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WO2020254577A1 - Extraction hydrotropique - Google Patents

Extraction hydrotropique Download PDF

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Publication number
WO2020254577A1
WO2020254577A1 PCT/EP2020/067101 EP2020067101W WO2020254577A1 WO 2020254577 A1 WO2020254577 A1 WO 2020254577A1 EP 2020067101 W EP2020067101 W EP 2020067101W WO 2020254577 A1 WO2020254577 A1 WO 2020254577A1
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WO
WIPO (PCT)
Prior art keywords
biological material
extract
biological
water
plant
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2020/067101
Other languages
English (en)
Inventor
Michael LAGUERRE
Antoine Charles BILY
Simona BIRTIC
Agathe MAZAUD
Raphael Lebeuf
Veronique NARDELLO-RATAJ
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Givaudan SA
Centre National de la Recherche Scientifique CNRS
Universite Lille 1 Sciences et Technologies
Ecole Centrale de Lille
Original Assignee
Givaudan SA
Centre National de la Recherche Scientifique CNRS
Universite Lille 1 Sciences et Technologies
Ecole Centrale de Lille
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Publication date
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Publication of WO2020254577A1 publication Critical patent/WO2020254577A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/10—Natural spices, flavouring agents or condiments; Extracts thereof
    • A23L27/11—Natural spices, flavouring agents or condiments; Extracts thereof obtained by solvent extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/02—Solvent extraction of solids
    • B01D11/0215—Solid material in other stationary receptacles
    • B01D11/0253—Fluidised bed of solid materials
    • B01D11/0257—Fluidised bed of solid materials using mixing mechanisms, e.g. stirrers, jets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/02—Solvent extraction of solids
    • B01D11/0288—Applications, solvents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D9/00—Crystallisation
    • B01D9/0004—Crystallisation cooling by heat exchange
    • B01D9/0013—Crystallisation cooling by heat exchange by indirect heat exchange
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D9/00—Crystallisation
    • B01D9/005—Selection of auxiliary, e.g. for control of crystallisation nuclei, of crystal growth, of adherence to walls; Arrangements for introduction thereof
    • B01D9/0054—Use of anti-solvent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B9/00—Essential oils; Perfumes
    • C11B9/02—Recovery or refining of essential oils from raw materials

Definitions

  • the present invention relates to methods for preparing biological extracts using hydrotropic agents, methods for purifying biological extracts formed using hydrotropic agents, the biological extracts obtained using the methods and uses and the use of the biological extracts, such as in food-stuffs, pharmaceuticals, flavours and fragrances, cosmetics, nutraceuticals and supplements, such as food supplements and sports supplements.
  • extracts that are to be used for human consumption are obtained using alcoholic based solvent or supercritical fluids (usually C0 2 ) due to their lack of toxicity.
  • Water is a natural solvent that is considered to be renewable; however, its strong polarity does not easily allow for the extraction of lipophilic/non-water-soluble molecules.
  • Hydrotropic agents are water-soluble organic compounds which, at and above a certain concentration, known as “MHC” (Minimum Hydrotropic Concentration), provide a significant increase in the solubility of organic compounds that are practically insoluble in water under normal conditions.
  • MHC Minimum Hydrotropic Concentration
  • MHC minimum hydrotropic concentration
  • the MHC can be determined using several physicochemical methods such as measurement of surface tension, conductivity, dynamic and static light scattering (RE. Coffman, DO. Kildsig. Self-association of nicotinamide in aqueous solution: Light scattering and vapor pressure osmometry studies, J. Pharm. Sci. (1996) 85(8): 848-853) or by plotting a solubilisation curve of a lipophilic compound (content of solubilised solute vs. hydrotrope concentration). Sudan Red, a lipophilic dye easily assayed by spectrophotometry, can be used as reference.
  • Hydrotropes are typically amphiphilic and may be ionic (anionic, cationic, zwitterionic) or non-ionic (resorcinol, nicotinamide, alkyl polyglycosides, etc.) and may have various structures, e.g. aromatic, aliphatic, or cyclic.
  • Hydrotropes with an amphiphilic molecular structure possess the ability to increase the solubility of sparingly soluble organic molecules in water (T. Hodgdon, E. Kaler, Hydrotropic solutions, Curr. Opin. Colloid. In. 12 (2007) 121-128). It is a molecular phenomenon whereby adding a second solute (hydrotrope) helps to increase the aqueous solubility of poorly soluble solutes (A. Saleh, L. El-Khordagui, Hydrotropic agents: a new definition, Int. J. Pharm. 24 (1985) 231-238).
  • hydrotropes typically contain both hydrophobic and hydrophilic fractions in them. In comparison to surfactant, they contain only a very small hydrophobic fraction (N. Kapadiya, I. Singhvi, K. Mehta, K. Gauri, D. Sen, Hydrotropy: a promising tool for solubility enhancement: a review, Int. J. Drug Dev. Res. 3 (201 1) 26-33).
  • biological extracts comprising lipophilic, hydrophobic, oil soluble and/or non-water-soluble compounds can be obtained by combining/mixing biological material (such as plant biological material, algal biological material, animal biological material and/or prokaryotic biological material) with an extraction solution comprising water and at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt.
  • biological material such as plant biological material, algal biological material, animal biological material and/or prokaryotic biological material
  • an extraction solution comprising water and at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt.
  • hydrotropic agent which is not a salt means that the at least one hydrotropic agent does not comprise alkali metal salts, or alkaline earth metal salts, or ammonium salts of aromatic or non aromatic carboxylates, sulfates or sulfonates, such as sodium xylene sulfonate, sodium para-toluene sulfonate, sodium cumene sulfonate, sodium butyl monoglycol sulfate, sodium butyl benzene sulfonate, or sodium salicylate.
  • alkali metal salts such as sodium xylene sulfonate, sodium para-toluene sulfonate, sodium cumene sulfonate, sodium butyl monoglycol sulfate, sodium butyl benzene sulfonate, or sodium salicylate.
  • alkali metals we mean principally sodium and potassium.
  • alkaline earth metals we mean principally magnesium and calcium
  • the at least one hydrotropic agent which is not a salt may also be refered to as a“non-salt hydrotropic agent”.
  • the present invention provides a method for providing a biological extract comprising: i) mixing biological material (such as plant biological material, algal biological material, animal biological material and/or prokaryotic biological material) with an extraction solution comprising water and at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt; and
  • the present invention also provides the use of an extraction solution comprising water and at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt, to provide a biological extract from biological material (such as plant biological material, algal biological material, animal biological material and/or prokaryotic biological material), wherein the use comprises:
  • biological material such as plant biological material, algal biological material, animal biological material and/or prokaryotic biological material
  • an extraction solution comprising water and at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt
  • the extraction solution does not comprise a soap compound.
  • soap compound is intended to mean a particular salt between a fatty acid containing more than six carbon atoms in its hydrophobic chain and a monovalent cation.
  • a soap compound is in particular a salt of a fatty acid and a monovalent cation that in an aqueous medium forms micelles.
  • the biological extract may be, for example, a liquid biological extract, a crude solid or semi-solid biological extract or a purified biological extract.
  • extraction solution comprising water and at least one hydrotropic agent is intended to mean an aqueous solution containing at least one hydrotropic agent at a concentration equal to or greater than the hydrotropic agent’s minimum hydrotropic concentration (MHC).
  • MHC minimum hydrotropic concentration
  • the MHC can be determined using several physicochemical methods such as measurement of surface tension, conductivity, dynamic and static light scattering (RE. Coffman, DO. Kildsig. Self-association of nicotinamide in aqueous solution: Light-scattering and vapor pressure osmometry studies, J. Pharm. Sci. (1996) 85(8): 848-853) or by plotting a solubilisation curve of a lipophilic compound (content of solubilised solute vs. hydrotrope concentration). Sudan Red, a lipophilic dye easily assayed by spectrophotometry, can be used as reference.
  • plant biological material is material that has been obtained from or is obtainable from plants, such as from plant roots and/or the aerial parts of the plant, such as leaves, flowers, stems, barks, fruits or seeds or their tissues.
  • the plant biological material may be obtained from the leaves of the plant.
  • algal biological material is material that has been obtained from or is obtainable from a macroalgal or a microalgal source, such as from seaweeds, freshwater algae or cultivated populations of single cell microalgal organisms of eukaryotic nature.
  • animal biological material is material that has been obtained from or is obtainable from an animal source, such as from secretions from the glands of mammals, i.e. musk.
  • prokaryotic biological material is material that has been obtained from or is obtainable from prokaryotic single cell organisms, such as bacteria.
  • the term “obtainable from” means that the plant and/or algal and/or animal and/or prokaryotic biological material may be obtained from a plant/algae/animal/prokaryote directly or may be isolated from the plant/algae/animal/prokaryote, or may be obtained from an alternative source, for example by chemical synthesis or enzymatic production.
  • the term“obtained” as used herein means that the extract is directly derived from the plant/algae/animal/prokaryote source.
  • liquid means a state of matter in which atoms or molecules within the liquid can move freely, while remaining in contact with one another, and will take the shape of its container.
  • a liquid will have a viscosity from about 1 cP at 20 °C to about 10,000 cP at 20 °C, such as from about 50 cP at 20 °C to about 5,000 cP at 20 °C.
  • the biological extract of the invention is enriched in compounds that have limited solubility in aqueous solutions, i.e. compounds that are lipophilic, hydrophobic, oil soluble and/or non-water-soluble.
  • the present invention also provides a method of providing lipophilic, hydrophobic, oil soluble and/or non-water-soluble compounds from biological material (such as plant biological material, algal biological material, animal biological material and/or prokaryotic biological material) comprising:
  • biological material such as plant biological material, algal biological material, animal biological material and/or prokaryotic biological material
  • an extraction solution comprising water and at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt
  • the present invention also provides the use of an extraction solution comprising water and at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt, to provide lipophilic, hydrophobic, oil soluble and/or non-water-soluble compounds from biological material (such as plant biological material, algal biological material, animal biological material and/or prokaryotic biological material), wherein the use comprises:
  • biological material such as plant biological material, algal biological material, animal biological material and/or prokaryotic biological material
  • an extraction solution comprising water and at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt
  • the term“enriched” means that the biological extract comprises about 0.05 % or more by weight of the extract compounds that have limited solubility in aqueous solutions, i.e. compounds that are lipophilic, hydrophobic, oil soluble and/or non-water-soluble.
  • the biological extract when the biological extract is a liquid biological extract provided by a method or use as defined herein comprising steps (i) and (ii), the extract may comprise about 0.05 % or more, about 0.1 % or more, about 1 % or more, about 2 % or more, or about 4 % or more by weight of the liquid extract of compounds that have limited solubility in aqueous solutions, i.e. compounds that are lipophilic, hydrophobic, oil soluble and/or non-water-soluble.
  • the term“lipophilic” means the compounds are able to dissolve in fats, oils, lipids, and non-polar solvents such as hexane or toluene.
  • non-polar solvents such as hexane or toluene.
  • at least about 90 % of the compound can dissolve in fats, oils, lipids, and non-polar solvents such as hexane or toluene, or at least about 95 % or at least about 99 % or about 100 %.
  • non-water soluble means the compounds have a solubility in water of less than about 1 g/L, such as less than about 0.5 g/L at 20 °C.
  • a solubility in water from about 0.001 g/L to about 1 g/L or are substantially insoluble in water.
  • the term“hydrophobic” means that the compounds have a very low solubility in water.
  • the compounds have a solubility in water of less than 1 g/L, such as less than 0.5 g/L at 20 °C.
  • a solubility in water from about 0.001 g/L to about 1 g/L or are substantially insoluble in water.
  • oil soluble means that the compounds have a high solubility in oil.
  • the compounds have a solubility in oil of from about 5 g/L or more, such as from about 10 g/L or more or from about 20 g/L or more.
  • hydrotropes in particular hydrotropes which are not a salt (i.e alkyl glycosides, alkyl glycerol ethers, alkyl polyethylene glycol ethers, dicarboxylic acids, nicotinamide or mixtures thereof), as used in the methods and uses of the present invention, interact with the structure molecules of the plant cells, i.e. molecules of the cell wall or the cell membrane. This interaction results in the release of compounds that would have otherwise been retained in polysaccharide-lignin networks that constituting cell walls.
  • the present invention also provides a method for providing a biological extract comprising: (i) mixing biological material (such as plant biological material, algal biological material, animal biological material and/or prokaryotic biological material) with an extraction solution comprising water and at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt; and
  • the present invention also provides the use of an extraction solution comprising water and at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt, to provide a biological extract from biological material (such as plant biological material, algal biological material, animal biological material and/or prokaryotic biological material), wherein the use comprises:
  • biological material such as plant biological material, algal biological material, animal biological material and/or prokaryotic biological material
  • an extraction solution comprising water and at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt
  • removing any undissolved biological material from the solution obtained in (i) to leave the biological extract wherein the extraction solvent releases lipophilic, hydrophobic, oil soluble and/or non-water-soluble compounds retained in the cells of the biological material (such as plant biological material, algal biological material, animal biological material and/or prokaryotic biological material).
  • the extraction solvent may release at least 10 %, at least 20 %, at least 40 % or at least 50 %, or at least 60 %, or at least 70 %, or at least 80 %, or at least 90 %, such as from about 10 % to about 80 % or from about 20 % to about 60 %, of lipophilic, hydrophobic, oil soluble and/or non-water-soluble compounds retained in the cells of the biological material (such as plant biological material, algal biological material, animal biological material and/or prokaryotic biological material), such as in the polysaccharide-lignin networks that constituting cell walls.
  • the biological material such as plant biological material, algal biological material, animal biological material and/or prokaryotic biological material
  • the present invention also provides a method for decreasing the density of cells and/or increasing cell porosity in biological material (such as plant biological material, algal biological material, animal biological material and/or prokaryotic biological material) comprising:
  • biological material such as plant biological material, algal biological material, animal biological material and/or prokaryotic biological material
  • an extraction solution comprising water and at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt
  • the present invention also provides the use of an extraction solution comprising water and at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt, to decrease the density of cells and/or increase cell porosity of biological material (such as plant biological material, algal biological material, animal biological material and/or prokaryotic biological material), wherein the use comprises:
  • biological material such as plant biological material, algal biological material, animal biological material and/or prokaryotic biological material
  • an extraction solution comprising water and at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt
  • Compounds that have limited solubility in aqueous solutions include, but are not limited to, phenolic compounds including phenolic acids (such as carnosic acid, rosmarinic acid, or caffeic acid), phenolic esters (such as gallate alkyl esters), phenolic diterpenes (such as carnosic acid and its derivatives), flavonoids (such as hesperidin or luteolin glucuronide), secoiridoids, curcuminoids (such as curcumin), stilbenes, phenolic alcohols, phenolic lipids (such as shogaol or ubiquinol), alkaloids, lipids, phenylpropanoids, coumarin (such as dimethoxycoumarin), organic acids (such as malic acid or tartaric acid), terpenoids including sesquiterpenoids, diterpenoids (
  • the biological extract may comprise one or more of the compounds listed above or mixtures thereof. These compounds may typically be natural biological flavourings and taste modifiers, fragrances, biocides, antimicrobials, proteins, enzymes, colourings, pigments, surfactants, antioxidants, vitamins and/or bioactive compounds of nutritional, cosmetic or pharmaceutical interest.
  • the biological material used in the method of the invention may be in the form of a liquid, such as fluid from the biological material, i.e. juice from a plant or fruit.
  • the biological material may be in the form of a solid, such as fresh or dried biological material, which may optionally be ground and/or mashed biological material, i.e. ground or mashed material obtained or obtainable from the biological material.
  • the biological material is not ground or not mashed.
  • the method of the invention (and the use of the hydrotropes agents that are not a salt) can be used also on non-ground materials with very good extraction rates. This is advantageus since it allows to perform an extraction of products without modifying the start material thus avoiding oxidation or degradation of some valuable products.
  • the biological material is not ground and/or not mashed leaves, petals (flowers), etc.
  • the biological material (such as fruits, roots, rind of the frutis, seeds etc), can be cutted in slices to allow the solvent (hydrotropes agents that are not a salt) enter the material.
  • the slices can be from about 0,1 cm to 1 cm.
  • the biological material is preferably plant biological material.
  • the plant biological material may be obtained from or obtainable from plant roots and/or plant aerial parts, such as the leaves, flowers, stems, barks, fruits and/or seeds, their tissues (such as the rind of the fruit) or mixtures thereof.
  • the plant biological material may be the leaves of the plant.
  • the plant biological material may be obtained from or obtainable from Lamiaceae (such as basil, mint, rosemary, sage, savory, marjoram, oregano, hyssop, thyme, lavender, perilla and mixtures thereof).
  • Lamiaceae such as basil, mint, rosemary, sage, savory, marjoram, oregano, hyssop, thyme, lavender, perilla and mixtures thereof.
  • Lamiaceae such as basil, mint, rosemary, sage, savory, marjoram, oregano, hyssop, thyme, lavender, perilla and mixtures thereof.
  • Lamiaceae such as basil, mint, rosemary, sage, savory, marjoram, oregano, hyssop, thyme, lavender, perilla and mixtures thereof.
  • the plant biological material may be rosemary and/or sage.
  • the plant biological material may be obtained from or obtainable from Curcuma (such as Curcuma longa) or Citrus genus (such as C. medica, C. reticulate , etc), Ginger (Zingiber officinale), Prunus genus (such as P. afrincana, P. armenicana, P. dulcis, P. avium, etc), (such as cherry flower) Gardenia jasminoides (such as gardenia fruits), Sellaginella genus, Olea europaea (such as Olive leaf), Equisetum, Crithmum (Sea fennel), Rose of Jericho, Saffron flower, etc.
  • Curcuma such as Curcuma longa
  • Citrus genus such as C. medica, C. reticulate , etc
  • Ginger Zingiber officinale
  • Prunus genus such as P. afrincana, P. armenicana, P. dulcis,
  • the biological extract obtained using the method of the invention may be enriched in carnosic acid and/or its derivatives and/or other lipophilic, hydrophobic, oil soluble and/or non-water-soluble compounds present in the biological material.
  • the biological extract may be enriched in carnosic acid, 12-methoxycarnosic acid, carnosol, rosmarinic acid, palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2-n6), linolenic acid (18:3-n3), oleanic acid, betulinic acid, ursolic acid, apigenin, luteolin glucuronide, luteolin-O-(O-acetyl) glucuronide isomer 1 , luteolin-O-(O-acetyl) glucuronide isomer 2, diosmetin, chlorophyll pigments, hinokione, rosmanol, epirosmanol, rosmadial, rosmaridiphenol, O- methylcarnosol, genkwanin, ladanein, cirsimaritin, artepillin, methyl-shogaol and
  • the extract when the biological extract is a liquid biological extract provided by a method or use as defined herein comprising steps (i) and (ii), the extract may comprise at least about 0.05 % by weight of the liquid extract of phenolic compounds, such as carnosic acid and/or derivatives thereof.
  • the biological extract is a crude solid or semi-solid biological extract provided by a method or use as defined herein comprising steps (i) to (v)
  • the extract may comprise at least about 2 % or about 5 % by weight of the dried solid or semi-solid extract of phenolic compounds, such as carnosic acid and/or derivatives thereof.
  • the biological extract is a purified biological extract provided by a method or use as defined herein comprising steps (i) to (vii)
  • the extract may comprise at least about 10 % by weight of the dried purified extract of carnosic acid and/or derivatives thereof.
  • the solvent is preferably a hydrotropic agent which is not a salt.
  • the solvent does not comprise sodium based hydrotropes.
  • the at least one hydrotropic agent in particular at least one hydrotropic agent which is not a salt, may be an alkyl glycoside, an alkyl glycerol ether, a dicarboxylic acid, nicotinamide, an alkyl polyethylene glycol ether of formula C.E j wherein / represents the number of carbon atoms of the alkyl chain length and j the number of ethylene oxides, or a mixture thereof.
  • Alkyl glycosides that may be used in the methods and uses described herein include, but are not limited to: amyl xyloside, heptyl glucoside and mixtures thereof.
  • Alkyl glycerol ethers that may be used in the methods and uses described herein include, but are not limited to: butyl glycerol ether, pentyl glycerol ether, isoamyl glycerol ether and mixtures thereof.
  • alkyl polyethylene glycol ethers of formula C j E j wherein
  • the alkyl group may be linear or branched, as for ⁇ 0 4 E 4 with an isobutyle chain or iC 5 E 2 with an isoamylic chain.
  • the alkyl group is linear.
  • the method of the invention may comprise or consist of:
  • plant biological material such as from a plant of the Lamiaceae species, i.e. rosemary and/or sage
  • extraction solution comprising water and at least one of citraconic acid, glutaric acid or a mixture
  • the present invention also provides biological extracts obtained by the above-mentioned methods and uses.
  • Dicarboxylic acids that may be used in the methods and uses described herein include, but are not limited to, citraconic acid, glutaric acid and mixtures thereof.
  • dicarboxylic acids that may be used in the methods and uses described herein include those comprising less than 6 carbon atoms.
  • dicarboxylic acids comprising 3 to 5 carbon atoms include those comprising 3 to 5 carbon atoms.
  • the present invention also provides the use of dicarboxylic acids (such as citraconic acid, glutaric acid and mixtures thereof) as a hydrotrope.
  • dicarboxylic acids such as citraconic acid, glutaric acid and mixtures thereof
  • the method of the invention may comprise or consist of:
  • the use of the invention may comprise the use of an extraction solution comprising water and at least one of citraconic acid, glutaric acid or a mixture thereof to provide a biological extract and/or lipophilic, hydrophobic, oil soluble and/or non-water-soluble compounds from plant biological material (such as from a plant of the Lamiaceae species, i.e. rosemary and/or sage), wherein the use comprises:
  • the present invention also provides biological extracts obtained by the above-mentioned methods and uses.
  • the concentration of the at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt, present in the extraction solution is at least the minimum hydrotropic concentration (MHC) of the at least one (non-salt) hydrotropic agent. For example, from about 1 to about 20 times, such as from about 2 to about 16 times or from about 4 to about 8 times the minimum hydrotropic concentration (MHC).
  • the at least one hydrotropic agent in particular at least one hydrotropic agent which is not a salt, may be present in the extraction solvent at a concentration of about 80 % or less by weight relative to the weight of the extraction solvent, for example, the hydrotropic agent may be present in the extraction solvent at a concentration of about 70 % of less or about 60 % or less by weight relative to the weight of the extraction solvent or about 50 % or less by weight relative to the weight of the extraction solvent, or about 40 % or less by weight relative to the weight of the extraction solvent or about 30 % or less by weight relative to the weight of the extraction solvent or about 20 % or less by weight relative to the weight of the extraction solvent.
  • the at least one hydrotropic agent in particular at least one hydrotropic agent which is not a salt, may be present in the extraction solvent at a concentration of from about 2 % or about 2.5 % to about 80 % by weight of the extraction solvent, such as from about 5 % to about 70 % by weight or from about 15 % to about 60 % by weight relative to the weight of the extraction solvent.
  • the at least one hydrotropic agent in particular at least one hydrotropic agent which is not a salt, may have a critical aggregation concentration (CAC) of at least 0.022 mol/L of water (i.e. 0.6 % w/v), or at least 0.16 mol/L of water (i.e. 3.6 % w/v).
  • CAC critical aggregation concentration
  • critical aggregation concentration means the concentration above which a hydrotrope starts self-aggregating or starts aggregating cooperatively with a solute.
  • the concentration of the at least one dicarboxylic acid may be from about 10 % to about 80 % by weight of the extraction solvent, such as from about 20 % or about 30 % or about 40 % or about 50 % to about 40 % or about 50 % or about 60 % or about 70 % by weight.
  • the biological material such as a plant of the Lamiaceae species, i.e. rosemary or sage
  • an extraction solvent comprising water and at least one alkyl glycoside and/or at least one alkyl glycerol ether (such as amyl xyloside, heptyl glucoside, butyl glycerol ether, pentyl glycerol ether and/or isoamyl glycerol ether)
  • the concentration of the at least one alkyl glucoside and/or at least one alkyl glycerol ether may be from about 10 % to about 40 % by weight of the extraction solvent, such as from about 15 % to about 30 % or from about 18 % to about 25 % by weight of the extraction solvent.
  • the biological material such as a plant of the Lamiaceae species, i.e. rosemary or sage
  • the concentration of the at least one alkyl polyethylene glycol ether may be from about 10 % to about 80 % by weight of the extraction solvent, such as from about 20 % or about 30 % or about 40 % or about 50 % to about 40 % or about 50 % or about 60 % or about 70 % by weight.
  • the biological material and the extraction solution comprising water and at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt
  • the biological material and the extraction solution comprising water and at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt
  • stirring such as magnetic or mechanical stirring.
  • Stirring may be conducted at any suitable revolution per minute (rpm), for example, the stirring may be done from about 1 rpm or about 10 rpm or about 50 rpm to about 500 rpm.
  • For mechanical stirring this may typically be done from about 1 rpm to 500 rpm, such as from about 10 rpm to about 200 rpm.
  • the biological material and the extraction solution comprising water and at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt, may be mixed at a temperature of from about 15 °C to about 100 °C, such as from about 20 °C to about 60 °C or from about 20 °C to about 80 °C or about 25 °C.
  • the biological material and the extraction solution comprising water and at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt, may be mixed at a pressure of from about 10 bar (1000 KPa) to about 1000 bar (100000 KPa) or from about 20 bar (2000 KPa) to about 100 bar (10000 KPa).
  • the biological material and the extraction solution comprising water and at least one hydrotropic agent may be mixed for a duration of from about 1 minute to about 5 hours, such as from about 2 hours, in particular at least one hydrotropic agent which is not a salt, from about 5 minutes to about 1 hour or from about 5 minutes to about 30 minutes.
  • the biological material and the extraction solution comprising water and at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt, may be mixed at a pH of less than 7, such as a pH of less than 5 or less than 3.
  • the pH may be from about 0 to about 7, or from about 1 to about 4, or from about 0 to about 3 or from about 1 to about 2.
  • a pH as defined above has an unexpected advantageous effect in that it allows for the extraction of specific lipophilic, hydrophobic, oil soluble and/or non-water- soluble compounds from biological material (i.e. the plant and/or algal and/or animal and/or prokaryotic biological material), in particular, plant biological material (such as phenolic compounds from plants of Lamiaceae species, i.e. rosemary and/or sage) that may otherwise be unstable at a pH outside of these ranges.
  • biological material i.e. the plant and/or algal and/or animal and/or prokaryotic biological material
  • plant biological material such as phenolic compounds from plants of Lamiaceae species, i.e. rosemary and/or sage
  • a pH within the range defined above allows for the extraction of the phenolic compound carnosic acid in preference to carnosol.
  • any solid biological material present in the solution obtained in step (i) may be removed by any means known in the art, for example by filtration, static or dynamic decantation, and/or centrifugation.
  • a method of the invention comprising steps (i) and (ii), may provide a liquid biological extract that is enriched in, for example comprises more than 0.05 % by weight of the extract, compounds that are not usually soluble in aqueous solutions as defined above.
  • the liquid biological extract may be enriched in, for example, comprise more than 0.05 % by weight of the extract, phenolic diterpenes, for example, carnosic acid and/or its derivatives and/or other lipophilic, hydrophobic, oil soluble and/or non-water-soluble compounds present in the biological material as defined previously.
  • the method of the invention may additionally comprise steps (iii) and (iv), wherein in step (iii) a flocculate and/or precipitate is obtained by adding water and/or cooling the solution obtained in step (ii) and in step (iv) the resulting solid material is collected.
  • a method of the invention comprising steps (i) to (iv) provides a crude solid biological extract. If required, the crude biological solid may be dried to reduce/remove any residual water present in the extract.
  • drying techniques such as, but not limited to, freeze-, spray-, oven-, heat- or vacuum-drying.
  • the present invention provides a method of providing a crude solid or semi solid biological extract comprising or consisting of:
  • the mixing may be at a pH less than 7, such as a pH of less than 5 or less than 3.
  • the pH may be from about 0 to about 7, or from about 1 to about 4, or from about 0 to about 3 or from about 1 to about 2;
  • step (iv) collecting the resulting solid material obtained in step (iii) from the solution; and v) optionally drying the solid material obtained in step (iv).
  • the present invention also provides the use of an extraction solution comprising water and at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt, to provide a crude solid or semi solid biological extract and/or lipophilic, hydrophobic, oil soluble and/or non-water-soluble compounds from biological material (such as plant biological material, algal biological material, animal biological material and/or prokaryotic biological material), wherein the use comprises:
  • the mixing may be at a pH less than 7, such as a pH of less than 5 or less than 3.
  • the pH may be from about 0 to about 7, or from about 1 to about 4, or from about 0 to about 3 or from about 1 to about 2;
  • step (iv) collecting the resulting solid material obtained in step (iii) from the solution; and v) optionally drying the solid material obtained in step (iv).
  • the biological material and at least one hydrotropic, in particular at least one hydrotropic agent which is not a salt, agent are as defined previously.
  • the water added in step (iii) reduces the concentration of the at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt, to near to or under the minimum hydrotropic concentration (MHC).
  • MHC minimum hydrotropic concentration
  • the amount of water required to achieve this will depend on the MHC of the at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt, present in the aqueous solution and the amount of at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt, present in the extraction solvent.
  • water may be added to dilute the concentration of the at least one hydrotrope, in particular at least one hydrotrope which is not a salt, by a factor of from about 2 to about 20, such as from about 3 to about 8 or from about 4 to about 6.
  • the MHC of the at least one hydrotrope in particular at least one hydrotrope which is not a salt
  • the concentration of the at least one hydrotrope, in particular at least one hydrotropic agent which is not a salt was 20% by weight of the extraction solvent.
  • Reducing the concentration of the at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt, to near to or under the MHC has the effect of causing any compounds present in the solution that are only soluble due to the presence of the at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt, to flocculate and/or precipitate from the solution. This solid material can then be collected.
  • the solid material may be collected using such techniques known in the art, such as filtration, static or dynamic decantation, and/or centrifugation.
  • step (v) the solid material may be dried using such techniques known in the art as previously defined.
  • the crude solid or semi-solid biological extract is enriched in, for example comprises about 2 % or more, about 4 % or more, about 5 % or more, 10 % or more, 20 % or more or 40 % or more by weight of the extract, compounds that are not usually soluble in aqueous solutions as defined above, i.e. compounds that are lipophilic, hydrophobic, oil soluble and/or non-water-soluble.
  • the biological material is plant material, such as from a plant of the Lamiaceae species, i.e.
  • the crude solid or semi-solid biological extract may be enriched in, for example comprise about 2 % or more, about 4 % or more, about 5 % or more phenolic diterpenes, for example, carnosic acid and/or its derivatives as defined above.
  • the crude solid or semi-solid biological extract may be enriched in, for example comprise about 2 % or more, about 4 % or more, about 5 % or more phenolic diterpenes, for example, carnosic acid and/or its derivatives as defined above.
  • compounds that are not usually soluble in aqueous solutions as defined above i.e. compounds that are lipophilic, hydrophobic, oil soluble and/or non- water-soluble.
  • step (iv) collecting the resulting solid material obtained in step (iii) from the solution; and v) optionally drying the solid material obtained in step (iv).
  • step (iii) water may be added to the solution obtained in step (ii) at any suitable speed.
  • water may be added at a speed of from about 0.01 mL/s (for example, dropwise) to about 5 mL/s.
  • step (iii) water may be added to the solution obtained in step (ii) at any temperature.
  • water may be added at a temperature of from about 15 °C to about 40 °C, such as from about 20 °C to about 30 °C.
  • the water may be added to the solution obtained in step (ii) with cooling.
  • water may be added to the solution obtained in step (ii) while the solution is being cooled to a temperature of from about 0°C or about 1°C to about 10°C, such as from about 2.5 °C to about 5 °C or water that has been pre-cooled to a temperature of from about 0 °C or about 1 °C to about 10 °C, such as from about 2.5 °C to about 5 °C may be used.
  • the solution may be stirred to induce flocculation/precipitation.
  • the solution may be stirred at any suitable speed.
  • the solution may be stirred at from about 130 rpm to about 500 rpm using a magnetic stirring or from about 1 rpm to about 500 rpm using a mechanical stirring.
  • step (iii) may be replaced with cooling the solution obtained in step (ii) causing solid material to precipitate (such as crystalize and separate from the solution.
  • the cooling of the solution obtained in step (ii) may be done at a temperature of from about 0 °C or about 1 °C to about 10 °C, such as from about 2.5 °C to about 5 °C.
  • the method of the invention may additionally comprise step (vi), wherein in step (vi) the crude solid biological extract is washed from about 1 to about 3 times or more, such as to about 10, or about 100 times, with water and the resulting solid material is collected.
  • a method of the invention comprising step (vi) provides a purified solid biological extract. If required, the purified biological solid may be dried to reduce/remove any residual water present in the extract.
  • drying techniques such as, but not limited to, freeze-, spray-, oven-, heat- or vacuum-drying.
  • the present invention provides a method of providing a purified solid biological extract comprising:
  • biological material such as plant biological material, algal biological material, animal biological material and/or prokaryotic biological material
  • an extraction solution comprising water and at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt
  • the mixing may be at a pH less than 7, such as a pH of less than 5 or less than 3.
  • the pH may be from about 0 to about 7, or from about 1 to about 4, or from about 0 to about 3 or from about 1 to about 2;
  • step (iii) collecting the solid material obtained in step (iii) from the solution;
  • step (iv) optionally drying the solid material obtained in step (iv);
  • step (iv) washing the solid obtained in step (iv) or (v) from about 1 to about 3 times with water and collecting the resulting solid material;
  • step (vi) optionally drying the solid material obtained in step (vi).
  • the present invention also provides the use of an extraction solution comprising water and at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt, to provide a purified solid biological extract and/or lipophilic, hydrophobic, oil soluble and/or non-water-soluble compounds from biological material (such as plant biological material, algal biological material, animal biological material and/or prokaryotic biological material), wherein the use comprises:
  • the mixing may be at a pH less than 7, such as a pH of less than 5 or less than 3.
  • the pH may be from about 0 to about 7, or from about 1 to about 4, or from about 0 to about 3 or from about 1 to about 2;
  • step (iv) collecting the resulting solid material obtained in step (iii) from the solution; and v) optionally drying the solid material obtained in step (iv);
  • step (iv) washing the solid obtained in step (iv) or (v) from about 1 to about 3 times with water and collecting the resulting solid material;
  • step (vi) optionally drying the solid material obtained in step (vi).
  • the biological material and at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt, are as defined previously.
  • the extract may comprise about 10 % or more, about 20 % or more, about 40 % or more, or about 60 % or more by weight of the dried purified extract of compounds that have limited solubility in aqueous solutions, i.e. compounds that are lipophilic, hydrophobic, oil soluble and/or non-water-soluble.
  • the extract may comprise about 10 % or more, about 20 % or more, about 40 % or more, or about 60 % or more by weight of the dried purified extract of compounds that have limited solubility in aqueous solutions, i.e. compounds that are lipophilic, hydrophobic, oil soluble and/or non-water-soluble.
  • the washing in step (vi) reduces the concentration of residual hydrotropic agent present in the extract.
  • the present inventors have found that the washing step (vi) does not reduce the concentration of lipophilic/non-water-soluble compounds present in the purified solid biological extract and may in fact result in an increase in the concentration of active compounds present and/or the extracts anti- microbial/anti-oxidant activity.
  • the biological material is plant material, such as from a plant of the Lamiaceae species, i.e.
  • rosemary and/or sage, and biological extract is enriched in carnosic acid and/or its derivatives, washing the extract does not significantly reduce the concentration of carnosic acid and/or its derivatives and/or other compounds of interest present within the purified extract, and may in fact result in an increase in the concentration of active compounds present and/or the extracts anti-microbial/anti-oxidant activity.
  • the biological extract obtained may comprise less than about 5 % by weight of the extract of the hydrotropic agent, in particular at least one hydrotropic agent which is not a salt, such as less than 2 % or less than 1 % or less than 0.1% of the hydrotropic agent, in particular at least one hydrotropic agent which is not a salt.
  • the biological extract obtained may be substantially free of hydrotropic agent.
  • the term“substantially free” means that the extract being described may contain small (for example up to 0.1 % or 0.01% by weight of the extract) of the hydrotropic agent, in particular at least one hydrotropic agent which is not a salt, provided that the presence of the hydrotropic agent, in particular at least one hydrotropic agent which is not a salt, does not affect the essential properties of the extract.
  • the method of the present invention may comprise or consist of:
  • the pH may be from about 0 to about 7, or from about 1 to about 4, or from about 0 to about 3 or from about 1 to about 2 ;
  • step (iv) collecting the resulting solid material obtained in step (iii) from the solution; and v) optionally drying the solid material obtained in step (iv)
  • step (iv) optionally washing the solid obtained in step (iv) or (v) from about 1 to about 3 times with water and collecting the resulting solid material;
  • step (vi) optionally drying the solid material obtained in step (vi).
  • the biological material i.e. plant and/or animal and/or prokaryotic biological material
  • the biological material may be dried and/ or ground e.g. into a powder, before being mixed with an extraction solution comprising water and at least one hydrotropic agent, in particular at least one hydrotropic agent which is not a salt.
  • the method of the invention may consist or consist essentially of the step described herein.
  • the present invention also provides a biological extract obtained using the method or uses described previously, which may be referred to hereafter as the“extract of the invention”.
  • the biological extract may be, for example, a liquid biological extract, a crude solid or semi-solid biological extract or a purified biological extract.
  • the biological extract may be enriched in compounds that have limited solubility in aqueous solutions, i.e. compounds that are lipophilic, hydrophobic, oil soluble and/or non-water-soluble.
  • the biological extract may comprise 0.05 % or more, 2 % or more or 10 % or more of compounds that have limited solubility in aqueous solutions, i.e. compounds that are lipophilic, hydrophobic, oil soluble and/or non-water-soluble.
  • the biological extract of the invention may comprise more than 15 %, or more than 20 %, or more than 40 % by weight of compounds that have limited solubility in aqueous solutions, i.e. compounds that are lipophilic, hydrophobic, oil soluble and/or non-water- soluble.
  • the biological extract of the invention may comprise less than about 5 % by weight of the extract of the extraction solvent, such as less than 2 % or less than 1 % or less than 0.1 % of the extraction solvent.
  • the biological extract may be substantially free of extraction solvent.
  • the biological extract of the invention may be used to provide phenolic compounds including phenolic acids, phenolic esters, phenolic diterpenes (such as carnosic acid and its derivatives), flavonoids, secoiridoids, curcuminoids, stilbenes and phenolic alcohols, alkaloids, lipids, phenylpropanoids, terpenoids including triterpenoids and carotenoids, and mixtures thereof, from the biological material (i.e. the plant and/or algal and/or animal and/or prokaryotic biological material).
  • the biological material i.e. the plant and/or algal and/or animal and/or prokaryotic biological material.
  • the biological extract may comprise one or more of the compounds listed above or mixtures thereof that may be used as natural biological flavourings and taste modifiers, fragrances, biocides, antimicrobials, proteins, enzymes, colourings, pigments, surfactants, antioxidants, vitamins, and/or bioactives of nutritional, cosmetic or pharmaceutical interest.
  • the biological extract may be high in compounds that provide anti-oxidant and/or anti-microbial activity (e.g. anti-bacterial activity).
  • the present invention provides a biological extract comprising antioxidants obtained from plant and/or algal and/or animal and/or prokaryotic biological material obtained by the methods described herein.
  • the present invention also provides the use of a biological extract obtained by the methods described herein as an anti-oxidant.
  • the anti-oxidant extract may be used in the compositions and/or products as described below.
  • the present invention also provides a biological extract comprising anti-microbial (e.g. anti-bacterial) compounds obtained from plant and/or algal and/or animal and/or prokaryotic biological material obtained by the methods described herein.
  • anti-microbial e.g. anti-bacterial
  • the present invention also provides the use of a biological extract obtained by the methods described herein as an anti-microbial (e.g. anti-bacterial).
  • an anti-microbial e.g. anti-bacterial
  • the anti-microbial extract e.g. anti-bacterial
  • the biological extract of the invention (which may be an anti-oxidant and/or anti-microbial (e.g. anti-bacterial)) may be used to provide a nutraceutical composition, a dietary or food product for humans or animals (such as functional food compositions, i.e. food, drink, feed or pet food or a food, drink, feed or pet food supplements), a nutritional supplement, a fragrance or flavouring, a pharmaceutical (pharmaceutical compositions or formulations), a veterinary composition, an oenological or a cosmetic formulation.
  • a nutraceutical composition e.e. food, drink, feed or pet food or a food, drink, feed or pet food supplements
  • a nutritional supplement i.e. food, drink, feed or pet food or a food, drink, feed or pet food supplements
  • a nutritional supplement i.e. food, drink, feed or pet food or a food, drink, feed or pet food supplements
  • a nutritional supplement i.e. food, drink, feed or pet food or a food, drink
  • the nutraceutical composition, dietary or food product for humans or animals may be administered orally or parenterally, or be for topical, rectal, nasal, auricular, vaginal and/or ocular application.
  • the present invention therefore provides a biological extract for use in nutraceutical compositions, dietary or food products for humans or animals (such as functional food compositions, i.e. food, drink, feed or pet food or a food, drink, feed or pet food supplements), nutritional supplements, fragrances or flavourings, pharmaceuticals (pharmaceutical compositions or formulations), veterinary compositions, oenological or cosmetic formulations.
  • the present invention also provides nutraceutical compositions, dietary or food products for humans or animals (such as functional food compositions, i.e. food, drink, feed or pet food or a food, drink, feed or pet food supplements), nutritional supplements, fragrances or flavourings, pharmaceuticals (pharmaceutical compositions or formulations), veterinary compositions, oenological or cosmetic formulations comprising the biological extract, and optionally one or more pharmaceutically/veterinary acceptable ingredients, such as excipients or carriers or (functional) food acceptable ingredients and mixtures thereof, as appropriate.
  • nutraceutical compositions i.e. food, drink, feed or pet food or a food, drink, feed or pet food supplements
  • pharmaceuticals pharmaceutical compositions or formulations
  • veterinary compositions oenological or cosmetic formulations comprising the biological extract
  • oenological or cosmetic formulations comprising the biological extract
  • pharmaceutically/veterinary acceptable ingredients such as excipients or carriers or (functional) food acceptable ingredients and mixtures thereof, as appropriate.
  • the biological extract may be combined with other biologically active compounds within a nutraceutical composition, a dietary or food product for humans or animals (such as a functional food composition, i.e. a food, a drink, a feed or pet food or a food, drink, feed or pet food supplement), a nutritional supplement, a fragrance or flavouring, a pharmaceutical (pharmaceutical composition or formulation), a veterinary composition, an oenological or cosmetic formulation.
  • references to pharmaceutically or veterinary acceptable excipients may refer to pharmaceutically or veterinary acceptable adjuvants, diluents and/or carriers as known to those skilled in the art.
  • Food acceptable ingredients include those known in the art (including those also referred to herein as pharmaceutically acceptable excipients) and can be natural or non-natural, i.e. their structure may occur in nature or not. In certain instances, they can originate from natural compounds and be modified before use (e.g. maltodextrin).
  • pharmaceutically or veterinary acceptable we mean that the additional components of the composition are generally safe, non-toxic, and neither biologically nor otherwise undesirable.
  • the additional components are generally sterile and pyrogen free.
  • Such components must be“acceptable” in the sense of being compatible with the extract of the invention and not deleterious to the recipients thereof.
  • pharmaceutically acceptable excipients includes any compound(s) used in forming a part of the formulation that is intended to act merely as an excipient, i.e. not intended to have biological activity itself.
  • extracts of the invention may be administered to a patient or subject (e.g. a human or animal patient or subject) by any suitable route, such as by the oral, rectal, nasal, pulmonary, buccal, sublingual, transdermal, intracisternal, intraperitoneal, or parenteral (including subcutaneous, intramuscular, intrathecal, intravenous and intradermal) route.
  • a patient or subject e.g. a human or animal patient or subject
  • any suitable route such as by the oral, rectal, nasal, pulmonary, buccal, sublingual, transdermal, intracisternal, intraperitoneal, or parenteral (including subcutaneous, intramuscular, intrathecal, intravenous and intradermal) route.
  • Extracts of the invention may be administered orally.
  • pharmaceutical or veterinary compositions according to the present invention may be specifically formulated for administration by the oral route.
  • compositions for oral administration include solid dosage forms such as hard or soft capsules, tablets, troches, dragees, pills, lozenges, powders and granules. Where appropriate, they can be prepared with coatings such as enteric coatings, or they can be formulated so as to provide controlled release of the active ingredient, such as sustained or prolonged release, according to methods well known in the art.
  • Liquid dosage forms for oral administration include solutions, emulsions, aqueous or oily suspensions, syrups and elixirs.
  • compositions e.g. pharmaceutical or veterinary or food compositions described herein, such as those intended for oral administration, may be prepared according to methods known to those skilled in the art, such as by mixing the components of the composition together.
  • compositions of the invention may contain one or more additional ingredients, such as food ingredients or pharmaceutical ingredients and excipients, such as sweetening agents, flavouring agents, colouring agents and preserving agents.
  • additional ingredients such as food ingredients or pharmaceutical ingredients and excipients, such as sweetening agents, flavouring agents, colouring agents and preserving agents.
  • the compositions of the invention may contain the active ingredient(s) in admixture with non-toxic pharmaceutically acceptable excipients (or ingredients) which are suitable for the manufacture of tablets.
  • excipients may, for example, be: inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, maltodextrin or alginic acid; binding agents, for example, starch, gelatine or acacia; or lubricating agents, for example magnesium stearate, stearic acid, talc and mixtures thereof.
  • inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate
  • granulating and disintegrating agents for example, corn starch, maltodextrin or alginic acid
  • binding agents for example, starch, gelatine or acacia
  • lubricating agents for example magnesium stearate, stearic acid, talc and mixtures thereof.
  • Solid compositions of the invention may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.
  • Liquid compositions of the invention may be contained within a capsule, which may be uncoated or coated as defined above.
  • Suitable pharmaceutical or veterinary carriers include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents.
  • solid carriers are lactose, terra alba, sucrose, cyclodextrin, maltodextrin, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, arabic gum, modified starch and lower alkyl ethers of cellulose, saccharose, silica and mixtures thereof.
  • liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water.
  • the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.
  • Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents.
  • solid carriers are lactose, terra alba, sucrose, cyclodextrin, maltodextrin, dextrin, talc, gelatine, agar, pectin, acacia, magnesium stearate, magnesium hydroxide; stearic acid, arabic gum, modified starch and lower alkyl ethers of cellulose, saccharose, silicon dioxide.
  • liquid carriers are syrup, vegetables oils, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water.
  • the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.
  • carrier may also refer to a natural product or a product originating from nature that has been transformed or modified so that it is distinct from the natural product from which it originated, such as maltodextrin.
  • extracts of the invention may be administered at varying doses (i.e. therapeutically effective doses, as administered to a patient in need thereof).
  • doses i.e. therapeutically effective doses, as administered to a patient in need thereof.
  • the skilled person will appreciate that the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to affect a therapeutic response in the mammal over a reasonable timeframe.
  • the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the age, condition, body weight, sex and response of the patient to be treated, and the stage/severity of the disease.
  • the pharmaceutical or veterinary or food compositions comprise an extract of the invention in a therapeutically effective amount.
  • the term “effective amount” is synonymous with “therapeutically effective amount”, “effective dose”, or “therapeutically effective dose” and when used in the present invention refers to the minimum dose of the extract of the invention necessary to achieve the desired therapeutic effect and includes a dose sufficient to reduce a symptom associated with inflammation. Effectiveness in treating the diseases or conditions described herein can be determined by observing an improvement in an individual based upon one or more clinical symptoms, and/or physiological indicators associated with the condition. An improvement in the diseases or conditions described herein also can be indicated by a reduced need for a concurrent therapy.
  • an effective amount of the extract of the invention will further depend upon factors, including, without limitation, the frequency of administration, the half-life of the extract of the invention, or any combination thereof.
  • the amount of the biological extract present in a nutraceutical composition, a dietary or food product for humans or animals will vary depending on the application.
  • the amount of biological extract present in a nutraceutical composition, a dietary or food product for humans or animals will be from about 0.0001 % to about 100 % by weight of the nutraceutical composition, dietary or food product for humans or animals (such as functional food compositions, i.e.
  • compositions of the invention may consist of or consist essentially of the extract of the invention, and optionally a carrier.
  • the diffusion of a molecule inside the plant requires both lipophilicity and mobility, the latter being directly related to the size of the molecule. This can be related to the structure and chemical composition of the cell barriers which are crossed during the diffusion. Indeed, during the diffusion across the phospholipid bilayer, the molecule crosses two types of environments: one mainly composed of the hydrophilic glycerol and phosphate functions and the other one composed of the hydrophobic alkyl chains of the phospholipids. Thus, very polar and apolar molecules require a lot of energy to cross the hydrophobic core and the hydrophilic layers respectively. A medium hydrophobicity and polarity is better suited to reduce the energy of the diffusion.
  • the molecule also needs to diffuse across the cell wall ( ⁇ 200 nm), mainly composed of polar cellulose fibers.
  • This layer is known to allow the diffusion of water, ions and water-soluble molecules such as plant hormones, but not proteins, hydrophobic molecules or particles with a diameter greater than 4 nm, which corresponds to the porosity of the matrix.
  • Vm molecular volume
  • CiEj hydrotropes
  • c) determine the optimum of efficiency for the hydrotropes by representing the isoconcentration curves as concentric circles with a center corresponding to the theoretical properties to maximize the efficiency of an hydrotrope, and the radius corresponding to a function of the concentration of extracted compound
  • the compound to be extracted is a lipophilic, hydrophobic, oil soluble and/or non-water-soluble compound.
  • the hydrotropic agent is in particular at least one hydrotropic agent which is not a salt (such as alkyl glycosides, alkyl glycerol ethers, alkyl polyethylene glycol ethers, dicarboxylic acids, nicotinamide or mixtures thereof)
  • the compound to be extracted is carnosic acid (CA) and the efficientcy is determined by the equation: (Equation 1)
  • Vm molecular volume (Vm)
  • FIG. 9b Influence of the number of washing steps on the composition of the rosemary extracts. Extractions were performed using a solution of amyl xyloside (C 5 Xyl) followed by a dilution with water by 4. Recovery of the precipitate was done by centrifugation.
  • C 5 Xyl amyl xyloside
  • FIG 10a Influence of the number of washing steps on the composition of the rosemary extracts. Extractions were performed using a solution of heptyl glucoside (C 7 GIU) followed by a dilution with water by 2.5. Recovery of the precipitate was done by centrifugation.
  • C 7 GIU heptyl glucoside
  • FIG. 10b Influence of the number of washing steps on the composition of the rosemary extracts. Extractions were performed using a solution of heptyl glucoside (C 7 GIU) followed by a dilution with water by 4. Recovery of the precipitate was done by centrifugation
  • Figure 15 Concentration of CA in the filtrate after 24 and 48 h of extraction from 1 g of whole leaves of rosemary in contact with 10 mL of solutions at 25 °C, at pH 2.
  • Figure 16. Thermal behavior of the solutions of amphiphiles without any solute (black bars) or in the presence of 2.5 g/L (dark grey) and after extraction, i.e. with CA and many other phytochemicals solubilized (light grey).
  • Figure 17 Evolution of the cloud point of an aqueous solution of 30 wt. % C ⁇ at pH 2 containing increasing amounts of camphor (C), carnosic acid (CA), rosmarinic acid (RA) and stearic acid (SA; not soluble above 10 g/L).
  • camphor C
  • CA carnosic acid
  • RA rosmarinic acid
  • SA stearic acid
  • FIG. 18 Kinetics of the extractions performed with aqueous solutions containing 30 wt. % of C 4 E I ( ⁇ ), C 4 E 2 ( ⁇ ) and C 4 E 3 ( ⁇ ) at 25 °C, pH 2 focused between 0 and 30.
  • the first step i.e. "washing step” is colored in dark grey
  • the second "diffusion” step in light grey
  • the third step i.e. the plateau, is in white.
  • FIG. 19 Analysis of the cross sections of rosemary before extraction (red background) and after 48 h of extraction with EtOH 96 %, C ⁇ , C 4 E 2 , C 4 E 3 . Trichomes are located inside the dotted lines. The width of the opening and the number of trichomes present in the opening have been measured by optical microscopy on 9 different leaves for each experiment.
  • Figure 20 Solubility of carnosic acid (CA) and ursolic acid (UA) in solutions of different glycosylated amphiphiles at 30 wt. % after stirring for 24 h at 25 °C, pH 2.
  • CA carnosic acid
  • U ursolic acid
  • Figure 21 Solubility curves of rosmarinic acid (RA) and ursolic acid (UA) in solutions of amyl xyloside (C 5 Xyl) at pH 2 and 25 °C.
  • Figure 24 Solubility curves of some fatty acids in solutions of caprylyl/capryl glycoside (C 8 -i 0 Gly) at pH 2 and 25 °C.
  • FIG. 25 (a) Concentration of CA extracted from whole rosemary leaves after 24 and 48 h of stirring by hydrotropic solutions at 30 wt. % and (b) kinetic rate between 1 and 8 h of extraction.
  • Figure 26 Effect of the pH of the extraction using C5Xyl as hydrotrope (30 wt. % in water) on the oxidative stability of carnosic acid. Insert: weight ratio beltween carnosic acid (CA) and carnosol, its oxidative product.
  • CA weight ratio beltween carnosic acid
  • Extractions were performed with Rosmarinus officinalis L. (rosemary) leaves harvested and dried in Morocco and Salvia officinalis (sage) leaves harvested and dried in eastern Europe. Different amphiphile molecules—whether hydrotropes or surfactants— were used.
  • Appyclean 6505 a concentrated solution of amyl xyloside (purity > 60 %), hereafter denoted“C 5 Xyl”, was purchased from Wheatoleo (ARD)
  • Sepiclear G7 a concentrated solution of heptyl glucoside (purity > 73 %), hereafter denoted“C 7 Glu” was purchased from Seppic
  • Appyclean 6781 a concentrated solution of caprylyl/capryl glycoside (purity > 60 %), hereafter denoted“C 8 -ioGly”, was purchased from Wheatoleo (ARD).
  • Simulsol SL4 a concentrated solution of butyl glucoside (purity: 62.7 wt. %), hereafter denoted “C4Glu”
  • Simulsol AS48 a concentrated solution of ethylhexyl polyglucoside (purity: 67.2 wt. %), hereafter denoted“bC8Glu”.
  • the commercially-available ethoxylated amphiphile Tween 80 (purity > 99 %) was purchased from Sigma Aldrich as well as citraconic acid (> 98 %), glutaric acid (99 %), and nicotinamide (> 98 %).
  • glycerol ethers amphiphiles which are not commercially available were used: (i) the 1-butyl glycerol ether, hereafter denoted [4.0.0], (ii) the 1-pentyl glycerol ether, hereafter denoted [5.0.0] and (iii) the isoamyl glycerol ether, hereafter denoted [ ⁇ 5.0.0].
  • the [4.0.0] was synthesized by a reaction between the corresponding alkyl glycidyl ether, i.e. butyl glycidyl ether and water according to the literature (R. Lebeuf, E. Illous, C. Dussenne, V. Molinier, E. Da Silva, M.
  • C E j amphiphiles Five alkyl polyethylene glycol ethers (C E j amphiphiles) were obtained from commercial sources: C 4 E ! (1-butoxy ethanol, purity > 99 %) and C 6 E 2 (diethylene glycol monohexyl ether, purity 95 %) were purchased from Sigma-Aldrich; C 4 E 2 (diethylene glycol monobutyl ether, purity > 99 %) from Acros Chemicals; C 4 E 3 (triethylene glycol monobutyl ether, purity > 99 %) and iC ⁇ (ethylene glycol monoisobutyl ether, purity > 99 %) from TCI.
  • CE j amphiphiles (C 5 E 2 , iC 5 E 2 , C 5 E 3 , C 6 E 3 , C 6 E 4 , C 8 E 3 , C 8 E 4 , C I0 E 4 and C I2 E 4 ) were synthesized with precursors purchased from Sigma-Aldrich (1-bromohexane, 1- bromooctane and 1-bromodecane), TCI (1-bromo-3-methylbutane), or Alfa Aesar (1- bromopentane, 1-bromododecane and polyethylene glycols). Their purity was assessed by NMR (3 97 wt. %) and GC analyses (> 99 %) and by comparing their cloud point with the reference value.
  • sodium xylene sulfonate (SXS) > 90 %), ethanol (96 %), propanol (> 99 %) and butanol (> 99 %) were supplied by Fluka, VWR, Alfa Aesar and TCI, respectively.
  • Hydrotrope amphiphiles, carnosic acid and its derivatives were all quantified by HPLC.
  • a Ci 8 column of type Uptisphere® 120 A from Interchim (particle size: 3pm; length: 150 mm) was used, preceded by a Ci 8 cartridge from Shimadzu.
  • Hydrotropes were separated from the rest of the solution by a 60:40 MeOH 1 (containing 0.1 % of TFA 2 ):water eluant whereas carnosic acid and its derivatives carnosol and 12-methyl carnosic acid were separated by a 80:20 MeOH (0.1 % TFA):water eluant.
  • Hydrotropes were detected by an ELSD detector and quantified thanks to a calibration curve which has previously been performed.
  • Carnosic acid and its derivatives were detected by a UV detector at 210 nm and quantified.
  • TFA Trifuoroacetic acid Hydrotropes and surfactants are both able to lower the surface tension between water and air.
  • the surface tension of three commercially-available glycosylated amphiphiles was measured with a K-100 tensiometer and the Wilhelmy rod method: C 5 Xyl, C 7 Glu, and C 8 -ioGly.
  • the surface tensions of the tested amphiphiles decrease until the“critical aggregation concentration” (CAC) of C 8 -ioGly, C 7 Glu and C 5 Xyl reaches 1.6 x 10 3 mol/L (i.e. 0.045 % w/v), 0.022 mol/L (i.e. 0.6 % w/v), 0.16 mol/L (i.e. 5.5 % w/v), resp.
  • CAC critical aggregation concentration
  • C 8.10 Gly has the best surface activity: its CAC is 10 times smaller than that of C 7 Glu and 100 times smaller than that of C 5 Xyl.
  • Amphiphile molecules such as C 8.10 Gly which have long alkyl chains and are able to self-aggregate in water to form micelles, are called “surfactants”. Amphiphile molecules with shorter alkyl chains such as C 7 Glu and C 5 Xyl are less surface active and are called“hydrotropes”.
  • the minimum hydrotropic concentration (MHC) of Tween 80, C 8.i0 Gly, C 7 GIU, C 5 Xyl and [4.0.0] solutions is 0.01 , 0.4, 2.8, 9, 14 % resp. for carnosic acid ( Figure 2).
  • Solubility of carnosic acid increases until it reaches values (S max ) of 14, 15, and 16 g/L resp. at Cmax for C 5 Xyl (30 %), Tween 80 (10 %), and C 7 Glu (20 %). Above the C max of amphiphiles, the solubility of carnosic acid reaches a plateau or even decreases. For C 8.10 Gly, the solubility of carnosic acid increases until 9 g/L at 4 % of amphiphile.
  • Alkyl polyethylene glycol ethers (C E j ) can be categorized as hydrotropes because of their capacity to self-assemble in water like surfactants, at higher concentration, but without forming well-defined aggregates such as micelles.
  • CiEj can exhibit a cloud point at temperature below 100 °C.
  • the temperature of liquid-liquid phase separation at which the cloud point is the lowest is called LCST (i.e. lower critical solution temperature). Salts can increase (salting in) or decrease (salting out) the cloud point, while hydrophobic solutes such as alkanes and long chain alcohols usually lower it.
  • Table 1 summarizes the main physicochemical properties of well-defined CiEj with i ranging from 4 to 12 and j from 1 to 4.
  • the long chain C 10 E 4 and C 12 E 4 are considered as true surfactants and have been included for comparison, as well as sodium xylene sulfonate (SXS) which is an archetypical anionic hydrotrope.
  • SXS sodium xylene sulfonate
  • log P and molecular volumes (Vm) were calculated with the CosmoQuick software. HLB were calculated according to Griffin’s equation.
  • C 4 E 2 lowers the surface tension of water to 28 mN/m compared to SXS which only reaches 52 mN/m (Table 1).
  • each CiE j was fixed at 30 wt. %, which is well above their CAC (Table 1).
  • CA is readily soluble in almost all hydrotropic systems leading to homogeneous single-phase solutions at 25 °C, except for iC 4 E 1 , C 6 E 2 and C 8 E 3 solutions, for which a two-phase system is obtained at 25, 50 and 75 °C due to a lower cloud point ( Figure 4).
  • iC 4 E 1 , C 6 E 2 and C 8 E 3 solutions for which a two-phase system is obtained at 25, 50 and 75 °C due to a lower cloud point ( Figure 4).
  • CA is solubilized in the upper CiEj-rich phase.
  • all CiE j form a single phase.
  • C I2 E 4 surfactant
  • the aqueous solution at 25 °C is very viscous due to the formation of liquid crystals. Indeed, at such temperature and high concentration, this surfactant forms a lamellar phase.
  • C I0 E 4 no liquid crystal was formed at room temperature but a lamellar phase appeared just above the CP (> 34 °C).
  • the short alkyl chain C,E j hydrotropes (i £ 8) do not form liquid crystals at 30 wt. % whatever the temperature.
  • the MHC of C 4 E 2 is 10 wt. % and a solution of 30 wt. % C 4 E 2 can solubilize more than 25 g/L of CA, which is more than ten times the average maximal amount of CA that can be extracted from rosemary.
  • amphiphiles presented in Examples 1 and 2 can be classified into two groups:
  • the required dilution factor to reach the MHC depends on the amphiphile. More precisely, it depends on two parameters: the hydrotrope concentration used to perform the extraction and its MHC. They are reported in Table 2. For the same theoretical carnosic acid concentration solubilized (i.e. 8 g/L, see Example 2), the required dilution factor for a solution of C 5 Xyl is only 2.5, whereas solutions of C 7 Glu and surfactants must be diluted with a higher amount of water (Table 2). For example, a Tween 80 solution requires a dilution factor of 300, which is technically and economically difficult to achieve, if not impossible, at an industrial level. Even at the laboratory scale, using Tween 80 as hydrotrope extractant imply that 21 L of water is used to dilute 70 ml_ of the recovered enriched solution.
  • hydrotropes present three advantages compared to surfactants: (i) they are more efficient to extract lipophilic, hydrophobic, oil soluble and/or non-water-soluble compounds, such as carnosic acid from biological material, such as rosemary (ii), they are fully compatible with classical precipitation techniques to recover the extracts, (iii) and they use much less amounts of water. Table 2. Conditions and results of extraction, dilution and recovery of the crude extract by filtration.
  • hydrotrope-containing extracts unwashed extracts
  • hydrotrope-free extracts refined extracts
  • the concentration of carnosic acid increased from 12.4 to 20.7 % (dilution by 2.5) or from 7.9 to 19.5 % (dilution by 4).
  • a homogeneous aqueous solution of 7 % of C 7 Glu was prepared. Ground rosemary leaves (10 g) were extracted with 100 ml_ of this solution for 30 min, and then, separated from the enriched solution by filtration. The extract was recovered by precipitation following the addition of water until a concentration of hydrotrope is reached below the MHC (2.8 %). This dark and sticky precipitate (i.e. the crude extract) was recovered by centrifugation, then freeze-dried and weighed.
  • the enriched solution was diluted with water by 2.5 and 860 mg of extract were recovered corresponding to a mass yield of 8.6 % (Fig 10a).
  • the enriched solution was diluted four times with water and only 210 mg of extract were obtained, corresponding to a mass yield of 2.1 % (Fig 10b).
  • the resulting extracts were analyzed and residual hydrotrope (resp. 79 and 18 % after dilutions by 2.5 and 4) was found.
  • Three successive washings by water were performed resulting in a drastic depletion of the hydrotrope from the extract. For instance, after three washing steps (dilution by 2.5), the hydrotrope proportion in the extract was reduced from 79 to 19 %.
  • a dilution factor of 4 was applied for the precipitation step, a total elimination of hydrotrope from the final extract was obtained after two washing steps.
  • the obtained hydrotrope- free extract appears as a light green powder with a fluid rheology. The total mass of the precipitate decreased with the number of washings due to the elimination of water-soluble“impurities”.
  • a homogeneous aqueous solution of C 5 Xyl (22.5 %) was prepared. 10 g of ground rosemary leaves were incubated at ambient temperature with 100 ml_ of this solution for 30 min with a 250 rpm-magnetic stirring and the enriched solution was separated from the plant parts by filtration. The filtrate was diluted with water by a factor 2.5. The resulting precipitate was magnetically stirred for 30 min and left to settle for another 30 min. The precipitate was recovered by filtration, then washed three times with water. Finally, 263 mg of a light green powder were collected (mass yield: 2.63 %).
  • the final extract contained 26 % of carnosic acid (HPLC quantification), and its derivatives, carnosol and methyl carnosic acid, were found at 5 and 12 % resp. in the refined extracts.
  • the extract was analyzed by 1 H NMR to make sure that the hydrotrope was totally eliminated by the washing procedure. Indeed, the protons of secondary hydroxyl groups (between 5.1 and 2.8 ppm) of the xylose of C 5 Xyl (Fig 11 (a)) are absent of the extract 1 H NMR spectrum (Fig 9.b.). Therefore, it can be considered that the hydrotrope had successfully been eliminated.
  • a second homogeneous aqueous solution of C 5 Xyl (30 %) was prepared. 10g of ground rosemary leaves were incubated at ambient temperature with 100 ml_ of this solution for 30 min at a 50 rpm (magnetic stirring) and the enriched solution was separated from the plant parts by filtration. The filtrate was diluted with water by a factor 4. The water was added slowly, i.e. at 0.1 mL/sec (dropwise). The resulting precipitate was magnetically stirred for 1 h and left to settle for another 1 h. The precipitate was recovered by centrifugation, then washed three times with water. Finally, 446 mg of a light green powder were collected (mass yield: 4.46 %). The final extract contained 24.6 % of carnosic acid (HPLC quantification), and its derivatives, carnosol and methyl carnosic acid, were found at 3 and 9 % resp. in the refined extracts.
  • a homogeneous aqueous solution of C 7 Glu (11.6 %) was prepared. Ten grams of ground rosemary leaves were incubated at ambient temperature with 100 ml_ of this solution for 30 min and the enriched solution was separated from the plant by filtration. The filtrate was diluted by water by a factor 4. The resulting precipitate was magnetically stirred for 30 minutes and let for settling for other 30 minutes. The precipitate was recovered by filtration, and then washed three times with water. Finally, 81 mg of a light green powder were collected (mass yield: 0.81 %). The final extract contained 16.4 % of carnosic acid and only 1 % of hydrotrope (HPLC quantification).
  • Hydrotropic extracts of rosemary obtained in examples 11 and 12 were assessed for their antioxidant activity in lipid matrices (sunflower oil fatty acid methyl esters, 2 mL) using the RapidOxy method at 110 °C and with a 300 kPa surpressure.
  • the antioxidant molecules oxidize during an induction period during which no significant oxidation of the oil occurs. At the end of the induction period, the oxidation of the oil exponentially increases because all antioxidants are consumed and therefore the oil is not protected.
  • An extract with acetone was also tested in comparison. This extract was obtained by extraction of 10 g of rosemary in 100 ml_ of acetone, and evaporation of acetone under vacuum in a rotative evaporator.
  • Hydrotropic extracts of rosemary obtained in examples 11 and 12 were assessed for their antioxidant activity in lipid matrices (sunflower oil) using the Rancimat method at 110 °C and with an air flow of 10 L/h.
  • the same concentration of extracts as those tested in the RapidOxy assay were tested in the Rancimat assay (i.e. 1000 ppm, 0.1 % w/v).
  • the antioxidant molecules oxidize in Rancimat apparatus during an induction period during which no significant oxidation of the oil occurs. At the end of the induction period, the oxidation of the oil exponentially increases because all antioxidants are consumed and therefore the oil is not protected. Each experiment was repeated three times independently.
  • the varying parameters were the stirring speed (50 or 500 rpm), the extraction time (15 or 30 min), the hydrotrope concentration (15 or 30 %) and the extraction temperature (25 or 60 °C), while the extraction performance was estimated through three responses: the carnosic acid concentration in the enriched solution and the dried final extract as well as the final mass yield (after precipitation).
  • the conditions of the precipitation and recovery of the extracts did not vary and were as follow: dilution with water to reach the MHC of C 5 Xyl (9 %), then magnetic stirring for 10 min at 350 rpm, followed by filtration on cellulose filter (2-3 pm) and lyophilization of the retentate.
  • Example 16 Feasibility of the precipitation and recovery processes using C 5 Xyl Table 6. Various feasible procedures of hydrotropic precipitation and extract recovery using C 5 Xyl.
  • the carnosic acid concentration in the residual water does not exceed 3 % of available carnosic acid and is somewhat similar (negligible) in all experiments (Table 6).
  • Table 6 shows values ranging from 4.2 to 7.9 % and from 3.2 to 17.4 %, respectively for the mass yield and the carnosic acid concentration in the final dried extract.
  • an extract with a significant concentration of carnosic acid has been obtained demonstrating that the process of the invention covers all the possible combinations/variations of the precipitation and recovery parameters.
  • Example 18 Extraction of rosemary without grinding with solution of 1 -butyl glycerol ether [4.0.0], 1-iso amyl glycerol ether [ ⁇ 5.0.0], and 1 -pentyl glycerol ether
  • Example 19 Extraction of rosemary with a solution of 1 -butyl glycerol ether [4.0.0], precipitation, and recovery of the crude extract by filtration
  • a homogeneous aqueous solution of 1-butyl glycerol ether (30 %) was prepared.
  • Ten grams of ground rosemary leaves (containing 2.72 % carnosic acid) were incubated at ambient temperature with 80 mL of this solution for 30 min and the enriched solution was separated from the plant by filtration.
  • the filtrate was analyzed by HPLC and the concentration of carnosic acid was found to be 2.9 g/L, which corresponds to a recovery rate of 85 %. This is surprisingly high, especially for a one-pass process.
  • Example 20 Extraction of sage with a solution of C 5 Xyl, precipitation, and recovery of the crude extract by filtration
  • a homogeneous aqueous solution of C 5 Xyl (22.5 %) was prepared.
  • Ten grams of common sage leaves (containing 1.85 % carnosic acid) were incubated at ambient temperature with 100 ml_ of this solution for 30 min and the enriched solution was separated from the plant by filtration.
  • the filtrate was analyzed by HPLC and the concentration of carnosic acid was found to be 1.3 g/L, which corresponds to a recovery rate of 57.5 %.
  • the filtrate was diluted by a factor 3.
  • the resulting precipitate was magnetically stirred for 30 min and let for settling for other 30 min.
  • the precipitate was recovered by filtration and freeze-dried.
  • 432 mg of a black pasty precipitate was collected (mass yield: 4.3 %), containing 20.4 % of carnosic acid (HPLC quantification), which corresponds to a final recovery rate of 47.7 %.
  • Example 21 60 °C-extraction of rosemary with a solution of citraconic acid, precipitation, and recovery of the crude extract by centrifugation
  • a homogeneous aqueous solution of a natural compound, citraconic acid was prepared at 70 %. Twenty grams of ground rosemary leaves (containing 3.17 % carnosic acid) were incubated at 60 °C with 200 mL of this solution for 30 min and the enriched solution was separated from the plant by centrifugation at 4200 rpm for 15 min. The supernatant was analyzed by HPLC and the concentration of carnosic acid was found to be 1.94 g/L, which corresponds to a recovery rate of 35.15 %. The supernatant was diluted by a factor of 14. The resulting precipitate was magnetically stirred for 15 min and let for settling for other 15 min.
  • Example 22 Room temperature-extraction of rosemary with a solution of citraconic acid, precipitation, recovery of the crude extract by centrifugation and washing of the crude extract
  • a homogeneous aqueous solution of a natural compound, citraconic acid was prepared at 70 %. Twenty grams of ground rosemary leaves (containing 3.17 % carnosic acid) were incubated at ambient temperature with 200 mL of this solution for 30 min and the enriched solution was separated from the plant by centrifugation at 4200 rpm for 15 min. The supernatant was analyzed by HPLC and the concentration of carnosic acid was found to be 2.01 g/L, which corresponds to a recovery rate of 36.09 %. The supernatant was diluted by a factor of 14. The resulting precipitate was magnetically stirred for 15 min and let for settling for other 15 min.
  • Example 23 Room temperature-extraction of rosemary with a solution of nicotinamide, precipitation, and recovery of the crude extract by centrifugation
  • a homogeneous aqueous solution of a natural compound, nicotinamide, was prepared at 40 %. Twenty grams of ground rosemary leaves (containing 3.17 % carnosic acid) were incubated at ambient temperature with 200 mL of this solution for 30 min and the enriched solution was separated from the plant by centrifugation at 4200 rpm for 15 min. The supernatant was analyzed by HPLC and the concentration of carnosic acid was found to be 0.53 g/L, which corresponds to a recovery rate of 9.90 %. The supernatant was diluted by a factor of 5. The resulting precipitate was magnetically stirred for 15 min and let for settling for other 15 min.
  • Example 24 Solubilization of hesperidin using an aqueous solution containing 40 % by weight nicotinamide
  • Example 25 Solubilization of curcumin using aqueous solution containing from 60 to 80 % by weight citraconic acid
  • curcumin was assessed in various aqueous solutions containing 60, 70, and 80 % citraconic acid in a similar manner to carnosic acid in Example 2. It was found to be 0.31 , 0.64, and 1.51 g/L, respectively.
  • Example 26 Solubilization of curcumin using an aqueous solution containing 60 % by weight C 5 Xyl
  • Example 27 Extraction of rosemary without grinding with aqueous solution of 30 % alkyl polyethylene glycol ethers (C E j )
  • Homogeneous aqueous solutions of alkyl polyethylene glycol ethers (CjE j ) were prepared for each of the fourteen C E j listed in Table 1. Their amphiphile concentration was set at 30 wt. % each (i.e. well above their CAC, Table 1). For comparison, 96 % ethanol, 100 % butanol as well as four aqueous solutions (30 % butanol; 30 % propanol; 30 % ethanol; and 30 % SXS) were also prepared. The aqueous solutions were acidified with 1 wt. % of phosphoric acid (i.e. pH around 2) to inhibit the oxidative degradation of CA into carnosol.
  • phosphoric acid i.e. pH around 2
  • SXS which is one of the first hydrotropes used for plant extraction, is not efficient at this concentration and pH compared to short CiE due to the low solubility of CA ( ⁇ to 0.8 g/L) in the SXS solution ( Figure 15).
  • CiE j Comparing CiE j between them, we can see that for a same alkyl chain length /, a decrease of the level of extracted CA is observed when the ethylene oxide number j increases, e.g. C 4 E I > C 4 E 2 > C 4 E 3 ; C 5 E 2 > C 5 E 3 ; C 6 E 2 > C 6 E 3 > C 6 E 4 , and C 8 E 3 > C 8 E 4 .
  • j is kept constant, a non-linear trend with the extraction efficiency is observed when increasing the alkyl chain length for the C,E 2 and C E 3 series. Indeed, they reach a maximum of efficiency for C 5 and C 6 respectively.
  • Example 28 Effect of solubilized carnosic acid and extracted phytochemicals from rosemary on the cloud point of aqueous solution of 30 % alkyl polyethylene glycol ethers (C E j )
  • the cloud point (CP) of hydrotropes is an important parameter for extraction of natural substances. For example, reaching the CP post-extraction can advantageously concentrate CA in a smaller phase which can constitute a unique purification procedure. Therefore, we measured the effect of solute concentration on CP.
  • Example 29 Kinetics parameters of the extraction of rosemary without grinding with aqueous solution of 30 % alkyl polyethylene glycol ethers (C E j )
  • the extraction process can be divided into three steps. First, a washing step takes place in the first minutes of extraction and corresponds to the solubilization of the most accessible CA in the rosemary leaves. Interestingly, the nature of the C j E j has no influence during this step and « 0.12 g/L of CA are extracted in 30 min. Then, there is a diffusion step of several hours during which CA diffuses from the inside of the plant to the solution, helped by the amphiphile which diffused into the interior of the plant and solubilized CA. Finally, until completion of extraction, the amount of extracted CA increases very slowly until reaching a plateau.
  • Example 30 Mode of action of alkyl polyethylene glycol ethers (C B j ) in the extraction of carnosic acid from rosemary
  • Trichomes secretory hairs which contain CA
  • Trichomes are cut/degraded after the extractions performed with the different CiE j .
  • trichomes have been counted by observing the opening and their density decreases after a treatment with hydrotropes, whereas it seems unchanged after a treatment with EtOH 96 % at ambient temperature.
  • CA should be more released into the hydrotropic solution.
  • the width of the opening on the lower side of leaves was measured and was found larger after treatment with CiE j but unchanged with EtOH. This suggests that hydrotropes penetrate inside the leaves and interact with the internal molecules of the cell wall or membrane.
  • EtOH can extract CA but does not de-structure the leaves, so it is able to diffuse through the barrier without destabilizing them.
  • Small hydrophilic head amphiphiles seem to have a hybrid behavior combining extraction mechanisms of alcohols with that of other CiE j .
  • C ⁇ degrade trichomes as the other C,E j do but also penetrate inside the plant matrix to extract CA as in the case of EtOH because even if the deterioration of the plant is similar to the other hydrotropes, the extraction is much quicker.
  • CA must diffuse across the barriers by another mechanism, with an efficiency similar to that of alcohols.
  • Example 31 Extraction of rosemary without grinding with a solution of 0 4 E 1 precipitation, and recovery of the crude extract by filtration
  • a homogeneous aqueous solution of the hydrotrope C ⁇ (40 %) was prepared. Two grams of whole rosemary leaves were incubated at room temperature and under magnetic stirring with 20 ml_ of this solution for 5 hours. The enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the CA recovery rate was found to be 24 %.
  • Example 32 A comparison of the solubility of carnosic, rosmarinic, and ursolic acids in aqueous solution of glycosylated amphiphiles
  • C5Xyl amyl xyloside
  • C7Glu heptyl glucoside
  • Example 33 A comparison of the solubility of alkyl gallate esters in aqueous solutions of C5Xyl
  • Figure 22 shows that C5Xyl can solubilize hydrophobic phenolic compounds such as gallate alkyl esters with a chain length ranging from 3 to 16, which correspond to log P ranging from 1.7 to 8.3. Therefore, the solubilization limit of C5Xyl is situated beyond log P 8.3, which is considerably hydrophobic.
  • hydrophobic phenolic compounds such as gallate alkyl esters with a chain length ranging from 3 to 16, which correspond to log P ranging from 1.7 to 8.3. Therefore, the solubilization limit of C5Xyl is situated beyond log P 8.3, which is considerably hydrophobic.
  • Example 34 A comparison of the solubility of fatty acids in aqueous solutions of C5Xyl
  • Figure 23 shows that C5Xyl can solubilize fatty acids containing 14 and 18 carbon atoms in their alkyl chain, which correspond to log P of 5.8 and 7.8, respectively. Furthermore, C5Xyl was unable to solubilize behenic acid with a very long chain length of 22 carbon atoms, corresponding to a log P of 9.9. Therefore, in this example, the solubilization limit of C5Xyl is situated between log P 7.8 and 9.9, which is in accordance with Example 33 obtained on alkyl gallate solutes and demonstrating that this limit is beyond log P 8.3. Even more precise is the limit obtained in Example 32, which is somewhere below log P 8.7. In conclusion, this limit should be located at log P of 8.5 for C5Xyl.
  • hydrotropic solutions of surfactant C 8/i oGly containing 20 g/L of solute were prepared as described above for hydrotrope C5Xyl. Solutions were stirred with a rotary agitator for 24 h. The solid phase was separated from the solution by a syringe filter (PTFE, 0.2 pm) and the solute was titrated in the filtrate by HPLC.
  • Figure 24 shows that, similarly to C5Xyl, the surfactant C 8/i oGly can solubilize fatty acids containing 14 and 18 carbon atoms in their alkyl chain, which correspond to log P of 5.8 and 7.8, respectively.
  • the tested surfactant was unable to solubilize substancial amounts of behenic acid with a very long chain length of 22 carbon atoms, corresponding to a log P of 9.9. Therefore, the solubilization limit of a surfactant such as C 8/10 Gly is relatively close to that of a hydrotrope such as C5Xyl.
  • Example 35 Extraction of rosemary without grinding with aqueous solutions of glycosylated amphiphiles at 30 wt. %.
  • Example 36 The pH-induced oxidative degradation of carnosic acid into carnosol during the extraction from rosemary leaves using C5Xyl as hydrotrope.
  • carnosic acid as an example of phenolic compounds.
  • Figure 26 shows that carnosic acid is oxidatively stable during extraction performed using C5Xyl as hydrotrope (30 wt. % in water) at pH below 3.5 approximately.
  • a signification oxidation of carnosic acid into its oxidation product, i.e. carnosol— which is much less antioxidant— occurs when the pH during the extraction procedure is increased at 5.5. Beyond the neutrality, this oxidation is complete and extraction of carnosic acid cannot be performed.
  • the extraction thus should be done at pH below 5, preferably below 3.5 and more preferably below 2.
  • the diffusion of a molecule inside the plant requires both lipophilicity and mobility, the latter being directly related to the size of the molecule. This can be related to the structure and chemical composition of the cell barriers which are crossed during the diffusion. Indeed, during the diffusion across the phospholipid bilayer, the molecule crosses two types of environments: one mainly composed of the hydrophilic glycerol and phosphate functions and the other one composed of the hydrophobic alkyl chains of the phospholipids. Thus, very polar and apolar molecules require a lot of energy to cross the hydrophobic core and the hydrophilic layers respectively. A medium hydrophobicity and polarity is better suited to reduce the energy of the diffusion.
  • the molecule also needs to diffuse across the cell wall ( ⁇ 200 nm), mainly composed of polar cellulose fibers.
  • This layer is known to allow the diffusion of water, ions and water-soluble molecules such as plant hormones, but not proteins, hydrophobic molecules or particles with a diameter greater than 4 nm, which corresponds to the porosity of the matrix.
  • the best multiple linear regression corresponding to this set of experiments is:
  • V m the molecular volume
  • log P the partition coefficient between octanol and water.
  • Example 39 Prediction equations of the ability of all studied hydrotropes/surfactants (30 wt. % in water) to extract carnosic acid from whole rosemary at room temperature during 48 hours - a mathematical model using the number of carbon atoms, the mass of the hydrophilic part, the molecular mass, and the CAC
  • the same set of 23 experiments is used as in Example 38, but the model now is calculated from all the 23 experiments, without test group.
  • Wth i number of carbons in alkyl chain
  • M hydro molar mass of the hydrophilic part (g/mol)
  • M molar mass of the molecule (g/mol)
  • CAC Critical Aggregation Concentration (g/L).

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Abstract

La présente invention concerne des procédés de préparation d'extraits biologiques faisant appel à des agents hydrotropes, des procédés de purification d'extraits biologiques formés à l'aide d'agents hydrotropes et l'utilisation des extraits biologiques, tels que dans des produits alimentaires, des produits pharmaceutiques, des arômes, des parfums, des produits cosmétiques, des nutraceutiques et des compléments, tels que des compléments alimentaires et des compléments de sport.
PCT/EP2020/067101 2019-06-20 2020-06-19 Extraction hydrotropique Ceased WO2020254577A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112409440A (zh) * 2021-01-04 2021-02-26 湖南德诺健康管理集团有限公司 一种以迷迭香油膏为原料生产熊果酸乳液的方法

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