 0 0 0 0 0 1 stop stop stop stop stop stop 2 0 0 0 0 0 0 stop stop stop stop stop stop 3 0 0 0 0 0 0 stop stop stop stop stop stop 2 1 0 0 0 0 0 0 stop stop stop stop stop stop 2 0 0 0 0 0 1 stop stop stop stop stop stop 3 0 0 0 0 0 1 stop stop stop stop stop stop 3 1 0 0 0 0 0 0 stop stop stop stop stop stop 2 0 0 0 0 0 0 stop stop stop stop stop stop 3 0 0 0 0 0 0 stop stop stop stop stop stop test 1 1 stop stop stop stop stop stop stop stop stop stop stop stop without 2 stop stop stop stop stop stop stop stop stop stop stop stop product 3 stop stop stop stop stop stop stop stop stop stop stop stop 2 1 stop stop stop stop stop stop stop stop stop stop stop stop 2 stop stop stop stop stop stop stop stop stop stop stop stop 3 stop stop stop stop stop stop stop stop stop stop stop stop 3 1 stop stop stop stop stop stop stop stop stop stop stop stop 2 stop stop stop stop stop stop stop stop stop stop stop stop 3 stop stop stop stop stop stop stop stop stop stop stop stop P: number of bites rep: repetition L: landing: number of mosquitoes landing on the treated area without biting stop = the test is stopped when more than 5 bites have been observed

(127) Under these test conditions, with the samples provided, the insect strains and methods considered, extracts 2 and 3 showed a repellent effect against the mosquito, with a persistence of 6 hours after application.

(128) Extract 1 showed a repellent effect against the mosquito, with a persistence of only 4 hours after application.

(129) Extract 2 of Euodia suaveolens Scheff, at a concentration of 35%, purified, bleached, and extract 3 comprising a mixture of 20%-menthoglycol and a 15%-extract of bleached Euodia suaveolens Scheff, thus show a true repellent effect, with a persistence of 6 hours against the Aedes aegypti mosquito.

(130) Test 3:

(131) A test is carried out with 4 compositions according to the invention:

(132) Extract 1: Codistilled extract of Euodia suaveolens Scheff, formulated at 0.3% in evodone;

(133) Extract 2: Codistilled extract of Euodia suaveolens Scheff, formulated at 2.36% in evodone;

(134) Extract 3: A composition comprising synthetic evodone, formulated at 3%;

(135) Extract 4: Oleoresin of crude, unrefined Euodia suaveolens Scheff, formulated at 0.26% in evodone.

(136) Under these test conditions, with the samples provided, and the insect strains and methods considered, the extracts showed a repellent effect against the mosquito, with the following periods of protection after application:

(137) 4 hrs with Extract 1;

(138) 6 hrs with Extract 2;

(139) 6 hrs with Extract 3;

(140) 6 hrs with Extract 4.

(141) A racemic mixture of synthetic evodone formulated at 3% (extract 3) has a repellent activity lasting 6 hours. Evodone can be used as an active agent to repel insects, and particularly mosquitoes. Evodone is responsible, at least in part, for the repellent activity of the extract of Euodia suaveolens Scheff according to the invention.

(142) Extract 2 of Euodia suaveolens Scheff comprising 2.36% evodone has a more effective repellent activity than extract 1 of Euodia suaveolens Scheff comprising 0.3% evodone and obtained under the same conditions by codistillation.

(143) The crude, unrefined oleoresin of Euodia suaveolens Scheff, formulated at 0.26% in evodone (extract 4), is as effective as extracts 2 and 3. Evodone is active as such and has a synergistic action in the crude unrefined oleoresin. Evodone may act synergistically in the crude extract according to the invention, with molecules such as other oxygenated terpenes, for instance menthalactone, or non-polar molecules present in the codistilled extracts.

(144) According to the invention, evodone has a repellent activity against mosquitoes. A concentration of 3% or even less of synthetic evodone in extracts of crude or refined Euodia suaveolens Scheff is as effective as a concentration of 30% of DEET.

(145) A person skilled in the art will select the cosmetically acceptable medium so that it does not interfere with the desirable properties of the extract and repellent compositions according to the invention.

(146) Of course, the invention is non limited to the embodiments and examples presented above and a person skilled in the art, through routine operations, may need to carry out other embodiments not exclusively described here, that broadly fall within the scope of the invention.