Use of bio-sourced lipid in a biphasic Centrifugal Partition Chromatography system

Abstract

The subject of the present invention relates to the use of at least one bio-sourced lipid in one of the solvents of a two-phase Centrifugal Partition Chromatography (CPC) purification system for the purification of at least one biomass extract.

Description

Title of the invention: Use of biosourced lipid in a biphasic Centrifugal Partition Chromatography system

[0001] The subject of the present invention relates to the use of at least one bio-sourced lipid in a biphasic Centrifugal Partition Chromatography (CPC) system. The invention relates in particular to the use of at least one bio-sourced lipid in a biphasic CPC system for fractionating an extract from a biomass. In this respect, the invention relates to the use of at least one bio-sourced lipid in a biphasic CPC system, both for purifying at least one compound from an extract from a biomass, but also for extracting at least one compound - in particular at least one undesirable compound - from an extract from a biomass. The invention also extends to a method for fractionating at least one extract from a biomass, the method comprising a step of Centrifugal Partition Chromatography in a biphasic system comprising at least one bio-sourced lipid.The invention also relates to any biomass extract - in particular any biomass extract depleted in at least one compound - in particular depleted in at least one undesirable compound - obtained as a result of such use and / or by the implementation of such a method.

[0002] Centrifugal partition chromatography (CPC) is a chromatography technique for partitioning a solute between two immiscible liquid phases of a two-phase system. CPC is based on the differences in partitioning a solute between two immiscible liquid phases. Each of these phases can be composed of two, three or more solvents. One of the two liquid phases is kept stationary in the column (rotor) by a constant centrifugal force field. This force field is generated by rotating a column composed of a cascade of partition cells connected to each other by channels etched on discs. The other liquid phase, called the mobile phase, percolates through the stationary phase using a pump. The solutes of a mixture are then separated according to their respective partition coefficients (Foucault AP, (1995), Solvent Systems in Centrifugal Partition Chromatography. In: Centrifugal Partition Chromatography. Ed., Marcel Dekker, New York, pp. 71-97). .

[0003] CPC has been used in particular to purify carotenoids from algae and yeasts. For example, Marchai et al. (“Centrifugal partition extraction of$- carotene from Dunaliella salina for efficient and biocompatible recovery of metabolites », Bioresource Technology, 2013; 134, 396-400) used a CPC system to purify [3-carotene from Dunaliella salina (D. salina) using ethyl oleate including 5% dichloromethane. Dichloromethane is not bio-sourced.

[0004] EP3647432 describes a method for purifying a microalgae pigment by CPC in an ethyl acetate / water system.

[0005] The objective of the present invention is to avoid the use of synthetic organic solvent in a two-phase system for purifying a biomass extract by CPC.

[0006] Thus, one of the objects of the present invention relates to a use of at least one biosourced lipid in one of the solvents of a two-phase Centrifugal Partition Chromatography (CPC) system, for a purification of at least one biomass extract.

[0007] The use of a bio-sourced lipid makes it possible to, at least in part, avoid the use of non-renewable fossil resources in a process for purifying at least one biomass extract, while allowing acceptable yields of purification by CPC.

[0008] Throughout the text, the expression "biphasic purification system" refers, according to certain embodiments, to a biphasic system allowing the obtaining of a substantially pure compound from a mixture of compounds, but also refers, according to other embodiments, to a biphasic system allowing the extraction of at least one compound - in particular at least one undesirable compound - (from) a mixture of compounds and so as to obtain a mixture of compounds substantially free from the extracted compound.

[0009] By "biomass", it is understood in the context of the invention, any material derived from a living organism. According to certain embodiments, said at least one biomass extract comprises (or consists of) an extract of a biomass derived from at least one organism of the eukaryote taxon. According to certain of these embodiments, said at least one biomass extract is an extract of a plant biomass. According to certain embodiments, said at least one biomass extract comprises (or consists of) an extract of a biomass derived from at least one organism of the bacteria taxon. According to certain embodiments, said at least one biomass extract comprises (or consists of) an extract of a biomass derived from at least one organism of the archaea taxon.The taxa of eukaryotes, bacteria and archaea are defined in the sense of the phylogenetic classification of living things (Guillaume Lecointre and Hervé Le Guyader, 2016, Phylogenetic classification of living things - Volume 1 - 4th edition. Belin Editions). According to the phylogenetic classification of living things, the taxon of bacteria includes more than 16,000 species, the taxon of archaea includes around 350 species and the taxon of eukaryotes includes around 1,900,000 species.

[0010] Throughout the text, the terms "substantially" and "at least substantially" usually indicate that a characteristic, structural or functional, should not be taken as marking an abrupt discontinuity, which would not have of physical meaning, but covers not only this structure or this function, but also slight variations of this structure or this function which produce, in the technical context considered, an effect of the same nature, if not of the same degree.

[0011] By “bio-sourced” product or lipid, it is understood in the context of the present invention a product or lipid entirely or partially derived from biomass. In the context of the present invention, the bio-sourced lipid is partially derived from biomass. According to certain advantageous embodiments, the bio-sourced lipid is essentially derived from biomass. According to certain advantageous embodiments, the bio-sourced lipid is exclusively derived from biomass. According to certain embodiments, the bio-sourced lipid is derived from a plant resource. According to some of these embodiments, the bio-sourced lipid is derived from a resource - in particular a plant resource - which is inexpensive and renewable.

[0012] In a particular embodiment, one - only - of the solvents of the biphasic purification system consists mainly - in particular exclusively - of at least one biosourced lipid. In one of these particular embodiments, one - only - of the solvents of the biphasic purification system consists (exclusively) of a single biosourced lipid. However, nothing prevents provision being made for one - only - of the solvents of the biphasic purification system to consist of a plurality of biosourced lipids in a mixture.

[0013] By "consisting mainly of", it is understood in the context of the present invention, an amount at least equal to 50%, in particular greater than 50%, by volume relative to the total volume of one of the solvents of the two-phase system, preferably greater than or equal to 75%, more preferably greater than or equal to 90%, even more preferably greater than or equal to 95%, such as greater than or equal to 97%, 98%, or even greater than or equal to 99% by volume relative to the total volume of the corresponding solvent of the two-phase system. In a particular embodiment, one of the solvents of the two-phase purification system consists exclusively of at least one biosourced lipid.In this embodiment, the biphasic purification system consists of a first solvent constituting a stationary phase and a second solvent constituting a mobile phase, the first solvent constituting the stationary phase being a vegetable oil, the second solvent constituting the mobile phase being a low molecular weight alcohol, the first solvent constituting the stationary phase and the second solvent constituting the mobile phase being in volume proportions of 1 / 1.

[0014] In certain embodiments of the present invention, the compound(s) to be purified comprise(s) or consist of any compound derived from a biomass extract. In these embodiments, the purified product is a product derived from a biomass and substantially pure. In certain other embodiments, the the desired product is an extract of a biomass, the desired product being a biomass extract at least substantially free of an undesirable compound of the biomass extract.

[0015] In certain preferred embodiments, the biomass is a terrestrial plant. According to these embodiments, the biomass extract is an extract derived from a terrestrial plant, i.e., a plant whose biotope extends to an emerged land. According to certain embodiments, the biomass is derived from a plant of the Embryophyta clade. According to certain embodiments, the biomass is derived from a plant of the Spermatophyta clade. According to certain embodiments, the plant is a terrestrial plant, excluding a Chlorophyta plant. According to certain embodiments, the plant is a terrestrial plant of the Chloroplastidae, excluding a Chlorophyta plant.

[0016] By "Embryophyte", it is understood in the context of the present invention and in the common sense, a terrestrial plant having the following specific derived characteristics: the female sexual organs are archegonia; the male sexual organs are antheridia; the cycle has a multicellular diploid phase; the skeletal walls of the spores contain sporopollenin; the epidermis is covered with a cuticle having stomata.

[0017] By "Spermatophyte" is meant in the context of the present invention and in the common sense, a terrestrial plant having the following specific derived characteristics: the stem has secondary growth, that is to say that a bifacial cambium produces the wood and phloem; ramifications are produced by buds in the axils of the leaves; the ovule is a set formed by the nucellus protected by one or two integument(s), and containing the female gametophyte; after fertilization, the ovule transforms into a seed; the spermatophyte plant is a plant with duplication of the entire genome.

[0018] In those embodiments of the present invention wherein the plant is a terrestrial plant, the plant extract comprising or consisting of a terrestrial plant extract, the compound(s) to be purified comprise(s) or consist(s) of any compound(s) derived from a terrestrial plant. In some embodiments, the plant extract comprises or consists of an extract of a terrestrial plant and the purified product is a product derived from a terrestrial plant and substantially pure. In certain other embodiments, the plant extract comprises or consists of an extract of a terrestrial plant and the desired product is an extract of a terrestrial plant, the desired product being at least substantially free of an undesirable compound of the terrestrial plant extract.

[0019] According to certain particular embodiments, the plant is a plant of the Cannabaceae family. According to certain particular embodiments, the plant is chosen in the group consisting of Cannabis sativa ssp. sativa, Cannabis sativa ssp. indica, Cannabis sativa ssp. ruderalis and their hybrids.

[0020] According to some embodiments, the plant is cannabis.

[0021] According to some embodiments, the plant extract comprises or consists of a mixture of cannabinoids. According to some embodiments, the plant extract comprises or consists of a mixture of cannabinoids and the purified product is a substantially pure cannabinoid. According to some other embodiments, the plant extract comprises or consists of a mixture of cannabinoids and the desired product is a plant extract comprises or consists of a mixture of cannabinoids, the desired product being at least substantially free of an undesirable compound of the terrestrial plant extract.

[0022] According to certain embodiments, the plant extract comprising or consisting of a mixture of cannabinoids and terpenoids (or isoprenoids) and / or flavonoids, the compound(s) to be purified comprise or consist of CBD from cannabis. In certain embodiments, the plant extract comprising or consisting of a mixture of cannabinoids and terpenoids (or isoprenoids) and / or flavonoids, the compound(s) to be purified comprise or consist of THC from cannabis.

[0023] In certain embodiments, the plant extract comprising or consisting of a mixture of cannabinoids and terpenoids (or isoprenoids) and / or flavonoids, the compound(s) to be purified comprise(s) or consist(s) of a plant extract substantially free of—in particular free of—THC. According to certain embodiments, the plant extract comprising or consisting of a mixture of cannabinoids, the compound(s) to be purified comprise or consist of CBD from cannabis. In certain embodiments, the plant extract comprising or consisting of a mixture of cannabinoids, the compound(s) to be purified comprise or consist of THC from cannabis. In certain embodiments, the plant extract comprising or consisting of a mixture of cannabinoids, the compound(s) to be purified comprises(comprise) or consists(consist) of a plant extract substantially free of THC - in particular free of THC.

[0024] Advantageously, the bio-sourced lipid comprises at least one vegetable oil. According to certain embodiments, the bio-sourced lipid comprises a vegetable oil. According to certain embodiments, the bio-sourced lipid comprises at least one vegetable oil selected from the group consisting of a sunflower oil, optionally a sunflower oil enriched with oleic acid (preferably enriched with bio-sourced oleic acid), an olive oil, a rapeseed oil, a sesame oil, a linseed oil, a hemp oil, a walnut oil, a hazelnut oil, a grape seed oil, a pumpkin seed oil, a jojoba oil, and of a coconut oil. In some other embodiments, the bio-based lipid comprises an animal oil. In some other embodiments, the bio-based lipid comprises an oil derived from a microorganism.

[0025] The bio-sourced lipid is accessible in industrial quantities. The bio-sourced lipid is accessible in industrial quantities with a stable composition.

[0026] Another subject of the present invention relates to a method for extracting at least one compound - in particular a method for purifying at least one active compound - from an extract of a biomass - in particular from an extract of all or only part of a plant, preferably a terrestrial plant -, a method in which at least one bio-sourced lipid is used in a two-phase system in Centrifugal Partition Chromatography (CPC). The present invention therefore relates to a method for purifying at least one active compound from an extract of a biomass in which at least one bio-sourced lipid is used in a two-phase system in Centrifugal Partition Chromatography (CPC).But the present invention also relates to a method for extracting at least one active compound from a biomass extract, in which at least one biosourced lipid is used in a two-phase system in Centrifugal Partition Chromatography (CPC), the active compound being an undesirable compound in the biomass extract.

[0027] According to certain embodiments, the present invention relates to a method for extracting a compound from a biomass - in particular a method for purifying an active compound from a plant extract -, comprising the following steps, in particular the following successive steps: a) suspending or dissolving a biomass - in particular at least one part of a plant - comprising said active compound so as to form an extract of said biomass - in particular an extract of said at least one part of a plant - comprising said compound - in particular said active compound -, b) purifying said compound using a biphasic liquid-liquid system for centrifugal partition chromatography (CPC), the biphasic liquid-liquid system comprising at least one bio-sourced lipid in one of the phases of the biphasic purification system, c) recovery of said compound in at least one of the phases of the two-phase purification system, in particular in said at least one bio-sourced lipid, and d) optionally separating the active compound from said at least one phase of the biphasic purification system by lyophilization, precipitation, crystallization, freezing, ultrafiltration, membrane separation, column chromatography, vaporization of said at least one purification solvent, or a combination thereof.

[0028] In certain embodiments, one of the phases of the biphasic system being formed of at least one biosourced lipid, in particular a single biosourced lipid, step c) consists of recovering the active compound in said at least one bio-sourced lipid, in particular in the single bio-sourced lipid.

[0029] According to certain other embodiments, the present invention relates to a method for extracting an undesirable compound from a biomass - in particular an undesirable compound from an extract of at least one part of a plant -, comprising the following steps, in particular the following successive steps: (a) suspending or dissolving a biomass, in particular at least one part of a plant, by which a biomass extract - in particular an extract of at least one part of a plant - comprising said undesirable compound is formed, b) extracting said undesirable compound from the biomass extract - in particular from the extract of said at least one plant part - using a two-phase liquid-liquid system for centrifugal partition chromatography (CPC), the two-phase liquid-liquid system comprising at least one bio-sourced lipid in one of the phases of the two-phase purification system, c) recovery of a plant extract substantially free of the undesirable compound, in at least one of the phases of the two-phase purification system, and d) optionally separating the plant extract substantially freed - in particular substantially free - from the undesirable compound and said at least one phase of the two-phase purification system by lyophilization, precipitation, crystallization, freezing, ultrafiltration, membrane separation, column chromatography, vaporization of said at least one purification solvent, or a combination thereof.

[0030] In certain embodiments of a method according to the present invention, the active compound is lipophilic. In certain of these embodiments of a method according to the present invention, the active compound is apolar.

[0031] By "lipophilic" is meant in the context of the present invention an active compound capable of being purified in a phase of the biphasic system comprising the biosourced lipid.

[0032] In certain other embodiments of a method according to the present invention, the active compound is hydrophilic. In certain of these other embodiments of a method according to the present invention, the active compound is polar.

[0033] By "hydrophilic" is meant in the context of the present invention an active compound capable of being purified in a phase of the biphasic system, immiscible with the phase of the biphasic system comprising the biosourced lipid.

[0034] In certain embodiments of a method according to the present invention, the plant extract substantially freed - in particular substantially free - of the compound undesirable is essentially hydrophilic and is obtained in a phase of the biphasic system not including the biosourced lipid.

[0035] According to certain embodiments, step a) is a suspension of the biomass - in particular of said at least one part of plant - in a liquid medium or a dissolution of the biomass - in particular of said at least one part of plant - in a liquid medium, said liquid medium consisting of one - in particular only one - of the solvents of the two-phase liquid-liquid system for centrifugal partition chromatography (CPC).

[0036] According to certain advantageous embodiments, step a) comprises suspending the biomass - in particular said at least one part of the plant - and macerating the biomass - in particular said at least one part of the plant - in a liquid medium or dissolving the biomass - in particular said at least one part of the plant - in a liquid medium, said liquid medium consisting of an alcoholic liquid medium forming one of the solvents of the two-phase liquid-liquid system for centrifugal partition chromatography (CPC).

[0037] In certain particular embodiments of a method according to the present invention, step a) is a suspension of the biomass - in particular of said at least one part of plant - in a hydroalcoholic medium or a dissolution of the biomass - in particular of said at least one part of plant - in a hydroalcoholic medium. In certain of these particular embodiments of a method according to the present invention, step a) is a suspension or a dissolution of the biomass - in particular of said at least one part of plant - in an alcoholic medium in which water is present in trace amounts only.

[0038] Indeed, an alcoholic medium may contain a quantity of water, captured by the alcoholic medium from the humidity in the air. However, nothing prevents the alcoholic medium from being formed from alcohol, in particular 100%, 99% or 95% ethanol.

[0039] Preferably, the alcohol chosen for such alcoholic or hydroalcoholic media according to the present invention is of low molecular weight, for example less than 100 g / mol, or even less than 50 g / mol.

[0040] Examples of such alcohols are methanol, ethanol, propan-1-ol, propan-2-ol, butan-1-ol, butan-2-ol, pentan-1-ol, pentan-2-ol, pentan-3-ol, alone or in mixtures.

[0041] Thus, depending on the nature of the active compound that one seeks to purify or extract from the biomass extract, in particular from the plant extract, and the purification or extraction strategy envisaged, the nature of the solvent of the alcoholic or hydroalcoholic liquid medium is adapted.

[0042] According to certain embodiments, the biomass comprises (or consists of) a biomass derived from at least one organism of the eukaryote taxon. According to In some of these embodiments, the biomass is a plant biomass. In some embodiments, the biomass comprises (or consists of) a biomass derived from at least one terrestrial plant. In some embodiments, the biomass comprises (or consists of) a biomass derived from at least one charophyte algae. In some embodiments, the biomass comprises (or consists of) a biomass derived from at least one animal. In some embodiments, the biomass comprises (or consists of) a biomass derived from at least one organism of the taxon bacteria. In some embodiments, the biomass comprises (or consists of) a biomass derived from at least one organism of the taxon archaea.

[0043] According to certain embodiments, the biomass comprises at least one (or consists of one) biomass chosen from the group formed by biomasses originating from at least one organism of the eukaryote taxon - in particular plant biomasses -, biomasses originating from at least one organism of the bacteria taxon and biomasses originating from at least one organism of the archaea taxon, alone or as a mixture of these biomasses.

[0044] According to certain embodiments of a method according to the invention, the biomass comprises or consists of all or part of a terrestrial plant. For example, in the method according to the present invention, said at least one part of terrestrial plant is selected from the group consisting of at least one part of root, at least one part of tuber, at least one part of rhizome, at least one part of stem, at least one part of bark, at least one part of leaf, at least one part of flower, and at least one part of fruit.

[0045] According to certain embodiments, the method according to the present invention comprises a step (prior to step a)) of mechanical transformation / fractionation of the biomass - in particular of said at least one part of plant -. Such mechanical transformation / fractionation can advantageously be obtained, for example by using a homogenizer, a grinder, a ball mill, bead beating, a mixer, ultrasound, a compression / expansion cycle, a microfluidizer, an expulsion press, and / or one or more freezing / thawing cycles.

[0046] In particular embodiments of a method according to the invention, one - only - of the solvents of the biphasic purification system consists mainly - in particular exclusively - of at least one biosourced lipid. In one of these particular embodiments, one - only - of the solvents of the biphasic purification system consists (exclusively) of a single biosourced lipid. However, nothing prevents provision being made for one - only - of the solvents of the biphasic purification system to consist of a plurality of biosourced lipids in mixture. Thus, in the method according to the present invention, the quantity of at least one bio-sourced lipid is at least equal to 50% by volume relative to the total volume of the corresponding solvent of the two-phase purification system, preferably greater than or equal to 75%, more preferably greater than or equal to 90%, even more preferably greater than or equal to 95%, such as greater than or equal to 97%, 98%, or even greater than or equal to 99% by volume relative to the total volume of the corresponding solvent of the two-phase purification system. In a particular embodiment, the corresponding solvent of the two-phase purification system consists exclusively of bio-sourced lipid.

[0047] In some of these embodiments of a method according to the invention, the two-phase purification system is formed of a first solvent constituting a stationary phase and a second solvent constituting a mobile phase, the first solvent constituting the stationary phase comprising a - in particular consisting of a - biosourced lipid. In some of these particular embodiments of a method according to the invention, said first solvent constituting the stationary phase comprises a - in particular consists of a - vegetable oil, and said second solvent constituting the mobile phase comprises - in particular consists of - at least one low molecular weight alcohol, the first solvent constituting the stationary phase and the second solvent constituting the mobile phase being in volume proportions of 1 / 1.

[0048] According to certain embodiments of a method according to the present invention, the biomass is derived from a terrestrial plant or is a terrestrial plant, i.e. a plant whose biotope extends to an emerged land. According to certain embodiments, the biomass is derived from a plant of the Embryophyta clade. According to certain embodiments, the biomass is derived from a plant of the Spermatophyta clade. According to certain embodiments, the plant is a terrestrial plant, excluding a Chlorophyta plant. According to certain embodiments, the plant is a terrestrial plant of the Chloroplastidae, excluding a Chlorophyta plant.

[0049] In these embodiments, the plant extract comprising or consisting of a terrestrial plant extract, the active compound(s) to be purified comprises or consists of any active compound of a terrestrial plant. Advantageously, in certain embodiments, the plant extract comprising or consisting of an extract of a terrestrial plant, the purified active compound is an active compound from a terrestrial plant which is substantially pure.

[0050] In certain other embodiments, the plant extract comprises or consists of an extract of a terrestrial plant and the desired product is an extract of a terrestrial plant, the desired product being at least substantially free of an undesirable compound of the terrestrial plant extract.

[0051] According to certain particular embodiments, the plant is a plant from the European Pharmacopoeia. According to certain particular embodiments, the plant is a plant from the Chinese Pharmacopoeia. According to certain particular embodiments, the plant is a plant from the Ayurvedic Pharmacopoeia. According to certain particular embodiments, the plant is a plant from the Indian Pharmacopoeia. According to certain particular embodiments, the plant is a plant from the American Pharmacopoeia. According to certain particular embodiments, the plant is a plant from the Japanese Pharmacopoeia. According to certain particular embodiments, the plant is a food plant. According to certain particular embodiments, the plant is a plant of industrial interest.

[0052] According to certain embodiments, the plant is chosen from the group consisting of plants of the Cannabaceae family, plants of the European pharmacopoeia, plants of the Chinese pharmacopoeia, plants of the Ayurvedic pharmacopoeia, plants of the Indian pharmacopoeia, plants of the American pharmacopoeia, plants of the Japanese pharmacopoeia, food plants, plants of cosmetic interest and plants of industrial interest.

[0053] According to certain particular embodiments, the plant is a plant of the Cannabaceae family. According to certain particular embodiments, the plant is selected from the group consisting of Cannabis sativa ssp. sativa, Cannabis sativa ssp. indica, Cannabis sativa ssp. ruderalis and their hybrids.

[0054] Preferably, the plant is cannabis.

[0055] In a particular embodiment of a method according to the present invention, the plant extract comprises or consists of THC and CBD derived from cannabis.

[0056] Advantageously, in certain embodiments of a method according to the invention, the plant extract comprises or consists of a mixture of cannabinoids, terpenoids (or isoprenoids) and / or flavonoids. Advantageously, in certain of these embodiments, the purified active compound is a substantially pure cannabinoid. In certain embodiments, the purified active compound is substantially pure CBD derived from cannabis.

[0057] In certain other embodiments, the plant extract comprises or consists of an extract of a terrestrial plant and the desired and obtained product is an extract of a terrestrial plant, the desired and obtained product being at least substantially free of an undesirable compound of the terrestrial plant extract. In certain embodiments, the plant extract comprises or consists of a mixture of cannabinoids, terpenoids (or isoprenoids) and / or flavonoids. the desired and obtained product is an extract of a terrestrial plant comprising CBD derived from cannabis and substantially free of THC. In certain embodiments, the extract of plant is a cannabis extract and the product sought and obtained is a cannabis extract comprising CBD derived from cannabis and substantially free of THC.

[0058] According to certain embodiments of a method according to the present invention, the bio-sourced lipid comprises a vegetable oil. According to certain embodiments of a method according to the invention, the bio-sourced lipid comprises a vegetable oil selected from the group consisting of a sunflower oil, optionally a sunflower oil enriched in oleic acid (bio-sourced), an olive oil, a rapeseed oil, a sesame oil, a linseed oil, a hemp oil, a walnut oil, a hazelnut oil, a grape seed oil, a pumpkin seed oil, a jojoba oil, and a coconut oil. According to certain other embodiments, the bio-sourced lipid comprises an animal oil. According to certain other embodiments, the bio-sourced lipid comprises an oil derived from a microorganism.

[0059] The subject of the present invention further relates to a product - in particular an active compound derived from a biomass extract or a biomass extract - in particular from a plant - freed from an undesirable compound of the biomass extract - in particular from a plant - capable of being obtained, in particular obtained, by the process according to the present invention, in particular characterized in that it has a desired level of purity.

[0060] Thus, the product obtained by the process according to the present invention, that is to say the at least substantially purified compound or the biomass extract - in particular the plant extract - substantially freed from at least one undesirable compound of the biomass extract - in particular the plant extract -, has the advantage of being free or substantially free of any trace of petro-sourced solvent.

[0061] Advantageously, the product obtained by the process according to the invention is an active (or active) product.

[0062] The description of the invention which follows with reference to the appended drawings, given as non-limiting examples, will make it clear what the invention consists of and how it can be implemented. In the appended figures:

[0063] [Fig-1] [Fig.l] is a representative fractogram of the quantitative analysis by HPLC-UV of cannabinoids from a cannabis extract, found in successive 10 mL fractions eluted at the outlet of the CPC column with a two-phase system formed of ethanol and sunflower oil enriched in oleic acid,

[0064] [Fig.2] [Fig.2] is a representative fractogram of the quantitative analysis by HPLC-UV of cannabinoids from a cannabis extract, found in the successive 10 mL fractions eluted at the outlet of the CPC column with a two-phase system formed of ethanol and refined olive oil,

[0065] [Fig.3] [Fig.3] is a representative fractogram of the quantitative analysis by HPLC-UV of cannabinoids of a cannabis extract, found in the fractions successive 10 mL eluted at the outlet of the CPC column with a two-phase system formed of ethanol and refined sesame oil,

[0066] [Fig.4] [Fig.4] is a representative fractogram of the quantitative analysis by HPLC-UV of cannabinoids from a cannabis extract, found in successive 10 mL fractions eluted at the outlet of the CPC column with a biphasic system formed of ethanol and jojoba oil,

[0067] [Fig.5] [Fig.5] is a representative fractogram of the quantitative analysis by HPLC-UV of cannabinoids from a cannabis extract, found in successive 10 mL fractions eluted at the outlet of the CPC column with a two-phase system formed of ethanol and linseed oil.

[0068] EXAMPLES

[0069] A cannabis extract comprising: - 4.21 + / - 0.08% cannabidiol (CBD), - 39.87 + / - 0.87% of the acid form of cannabidiol (CBD-a), - 8.48 + / - 0.19% of tetrahydrocannabinol (d9-THC), and - 18.25 + / - 0.40% of the acid form of tetrahydrocannabinol (d9-THC-a), is solubilized in anhydrous ethanol (EtOH). The mixture is subjected to a CPC purification step for the purpose of THC remediation (THC and THC-a) and production of a CBD / CBD-a composition having a THC / THC-a level of less than 0.05% (by mass). The production of the CBD / CBD-a composition is carried out with a high yield.

[0070] The CPC purification is carried out with a SCPC-250+1000 centrifugal partition chromatography apparatus (Gilson Purifications SAS) equipped with a chromatographic column with an effective working volume of 235 mL and composed of 848 chromatographic cells. The chromatography apparatus is equipped with two solvent inlet ports, each inlet port corresponding to one of the solvents of the two-phase system. The chromatographic column is pre-filled with the mobile phase and then rotated at a speed of 1500 rpm and maintained at this speed throughout the purification. The control, pumping, detection and collection of the fractions are carried out with a multi-purpose device (Spot Prep 2, Gilson Purifications SAS). The mobile phase consisting of anhydrous ethanol (EtOH) is carried into the column with a flow rate of 5 mL / min in ascending mode (AM).500 mg of extract to be purified are solubilized in 10 mL of the mobile phase and are introduced into an injection loop with a volume of 30 mL. Detection is carried out at a wavelength of 228 nm with a self-triggering detector (DAD) and each collected fraction has a volume of 10 mL. The content of the fractions in CBD-A, CBD, d9THC-A and d9THC is determined by HPLC-UV. Fractions devoid of THC are selected.

[0071] The analysis of each successive 10 mL fraction leaving the CPC column is carried out by HPLC-UV in order to calculate: - the partition coefficient (KD) of each of the solutes between the mobile (EtOH) and stationary (oil) phases as the ratio of the area under the absorbance curve of the analyte in the lower phase (oil) and the area under the absorbance curve of the analyte in the upper phase (EtOH), and - the selectivity S estimated as the ratio of the partition coefficient (KD / d9-THC) of tetrahydrocannabinol (d9-THC) as determined above, and the partition coefficient (KD) of cannabidiol (CBD) as determined above.

[0072] Biphasic system comprising a sunflower oil enriched with oleic acid as a biosourced lipid. The concentration of the extract introduced into the column is 524 mg / 10 mL of mobile phase (EtOH). The fatty acid composition of oleic acid enriched sunflower oil is as follows: -Oleic acid (C18:l(co-9)): 89.4% -Palmitic acid (C16:0): 3.7% -Linoleic acid (C18:2(co-6)): 2.9% -Stearic acid (Cl8:0): 2.2% -Trans-vaccenic acid (C18:l(co-7)): 1.2%. The dynamic viscosity (p) of oleic acid enriched sunflower oil is 40.09 mPa.s. The ascending partition coefficient (AMDC) of each of the cannabinoids CBD, CBD-a, d9THC and d9THC-a is given in Table 1 below: KD AM CBD CBD-a d9THC d9THC-a 0.2214 0.1476 0.6401 0.6758 Table 1 The selectivity (S) established according to the formula S = KD(AM, d9THC) / KD(AM, CBD) is 2.8913. The retention of the stationary phase is 64% and represents the percentage by volume (%, v / v) of stationary phase remaining in the column after equilibration of the chromatographic apparatus. The overall coefficient (kOa) is 0.027 s 1 and corresponds to the product kOa = NUT x (Q / Vc) in which NUT is the number of transfer units, Q is the elution volume flow rate and Vc is the useful volume of the column. The descriptive fractogram of the purification of cannabinoids by CPC with a biphasic system EtOH / oleic acid-enriched sunflower oil is shown in [Fig.l] in which the curve identified by solid squares (■) corresponds to the elution of cannabidiol (CBD), the curve identified by solid triangles (A) corresponds to the elution of tetrahydrocannabinol (d9-THC), the curve identified by crosses (x) corresponds to the elution of the acid form of tetrahydrocannabinol (d9-THC-A) and the smooth curve (without symbol) corresponds to the elution of the acid form of cannabidiol (CBD-A). The CBD-a restitution yield is greater than 98% of CBD-a restored and the CBD restitution yield is greater than 90% of CBD restored. The productivity of the purification method is 1.33 g of extract / h / L (liter of column).

[0073] Biphasic system comprising refined olive oil as biosourced lipid. The concentration of the extract introduced into the column is 482 mg / 10 mL of mobile phase (EtOH). The fatty acid composition of refined olive oil is as follows: ^Oleic acid (Cl8:l(co-9)): 74.1% - Palmitic acid (C16:0): 12.5% ^Linoleic acid (C18:2(co-6)): 8.4% ^Stearic acid (Cl8:0): 3.6%. The dynamic viscosity (p) of refined olive oil is 32.63 mPa.s. The ascending partition coefficient (AMDC) of each of the cannabinoids CBD, CBD-a, d9THC and d9THC-a is given in Table 2 below: KD AM CBD CBD-a d9THC d9THC-a 0.385 0.283 1.015 1.261 Table 2 The selectivity (S) established according to the formula S = KD(AM, d9THC) / KD(AM, CBD) is 2.633. The retention of the stationary phase is 56% and the overall coefficient (kOa) is 0.033 s1. The CBD-a restitution yield is greater than 97% of CBD-a restored and the CBD restitution yield is greater than 86% of CBD restored. The productivity of the purification method is 1.3 g of extract / h / Liter of column. The descriptive fractogram of the purification of cannabinoids by CPC with a biphasic EtOH / refined olive oil system is represented in [Fig.2] in which the curve identified by solid squares (■) corresponds to the elution of cannabidiol (CBD), the curve identified by solid triangles (A) corresponds to the elution of tetrahydrocannabinol (d9-THC), the curve identified by crosses (x) corresponds to the elution of the acid form of tetrahydrocannabinol (d9-THC-A) and the smooth curve (without symbol) corresponds to the elution of the acid form of cannabidiol (CBD-A).

[0074] Biphasic system comprising a refined sesame oil as a bio-sourced lipid. The concentration of the extract introduced into the column is 488 mg / 10 mL of mobile phase (EtOH). The fatty acid composition of refined sesame oil is as follows: ^Oleic acid (Cl8:l(co-9)): 40.3% - Palmitic acid (Cl6:0): 9.4% ^Linoleic acid (C18:2(co-6)): 44.3% ^Stearic acid (Cl8:0): 4.3%. The dynamic viscosity (p) of sesame oil is 45.66 mPa.s. The ascending partition coefficient (KDAM) of each of the cannabinoids CBD, CBD-a, d9THC and d9THC-a is given in Table 3 below: KD AM CBD CBD-a d9THC d9THC-a 0.337 0.229 1.185 1333 Table 3 The selectivity (S) established according to the formula S = KD(AM, d9THC) / KD(AM, CBD) is 3.519. The retention of the stationary phase is 62% and the overall coefficient (kOa) is 0.042 s1. The CBD-a restitution yield is greater than 98% of CBD-a restored and the CBD restitution yield is greater than 94% of CBD restored. The productivity of the purification method is 1.3 g of extract / h / Liter of column. The descriptive fractogram of the purification of cannabinoids by CPC with a biphasic system EtOH / refined sesame oil is represented in [Fig.3] in which the curve identified by solid squares (■) corresponds to the elution of cannabidiol (CBD), the curve identified by solid triangles (A) corresponds to the elution of tetrahydrocannabinol (d9-THC), the curve identified by crosses (x) corresponds to the elution of the acid form of tetrahydrocannabinol (d9-THC-A) and the smooth curve (without symbol) corresponds to the elution of the acid form of cannabidiol (CBD-A).

[0075] Biphasic system comprising jojoba oil as biosourced lipid. The concentration of the extract introduced into the column is 494 mg / 10 mL of mobile phase (EtOH). The fatty acid composition of jojoba oil is as follows: ^Gadoleic acid (C20: l(co-9)): 71.35% - Erucic acid (C22:l(co-9): 15.04% - Oleic acid (C18:l(co-9)): 8.77%. The dynamic viscosity (q) of jojoba oil is 21.88 mPa.s. The ascending partition coefficient (KDAM) of each of the cannabinoids CBD, CBD-a, d9THC and d9THC-a are given in Table 4 below: KD AM CBD CBD-a d9THC d9THC-a 0.378 0.495 0.914 1.230 Table 4 The selectivity (S) established according to the formula S = KD(AM, d9THC) / KD(AM, CBD) is 2.420. The retention of the stationary phase is 55% and the overall coefficient (kOa) is 0.044 s1. The CBD-a restitution yield is greater than 97% of CBD-a restored and the CBD restitution yield is greater than 90% of CBD restored. The productivity of the purification method is 1.7 g of extract / h / Liter of column. The descriptive fractogram of the purification of cannabinoids by CPC with a biphasic EtOH / jojoba oil system is shown in [Fig.4] in which the curve identified by solid squares (■) corresponds to the elution of cannabidiol (CBD), the curve identified by solid triangles (A) corresponds to the elution of tetrahydrocannabinol (d9-THC), the curve identified by crosses (x) corresponds to the elution of the acid form of tetrahydrocannabinol (d9-THC-A) and the smooth curve (without symbol) corresponds to the elution of the acid form of cannabidiol (CBD-A).

[0076] Biphasic system comprising linseed oil as biosourced lipid. The concentration of the extract introduced into the column is 489 mg / 10 mL of mobile phase (EtOH). The fatty acid composition of jojoba oil is as follows: ^Alpha-linolenic acid (C18:3(co-3)): 60.6% - Oleic acid (C18:1(co-9)): 15.7%. - Palmitic acid (C16:0): 5.3% sOleic acid (C18:2(co-6)): 13.9% ^Stearic acid (Cl8:0): 4.4%. The dynamic viscosity (q) of linseed oil is 28.48 mPa.s. The ascending partition coefficient (KDAM) of each of the cannabinoids CBD, CBD-a, d9THC and d9THC-a is given in Table 5 below: KD AM CBD CBD-a d9THC d9THC-a 0.481 0.317 1.289 1.358 Table 5 The selectivity (S) established according to the formula S = KD(AM, d9THC) / KD(AM, CBD) is 2.682. The retention of the stationary phase is 56% and the overall coefficient (kOa) is 0.058 s. The CBD-a restitution yield is greater than 98% of CBD-a restored and the CBD restitution yield is greater than 95% of CBD restored. The productivity of the purification method is 1.5 g of extract / h / Liter of column. The descriptive fractogram of the purification of cannabinoids by CPC with a biphasic EtOH / linseed oil system is shown in [Fig.5] in which the curve identified by solid squares (■) corresponds to the elution of cannabidiol (CBD), the curve identified by solid triangles (A) corresponds to the elution of tetrahydrocannabinol (d9-THC), the curve identified by crosses (x) corresponds to the elution of the acid form of tetrahydrocannabinol (d9-THC-A) and the smooth curve (without symbol) corresponds to the elution of the acid form of cannabidiol (CBD-A).

Claims

Claims

1. Use of at least one bio-sourced lipid in one of the solvents of a two-phase Centrifugal Partition Chromatography (CPC) purification system for purification of at least one biomass extract.

2. Use according to claim 1, characterized in that one of the solvents of the two-phase purification system consists mainly of at least one bio-sourced lipid.

3. Use according to claim 1 or 2, characterized in that the biomass is a terrestrial plant.

4. Use according to claim 3, characterized in that the plant is a plant of the Cannabaceae family.

5. Use according to one of claims 3 or 4, characterized in that the plant extract comprising or consisting of a mixture of cannabinoids and terpenoids and / or flavonoids, the compound(s) to be purified comprise or consist of CBD from cannabis.

6. Use according to one of claims 3 or 4, characterized in that the plant extract comprising or consisting of a mixture of cannabinoids and terpenoids and / or flavonoids, the compound(s) to be purified comprises(comprise) or consists(consist) of a plant extract freed from THC.

7. Use according to any one of the preceding claims, characterized in that the bio-sourced lipid comprises at least one vegetable oil, in particular at least one vegetable oil chosen from the group consisting of a sunflower oil, optionally a sunflower oil enriched with oleic acid, an olive oil, a rapeseed oil, a sesame oil, a linseed oil, a hemp oil, a walnut oil, a hazelnut oil, a grape seed oil, a pumpkin seed oil, a jojoba oil and a coconut oil.

8. Method for extracting at least one compound from a biomass extract, in which at least one bio-sourced lipid is used in a two-phase system in Centrifugal Partition Chromatography (CPC).

9. Method according to claim 8, characterized in that it comprises the following successive steps: a) suspending or dissolving a biomass comprising said compound so as to form an extract of said biomass comprising said compound, b) purifying said compound using a two-phase liquid-liquid system for centrifugal partition chromatography (CPC), the two-phase liquid-liquid system comprising at least one bio-sourced lipid in one of the phases of the two-phase purification system, c) recovering said compound in at least one of the phases of the two-phase purification system, in particular in said at least one bio-sourced lipid, and d) optionally separating said compound from said at least one phase of the two-phase purification system by lyophilization, precipitation, crystallization, freezing, ultrafiltration, membrane separation, column chromatography, vaporization of said at least one purification solvent, or a combination thereof.

10. Method according to one of claims 8 or 9, characterized in that said compound is lipophilic.

11. Method according to one of claims 8 to 10, characterized in that step a) is a suspension of said biomass in a liquid medium or a dissolution of said biomass in a liquid medium, said liquid medium consisting of one of the solvents of the two-phase liquid-liquid system for centrifugal partition chromatography (CPC).

12. Method according to any one of claims 8 to 11, characterized in that step a) is a suspension of said biomass in a liquid medium or a dissolution of said biomass in a liquid medium, said liquid medium consisting of an alcoholic liquid medium forming one of the solvents of the two-phase liquid-liquid system for centrifugal partition chromatography (CPC).

13. Method according to any one of claims 8 to 12, characterized in that the biomass comprises at least one biomass chosen from the group formed by biomasses originating from at least one organism of the eukaryote taxon, biomasses originating from at least one organism of the bacteria taxon and biomasses originating from at least one organism of the archaea taxon, alone or as a mixture of these biomasses.

14. Method according to any one of claims 8 to 13, characterized in that the biomass comes from a terrestrial plant.

15. Method according to any one of claims 8 to 14, characterized in that it comprises a prior step of mechanical transformation / fractionation of the biomass.

16. Method according to any one of claims 8 to 15, characterized in that one of the solvents of the two-phase purification system is predominantly at least one bio-sourced lipid.

17. Method according to any one of claims 8 to 16, characterized in that said biomass comprises or consists of all or part of a terrestrial plant, in particular chosen from the group formed by at least one part of root, at least one part of tuber, at least one part of rhizome, at least one part of stem, at least one part of bark, at least one part of leaf, at least one part of flower and at least one part of fruit.

18. Method according to any one of claims 14 to 17, characterized in that the plant is a plant of the Cannabaceae family, in particular a plant chosen from the group formed by Cannabis sativa ssp. sativa, Cannabis sativa ssp. indica, Cannabis sativa ssp. ruderalis and their hybrids.

19. A method according to claim 18, characterized in that the plant extract comprises or consists of THC and CBD from cannabis.

20. Method according to any one of claims 8 to 19, characterized in that the bio-sourced lipid comprises a vegetable oil, in particular a vegetable oil chosen from the group consisting of a sunflower oil, optionally a sunflower oil enriched with oleic acid (bio-sourced), an olive oil, a rapeseed oil, a sesame oil, a linseed oil, a hemp oil, a walnut oil, a hazelnut oil, a grape seed oil, a pumpkin seed oil, a jojoba oil, and a coconut oil.

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