WO2022162291A1

WO2022162291A1 - Non-invasive method for extracting compounds from at least one aerial part of a plant

Info

Publication number: WO2022162291A1
Application number: PCT/FR2022/000006
Authority: WO
Inventors: Leon QUTISHAT
Original assignee: Fl Nova
Priority date: 2021-01-26
Filing date: 2022-01-26
Publication date: 2022-08-04
Also published as: FR3119095A1

Abstract

The invention relates to a non-invasive method for extracting compounds from at least one aerial part of a land plant, comprising a step of covering the at least one aerial part of a land plant with an impermeable or semi-impermeable material, a step where the at least one aerial part of the land plant is left covered for a period of at least 3 hours, at a temperature greater than 0°C, enabling the generation of a solution on the walls of the material, and a step of recovering this solution comprising the extracted compounds.

Description

Title of the invention: Method for the non-invasive extraction of compounds from at least one aerial part of a plant

This patent application relates to a non-invasive method for extracting compounds from at least one aerial part of a terrestrial plant, comprising a step of covering said at least one aerial part of a terrestrial plant with a waterproof or semi-waterproof material, a step in which said at least one aerial part of the terrestrial plant is left covered for a period of at least 3 hours, at a temperature above 0°C, allowing the generation of a solution on the walls of the material, and a step for recovering this solution, comprising the extracted compounds.

[0002] Many methods exist today for extracting compounds from plants. Most of these methods are invasive, and therefore do not allow the reproducibility of the extraction on the same part of the plant, or simply the same plant.

[0003] US6254896 discloses a method of collecting an extract or essence from a flower or other part of a living, undamaged plant, the part of the plant being placed in a container of extraction which can be plastic or any other impermeable material and the extraction takes place at natural room temperature for about 3 hours to 2 weeks. However, this document describes that said extraction vessel comprises an extraction solvent (water, oil, etc.) and the bringing into contact of the extraction solution with the part of the living plant.

[0004] Other documents describe non-invasive extraction methods, but each time using a solvent to extract the compounds from the plants.

[0005] The use of solvent can damage the submerged part of the plant, as well as the extracted components, or complicate the extraction process by requiring an additional extraction step aimed at eliminating the solvent in order to concentrate the solution in the desired compounds.

[0006] Thus, there remains the need for a simplified process facilitating the extraction of compounds contained in plants, in particular without having recourse to a solvent.

[0007] The Applicant has solved this technical problem thanks to the different steps of the method according to the invention, allowing the extraction of compounds without damaging the plant, and without using a solvent outside the plant. This makes it possible to avoid, at the time of isolating the extracted compounds, the use of a stage which can be complex for removing the solvent, and to limit the deterioration of the compounds extracted from plants. This also allows, thanks to the non-deterioration of the plant, to be able to produce these compounds continuously, without having to cut and/or kill the plant or a part thereof, and is thus an important criterion from an ecological point of view. Furthermore, the method according to the invention allows the extraction of molecules which are not obtained by the existing methods.

Thus, according to a first aspect, the invention relates to a method for the non-invasive extraction of compounds from at least one aerial part of a terrestrial plant comprising: a. a step of completely covering said at least one aerial part of a terrestrial plant with a waterproof or semi-waterproof material, a space remaining between said at least one aerial part of a terrestrial plant and said material; b. a step where said at least one aerial part of a terrestrial plant is left covered according to step a. for a period of at least 3 hours, at a temperature above 0°C, allowing the generation of a solution on the walls of said impermeable or semi-impermeable material; vs. a step of recovering said solution from the walls of the material, said solution comprising the extracted compounds.

By extraction is understood according to the invention that the method makes it possible to obtain compounds in solution which were present in the plant.

By plant is meant a photosynthetic and autotrophic organism, characterized by plant cells, and forming part of the eukaryotic kingdom.

[0011] Land plants are understood to mean embryophytes or cormophytes which grow on dry land. This super-division of the plant kingdom includes the groups of bryophytes (mosses), pteridophytes (ferns) and spermatophytes (seed plants). Green algae, red algae and glaucophytes are excluded from this designation of terrestrial plants.

[0012] By aerial parts of the terrestrial plant, or vegetative apparatus, are meant the "visible" parts of the plant in its natural state, namely therefore the stem, the buds, the leaves, the flowers, the fruits, the branches.

By at least one aerial part of a terrestrial plant is understood that at least one part of the plant is covered, or several aerial parts of the plant, or all of the aerial parts of the terrestrial plant.

Preferably, said at least one aerial part of a terrestrial plant is chosen from flowers, leaves, stems, branches, fruits, buds or all of the aerial parts of the terrestrial plant.

[0015] By “non-invasive” is meant that the living plant or part of the plant is not damaged, and therefore continues its life without being altered. When it is a part of the plant, it is not separated from the rest of the plant.

By compound is meant a chemical substance.

[0017] By "covering step" is understood that said at least one aerial part or all of the aerial parts of the terrestrial plant is enclosed, in other words covered, by the material according to the invention, so that it is no longer directly exposed to the open air.

[0018] By “tight material” is meant according to the invention a material which is hermetic, and which therefore does not allow fluids and gases to pass.

By "semi-tight material" is understood according to the invention a material which makes it possible to limit the passage of fluids and gases to a certain threshold, making it hermetic for gases or fluids exceeding this threshold.

Preferably, said waterproof or semi-waterproof material is chosen from glass, plastic, synthetic or natural fabric, clay, leather, or metal. Preferably, said plastic is polyethylene terephthalate.

[0021] “Polyethylene terephthalate” means the plastic more commonly referred to as PET. It is used in particular for the manufacture of plastic bottles.

[0022] "Space remaining between said at least one aerial part of a terrestrial plant and said material" means according to the invention that a space is left between said at least one aerial part of the plant or all the aerial parts of the terrestrial plant and said material, that is to say that the material is not "clad" to the part of the plant.

By "solution" is meant the solution obtained by the method according to the invention, on the walls of said material, which may be the result of the transpiration of the terrestrial plant or of said at least part of the terrestrial plant.

[0024] By “recovery step” is meant a step making it possible to collect the drops of solution present on the walls of the material. This recovery step can be performed in any way that one skilled in the art would be able to perform.

The higher the temperature, the better the efficiency of the process according to the invention.

Preferably, the temperature in step b. does not exceed 50°C.

According to a preferred embodiment, the temperature in step b. is between 5°C and 40°C, more preferably between 10°C and 35°C, even more preferably between 20°C and 35°C.

Preferably, the maximum duration of step b. is two weeks.

According to a preferred embodiment, the duration of step b. is between 12 hours and two weeks, more preferably between 1 day and 10 days.

Preferably, said material is in the form of a bell, a film, a bag, a pipe, or a cylinder, surrounding said at least one aerial part of a plant or the entire terrestrial plant.

[0031] According to one embodiment, said material is a glass or PET bell jar surrounding the entire plant or the entire plant.

[0032] According to one embodiment, said material is a PET bell surrounding one or more flowers or leaves of the plant or the whole plant.

According to one embodiment, said material is a PET bag surrounding one or more flowers or leaves of the plant, or the whole plant.

According to one embodiment, said material is a plastic film surrounding flowers or leaves of the plant, or the entire plant.

Preferably, said extracted compounds comprise essential oils and/or volatile compounds and/or terpins, and/or flavonoids and/or alkaloids, and/or proteins and/or fatty acids and/or sugars.

According to one embodiment, said compounds obtained comprise organic compounds: aliphatic hydrocarbons, aromatic hydrocarbons, alcohols, amines, amides, carboxylic acids, esters, quinones, anthraquinone, flavonoids, alkaloids, ester, amines, saponin, fatty acid , terpines, 2-benzo-pyrans, 4,5-di substituted thiazoles, acyclic monoterpenoids, aldehydes, alpha, beta-unsaturated carbonyl compounds, anisoles, aromatic monoterpenoids, benzene and substituted derivatives, benzodi-oxoles, benzofurans, acid esters benzoic acid, benzoyl derivatives, bicyclic monoterpenoids, branched alkanes, branched unsaturated hydrocarbons, C-nitro compounds, caprolactams, amides of carboxylic acids, esters of carboxylic acids, chlorobenzenes, cumenes, cycloalkanes, dialkyl ethers, acids dicarboxylics and derivatives, dimethoxybenzenes, fatty acid esters, fatty acid methyl esters, fatty alcohol esters, fatty alcohols, hemiacetals, indoles, acids jasmonics, ketones, long chain fatty alcohols, m-xylenes, menthane mono-terpenoids, methoxyphenols, methylpyridines, n-vinylaziridines, other non-metallic sulphides, oxanes, phenol ethers, phenylpropanes, phenylpropenes, hydrocarbons polycyclics, polyols, primary alcohols, secondary alcohols, sesquiterpenoids, styrenes, sulfones, sulfonyl chlorides, tertiary alcohols, tertiary amines, tetraalkylammonium salts, tetrahydrofurans, toluenes, trihalomethanes, vinyl chlorides.

These compounds can in particular be extracted from peppermint.

The compounds obtained and their concentrations depend on the terrestrial plant used and on said at least part of the terrestrial plant used, as well as on the stress and temperature conditions of the plant.

[0039] Preferably, step c. recovery of the solution is carried out:

- by stirring said material, to make the drops of solution fall into a container; and or

- thanks to a pump sucking said solution on the walls of said material, or thanks to at least one pipe connected to said material, into which said solution falls, said at least one pipe being connected to at least one container; and or

- Thanks to at least one valve for collecting said solution.

[0040] The pipes and valves are located at the bottom of the material, so that the drops fall by gravity.

The extracted compounds, included in said solution, can be used directly.

This solution can thus be used as floral water, or in cosmetic formulations as a flavoring or perfuming agent or for the therapeutic virtues of the compounds contained in the solution.

According to one embodiment, said method further comprises a step d. isolation of said extracted compounds.

According to one embodiment, said step d. isolation of said extracted compounds is carried out by distillation of the solution obtained in step c.

According to one embodiment, said step d. Isolation of said extracted compounds is carried out by extraction with an organic solvent and then evaporation of this solvent.

This use of solvent at this stage is less problematic than at the time of the extraction of the compounds, because it does not degrade the compounds which would be degraded if they were extracted directly with solvent, and thus does not degrade the plant.

According to one embodiment, said step d. isolation of said extracted compounds is carried out by chromatography. This chromatographic step makes it possible to isolate by group of compounds or by specific compounds.

According to one embodiment, said step d. insulation is carried out by solid phase microextraction (SPME), preferably on a DVB/CAR/PDMS fiber previously conditioned according to the manufacturer's recommendations, according to the following conditions: incubation: 10min at 60°C, extraction: 10min at 60°C, desorption: 2min at 250°C.

[0049] Step d. makes it possible to concentrate compounds that were present from step c. but not detectable in the analysis (for example by chromatography), by threshold effect. This is found in example 2, or the different concentrations between sample 1 and 2, the latter resulting from a distillation step carried out on sample 1. Compounds which were not detected in sample 1 are detected for sample 2, and compounds that were present in sample 1 are no longer detected in sample 2, because they were evaporated during the distillation.

According to one embodiment, said land plant is chosen from rose, rosemary, peppermint, lemon balm.

tricks

[0051] [Fig 1] is a diagram representing the method according to the invention on an aerial part of the plant, the leaves. In the diagram are schematically represented the material surrounding the leaves, the plant and the drops of solution on the walls of the material.

[0052] [Fig 2] is a diagram representing the method according to the invention on the whole aerial parts of the terrestrial plant. In the diagram are schematically represented the material surrounding the plant, the plant and the drops of solution on the walls of the material.

[0053] [Fig 3] is a diagram representing the method according to the invention on an aerial part of the plant, the leaves, said material comprising a valve making it possible to recover the solution. In the diagram are schematically represented the material surrounding the leaves, the plant, a valve and the drops of solution.

[0054] [Fig 4]: chromatogram of the lemon balm sample detected by UHPLC-UV at 280 nm and 330 nm.

[0055] [fig 5]: chromatograms of rosmarinic acid (a), ferulic acid + rosmarinic acid (b) and a lemon balm extract (c) at 330 nm.

[0056] [fig 6: chromatogram of sample 1 obtained by spme GC-MS

[0057] [fig 7]: chromatogram of sample 2 obtained by spme GC-MS

Examples

Example 1: Method for the non-invasive extraction of compounds from at least one aerial part of a terrestrial plant:

The method according to the invention is used to extract compounds from plants.

Table 1 summarizes the different parameters of the methods used in examples 2 and 3:

Table 1: Perimeters of the solutions used in Examples 2 and 3.

Sample 2 of Example 2 is obtained by distillation of Sample 1. Similarly, the rosemary sample distilled according to Example 2 is obtained by distillation of the rosemary sample of example 2.

Example 2: Analysis of solutions obtained according to the method of the invention, from lemon balm (sample of lemon balm) and peppermint (samples 1 and 2):

A sample of lemon balm solution obtained using the method according to the invention, as indicated in example 1, was centrifuged to remove the particles and the supernatant was analyzed by high performance liquid chromatography coupled with a diode array detector (HPLC-UV).

Two samples of peppermint solution obtained using the method according to the invention, as indicated in Example 1, were diluted to ^{1/5 th} in distilled water. In order to optimize the extraction of the compounds by solid phase micro-extraction (SPME), 2g of salt (NaCl) were added to each sample. An extraction control was also added to each sample, 3-octanol, at a concentration of 75 μg/L. The two solution samples were analyzed by gas chromatography coupled to a mass spectrometer (GC-MS). Sample 2 is the result of the distillation of sample 1.

[0066] HPLC-UV analyzes

The HPLC-UV analysis was carried out following a European Pharmacopoeia protocol for the analysis of lemon balm extracts. The protocol indicates UV detection at 330 nm, corresponding to the reference wavelength for the analysis of lemon balm extracts. Detection at 280 nm (reference wavelength for phenolic compounds) was also used.

Figure 4 shows the chromatograms of the lemon balm sample analyzed by HPLC-UV and detected at 280 nm and 330 nm.

[0069] Detection at 330 nm does not make it possible to detect compounds. Detection at 280 nm shows some very low intensity peaks not corresponding to any of the compounds available in our database. These peaks correspond probably to the background noise of the analysis.

For comparison, an HPLC-UV analysis of lemon balm extract had already been carried out in the past. The sample was extracted with EtOH and the analysis shows the presence of phenolic compounds, in particular rosmarinic acid, in the ethanolic extract of lemon balm.

[0071] Figure 5 shows an analysis of lemon balm extract with a chromatogram of the rosmarinic acid standard (a), a chromatogram of the ferulic acid and rosmarinic acid standards (b) and a chromatogram of a lemon balm extract (vs). The chromatogram (c) shows that rosmarinic acid is indeed present in the lemon balm extract and is visible at 330 nm.

The GC-MS analysis (Agilent 7890B/MS single quadrupole) was carried out on a DB-Wax column (60m x 320pm x 0.5pm) previously conditioned according to the manufacturer's recommendations. The compounds of the two samples were extracted by SPME on a DVB/CAR/PDMS fiber previously conditioned according to the manufacturer's recommendations, according to the following conditions: incubation: 10min at 60°C, extraction: 10min at 60°C, desorption : 2min at 250°C.

The compounds are identified by comparison of the mass spectra with the NIST14 base (MS) and the retention indices (RI). A compound is putatively identified when its similarity (the match factor) is greater than 80% and/or its retention index is between +/-30 compared to the literature.

[0074] GC-MS analyzes

The GC-MS analysis (Agilent 7890B/MS single quadrupole) was performed on a DB-Wax column (60m x 320pm x 0.5pm) previously conditioned according to the manufacturer's recommendations. The compounds of the two samples were extracted by SPME on a DVB/CAR/PDMS fiber previously conditioned according to the manufacturer's recommendations, according to the following conditions: incubation: 10min at 60°C, extraction: 10min at 60°C, desorption : 2min at 250°C.

The chromatogram of sample 1 (Figure 6) shows the compounds detected by GC-MS. Table 2 indicates the compounds identified, the aromatic descriptors as well as the concentrations relative to the internal standard (3-octanol 75 pg/L).

Table 2: Compounds of sample 1

0079] The chromatogram of sample 2, Figure 7, shows the compounds detected by GC-MS. Table 3 indicates the identified compounds, the aromatic descriptors as well as the concentrations relative to the internal standard (3 -octanol

75pg/L).

[0080] Table 3: Composition of sample 2

[0081] Assessment:

[0082] HPLC-UV analyzes

The analysis of the lemon balm sample by HPLC-UV shows the presence of phenolic compounds, in particular rosmarinic acid, in the ethanolic extract.

[0084] GC-MS analyzes

The SPME GC-MS analysis of the samples made it possible to identify 110 compounds in sample 1 and 164 compounds in sample 2.

This demonstrates that the distillation step used to obtain sample 2, from sample 1 resulting from the process according to steps a, b and c, allows to further concentrate certain compounds, and to allow their appearance (after exceeding the concentration limit threshold) in the analysis by GC-MS.

Compounds which were present in sample 1 are no longer detected in sample 2, because they have been evaporated during the distillation.

The method according to the invention thus makes it possible to extract compounds from plants, without being invasive.

Example 3: Analysis of compounds extracted from rosemary, rose, distilled rosemary, peppermint and lemon balm:

The same study carried out in Example 2 concerning the two samples, this time was carried out on samples of solution obtained by means of the method according to the invention (parameters explained in Example 1), from Rosemary, of Rose, Distilled Rosemary, Peppermint and Lemon Balm.

Samples 1, 2, 4, and 5 of this example were obtained according to the method of the invention with steps a, b and c.

Sample 3 is a sample obtained by the distillation of sample 1 of this example.

The compounds obtained from these plants are indicated in Table 3.

Table 3: Composition of the various samples (Concentration pg/L 3-octanol equivalent)

Claims

[Claim 1] A method of non-invasively extracting compounds from at least an aerial part of a terrestrial plant comprising: a. a step of completely covering said at least one aerial part of a terrestrial plant with an impermeable or semi-impermeable material, a space remaining between said at least one aerial part of a terrestrial plant and said material; b. a step where said at least one aerial part of a terrestrial plant is left covered according to step a. for a period of at least 3 hours, at a temperature above 0°C, allowing the generation of a solution on the walls of said impermeable or semi-impermeable material; vs. a step of recovering said solution from the walls of the material, said solution comprising the extracted compounds; d. a step of isolating said extracted compounds.

[Claim 2] Extraction method according to the preceding claim, characterized in that the said impermeable or semi-impermeable material is chosen from among glass, plastic, synthetic or natural fabric, clay, leather, or metal.

[Claim 3] Extraction method according to the preceding claim, characterized in that said plastic is polyethylene terephthalate.

[Claim 4] Extraction method according to any one of the preceding claims, characterized in that the said material is in the form of a bell, a film, a bag, a pipe, or a cylinder, surrounding the said at least one aerial part of 'a plant.

[Claim 5] Extraction method according to any one of the preceding claims, said at least one aerial part of a terrestrial plant being chosen from flowers, leaves, stems, branches, fruits, buds, or all the aerial parts of the terrestrial plant.

[Claim 6] Extraction method according to any one of the preceding claims, characterized in that the said extracted compounds comprise essential oils, and/or volatile compounds and/or terpins, and/or flavonoids and/or alkaloids and/or proteins and/or fatty acids and/or sugars.

[Claim 7] Extraction method according to any one of the preceding claims, characterized in that step c. recovery of the solution is carried out:

- Thanks to at least one valve for collecting said solution.

[Claim 8] Extraction method according to any one of the preceding claims, characterized in that step d. is carried out by distillation of the sample obtained in step c.

[Claim 9] Extraction method according to any one of claims 1 to

7, characterized in that step d. isolating said compounds extracted in step c. is carried out using an organic solvent, or by chromatography, or by solid phase micro-extraction.

[Claim 10] Extraction method according to any one of the preceding claims, characterized in that the said terrestrial plant is chosen from among rose, rosemary, peppermint, lemon balm.