FR2673639A1 - Fractions enriched in fatty acid and extracted from Morinda and insecticidal compositions containing the said fractions and/or fatty acids - Google Patents

Abstract

Fractions enriched in fatty acids, obtained from ripe fruit of the genus Morinda by a process including an extraction stage and a separation stage. These fractions and compounds correspond to the formula RX in which R denotes an optionally substituted C5-C9 alkyl or alkylene group and X denotes an optionally esterified alcohol, acid or aldehyde group, can be employed for the destruction or neutralisation of insects. Compositions containing these fractions or these compounds are particularly effective against certain species of Drosophila, ants and cockroaches.

Description

The present application relates to fractions enriched with fatty acids extracted from fruits of the genus Morinda
It also relates to the use of these fractions and / or fatty acids or fatty acid derivatives in insecticidal compositions
Drosophila flies, well known to geneticists, can in some cases become pests for fruit and vegetable stocks and also in the wine industry. In some cases, up to 50% of production is damaged by Drosophila larvae
In the genus Drosophila, the species D.

melanogaster are most involved in the damage caused to agricultural production
There are three species close to D. melanogaster
D. simulans, which is a cosmopolitan species, and D.

mauritiana and D. sechellia which are island Drosophila from the Indian Ocean.

D. sechellia seems to be able to develop only on the fruit of a shrub: Morinda citrifolia. This member of the coffee tree family has a very wide distribution from the Mascarene Islands to Southeast Asia with recent populations described in Hawaii, Moorea and
Guadeloupe. These latter stands are said to be due to imports by populations originating in China and Southeast Asia. This shrub is used for its dyeing properties and in traditional medicine as a hypotensive, antiseptic and laxative
During fruit collections in the islands
Seychelles, it has been noted that the only Drosophila emerging from ripe fruits are of the species D. sechellia, while D. melanogaster and especially D. simulans exist in the archipelago. against a greater number of species including
D. simulans.

Experiments after this observation have again shown that the pulp of Morinda was toxic, the skin and the seeds do not seem to cause mortality in Drosophila (DEA thesis by Luc Legal
Morinda Citrifolia, Comparative study of the tolerance and behavior of three species of the melanogaster subgroup including
D. sechellia; University of Paris VI, October 1988)
It has also been shown that the toxins responsible for Drosophila mortality are not very volatile, making contact between the insect and the pulp compulsory in order to obtain maximum mortality and that only the ripe Morinda fruit could present toxicity. , the rotten fruit and the green fruit not being toxic
Other preliminary experiments also tend to indicate that the toxin is soluble in a solvent of moderate polarity (Legal et al., Llth European Drosophila
Research Conference, Marseille, September 5-8, 1989)
Thus, it has been shown that the ripe fruits of
Morinda have a certain toxicity vis-à-vis specific species of Drosophila, but the low activity of the pulp preparations, does not allow an industrial application of these observations.
The Applicant has therefore endeavored to isolate fractions having significant toxicity and to highlight the active ingredients
First, it showed that the active ingredients could be isolated by a simple extraction process
It has surprisingly shown that these fractions and active ingredients are toxic to many species of insects
According to the invention, it has also been found that certain fatty acids and their derivatives could be applied as insecticides
The present invention therefore relates to fractions enriched with fatty acids which can be obtained from fruits of the genus Morinda by a process comprising at least the following steps
- extraction with a solvent of average polarity of
fruit pulp or crushed whole fruit
followed by filtration and evaporation
- decantation of the dry extract obtained in a medium
aqueous comprising at least one non-polar solvent, and
- separation of the organic and aqueous phases.

 The solvent of medium polarity preferably used for the extraction is acetone, but other solvents such as dioxane, ethyl acetate, dichloromethane, dimethyl sulfoxide can be used, as well as combinations of solvents allowing to obtain a mixture of medium polarity, although the best results have been obtained with acetone.

The decantation step can be carried out in an aqueous medium comprising as an apolar solvent hexane and more preferably in an aqueous medium comprising acetone and hexane
The amounts of water, hexane and acetone in the settling medium can advantageously be substantially equal in volume
Without this theory implying any limitation of the present invention, it is believed that acetone has a role in disaggregating the tissues of the fruit while hexane is a solvent for the active principles responsible for the toxicity of the fruit.
It has been found, according to the invention, that the fractions enriched in fatty acids, as described above are valuable products as insecticides or as active agents of insecticides
Some fatty acids have also been found to have insecticidal properties
The invention therefore also relates to an insecticide composition containing
- an effective amount of a fraction, as mentioned above and in particular the organic fraction and / or
an effective amount of a compound, of a mixture of compounds, of derivatives of these compounds, or of complexes comprising these compounds, said compounds corresponding to the formula RX in which
* R represents an alkyl or alkylene group in
C5 to C9 optionally substituted
* X represents a possible alcohol group le-
esterified, acid or aldehyde
The group R can be linear or branched.

Preferably R comprises 7 carbon atoms in which case said compound can be octanoic acid or octanol
Said compound can also be hexanoic acid, heptanoic acid, nonanoic acid or decanoic acid or one of their derivatives.

 The compositions according to the invention may contain additives customary in insecticide compositions and in particular additives making it possible to produce insecticide formulations in the form of liquids, paste gels, powders, solids (blocks, plates, etc.), aerosols.

The amounts of these compounds will be adjusted by a person skilled in the art according to the precise use which will be made of them and in particular the mode of administration, the volumes and / or surfaces to be treated as well as the insects to be destroyed or neutralized.
Preferably, the amounts of these compounds are in a range from 0.1 to 500 g / kg of the composition and in particular in a range between 0.1 and 250 g / kg of the composition.

 Another object of the present invention is the use for the destruction or neutralization of insects of a fraction as previously described and in particular of the organic fraction and / or of one or more compounds corresponding to the formula RX such as previously defined.

The present invention is illustrated without however being limited by the following application examples, with reference to the appended drawings in which
Figure 1 shows the protocol for extracting toxins from crushed fruit
FIG. 2 represents the kinetics of fly mortality in the presence of 150 μg of acetone extract, the percentage of mortality being indicated on the ordinate, and the time on the abscissa
Figures 3 and 4 show the gas chromatography spectra of the acetone extract and the hexane fraction, respectively.
FIGS. 5a and 5b represent the gas chromatography spectra of hexane fractions of ripe fruit and of rotten fruit respectively.

 Figures 6a and 6b show the chromatography peaks between the graduations 1000 and 2500 the peaks not corresponding to the fatty acids having been eliminated from Figure 6a by computer processing, in order to obtain Figure 6b.

Figures 7 and 8 are respectively electron impact and methane chemical ionization mass spectra of octanoic acid.
Figures 9 and 10 show the mortality kinetics of D. mauritania and D. melanogaster respectively in the presence of octanoic acid and pulp of
Morinda
Figures 11 and 12 illustrate the mortality of D.

melanogaster respectively in the presence of different doses of octanoic acid and Morinda pulp
Figure 13 shows the comparative effect of decanoic, octanoic and hexanoic acids on male and female D. melanogaster flies
Figure 14 shows the dose effect on D.

octanoic acid sechellia
Figures 15 and 16 illustrate the effect of octanoic acid on different species of ants
In Figures 9 to 16, the percentage of mortality is expressed on the ordinate, time being expressed on the abscissa
EXAMPLE 1 Extraction of toxic agents
In order to isolate the active ingredient (s) responsible for the toxicity of the Morinda fruit, solvents of different polarities were tested
The extraction was followed by direct estimation of the toxicity of each fraction
The acetone extraction protocol is summarized in Figure 1. 100 g of crushed whole Morinda fruit are extracted with 100 ml of acetone for 18 hours then are filtered and evaporated
6.1 g of dry extract are obtained which are then resuspended in an aqueous solution containing 1/3 of water, 1/3 of acetone and 1/3 of hexane
After decanting for one hour, the hexane fraction (170 mg) and the acetone + water fraction (4.5 g) are separated.
These fractions are evaporated and the toxicity tests are carried out.

 The mortality kinetics of the acetone extract are illustrated in FIG. 2.

 Female flies D. mauritania (mau), D.

melanogaster of Cs and Ta strains, as well as flies from crosses between D. mauritania and D. sechellia (mause and semau) were placed in the presence of 150 μg of acetone extract in a petri dish.

 The results indicate a very clear effect of the acetone extract on the mortality of D. mauritania and of the Cs strain in particular, but a virtual absence of effect on the Ta strain.

The respective toxicities of the hexane and acetone + water fractions were evaluated comparatively. It appears that only the hexane fraction has significant toxicity.
The figures expressed above represent the averages of the quantities of the products obtained by working on 10 ripe fruits.

 This extraction can also be made from Morinda pulp.

 The extracts obtained at different stages of the extraction process are analyzed by gas chromatography (nitrogen) with a flame ionization detector (Figures 3 to 6).

These analyzes were carried out using apolar columns of type OV1 or DB5 with a length of 50 meters (SGE, Villeneuve St-Georges) which are silica columns on which are grafted methyl groups at about 80%
Certain extracts are also analyzed by mass spectrometry in electronic impact or in chemical ionization with methane (Figures 7 and 8
FIG. 3 represents a gas chromatographic analysis of a total extract of ripe fruit after extraction with acetone. A large number of massive peaks which are difficult to separate are observed after a fairly well resolved start of the spectrum.
The following peaks have been identified
at graduation 1100: butanoic acid
at graduation 1450: hexanoic acid
at graduation 1750: octanoic acid
at graduation around 1900: nonanoic acid
at the 2000 graduation: decanoic acid
The octanoic acid peak represents 30 to 40% of the surface area of the total fatty acid peaks
FIG. 4 represents the profile of gas chromatography of the hexanoic fraction obtained by treatment of the extract analyzed in FIG. 3.

There is a sharp decrease in the number of observable products and a disappearance of many products leaving after the 2000 graduation.

The area of the octanoic acid peak represents on average 70% of the total area of the fatty acid peaks
FIGS. 5a and 5b respectively represent spectra carried out under the same conditions as above of hexane fractions of ripe fruit and of rotten fruit.

The figures given indicate the probable molecular weights and the corresponding species
There is a very clear decrease in the size of the peaks corresponding to hexanoic, octanoic and decanoic acids in particular, in the case of rotten fruit
Figures 6a and 6b correspond to the peaks between the graduations 1000 and 2500 obtained on a ripe fruit.

The signal of FIG. 6a has been processed by a computer in order to eliminate the peaks which do not correspond to the fatty acids.
This treatment was made possible thanks to the coupling of gas chromatography with mass spectrometry. Each significant peak obtained in chromatography is automatically analyzed by spectrometry in order to determine its molecular mass and to verify that it has an acid function.

 Indeed, as shown in FIG. 7, the electron impact mass spectra of fatty acids, in this case the chromatographic peak of octanoic acid, have a degradation peak characteristic of the acid function at 60.

FIG. 8 which is the mass spectrum in chemical ionization with methane of the chromatography peak of octanoic acid reveals a peak of molecular mass 145 corresponding to non-degraded octanoic acid
From a solution of pure octanoic acid of known concentration and a previously weighed extract, it is possible to obtain a correct evaluation by comparison of the surfaces of the gas chromatography peaks corresponding to the products studied.

Octanoic acid which is the majority acid is present in a fairly reproducible quantity and at an average value of 45 Hg per gram of ripe fruit before extraction. The other two acids (hexanoic and decanoic) are in much smaller and variable amounts (10 to 20 times less than for octanoic acid). It should also be noted that, in certain cases, significant quantities of methyl but also ethyl esters of these acids exist among the products identified in the fruit.
Preliminary experiments have shown that among the solvents that can be used, acetone in its pure state is the solvent best suited to recover the maximum of toxic activity from fruit extracts
It should be noted that since acetone is partially miscible with water, the extract produced with acetone is in fact a mixture of the water coming from the fruit and the extraction acetone.
Other solvents were tested (methanol ethanol, ether, hexane, water) with variable efficiency but in all cases less than that described in this example. Extracts made with solvents of high polarity (methanol) or very non-polar (hexane) were the least effective
EXAMPLE 2 Action of macaws acids on
Drosophila.

All the toxicity tests described in this example and the following examples are carried out by depositing samples of pulp or fatty acid solution at the bottom of a petri dish, and covering them with a thin sheet of absorbent paper.
The insects are then introduced into each petri dish, and the number of dead insects is recorded as a function of time.
FIG. 9 shows the mortality kinetics of male flies Drosophila mauritania (mau) and D. melanogaster (me) in the presence of 50 μg of octanoic acid
Figure 10, for its part, relates to the mortality kinetics of male D. melanogaster (me), D. simulans (si) and D. mauritiana (mau) flies in the presence of 1.5 g of pulp.
The comparison of these two figures shows that the same kinetics of mortality are obtained with an amount of 50 μg of octanoic acid for these strains sensitive to
Morinda with an amount of 1.5 g of pulp
Figure 11 shows the mortality kinetics of D. melanogaster (me) flies in the presence of different concentrations of octanoic acid
Figure 12 is a comparative figure showing the effect of the amount of pulp present in the can
Petri dish on the mortality of male or female D. Melanogaster flies, after 20 minutes of treatment
The comparison of these two curves shows that in both cases there is a dose effect of the pulp or octanoic acid on the mortality of flies
Other fatty acids have also been tested for their mortality against D. melanogaster
For this purpose, 50 μg of decanoic acid (deca), octanoic acid (octa) or hexanoic acid (hexa) were placed in petri dishes. The kinetics of fly mortality have been noted in the figure 13 which clearly shows that decanoic and hexanoic acids have very reduced effects on fly mortality compared to octanoic acid
It should be noted that the hexanoic and decanoic acids nevertheless induce a loss of coordination in the movement of Drosophila, which suggests that they could participate in a neurotoxic action contributing to the overall toxicity of Morinda on Drosophila.
The effect of octanoic acid was also tested on the D.sechellia strain, which is not sensitive to extracts of ripe fruit.

Figure 14 illustrates the dose effect of octanoic acid on the mortality of D. sechellia, the doses being 150 pg, 200; ig, 250 Hg and 500 Hg per petri dish
By comparison with the preceding figures, it is noted that a dose 5 to 6 times higher compared to the other strains is required, in order to observe a beginning of sensitivity to octanoic acid
EXAMPLE 3 Effect of Octanoic Acid on Other Insect Species
Various species of ants have also been tested (Figures 15 and 16) for their kinetics of mortality in the presence of octanoic acid.
Lasius ants, which are the black ants usually found in homes, are particularly sensitive to octanoic acid as shown in Figure 15
The ant species Myrmica, Pheidole and M.rubra have also been tested and have been shown to be sensitive to octanoic acid (Figures 15 and 16).

A clear dose-response effect could be observed on other silvicultural species
We can therefore perfectly control the toxicity and harmfulness of the product. Video recording of symptoms of mortality in these insects suggests neurotoxic action of octanoic acid
Octanoic acid has been tested on the German cockroach (Blattela germanica). The kinetics of mortality were carried out with doses of acid between 20g and 1000 pg per cm2, in Petri dishes whose size is larger than those used in the tests on the Drosophila fly. These doses make it possible to obtain 100% mortality in an average time of 15 to 30 minutes
Preliminary tests on American cockroaches also suggest sensitivity to octanoic acid
In this species, the sublethal dose effect of the product causing specific paralysis of the posterior part of the insect was noted
Another advantage of octanoic acid is its very low toxicity on vertebrates. In rats by ingestion, mortality occurs only for doses close to 10 g / kg (Merck Index 11th edition, n 1765), which allows this product to be used in large quantities without danger for higher organisms
Octanol has also been tested at doses equivalent to octanoic acid. Almost identical toxic activity has been shown in D. melanogaster. This result is important for industrial applications, the alcohol being less reactive than the acid, compared to the surfaces treated with the insecticidal compositions (for example concrete).

The compounds according to the invention in C8 therefore seem to cause a very high mortality with respect to insects, the derivatives in C6, C7, Cg and C10 seem rather to have a paralyzing activity.
The presence of these two types of products in insecticide compositions would therefore make it possible to obtain a synergistic action of their two activities.

It should be noted, however, that the methyl ester of octanoic acid used at doses equivalent to those used for the acid does not seem to have significant activity on Drosophila
The tests reported above show that octanoic acid has several advantages, among others
* Specific toxicity for insects by a neurotoxic action. (It is assumed that the carbon chain of this molecule allows rapid penetration through the lipid cuticle of insects
* A relative volatility of the molecule which allows it to be used according to whether it will be coupled or not with compounds retaining or not the product and which moreover allows a varied and controllable use over time
* Low toxicity for higher animals and a fortiori for humans

Claims (16)

 1. Fractions enriched in fatty acids which can be obtained from fruits of the genus Morinda by a process comprising at least the following steps  - extraction with a medium polarity solvent of the pulp of fruit or crushed whole fruit, followed by filtration and evaporation  decanting of the dry extract obtained in an aqueous medium comprising at least one non-polar solvent, and  - separation of organic and aqueous phases  2. Fractions according to claim 1, characterized in that the solvent of medium polarity is acetone  3. Fractions according to one of claims 1 or 2, characterized in that the non-polar solvent is hexane  4.Fractions according to any one of claims 1 to 3, characterized in that the decantation step is carried out in an aqueous medium comprising acetone and hexane  5. Fractions according to claim 4, characterized in that the amounts of water, hexane and acetone in the settling medium are substantially equal in volume  6.Insecticide composition containing  - an effective amount of a fraction according to any one of claims 1 to 5, and / or  an effective amount of a compound, of a mixture of compounds, of derivatives of these compounds or of complexes comprising these compounds, said compounds corresponding to the formula R-X in which  * R represents an optionally substituted C5-C9 alkyl or alkylene group  * X represents an optionally esterified alcohol, acid or aldehyde group  7. Composition according to claim 6 characterized in that the group R is linear  8. Composition according to any one of claims 6 and 7, characterized in that the group R is preferably a C7 alkyl group.  9. Composition according to claim 8 characterized in that said compound is octanoic acid or octanol  10. Composition according to any one of claims 6 and 7, characterized in that said compound is hexanoic acid, heptanoic acid, nonanoic acid, decanoic acid or one of their derivatives  11. Composition according to any one of claims 6 and 7, characterized in that it contains octanoic acid or octanol and one or more compounds of formula R-X in which R is a C5 alkyl group, C6 or C9.  12. Composition according to any one of claims 6 to II, characterized in that it contains the usual additives of insecticidal compositions and in particular additives making it possible to produce insecticidal formulations in the form of liquids, gel pastes, powders, solids , aerosols.  13. Composition according to one of claims 6 to 12, characterized in that it contains amounts of RX compounds included in a range from 0.1 to 500 g / kg and preferably 0.1 to 250 g / kg of the composition  14. Use for the destruction or neutralization of insects of a fraction according to one of claims 1 to 5.  15. Use for the destruction or neutralization of insects of one or more compounds, derivatives of these compounds or complexes comprising these compounds, said compounds corresponding to the formula R-X in which  * R represents an alkyl or alkylene group in C5 to Cg optionally substituted  * X represents an optionally esterified alcohol, acid or aldehyde group  16. Use according to claim 15, in which the compound R-X is as defined in any one of claims 7 to 11.