FR3058722A1 - PROCESS FOR EXTRACTING TERPENIC COMPOUNDS - Google Patents

Abstract

The subject of the invention is a process for extracting terpene compounds from a starting composition comprising: (a) extracting the starting composition in a solvent comprising an alkyl lactate, (b) recovering the liquid fraction obtained, (c) diluting it with water, (d) optionally recovering a colloid containing the terpene compounds, (e) freezing the mixture and (f) optionally thawing it to obtain the terpene compounds which have not been recovered previously. This process makes it possible to extract iridals from plants of the Iridaceae family.

Description

© Publication number: 3,058,722 (to be used only for reproduction orders) (© National registration number: 16 61128 ® FRENCH REPUBLIC

NATIONAL INSTITUTE OF INDUSTRIAL PROPERTY

COURBEVOIE © Int Cl ⁸ : C 07 C 403/00 (2017.01), C 11 B 9/00

A1 PATENT APPLICATION

©) Date of filing: 17.11.16. © Applicant (s): UNIVERSITE DE BORDEAUX Eta- (© Priority: basement public— FR and SAYOUS VICTOR— FR. @ Inventor (s): SAYOUS VICTOR, WAFFO TEGUO PIERRE and MERILLON JEAN-MICHEL. (43) Date of public availability of the request: 18.05.18 Bulletin 18/20. ©) List of documents cited in the report preliminary research: Refer to end of present booklet (© References to other national documents ® Holder (s): UNIVERSITE DE BORDEAUX Etablisse- related: public, SAYOUS VICTOR. ©) Extension request (s): © Agent (s): CABINET BECKER ET ASSOCIES.

PROCESS FOR THE EXTRACTION OF TERPENIC COMPOUNDS.

FR 3 058 722 - A1 \ bj) The subject of the invention is a process for extracting terpene compounds from a starting composition, comprising a step consisting in: (a) extracting the starting composition in a solvent comprising an alkyl lactate, (b) recovering the liquid fraction obtained, (c) diluting it with water, (d) optionally recovering a colloid containing the terpene compounds, (e) freezing the mixture and (f) optionally thaw, to obtain the terpene compounds which would not have been recovered previously.

This process makes it possible in particular to extract iridals from plants of the Iridaceae family.

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PROCESS FOR THE EXTRACTION OF TERPENIC COMPOUNDS

OBJECT OF THE INVENTION

The present invention relates to a method for extracting terpene compounds from a starting composition such as a plant material, in particular from parts of plants of the Iridaceae family, using a green solvent comprising an alkyl lactate.

BACKGROUND OF THE INVENTION

Terpenoids are compounds of plant origin, sought after for their odoriferous and pharmaceutical properties, in particular anti-neoplastic and anti-bacterial. They are generally extracted from plants using organic solvents such as hexane, dichloromethane or ethers.

Organic solvents, however, present toxicity problems and involve significant reprocessing costs to limit their environmental impact. It would therefore be useful to have a terpenoid extraction process which is more environmentally friendly but has the same efficiency as the processes using organic solvents, in particular an extraction yield at least as high.

An example of a process using a less toxic solvent for the extraction of iridal-type triterpenoids is described in documents FR 2 746 645 and WO 97/36604. It includes bringing a ground portion of iris plant parts into contact with 70% (v / v) ethanol. However, this process does not make it possible to selectively extract the iridals and it must necessarily comprise a subsequent stage of dilution in water of the ethanolic phase possibly concentrated, then of washing the resulting aqueous phase with a solvent such as ether diethyl, the ethereal phase then being recovered, evaporated to dryness, then taken up in ethanol, in order to obtain iridals with acceptable purity. A similar process for extracting triterpenes from iris is described in document FR 2 620 702.

Ethyl lactate is a green solvent which constitutes an interesting alternative to organic solvents such as hexane, insofar as it is biodegradable, renewable, non-polluting and non-carcinogenic. However, its use in the field of phytochemistry encounters a significant drawback, insofar as it is not volatile. It is therefore generally separated by distillation from the extracted compounds. However, prolonged heating at high temperature is likely to degrade the compounds sensitive to oxidation and negatively affects the economy of the extraction process. The use of ethyl lactate as an extraction solvent has therefore to date been limited to the extraction of phytosterols (US Pat. No. 7,368,138) and / or to processes involving treatment of the plant material using an antioxidant, such as a tocopherol, intended to prevent the degradation of sensitive compounds such as carotenoids, which are tetraterpenoids (US-7,572,468). The application of ethyl lactate to the extraction of other terpene compounds, in particular triterpenoids, therefore requires adaptation of conventional extraction methods.

In this context, the Applicants have developed a simple, inexpensive and effective process for the extraction of terpene compounds using alkyl lactate.

SUMMARY OF THE INVENTION

The subject of the invention is a process for extracting terpene compounds from a starting composition, comprising the successive stages consisting in:

a) extracting the starting composition using a physical process, in a solvent comprising an alkyl lactate,

b) treating the suspension obtained to eliminate the solid fraction and recover the liquid fraction,

c) dilute the liquid fraction with water to obtain a mixture containing water and alkyl lactate and a colloid containing terpene compounds,

d) possibly recovering the colloid,

e) freezing the mixture optionally devoid of the colloid to obtain a heavy phase containing the alkyl lactate, a light phase containing water in solid form and optionally an intermediate phase containing the terpene compounds which have not been recovered at 1 'step d),

f) optionally, melting said frozen mixture, and

g) recovering the heavy phase and, where appropriate, said intermediate phase.

DETAILED DESCRIPTION

The method according to the invention aims to extract terpene compounds contained in a starting composition.

By terpene compounds is meant both terpenes, which are hydrocarbons derived from isoprenic units with five carbon atoms, assembled to form linear or cyclic structures, and terpenoids (or isoprenoids), which are lipophilic organic compounds derived from cyclic terpenes by the addition of one or more functional groups such as an alcohol or an aldehyde. Terpenes and terpenoids are odoriferous compounds. Examples of terpene compounds include bornane, limonene, pinene, myrcene, eugenol, bisabolane, iridals, citral, menthol, eucalyptol, camphor, linalol and bisabolol. The preferred terpene compounds according to the invention are the cyclic triterpenoids (C30), advantageously the iridals and more preferably the bicycloiridals.

The starting composition, containing these terpene compounds, can be a mixture of compounds obtained by chemical synthesis or preferably a vegetable material. In the latter case, it is advantageously in solid form or in the form of a dispersion. By plant material is meant a whole plant or advantageously a part of a plant, in particular comprising or consisting of at least one of its aerial organs (buds, stems, leaves, flowers) and / or underground (roots, rhizomes). The plant considered can be any plant producing terpene compounds. Examples of such plants include in particular those belonging to the families of iridaceae, asteraceae, apiaceae, myrtaceae (such as eucalyptus and clove), as well as cinnamon, ginger, rosemary, mint, lavender, chamomile, thyme, coriander and cannabis, without this list being exhaustive.

It is preferred according to the invention to use a part of a plant from the Iridaceae family, preferably the rhizomes and / or the leaves of plants from the Iridaceae family, more particularly those of the genus Iris and preferably the species Iris pallida, Iris germanica, Iris florentinct. Iris versicolor, Iris sibirica and Iris pseudacorus. According to a particularly preferred embodiment of the invention, a part of a plant of the species Iris pallida is used.

In one embodiment, the plant material is washed with water, peeled, crushed, ground and / or dried before being used in the process according to the invention.

In the first step of the process according to the invention, the starting composition is brought into contact with an extraction solvent comprising an alkyl lactate, generally a C1-C3 alkyl lactate and preferably methyl lactate or d ethyl, better, ethyl lactate. In one embodiment, the extraction solvent further comprises ethanol, in a volume ratio of ethanol to alkyl lactate of between 5 and 80% and preferably between 20 and 50%. According to a preferred embodiment of the invention, the extraction solvent does not contain any solvent other than alkyl lactate and optionally ethanol.

This contacting step can be carried out by any suitable physical process and in particular by maceration, mixing with ultrasound or passage in the microwave of the mixture of the starting composition with the alkyl lactate. This step is generally carried out with stirring, at a temperature of 0 to 40 ° C, preferably from 20 to 25 ° C, for a period ranging for example from 20 to 72 hours, in particular from 40 to 60 hours. The amount of solvent used must be sufficient to wet the starting composition and can for example be between 1 and 2 liters of solvent per kilogram of starting composition.

The suspension obtained is then treated to remove the solid fraction and recover the liquid fraction. This treatment step can be carried out by filtration, centrifugation or using a membrane, preferably by filtration. In the case where ethanol is used as co-solvent, the process further comprises a step b ') of evaporation of the ethanol, preferably at a temperature below 40 ° C, more preferably at a temperature of 28 to 35 ° C, after step b) of filtration and before step c) described below.

The liquid fraction contains the desired terpene compounds, dissolved in the alkyl lactate. Since the alkyl lactate cannot be evaporated, the inventors have developed a simple and inexpensive method for breaking the solubility of terpene in alkyl lactate, consisting in diluting the liquid fraction with water, so increasing the polarity of this fraction and thus obtaining a homogeneous mixture containing water and alkyl lactate and a colloid containing the desired terpene compounds, which are not soluble in the mixture of water and lactate d 'alkyl. In this step, which can be carried out at room temperature, the volume ratio of water to the liquid fraction is generally between 10: 1 and 50: 1, for example between 10: 1 and 20: 1.

The colloid can then be recovered by any suitable means, in particular by filtration or by the use of flocculating agents or even by centrifugation. Alternatively, the terpene compounds may not be recovered at this stage, but after a freeze / thaw step which will be described below. According to yet another variant, part of the terpene compounds can be recovered in this step and another part in the rest of the process, as described below.

Thus, the method according to the invention comprises the following embodiments, which are implemented at the end of step c):

a process comprising the successive stages consisting in: (d) recovering the colloid, (e) freezing the mixture devoid of the colloid to obtain a heavy phase containing the alkyl lactate and a light phase containing water in solid form, (g) recovering the heavy phase;

a process comprising the successive stages consisting in: (e) freezing the mixture containing the colloid to obtain a heavy phase containing the alkyl lactate, a light phase containing water in solid form and an intermediate phase containing the terpene compounds , (f) melting said frozen mixture, and (g) recovering the heavy phase and said intermediate phase;

a process comprising the successive stages consisting in: (d) recovering the colloid, (e) freezing the mixture devoid of the colloid to obtain a heavy phase containing the alkyl lactate, a light phase containing water in solid form and an intermediate phase containing the terpene compounds which were not recovered in step (d), (f) melting said frozen mixture, and (g) recovering the heavy phase and said intermediate phase.

The process according to the invention then comprises a step of freezing the mixture (devoid or not of the colloid) to obtain a heavy phase containing ethyl lactate, a light phase containing water in solid form and optionally an intermediate phase containing terpene compounds which were not recovered in the previous step. It is understood that the freezing step makes it possible to separate the alkyl lactate from the water and / or the desired compounds, by overcoming the problems linked to the low volatility of the alkyl lactate. This step is carried out at a temperature of -5 to -30 ° C, preferably from -10 to -20 ° C, for a sufficient time (for example from 1 to 10 days) to observe a phase separation between the ethyl lactate, which will constitute the heavy phase of the mixture, and water, less dense than the alkyl lactate and which will constitute the light phase of the mixture.

In the case where the intermediate phase is absent, it is possible, at the end of this step, to recover the alkyl lactate directly in order to be able to recycle it in the process, for example using a bottom tap tank. In the case where an intermediate phase is present, the process comprises an additional step consisting in melting the mixture, generally bringing it to room temperature, so as to be able to recover the alkyl lactate, then the intermediate phase containing the terpene compounds research. If necessary, one or more freeze / thaw cycles can be used to form the intermediate phase.

In addition, the steps of freezing / thawing / recovering the alkyl lactate (steps e) to g) of the process according to the invention) can thus be repeated in order to obtain a purified heavy phase and optionally increase the level of terpene compounds recovered.

This method can also comprise a step consisting in mixing the colloid recovered in step d) with the intermediate phase resulting from step g).

The yield of the extraction process according to the invention, as well as the chemical nature of the terpene compounds obtained will depend on the starting composition, in particular on the plant material, used. In the case of plants of the genus Iris, the iridals may moreover be in reduced form, or if necessary in oxidized, or even dehydrated form, leading to cyclic or polymerized structures. In particular, the side chain of the iridals may contain double bonds resulting from dehydration or dehydrogenation of the natural iridals, or may be hydroxylated. They can also be obtained in hydroxylated or esterified form, for example with fatty acids, or etherified.

In the case where the plant material belongs to the genus Iris, it is thus possible to selectively obtain one or more iridals chosen from: monocycloiridals, more especially comprising iridal (21-deoxy-iridogermanal), 16-0-acetyl -isoiridogermanal, 17hydroxyiridal, 26-hydroxyiridal, 17,26-dihydroxyiridal, 21-hydroxyiridal (iridogermanal), 10-deoxy-21-hydroxyiridal (10-deoxy-iridogermanal), 10-deoxy-17hydroxyiridal, l 'iriversical, 16,17-didehydro-26-acetoxy-iriversical, 16,17-didehydro3058722 iridal, 16,17-didehydro-26-hydroxy-iridal and 17,29-didehydroiridal; bicycloiridals comprising in particular iriflorental, iripallidal, Γα-irigermanal, γ-irigermanal and deoxyiripallidal; and spiranic derivatives such as spiroiridal, 29-hydroxy-spiroiridal, 29acétoxyspiroiridal, belamcandal and 28-deacetyl-belamcandal; and their mixtures.

The method according to the invention more particularly makes it possible to extract bibycloiridals, such as iriflorental and iripallidal.

The terpene compounds can then be purified using any suitable technique, in particular by high performance liquid chromatography (HPLC).

The iridals can advantageously be used as active pharmaceutical ingredients, in particular in the treatment of cancers or malaria. In these applications, they are generally included in an effective amount in a composition comprising a pharmaceutically acceptable medium, suitable in particular for administration by the oral route or by intravenous, subcutaneous or intramuscular injection.

As a variant, the iridals can be converted into irones according to any process known to those skilled in the art, in order to be used as constituents of perfumes in the cosmetic or perfumery industry or as food flavorings. The ironies indeed present a characteristic smell of violet which is much sought after.

The conversion of iridals into irones, in particular into a-irone, β-irone and / or γ-irone, can in particular be carried out microbiologically, in particular enzymatically, in the presence of a peroxidizing system comprising hydrogen peroxide and a peroxidase or oxygen and a lipoxygenase, optionally in the presence of an enzyme substrate (as described in particular in EP 0 443 926), or by subjecting the iridals to the action of a microorganism of the genus Botryotinia or Sclerotinia or alternatively by permanganic oxidation (as described in particular in FR 2 620 702). The desired irone can then be obtained by steam entrainment, hydrodistillation or extraction using a supercritical fluid or a solvent immiscible with water.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 represents the HPLC chromatogram at 242 nm of the dlris pallida extract obtained according to Example 1, by extraction with ethyl lactate, as well as the UV-visible spectra a) to c) of the compounds at 14.996 min, 16.671 min and 16.244 min, respectively.

EXAMPLES

The invention will be better understood in the light of the following example, which is given purely by way of illustration and is not intended to limit the scope of the invention, defined by the appended claims.

Example: Extraction of iridals

An extract enriched in iridals was prepared from rhizomes of iris pallida in accordance with the process according to the invention.

To do this, 1.5 kg of rhizomes ülris pallida were washed with water and ground. This ground material was macerated in a mixture of 2 L of ethyl lactate and ethanol (60/40, v / v), with stirring, for 48 h at room temperature. The mixture was then filtered and drained to remove the solid fraction and the liquid fraction was collected. The alcohol was then evaporated in vacuo using a rotary evaporator. After evaporation, there remained 1.2 L of an organic phase based on ethyl lactate containing the desired compounds. This organic phase was diluted and homogenized in 15 L of water, in a container comprising a tap at the bottom of the tank. The formation of a whitish colloidal precipitate, comprising the desired compounds, has been observed. The mixture of water and ethyl lactate was then placed at -20 ° C for 2 days, until the water was frozen and the ethyl lactate had decanted at the bottom of the tank. The mixture was then thawed and then refrozen for 2 days. A total of 5 freeze / thaw cycles were performed. During the last thawing, a three-phase system was observed, consisting of an upper part composed of clear water, an intermediate phase containing the precipitated iridals and a lower part containing ethyl lactate.

The ethyl lactate was removed by means of the tank bottom valve, then the upper and intermediate phases were filtered and centrifuged so as to recover all of the aggregates formed. The material thus recovered was taken up with a minimum volume of ethanol, so as to clean the filters and equipment. Finally, after addition of water and evaporation of the alcohol, the desired compounds were lyophilized. 12g of a powder rich in iridals was thus obtained, ie a yield of 0.8% from the rhizomes.

Characterization of the extract

The extract obtained as described above was analyzed by HPLC.

The analysis was carried out using an Agilent®1100 HPLC chain equipped with a degasser, a low-pressure quaternary pump, an automatic changer, a UV-Visible10 DAD detector (200 nm at 800 nm) and a Prontosil® Cis column (250 x 4.0 mm; 5pm, 120Â). The mobile phase consisted of water (solvent A) and acetonitrile (solvent B). The elution was carried out at 1 ml / min according to the gradient described in Table 1. 10 μL of a methanolic solution of the extract at 2 mg / ml were injected. Detection was carried out at 242 nm and 280 nm.

T % B (min) (acetonitrile) 0 60 5 60 8 68 13 68 16 69 21 69 24 71 29 71 32 100 37 100 40 60 45 60

Table 1

In Figure 1, the peaks of the compounds between 14 and 17 min are all compounds of the family of bicycloiridals. These bicycloiridals have an absorption maximum close to 240 nm.

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The peak at retention time of 14.996 min corresponds to iriflorental and that at 16.761 to iripallidal. The ^3rd peak at 16.244 min may correspond to an isomer of previous bicycloiridals. The dosage on the raw extract indicates that it is composed of 15% iriflorental and 17.6% iripallidal, a total for the two bicycloiridals of 33.6%.

Claims (9)

1. Method for extracting terpene compounds from a starting composition, comprising the successive stages consisting in: a) extracting the starting composition using a physical process, in a solvent comprising an alkyl lactate, b) treating the suspension obtained to eliminate the solid fraction and recover the liquid fraction, c) dilute the liquid fraction with water to obtain a mixture containing water and alkyl lactate and a colloid containing terpene compounds, d) possibly recovering the colloid, e) freezing the mixture optionally devoid of the colloid to obtain a heavy phase containing the alkyl lactate, a light phase containing water in solid form and optionally an intermediate phase containing the terpene compounds which have not been recovered at 1 'step d), f) optionally, melting said frozen mixture, and g) recovering the heavy phase and, where appropriate, said intermediate phase. 2. Method according to claim 1, characterized in that steps e) to g) are repeated to obtain a purified heavy phase. 3. Method according to claim 1 or 2, characterized in that the solvent further comprises ethanol, in a volume ratio of ethanol to alkyl lactate of between 5 and 80%, and in that the process further comprises a step b ') of evaporation of the ethanol, preferably at a temperature below 40 ° C, between steps b) and c). 4. Method according to any one of claims 1 to 3, characterized in that it further comprises a step consisting in mixing the colloid recovered in step d) with the intermediate phase resulting from step g). 5. Method according to any one of claims 1 to 4, characterized in that the alkyl lactate is chosen from C1-C3 alkyl lactates, preferably from methyl lactate and ethyl lactate, more preferably ethyl lactate. 6. Method according to any one of claims 1 to 5, characterized in that the starting composition consists of a plant material chosen from plant parts, preferably rhizomes and / or leaves, plants of the Iridaceae family. 5 7. Method according to claim 6, characterized in that the plants are chosen from those of the genus Iris and preferably from the species Irispallida, Iris permanica. Iris florentina. Iris versicolor, Iris sibirica and Iris pseitdctconts, more preferably Iris pallida. 8. Method according to any one of claims 1 to 7, characterized in that the compounds 10 terpenics are cyclic triterpenoids. 9. Method according to any one of claims 6 and 7, characterized in that the terpene compounds are iridals, preferably bicycloiridals such as iripallidal and iriflorental. Ο