EUTECTIC EXTRACTION OF SOLIDS

Abstract

The present in relates to methods and uses for preparing biological extracts using Deep Eutectic Solvents (DES) as hydrotropic agents, methods for purifying biological extracts formed using Deep Eutectic Solvents (DES) as hydrotropic agents, the biological extractions obtained using the methods and uses and the use of the biological extracts, such as in food-stuffs, flavours and fragrances, pharmaceuticals, cosmetics, nutraceuticals and supplements, such as food supplements and sports supplements.

Claims

1. A method for providing a solid biological extract comprising: i) mixing biological material with an extraction solution comprising water and a Deep Eutectic Solvent (DES); ii) removing any undissolved biological material from the solution obtained in (i); iii) obtaining a flocculate and/or precipitate by adding water to and/or cooling the solution obtained in step (ii); iv) collecting the resulting solid material obtained in step (iii) from the solution; and v) optionally drying the solid material obtained in step (iv).

2. The method for providing the solid biological extract of claim 1: wherein the extraction solution of step i) is free of water.

3. The method according to claim 1, wherein the solid biological extract comprises at least 2% by weight of lipophilic, hydrophobic, oil soluble and/or non-water-soluble compounds.

4. The method according to claim 3, wherein the lipophilic, hydrophobic, oil soluble and/or non-water-soluble compounds comprise one or more of phenolic compounds including phenolic acids, phenolic esters, phenolic diterpenes, flavonoids, secoiridoids, curcuminoids, bixin, capsaicinoids, cannabinoids, pyranoanthocyanins, stilbenes, phenolic alcohols, phenolic lipids, sylimarins, alkaloids, lipids, phenylpropanoids, coumarin, organic acids, terpenoids including monoterpenoids, sesquiterpenoids, diterpenoids, saponins, lignans, anthraquinone, glucosinolates, sulforaphane and isothiocyanates, triterpenoids, sapogenins or carotenoids, and mixtures thereof, from the biological material.

5. The method according to claim 1, wherein the solid biological extract comprises at least 2% by weight carnosic acid and/or its derivatives and/or other lipophilic, hydrophobic, oil soluble and/or non-water-soluble compounds present in the biological material.

6. (canceled)

7. (canceled)

8. The method according to claim 1, wherein the biological material is a plant biological material.

9. The method according to claim 8, wherein the plant biological material is obtained or obtainable from the plant roots, the aerial parts of a plant or a mixture thereof.

10. The method according to claim 8, wherein the plant biological material is obtained from or obtainable from at least one plant of the Lamiaceae species.

11. (canceled)

12. (canceled)

13. The method according to claim 1, wherein the DES is obtained by combining at least two compounds selected from methylamines, organic acids, sugars, polyols, amino acids, and urea.

14. The method according to claim 13, wherein: a. the methylamines are selected from N-trimethylamine oxide (TMAO), betaine, glycerophosphocholine, carnitine, homarine, choline chloride, and methyl sulfonium solutes including dimethylsulfonopropionate (DMSP) and derivatives thereof, for example, their halide forms; b. the organic acids are selected from levulinic acid, lactic acid, malic acid, maleic acid, pyruvic acid, fumaric acid, succinic acid, citric acid, citraconic acid, glutaric acid, glycolic acid, acetic acid, aconitic acid, tartaric acid, ascorbic acid, malonic acid, oxalic acid, glucuronic acid, neuraminic acid, sialic acid, shikimic acid, phytic acid, galacturonic acid, iduronic acid, hyaluronic acid, hydroxycitric acid, lactone derivatives and derivatives thereof; c. the sugars are selected from trehalose, glucose, sucrose, lactose, ribose, fructose, galactose and derivatives thereof; d. the polyols are selected from glycerol, erythritol, mannitol, sorbitol, xylitol, ethylene glycol, propylene glycol, ribitol, aldonitol, propanediol, inositol, pentylene glycol, and derivatives thereof; and e. the amino acids are selected from glycine, proline, taurine, lysine, and derivatives thereof.

15. The method according to claim 1, wherein the DES is: urea and betaine, glycerol and betaine, pyruvic acid and betaine, choline chloride and urea, glycerol and choline chloride, malic acid and choline chloride, levulinic acid and betaine, lactic acid and betaine, sorbitol and levulinic acid, betaine and sorbitol, proline and levulinic acid, betaine and proline, betaine and glucose, proline and glucose, lysine and levulinic acid, glycerol and sorbitol, glycerol and lactic acid, glucose and levulinic acid, xylitol and levulinic acid, sorbitol and lactic acid, urea and betaine HCl, or glycerol and levulinic acid.

16. The method according to claim 1, wherein the concentration of the DES in the extraction solution used in step (i) is at least the minimum hydrotropic concentration (MHC) of the DES.

17. (canceled)

18. (canceled)

19. (canceled)

20. The method according to claim 1, further comprising: vi) washing the solid obtained in step (iv) or (v) from about 1 to about 10 times or more with water and collecting the resulting solid material, and optionally drying the solid material.

21. The solid biological extract obtained or obtainable by the method as defined in claim 1.

22. (canceled)

23. The solid biological extract according to claim 21, wherein the solid biological extract comprises at about 2% or more by weight of the solid biological extract of lipophilic, hydrophobic, oil soluble and/or non-water-soluble compounds or about 10% or more by weight of the extract of lipophilic, hydrophobic, oil soluble and/or non-water-soluble compounds.

24. (canceled)

25. (canceled)

26. The solid biological extract according to claim 21, wherein the solid biological extract comprises about 2% or less by weight of the solid biological extract of DES or about 0.04% or less by weight of the solid biological extract of DES.

27. (canceled)

28. (canceled)

29. A method of utilizing the solid biological extract according to claim 21 as an anti-oxidant, anti-microbial, anti-inflammatory, as colour or pigments, as vitamins, as surfactant, as flavouring agent, as fragrance and/or as taste modifiers.

30. (canceled)

31. A nutraceutical composition, a dietary or food product for humans or animals, a nutritional supplement, a fragrance or flavouring, a pharmaceutical, a veterinary composition, or an oenological or cosmetic formulation comprising the solid biological extract according to claim 21, and optionally a pharmaceutically/veterinary acceptable ingredient.

32. The method according to claim 2, wherein the DES is obtained by combining at least two compounds selected from methylamines, organic acids, sugars, polyols, amino acids and urea.

33. The method according to claim 32, wherein: f. the methylamines are selected from N-trimethylamine oxide (TMAO), betaine, glycerophosphocholine, carnitine, homarine, choline chloride, and methyl sulfonium solutes including dimethylsulfonopropionate (DMSP) and derivatives thereof, for example, their halide forms; g. the organic acids are selected from levulinic acid, lactic acid, malic acid, maleic acid, pyruvic acid, fumaric acid, succinic acid, citric acid, citraconic acid, glutaric acid, glycolic acid, acetic acid, aconitic acid, tartaric acid, ascorbic acid, malonic acid, oxalic acid, glucuronic acid, neuraminic acid, sialic acid, shikimic acid, phytic acid, galacturonic acid, iduronic acid, hyaluronic acid, hydroxycitric acid, lactone derivatives and derivatives thereof; h. the sugars are selected from trehalose, glucose, sucrose, lactose, ribose, fructose, galactose, and derivatives thereof; i. the polyols are selected from glycerol, erythritol, mannitol, sorbitol, xylitol, ethylene glycol, propylene glycol, ribitol, aldonitol, propanediol, inositol, pentylene glycol, and derivatives thereof; and j. the amino acids are selected from glycine, proline, taurine, lysine, and derivatives thereof;

34. The method according to claim 2, wherein the DES is: urea and betaine, glycerol and betaine, pyruvic acid and betaine, choline chloride and urea, glycerol and choline chloride, malic acid and choline chloride, levulinic acid and betaine, lactic acid and betaine, sorbitol and levulinic acid, betaine and sorbitol, proline and levulinic acid, betaine and proline, betaine and glucose, proline and glucose, lysine and levulinic acid, glycerol and sorbitol, glycerol and lactic acid, glucose and levulinic acid, xylitol and levulinic acid, sorbitol and lactic acid, urea and betaine HCl, or glycerol and levulinic acid.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0255] FIG. 1. Effect of different betaine:urea mixtures on the solubility of the dye “disperse red 13” in aqueous solution at room temperature as measured by the absorbance at 525 nm. All solutions have been prepared in distilled water without any control of the pH. Results are expressed as the mean±Sd of a triplicate experiment (independent). Insert: S.sub.max as a function of the urea-to-betaine molar fraction (%).

[0256] FIG. 2. Effect of different betaine:urea mixtures on the solubility of disperse red 13 in aqueous solution at room temperature as measured by the absorbance at 525 nm. All solutions have been prepared in pH 7 phosphate buffer solution. When necessary, the final hydrotropic solution have been adjusted to pH 7.0 with concentrated HCl. Added HCl volumes were less than 1% of the total volume in all tested mixtures, so the concentrations were not corrected. Results are expressed as the mean±Sd of a triplicate experiment (independent) insert; S.sub.max as a function of the urea-to-betaine molar fraction (%).

[0257] FIG. 3. Effect of different choline chloride:urea mixtures on the solubility of disperse red 13 in aqueous solution at room temperature as measured by the absorbance at 525 nm. All solutions have been prepared in distilled water without any control of the pH. Results are expressed as the mean±Sd of a triplicate experiment (independent), insert: S.sub.max as a function of the urea-to-choline chloride molar fraction (%).

[0258] FIG. 4. S.sub.max of carnosic acid as a function of the urea-to-betaine molar fraction (%).

[0259] FIG. 5. S.sub.max of hesperidin as a funtion of the urea-to-betaine molar fraction (%).

[0260] FIG. 6. S.sub.max of curcumine as a function of the urea-to-betaine molar fraction (%).

[0261] FIG. 7 Recovery rate of carnosic acid (CA) extraction from ground rosemary leaves (A), CA content in the final crude (non-washed) extracts (B), and mass yield (C) obtained by using a 1:1 or a 2:1 urea:betaine deep eutectics at room temperature (RT) for 0.5, 1, 2, 3, 7, or 16 hours. All solvents comprise 950 g/L of DES in water. For all extractions, the plant:solvent weight ratio was 1:10 (denoted 10 M) and the precipitation was obtained using nine volumes of water at room temperature.

[0262] FIG. 8. Recovery rate of carnosic acid (CA) extraction from ground rosemary leaves (A), CA content in the final purified (washed) extracts (B), and mass yield (C) obtained by using a 1:1 or a 2:1 urea:betaine deep eutectics at room temperature (RT) for 0.5, 1, 2, or 3 hours. All solvents comprise 950 g/L of DES in water. The plant:solvent weight ratios were 1:10 (denoted 10 M), 1:15 (denoted 15 M), or 1:20 (denoted 20 M), and the precipitation was obtained using nine volumes of water at room temperature. The extracts were washed 1, 2 or 5 times.

[0263] FIG. 9. Recovery rate of carnosic acid (CA) extraction from ground rosemary leaves (A), CA content in the final crude (non-washed) extracts (B), and mass yield (C) obtained by using a 1:2 betaine:levulinic acid deep eutectics containing 10% of water at room temperature (RT) for 1 hour. The plant:solvent weight ratios were 1:10 (denoted 10 M) or 1:15 (denoted 15 M) and the precipitation was obtained for both conditions using nine volumes of water at room temperature.

[0264] FIG. 10. Recovery rate of carnosic acid (CA) extraction from ground rosemary leaves (A), CA content in the final purified (washed) extracts (B), and mass yield (C) obtained by using 1:2 betaine:levulinic acid deep eutectics containing 5, 10, 20, 25, or 30% of water at room temperature (RT) for 0.25, 0.5, 1, 2, or 3 hours. The plant:solvent weight ratios were 1:6 (denoted 6 M), 1:8 (denoted 8 M), 1:10 (denoted 10 M), or 1:15 (denoted 15 M). The precipitation was obtained using 1.5, 2, 3, 4, or 9 volumes of water at room temperature or at 4° C.

[0265] FIG. 11. Recovery rate of carnosic acid (CA) extraction from ground rosemary leaves (A). CA content in the final crude (non-washed) extracts (B), and mass yield (C) obtained by using a 1:1 or a 1:2 betaine:lactic acid deep eutectics containing 10% of water at room temperature (RT) for one hour. The plant:solvent weight ratios were 1:10 (denoted 10 M), or 1:15 (denoted 15 M) and the precipitation was obtained using nine volumes of water at room temperature.

[0266] FIG. 12. Recovery rate of carnosic acid (CA) extraction from ground rosemary leaves (A), CA content in the final purified (washed) extracts (B), and mass yield (C) obtained by using 1:2 or 1:3 betaine:lactic acid deep eutectics containing 5, 10, 20, or 25% of water at room temperature (RT) for one hour. The plant:solvent weight ratios were 1.8 (denoted 8 M), 1:10 (denoted 10 M), or 1:15 (denoted 15 M) and the precipitation was obtained using 4 or 9 volumes of water at room temperature. The first sample of the figure (from left) has been obtained after filtration of the precipitation waters on a 2 μm filter. The others have been centrifuged.

[0267] FIG. 13. Recovery rate of carnosic acid (CA) extraction from ground rosemary leaves (A), CA content in the final crude (non-washed) or purified (washed) extracts (B), and mass yield (C) obtained by using 1:2 betaine:glycerol deep eutectics containing 20 or 25 % of water at room temperature (RT) for one hour. The plant:solvent weight ratio was 1:10 (denoted 10 M) and the precipitation was obtained using 4 or 9 volumes of water at room temperature.

[0268] FIG. 14. Recovery rate of carnosic acid (CA) extraction from ground rosemary leaves (A). CA content in the final purified (washed) extracts (B), and mass yield (C) obtained by using 1:1, 1:2, or 2:1 betaine:sorbitol deep eutectics containing 20, 30, or 50% of water at room temperature (RT) for 0.5, 1 or 2 hours. The plant:solvent weight ratios were 1:8 (denoted 8 M), or 1:10 (denoted 10 M) and the precipitation was obtained using four volumes of water at room temperature.

[0269] FIG. 15. Recovery rate of carnosic acid (CA) extraction from ground rosemary leaves (A). CA content in the final purified (washed) extracts (B), and mass yield (C) obtained by using 1:1 or 1:2 betaine:sorbitol deep eutectics containing 20 or 50% of water at 60° C. for 0.5 hour. The plant:solvent weight ratios were 1:8 (denoted 8 M), or 1:10 (denoted 10 M) and the precipitation was obtained using four volumes of water at room temperature.

[0270] FIG. 16. Recovery rate of carnosic acid (CA) extraction from ground rosemary leaves (A). CA content in the final purified (washed) extracts (B), and mass yield (C) obtained by using 1.1 sorbitol:levulinic acid deep eutectics containing 20 or 30% of water at room temperature (RT) for 0.5 hour. The plant:solvent weight ratio was 1:10 (denoted 10 M) and the precipitation was obtained using four volumes of water at room temperature.

[0271] FIG. 17. Recovery rate of carnosic acid (CA) extraction from ground rosemary leaves (A), CA content in the final purified (washed) extracts (B), and mass yield (C) obtained by using a 1:2 betaine:proline deep eutectics containing 30% of water at room temperature (RT) for 0.5, 1, or 2 hours. The plant:solvent weight ratio was 1:10 (denoted 10 M) and the precipitation was obtained using four volumes of water at room temperature.

[0272] FIG. 18. Recovery rate of carnosic acid (CA) extraction from ground rosemary leaves (A), CA content in the final purified (washed) extracts (B), and mass yield (C) obtained by using a 1:2 betaine:proline deep eutectics containing 30% of water at 60° C. for 0.5 or 2 hours. The plant:solvent weight ratio was 1:10 (denoted 10 M) and the precipitation was obtained using four volumes of water at room temperature.

[0273] FIG. 19. Recovery rate of carnosic acid (CA) extraction from ground rosemary leaves (A), CA content in the final purified (washed) extracts (B), and mass yield (C) obtained by using 1:1, 1:2, 2:1, or 3:1 proline:glucose deep eutectics containing 20 or 30% of water at 60° C. or room temperature (RT) for 0.5, 1, or 2 hours. The plant:solvent weight ratio was 1:10 (denoted 10 M) and the precipitation was obtained using four volumes of water at room temperature.

[0274] FIG. 20. Recovery rate of carnosic acid (CA) extraction from ground rosemary leaves (A), CA content in the final purified (washed) extracts (B), and mass yield (C) obtained by using 1:1, 1:2, 2:1, and 3:1 betaine:glucose deep eutectics containing 20, 30, or 50% of water at 60° C. for 0.5, 1, or 2 hours. The plant:solvent weight ratio was 1:10 (denoted 10 M) and the precipitation was obtained using four volumes of water at room temperature.

[0275] FIG. 21. Recovery rate of carnosic acid (CA) extraction from ground rosemary leaves (A), CA content in the final purified (washed) extracts (B), and mass yield (C) obtained by using a 1:2 betaine:pyruvic acid deep eutectics containing 25% of water at room temperature (RT) for 0.5 hour. The plant:solvent weight ratios were 1:8 (denoted 8 M), 1.10 (denoted 10 M), or 1:15 (denoted 15 M) and the precipitation was obtained using four volumes of water at room temperature.

[0276] FIG. 22. Recovery rate of carnosic acid (CA) extraction from ground rosemary leaves (A), CA content in the final crude (non-washed) or purified (washed) extracts (B), and mass yield (C) obtained by using a 2:1 urea:choline chloride deep eutectics free of exogenously added water, a 1:1 glycerol:lactic acid deep eutectics containing 20% of water, a 1:1 glycerol:levulinic acid deep eutectics containing 20% of water, a 1:1 sorbitol:lactic acid deep eutectics containing 20% of water, a 1:1 xylitol:levulinic acid deep eutectics containing 20% of water, a 1:2 glucose:levulinic acid deep eutectics containing 30% of water, a 1:1 proline:levulinic acid deep eutectics containing 20% of water, and a 1:1 lysine:levulinic acid deep eutectics containing 20% of water. Extractions have been performed at 60° C. or room temperature (RT) for 5 or 1 hour. The plant:solvent weight ratio was 1:10 (denoted 10 M) for all solvents and the precipitation was obtained using 4or 9 volumes of water at room temperature.

[0277] FIG. 23. Recovery rate of carnosic acid (CA) extraction from ground rosemary leaves using a 2:1 urea:betaine HCl deep eutectics free of exogenously added water, betaine:glycerol deep eutectics at various molar ratio (1:1, 1:2, or 2:1) and containing 10, 20, or 30% of water, 1:1 malic acid:choline chloride deep eutectics containing 20 or 30% of water, glycerol:choline chloride deep eutectics at various molar ratio (1:1, 1:2, or 2:1) and containing 0, 10, or 20% of water, a 1:1 glycerol:sorbitol deep eutectics containing 20% of water, a 1:2 lysine:levulinic acid deep eutectics containing 20% of water, and a 1:1 betaine:proline deep eutectics containing 30% water. All extractions have been performed at room temperature (RT) for 0.5 or 1 hour with a piant:solvent weight ratio of 1:10 (denoted 10 M).

[0278] FIG. 24. Antioxidant activity measured by the Rancimat assay of crude (non-washed) and purified (washed) extracts of rosemary obtained by using urea:betaine deep eutectics.

[0279] FIG. 25. Antioxidant activity measured by the Rancimat assay of purified (washed) extracts of rosemary obtained by using betaine:levulinic acid deep eutectics.

[0280] FIG. 26. Linear positive correlation between the protection factor measured using the Rancimat assay and the carnosic acid content in the final extract (%).

[0281] FIG. 27. Effect of the content of salt (NaCl) in water on the carnosic acid (CA) hydrosolubility expressed as the CA content in the supernatant.

[0282] FIG. 28. Active recovery rate (A), content in the final extracts (B) and mass yield (C) obtained after extraction for 30 min of ground turmeric roots using different DES and temperatures (room temperature, RT or 60° C.). Solvents used were 1:1 sorbitol:levulinic acid containing 20% of water, 1:1 proline:levulinic acid containing 20% of water, 1:2 betaine:glycerol containing 30% of water, 1:2 betaine:levulinic acid containing 25% of water, and 1:2 betaine:lactic acid containing 10% of water. For all solvents, the plant:solvent weight ratio was 1:10 and the precipitation was obtained using four volumes of water at room temperature.

[0283] FIG. 29. Hesperidin recovery rate during extraction at room temperature for 30 min of ground orange peel using different DES. Solvents used were 1:1 sorbitol:levulinic acid containing 20% of water, 1:1 proline:levulinic acid containing 20% of water, 1:2 betaine:glycerol containing 30 of water, 1:2 betaine:levulinic acid containing 25% of water, and 1:2 betaine:lactic acid containing 10% of water. For all solvents, the plant:solvent weight ratio was 1:10.

EXAMPLES

[0284] The present invention will be further described by reference to the following non-limiting examples.

[0285] Material and Methods

[0286] Solubilization of the Dye ‘Red Disperse 13’

[0287] Urea (≥98%, U5378, Sigma) and betaine (≥99%, 61962, Sigma) were of analytical grade. Care was taken to avoid any moisture by storing urea and betaine in a desiccator with silica. For choline chloride such caution was insufficient to avoid moisture and it was recrystallized with ethanol and stored in a desiccator with P.sub.2O.sub.5 as desiccant.

[0288] The desired amounts of betaine and/or urea powders were dissolved in distilled water or phosphate buffer solution (PBS, 67 mM) at pH 7.0 to form a 100 mL solution.

[0289] The concentration of urea in the urea:betaine mixtures in water or PBS ranged from 0.025 to 8.3 M.

[0290] The concentrations of pure urea solution in water or PBS ranged from 0.025 to 10.1 M, the latter value corresponding to the saturation level of urea.

[0291] Finally, the concentrations of pure betaine in water or PBS ranged from 0.025 to 5.4 M, this latter corresponding to the saturation level of betaine.

[0292] The solutions of urea and/or betaine in PBS were adjusted to provide a pH of 7.0 using concentrated HCl. The volume of added HCl was taken into account when calculating the final molar concentration of betaine and/or urea.

[0293] 10 mL of the osmolyte solution was added to a vial containing 5 mg red disperse 13 in excess (final concentration: 364827, Sigma), then magnetically stirred (500 rpm) for 16 hours and filtrated (0.45 μm). The concentration of red disperse 13 in the filtrate is measured at 525 nm (Shimadzu UV-1800, Japan).

EXAMPLE

Example 1

Solubilization Properties of Betaine:Urea Mixtures for Disperse Red 13 in Non-Buffered Aqueous Media

[0294] Betaine is a trimethylated form of glycine discovered for the first time in sugar beet juice. It is en abundant natural resource which can play the role of a hydrogen-bond acceptor through the two oxygen atoms of the carboxylate group (COO). Urea is known as a strong hydrogen-bond donor through the primary amine groups.

[0295] In this Example, anhydrous urea:betaine (U:B) mixtures at different molar ratios were first prepared, then solubilized in distilled water at different concentrations ranging from 1.5 g/L to the saturation level which was 607, 813, 985, 957, 795, 741, and 633 g/L for the U:B ratios 1:0, 3:1, 2:1, 1:1, 1:2, 1:3, and 0:1, respectively.

[0296] These liquid mixtures were mixed with an excess of disperse red 13 used as a hydrophobic probe to screen the solubilization properties of the solvents. It can be observed from FIG. 1 that the disperse red 13 absorbance increases exponentially 525 nm approx.) with increasing the deep eutectic concentration in water. This clearly demonstrates that the eutectic composition (1:2, betaine:urea) and the others (called peritectic mixtures instead of eutectic mixtures) behave as hydrotropes in water.

[0297] The MHC decreases as follows: 1:3 U:B>1:2 U:B>1:1 U:B>3:1 U:B>urea.

[0298] More importantly, the highest S.sub.max (the maximal solubilization capacity of a given solute by a hydrotrope) was observed for the 1:2 betaine:urea ratio, which corresponds to the eutectic composition of the binary system. This is best observed in the FIG. 1 insert which plots S.sub.max as a function of the urea molar fraction to betaine (%).

[0299] This means that, among all mixtures tested in this Example, the solvent with the highest capacity to solubilize the disperse red 13 was a 2:1 U:B solution at concentration of 985 g/L of water (saturation level or C.sub.max).

[0300] In this example, taking into account that there is no plateau in the hydrotropy of urea and U:B mixtures, the maximal achievable concentrations (saturation level) of deep eutectics in water (C.sub.max, x-axis, FIG. 1) correspond to the maximal achievable solubilizations of red disperse 13 (S.sub.max, y-axis, FIG. 1). In other words, the C.sub.max is the hydrotrope concentration enabling to reach the S.sub.max for a given solute. Here, the higher the concentration in deep eutectics, the higher the concentratton of solubilized disperse red 13 (colligative effect). The fact that the optimized S.sub.max is critically observed for the eutectic composition is an unprecedented finding.

[0301] Furthermore, although urea alone displays a hydrotropic behavior, this is not the case of betaine alone which increased the disperse red 13 too modestly to be qualified as such. The addition of urea to betaine in specific proportions (the eutectic composition, i.e. here 2:1 UB) thus lead to a strong synergistic effect of the corresponding mixture for solubilizing disperse red 13.

Example 2

Solubilization Properties of Betaine:Urea Mixtures for Disperse Red 13 in Buffered and pH 7-Adjusted Aqueous Media

[0302] To verify that the criticality of the U:B molar ratio is not simply due to a pH effect (pH varies depending on the ratio between urea and betaine and also on their concentration in the aqueous solution), a second series of experiments was conducted by solubilizing disperse red 13 in the same U:B mixtures but in a pH=7 phosphate buffer.

[0303] The high deep eutectic concentrations required to adjust the pH to 7 with a concentrated HCl solution because the buffering effect of the buffer was not always sufficient to counter the betaine-induced increase of pH. Added HCl volumes were less than 1% of the total volume in all tested mixtures, so the concentrations were not corrected.

[0304] The results obtained were close to those seen in Example 1 with distillated water (uncontrolled pH) demonstrating that the deep eutectic hydrotopy is not primarily determined by the pH, but is rather controlled by the critical molar ratio between both components (FIG. 2).

[0305] Further experiments were thus conducted in distillated water. As previously observed, the eutectic composition (2:1 UB) at the saturation level in water offer the best conditions to solubilize the disperse red 13 dye. The exact same synergy as in Example 1 is also observed at pH 7 between urea and betaine.

Example 3

Solubilization Properties of Choline Chloride Urea Mixtures for Disperse Red 13 in Non-Buffered Aqueous Media

[0306] Choline chloride (ChCl) is a methylamine salt which can be either extracted from biomass or readily synthesized from fossil reserves through a very high atom economy process. In combination with hydrogen bond donors such as urea at a molar ratio of 2:1, urea:ChCl (UC); ChCl could produce a deep eutectic solvent that is liquid at 12 ° C. (FIG. 3).

Example 4

Solubilization Properties of Betaine:Urea Mixtures for Carnosic Acid in Non-Buffered Aqueous Media

[0307] In this Example, anhydrous urea:betaine (U:B) mixtures at different molar ratios were first prepared, then solubilized in distilled water at the saturation level; i.e. 607, 813, 985, 957, 795, 741, and 633 g/L for the U:B ratios 1:0. 3:1. 2:1, 1:1, 1:2, 1:3, and 0:1, respectively. These liquid mixture were mixed with an excess of carnosic acid, a diterpene antioxidant used as a hydrophobic probe to screen the solubilization properties of the different solvents.

[0308] It can be observed from FIG. 4 that the highest carnosic acid S.sub.max (the maximal solubilization capacity of carnosic acid by a deep eutectics) was observed for the 1:2 betaine:urea ratio, which corresponds to the eutectic composition of the binary system. This means that, among all mixtures tested in this Example, the solvent with the highest capacity to solubilize carnosic acid is a 2:1 U:B solution at concentration of 985 g/L of water (saturation level or C.sub.max). Furthermore, a net synergy was obtained for the 2:1 U:B mixture.

Example 5

Solubilization Properties of Betaine Urea Mixtures for Hesperidin in Non-Buffered Aqueous Media

[0309] In this Example, anhydrous urea:betaine (U:B) mixtures at different molar ratios were first prepared, then solubilized in distilled water at the saturation level, i.e. 607, 813, 985, 957, 795, 741, and 633 g/L for the U:B ratios 1:0, 3:1, 2:1, 1:1, 1:2, 1:3, and 0:1, respectively.

[0310] These liquid mixtures were mixed with an excess of hesperidin, a flavonoid bioactive used as a hydrophobic probe to screen the solubilization properties of the different solvents.

[0311] It can be observed from FIG. 5 that the highest hesperidin S.sub.max (the maximal solubilization capacity of hesperidin by a deep eutectics) was observed for both the 1:2 and the 1:1 betaine:urea ratio, which corresponds to a composition nearby, or exactly at, the eutectic composition of the binary system.

[0312] This means that, among all mixtures tested in this Example, the solvents with the highest capacity to solubilize hesperidin are solutions with (i) a 2:1 U:B solution at concentration of 985 g/L of water and (ii) a 1:1 U:B solution at concentration of 957 g/L of water (saturation level or C.sub.max in both cases). Furthermore, a net synergy was obtained for these two solvents compared to saturated solutions of pure betaine or pure urea.

Example 6

Solubilization Properties of Betaine:Urea Mixtures for Curcumin in Non-Buffered Aqueous Media

[0313] In this Example, anhydrous urea:betaine (U:B) mixtures at different molar ratios were first prepared, then solubilized in distilled water at the saturation level, i.e. 607, 813, 985, 957, 795, 741, and 633 g/L for the U:B ratios 1:0, 3:1, 2:1, 1:1, 1:2, 1:3, and 0:1, respectively. These liquid mixtures were mixed with an excess of curcumin, a phenolic bioactive and pigment used as a hydrophobic probe to screen the solubilization properties of the different solvents.

[0314] It can be observed from FIG. 6 that the highest curcumin S.sub.max (the maximal solubilization capacity of curcumin by a deep eutectics) was observed for the 1:1 urea:betaine ratio, which corresponds to a composition nearby the eutectic composition of the binary system. This means that, among all mixtures tested in this Example, the solvents with me highest capacity to solubilize hesperidin is a 1:1 U:B solution at concentration of 957 g/L of water (saturation level or C.sub.max). Furthermore, a net synergy was obtained for this solvent compared to saturated solutions of pure betaine or pure urea. In fact, all other combinations also exhibit a synergistic effect regarding their solubilization properties.

Example 7

Extraction of Rosemary Using Urea:Betaine Deep Eutectics, Cake Removal by Centrifugation then Filtration, Separation of the Extract from the Deep Eutectics Using Water as an Anti-Solvent, and Recovery of the Crude Extract by Centrifugation

[0315] Homogeneous aqueous solutions of 2:1 urea:betaine (UB2/1_950 g/L) and 1.1 urea:betaine (UB1/1_950 g/L) mixtures both near the saturation level (950 g/L) were prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 200 mL of this deep eutectic solution (plany:solvent: 1:10) for 0.5, 1, 2, 3, 7, or 16 hours and the enriched solution was separated from the plant by centrifugation followed by a filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be comprised between 27 and 43% (FIG. 7a). Then, the filtrate was diluted by 9 volumes of water. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the deep eutectics from the extract. The resulting precipitate was magnetically stirred for 15 minutes and left to settle for another 15 minutes. The precipitate was recovered by centrifugation and dried overnight at 45° C. in a vacuum oven. Finally, dried pellets (hereafter referred to as ‘crude extract’) was collected. They contained between 11 and 19% of carnosic acid (HPLC quantification) (FIG. 7b). The final mass yield was between 1.2 and 4.2% (FIG. 7c).

Example 8

Extraction of Rosemary Using Urea:Betaine Deep Eutectics, Cake Removal by Centrifugation then Filtration, Separation of the Extract from the Deep Eutectics Using Water as an Anti-Solvent, Recovery of the Crude Extract by Centrifugation, and Removal of Residual Deep Eutectics by a Washing Procedure with Water

[0316] Here the process followed in Example 7 was reproduced with an additional washing procedure at the end. Homogeneous aqueous solutions of 2:1 urea:betaine (UB2/1_950 g/L) and 1:1 urea:betaine (UB1/1_950 g/L) mixtures both near the saturation level (950 g/L) were prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 200 mL of this deep eutectic solution (plant:solvent 1:10) for 0.5, 1, 2, or 3 hours and the enriched solution was separated from the plant by centrifugation followed by a filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be composed between 28 and 65% (FIG. 8a). Then, the filtrate was diluted by 9 volumes of water. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the deep eutectics from the extract. The resulting precipitate was magnetically stirred for 15 minutes and left to settle for another 15 minutes.

[0317] Then, an additional washing procedure using 200 mL of water was applied to remove the residual deep eutectics from the extract (hereafter referred to as ‘solvent-free eutectic extract’ or ‘washed extract’) and further increase the carnosic acid concentration. The solvent-free eutectic extract that has been dried overnight at 45° C. in a vacuum oven finally contained between 17 and 33 of carnosic acid (HPLC quantification) ((FIG. 8b), which represent an important improvement of the carnosic acid content compared to the process without the washing procedure described in Example 7. The final mass yield concomitantly decreased with values ranging from 0.8 to 2.4% (FIG. 8c), which is still well acceptable regarding industrial standards.

Example 9

Chemical Characterization of the Rosemary Extracts Obtained in Examples 7 (Crude Extract) and 8 (Purified Extract) Using a 2:1 Urea:Betaine Mixture Near the Saturation Level (950 g/L) at Room Temperature for 2 Hour

[0318] Table 1 shows some of the identified compounds found in a eutectic rosemary extract before (crude extract corresponding to sample ‘UB2/1_950 g/L_10M_RT_2h_10vol_crude’ depicted in FIG. 7) and after (purified extract corresponding to sample ‘UB2/1_950 g/L_10M_RT_2h_10vol_1washed’ depicted in FIG. 8) the application of the washing procedure. The sign + means presence, and the sign − means absence.

TABLE-US-00001 TABLE 1 Identified compounds in eutectic rosemary extracts before (crude extract) and after (purified extract) the application of the washing procedure Identified compounds in the Chemical Crude Purified eutectic extracts Formula family extract extract Gluconic acid C.sub.6H.sub.12O.sub.7 Organic acid + − Malic acid C.sub.4H.sub.6O.sub.5 Organic acid + − Tartaric acid C.sub.4H.sub.6O.sub.4 Organic acid + − Salycilic acid C.sub.7H.sub.6O.sub.3 Phenolic acid + − Caffeic acid C.sub.9H.sub.8O.sub.4 Phenolic acid + − Nepitrin C.sub.22H.sub.22O.sub.12 Flavonoid + − Rosmarinic acid C.sub.18H.sub.16O.sub.8 Phenolic acid + − Luteolin glucuronide C.sub.21H.sub.18O.sub.12 Flavonoid + − Luteolin 3-O-(o-acetyl) D C.sub.23H.sub.20O.sub.13 Flavonoid + + glucuronide isomer I Luteolin 3-O-(o-acetyl) D C.sub.23H.sub.20O.sub.13 Flavonoid + + glucuronide isomer II Luteolin 3-O-(o-acetyl) D C.sub.23H.sub.20O.sub.13 Flavonoid + − glucuronide isomer III Scutellarein C.sub.15H.sub.10O.sub.6 Flavonoid + − Nepetin/isorhamnetin C.sub.16H.sub.12O.sub.7 Flavonoid + + Diosmetin C.sub.16H.sub.12O.sub.6 Flavonoid + + Hispidulin C.sub.16H.sub.12O.sub.6 Flavonoid + + Cirsimaritin C.sub.17H.sub.14O.sub.6 Flavonoid + + Apigenin C.sub.15H.sub.10O.sub.5 Flavonoid + + Dimethoxycoumarin C.sub.11H.sub.10O.sub.4 Coumarin + + Rhamnazin/dimethylquercetin C.sub.17H.sub.14O.sub.7 Flavonoid + + Rosmanol C.sub.20H.sub.26O.sub.5 Phenolic + + diterpene Cirsimaritin isomer C.sub.17H.sub.14O.sub.6 Flavonoid + + Epiisorosmanol C.sub.20H.sub.26O.sub.5 Phenolic + + diterpene Epirosmanol C.sub.20H.sub.26O.sub.5 Phenolic + + diterpene Hydroxycryptotanshinone C.sub.19H.sub.20O.sub.4 Diterpenoid − + Genkwanin C.sub.16H.sub.12O.sub.5 Flavonoid + + Epirasmanol isomer C.sub.20H.sub.26O.sub.5 Phenolic − + diterpene Gingerol C.sub.17H.sub.26O.sub.4 Phenolic lipid − + Rosmadial C.sub.20H.sub.24O.sub.5 Phenolic + + diterpene Epirosmanol methyl ether C.sub.21H.sub.28O.sub.5 Phenolic + + diterpene Ubiquinol C.sub.18H.sub.28O.sub.4 Phenolic lipid − + para-Miltioic acid C.sub.19H.sub.24O.sub.5 Diterpenoid − + Epirosmanol methyl ether C.sub.21H.sub.28O.sub.5 Phenolic + + diterpene Carnosol C.sub.20H.sub.26O.sub.4 Phenolic + + diterpene Carnosol isomer C.sub.20H.sub.26O.sub.4 Phenolic + + diterpene Rosmadial isomer C.sub.20H.sub.24O.sub.5 Phenolic + + diterpene Rosmaridiphenol C.sub.20H.sub.28O.sub.3 Phenolic + + diterpene Carnosic acid C.sub.20H.sub.28O.sub.4 Phenolic + + diterpene 12-Methoxycarnosic acid C.sub.21H.sub.30O.sub.4 Phenolic + + diterpene 5,6,7,10-Tetrahydro-7- C.sub.19H.sub.26O.sub.3 Phenolic − + hydroxyrosmariquinone diterpene Shogaol C.sub.20H.sub.30O.sub.3 Phenolic lipid + + Ursolic acid C.sub.30H.sub.48O.sub.3 Triterpene + +

Example 10

Extraction of Rosemary Using Betain:Levulinic Acid DES, Cake Removal by Filtration, Separation of the Extract from the Deep Eutectics Using Water as an Anti-Solvent, and Recovery of the Crude Extract by Centrifugation

[0319] A homogeneous solution of 1:2 betaine:levulinic acid_mixture containing 10% of water (BLe1/2_10% w) was prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 200 mL (plant:solvent:1:10) or 300 mL (plant:solvent 1:15) of this deep eutectic solution for one hour and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be comprised between 73 and 76% (FIG. 9a). Then, the filtrate was diluted by 9 volumes of water. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the deep eutectics from the extract. The resulting precipitate was magnetically stirred for 15 minutes and left to settle for another 15 minutes. The precipitate was recovered by centrifugation and freeze-dried overnight. Finally, dried pellets (hereafter referred to as ‘crude extract’) was collected. They contained between 29 and 31% of carnosic acid (HPLC quantification) (FIG. 9b). The final mass yield was between 6.3 and 11% (FIG. 9c).

Example 11

Extraction of Rosemary Using Betaine:Levulinic Acid DES, Cake Removal by Filtration, Separation of the Extract from the Deep Eutectics Using Water as an Anti-Solvent, Recovery of the Crude Extract by Centrifugation, and Removal of Residual Deep Eutectics by a Washing Procedure with Water

[0320] Here the process followed in Example 10 was reproduced with an additional washing procedure at the end. Homogeneous solutions of 1:2 betaine:levulinic acid mixtures containing 5% (BLel1/2_5%w), 10% (BLe1/2_10% w), 20% (BLe1/2_20% w), 25% (BLe1/2_25% w), and 30% (BLe1/2_30% w) of water were prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 120 mL (plant:solvent: 1:6), 160 mL (plant:solvent: 1:8), 200 mL (plant:solvent: 1:10), or 300 mL (plant:solvent: 1:15) of this deep eutectic solution for 0.26, 0.5, 1, 2, or 3 hours and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be comprised between 63 and 82% (FIG. 10a). Then, the filtrate was diluted by 1.5, 2, 3, 4, and 9 volumes of water at 4 or 10° C. or at room temperature. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the deep eutectics from the extract. The resulting precipitate was magnetically stirred for 15 minutes and left to settle for another 15 minutes.

[0321] Then, an additional washing procedure using the same volume of water as the volume or solvent used for the extraction (120, 160, 200, or 300 mL) was applied to remove the residual deep eutectics from the extract and further increase the carnosic acid concentration. The solvent-free eutectic extract that has been freeze-dried overnight finally contained between 36 and 45% of carnosic acid (HPLC quantification). (FIG. 10b), which represents an important improvement of the carnosic acid content compared to the process without the washing procedure described in Example 10. The final mass yield concomitantly decreased with values ranging from 2.7 to 4.5% (FIG. 10c), which is still well acceptable regarding industrial standards.

Example 12

Extraction of Rosemary Using Betaine:Lactic Acid DES, Cake Removal by Filtration, Separation of the Extract from the Deep Eutectics Using Water as an Anti-Solvent and Recovery of the Crude Extract by Centrifugation

[0322] Homogeneous solutions of 1:2 betaine:lactic acid (BLa1/2_10% w) and 1:1 betaine:latic acid (BLa1/1_10% w) mixtures each containing 10% of water were prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 200 mL (plant:solvent 1:10) or 300 mL (plant:solvent: 1:15) of these deep eutectic solutions for one hour and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be comprised between 32 and 80% (FIG. 11a). Then, the filtrate was diluted by 9 volumes of water. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the deep eutectics from the extract. The resulting precipitate was magnetically stirred for 15 minutes and left to settle for another 15 minutes. The precipitate was recovered by centrifugation and freeze-dried overnight. Finally, dried pellets were collected. They contained between 23 and 40% of carnosic acid (HPLC quantification) (FIG. 11b). The final mass yield was between 1.7 and 7.5% (FIG. 11c).

Example 13

Extraction of Rosemary Using Betame:Lactic Acid DES, Cake Removal by Filtration, Separation of the Extract from the Deep Eutectics Using Water as an Anti-Solvent Recovery of the Crude Extract by Centrifugation, and Removal of Residual Deep Eutectics by a Washing Procedure with Water

[0323] Here the process followed in Example 12 was reproduced with an additional washing procedure at the end. Homogeneous solutions of 1:2 betaine:lactic acid mixtures containing 5% (BLa1/2_5% w), 10% (BLa1/2_10% w), 20% (BLa1/2_20% w), and 25% (BLa1/2_25% w) % of water and a solution of 1:3 betaine:lactic acid mixture containing 10% of water (BLa1/3_10% w) were prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 160 mL (plant:solvent 1:8), 200 mL (plant:solvent 1:10), or 300 mL (plant:solvent 1:15) of this deep eutectic solution for one hour and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be compnsed between 40 and 80% (FIG. 12a). Then, the filtrate was diluted by 4 or 9 volumes of water at room temperature. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the deep eutectics from the extract. The resulting precipitate was magnetically stirred for 15 minutes and left to settle for another 15 minutes. Then, an additional washing procedure using the same volume of water as the volume of solvent used for the extraction (160, 200, or 300 ml) of water was applied to remove the residual deep eutectics from the extract and further increase the carnosic acid concentration. The solvent-free eutectic extract that has been freeze-dried overnight finally contained between 33 and 41% of carnosic acid (HPLC quantification) (FIG. 12b). The final mass yield ranged from 0.8 to 4.6% (FIG. 12c), which is still well acceptable regarding industrial standards.

Example 14

Extraction of Rosemary Using Betaine:Glycerol DES, Cake Removal by Filtration, Separation of the Extract from the Deep Eutectics Using Water as an Anti-Solvent, and Recovery of the Crude Extract by Centrifugation

[0324] Homogeneous solutions of 1:2 betaine:glycerol mixture containing 20% of water (BGly1/2_20% w) were prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 200 mL (10 M) of this deep eutectic solutions for one hour and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be 40% (FIG. 13a, sample ‘BGly1/2_20% w_10M_RT_1h_9vol_crude’). Then, the filtrate was diluted by 9 volumes of water. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the deep eutectics from the extract. The resulting precipitate was magnetically stirred for 15 minutes and left to settle for another 15 minutes. The precipitate was recovered by centrifugation and freeze-dried overnight. Finally, dried pellets were collected. They contained 17% of carnosic acid (HPLC quantification) (FIG. 13b). The final mass yield was 2.4% (FIG. 13c).

Example 15

Extraction of Rosemary Using Betame:Glycerol DES, Cake Removal by Filtration, Separation of the Extract from the Deep Eutectics Using Water as an Anti-Solvent Recovery of the Crude Extract by Centrifugation, and Removal of Residual Deep Eutectics by a Washing Procedure with Water

[0325] Here the process followed in Example 14 was reproduced with an additional washing procedure at the end. Homogeneous solutions of 1:2 betaine:glycerol mixtures containing 20% (BGly1/2_20% w) and 25% (BGly1/2_25% w) of water were prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 200 ml of this deep eutectic solution for one hour and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be comprised between 22 and 43 % (FIG. 13a). Then, the filtrate was diluted by 4 or 9 volumes of water at room temperature. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the deep eutectics from the extract. The resulting precipitate was magnetically stirred for 15 minutes and left to settle for another 15 minutes.

[0326] Then, an additional washing procedure using 200 mL of water was applied to remove the residual deep eutectics from the extract and further increase the carnosic acid concentration. The solvent-free eutectic extract that has been freeze-dried overnight finally contained between 29 and 40% of carnosic acid (HPLC quantification) (FIG. 13b). The final mass yield ranged from 0.2 to 0.8% (FIG. 13c), which is still well acceptable regarding industrial standards.

Example 16

Extraction of Rosemary Using Betaine:Sorbitol DES at Room Temperature, Cake Removal by Filtration, Separation of the Extract from the Deep Eutectics Using Water as an Anti-Solvent, Recovery of the Crude Extract by Centrifugation, and Removal of Residual Deep Eutectics by a Washing Procedure with Water

[0327] Homogeneous solutions of 1:2 betaine:sorbitol mixtures containing 20% (BS1/2_20% w), 30% (BS1/2_30% w) and 50% (BS1/2_50% w) of water were prepared as well as solutions of 1:1 betaine:sorbitol mixtures containing 20% (BS1/1_20% w), 30% (BS1/1_30% w) and 50% (BS1/1_50% w), and 2:1 betaine:sorbitol mixtures containing 30% (BS2/1_30% w). Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 160 mL (plant:solvent: 1:8), 200 mL (plant:solvent: 1:10); and 300 mL (plant:solvent: 1.15) of this deep eutectic solution for 0.5, 1, and 2 hour and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be comprised between 3.9 and 13.1% (FIG. 14a). Then, the filtrate was diluted by 4 volumes of water at room temperature. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the deep eutectics from the extract. The resulting precipitate was magnetically stirred for 15 minutes and left to settle for another 15 minutes.

[0328] Then, an additional washing procedure using the same volume of water as the volume of solvent used for the extraction (160, 200, or 300 mL) was applied to remove the residual deep euterctics from the extract and further increase the camosic acid concentration. The solvent-free eutectic extract that has been freeze-dried overnight finally contained between 1 and 18% of carnosic acid (HPLC quantification) (FIG. 14b). The final mass yield ranged from 0.1 to 1.7% (FIG. 14c), which is still well acceptable regarding industrial standards.

Example 17

Extraction of Rosemary Using Betaine:Sorbitol DES at 60° C. Cake Removal by Filtration, Separation of the Extract from the Deep Eutectics Using Water as an Anti-Solvent, Recovery of the Crude Extract by Centrifugation, and Removal of Residual Deep Eutectics by a Washing Procedure with Water

[0329] Homogeneous solutions of 1:2 betaine:sorbitol mixtures containing 50% of water (BS1/2_50% w) and solutions of 1:1 betaine:sorbitol mixtures containing 20% (BS1/1_20% w), and 50% (BS1/1_50% w) of water were prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at 60° C. with 160 mL (plant:solvent 1:8) or 200 mL (plant:solvent 1:10) of this deep eutectic solution for 0.5, 1, or 2 hours and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be comprised between 3.9 and 13.1% (FIG. 15a). Then, the filtrate was diluted by 4 volumes of water at room temperature. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the deep, eutectics from the extract. The resulting precipitate was magnetically stirred for 15 minutes and left to settle for another 15 minutes.

[0330] Then, an additional washing procedure using the same volume of water as the volume of solvent used for the extraction (160 or 200 mL) was applied to remove the residual deep eutectics from the extract and further increase the carnosic acid concentration. The solvent-free eutectic extract that has been freeze-dried overnight finally contained between 1 and 18% of carnosic acid (HPLC quantification) (FIG. 15b). The final mass yield ranged from 0.1 to 1.7% (FIG. 15c), which is still well acceptable regarding industrial standards.

Example 18

Extraction of Rosemary Using Sorbitol:Levulinic Acid DES, Cake Removal by Filtration, Separation of the Extract from the Deep Eutectics Using Water as an Anti-Solvent, Recovery of the Crude Extract by Centrifugation, and Removal of Residual Deep Eutectics by a Washing Procedure with Water

[0331] Homogenous solutions of 1:1 sorbitol:levulinic acid mixtures containing 20% (SLe1/1_20% w) or 30% (SLe1/1_30% w) of water were prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 200 mL (plant solvent: 1:10) of this deep eutectic solution for 0.5 hour, and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be 58 and 27%, respectively (FIG. 16a) Then, the filtrate was diluted by 4 volumes of water at room temperature. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the deep eutectics from the extract. The resulting precipitate was magnetically stirred for 15 minutes and left to settle for another 15 minutes.

[0332] Then, an additional washing procedure using 200 mL of water was applied to remove the residual deep eutectics from the extract and further increase the carnosic acid concentration. The solvent-free eutectic extract that has been freeze-dried overnight finally contained 36% of carnosic acid for SLe1/1_20% w and 31% for SLe1/1_30% w (HPLC quantification) (FIG. 16b). The final mass yield was found to be 1.1 and 0.5, respectively (FIG. 16c), which is still well acceptable regarding industrial standards.

Example 19

Extraction of Rosemary Using Betaine:Proline DES at Room Temperature, Cake Removal by Filtration, Separation of the Extract from the Deep Eutectics Using Water as an Anti-Solvent, Recovery of the Crude Extract by Centrifugation, and Removal of Residual Deep Eutectics by a Washing Procedure with Water

[0333] A homogenous solution of 1:2 betaine:proline mixture containing 30% of water was prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 200 mL (plant:solvent: 1:10) of this deep eutectic solution for 0.5, 1, or 2 hours, and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be between 14 and 24% (FIG. 17a). Then, the filtrate was diluted by 4 volumes of water at room temperature. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the deep eutectics from the extract. The resulting precipitate was magnetically stirred for 15 minutes and left to settle for another 15 minutes.

[0334] Then, an additional washing procedure using 200 mL of water was applied to remove the residual deep eutectics from the extract and further increase the carnosic acid concentration. The solvent-free eutectic extract that has been freeze-dried overnight finally contained between 10 and 16% of carnosic acid (HPLC quantification) (FIG. 17b). The final mass yield was found to be between 0.1 and 1.7 (FIG. 17c), which is still well acceptable regarding industrial standards.

Example 20

Extracton of Rosemary Using Betaine:Proline DES at 60° C. Cake Removal by Filtration, Separation of the Extract from the Deep Eutectics Using Water as an Anti-Solvent Recovery of the Crude Extract by Centrifugation, and Removal of Residual Deep Eutectics by a Washing Procedure with Water

[0335] A homogenous solution of 1:2 betaine:proline mixture containing 30% of water was prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at 60° C. with 200 mL (plant:solvent; 1:10) of this deep eutectic solution for 0.5, or 2 hours, and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be 38% for the extraction performed after 2 hours (FIG. 18a). Unfortunately, the recovery rate has not been evaluated for the extraction performed after 0.5 hour.

[0336] Then, the filtrate was diluted by 4 volumes of water at room temperature. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the deep eutectics from the extract. The resulting precipitate was magnetically stirred for 15 minutes and left to settle for another 15 minutes.

[0337] Then, an additional washing procedure using 200 mL of water was applied to remove the residual deep eutectics from the extract and further increase the carnosic acid concentration. The solvent-free eutectic extract that has been freeze-dried overnight finally contained 21% (0.5 h) and 20% (2 h) of carnosic acid HPLC quantification) (FIG. 18b). The final mass yield was found to be 2.9% in both cases (FIG. 18c).

Example 21

Extraction of Rosemary Using Proline:Glucose DES, Cake Removal by Filtration, Separation of the Extract from the Deep Eutectics Using Water as an Anti-Solvent, Recovery of the Crude Extract by Centrifugation, and Removal of Residual Deep Eutectics by a Washing Procedure with Water

[0338] Homogenous solutions of 1:1 proline:glucose mixtures containing 20% (PGlu1/1_20% w) or 30% (PGlu1/1_30% w) of water were prepared as well as 1:2 proline:glucose (PGlu1/2_20% w), 2:1 proline:glucose (PGlu2/1_20% w), and 3:1 proline:glucose (PGlu3/1_20% w) mixtures each containing 20% of water. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature, 60° C, or 100° C. with 200 mL (plant:solvent: 1:10) of this deep eutectic solution for 0.5, 1, or 2 hours, and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be between 6 and 22% (FIG. 19a). Then, the filtrate was diluted by 4 volumes of water at room temperature. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the deep eutectics from the extract. The resulting precipitate was magnetically stirred for 15 minutes and left to settle for another 15 minutes.

[0339] Then, an additional washing procedure using 200 mL of water was applied to remove the residual deep eutectics from the extract and further increase the carnosic acid concentration. The solvent-free eutectic extract that has been freeze-dried overnight finally contained between 4 and 23% of carnosic acid (HPLC quantification) (FIG. 19b). The final mass yield was found to be between 0.3 and 3% (FIG. 19c), which is still well acceptable regarding industrial standards.

Example 22

Extraction of Rosemary Using Betaine:Glucose DES, Cake Removal by Filtration, Separation of the Extract from the Deep Eutectics Using Water as an Anti-Solvent Recovery of the Crude Extract by Centrifugation, and Removal of Residual Deep Eutectics by a Washing Procedure with Water

[0340] Homogenous solutions of 1:2 betaine:glucose mixtures containing 30% (BGlu1/2_30% w) or 50% (BGlu1/2_50% w) of water were prepared as well as 1:1 betaine:glucose (BGlu1/2_20% w), 2:1 betaine:glucose (BGlu2/1_20% w), and 3:1 betaine:glucose (BGlu3/1_20% w) mixtures each containing 20% of water. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at 60° C. with 200 mL (plant:solvent 1:10) of this deep eutectic solution for 0.5, 1, or 2 hours, and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be between 1 and 35% (FIG. 20a). Then, the filtrate was diluted by 4 volumes of water at room temperature. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the deep eutectics from the extract. The resulting precipitate was magnetically stirred for 15 minutes and left to settle for another 15 minutes.

[0341] Then, an additional washing procedure using 200 mL of water was applied to remove the residual deep eutectics from the extract and further increase the carnosic acid concentration. The solvent-free eutectic extract that has been freeze-dried overnight finally contained between 1 and 22% of carnosic acid (HPLC quantification) (FIG. 20b). The final mass yield was found to be between 1.1 and 1.7% (FIG. 20c).

Example 23

Extraction of Rosemary Using Betaine:Pyruvic Acid DES, Cake Removal by Filtration, Separation of the Extract from the Deep Eutectics Using Water as an Anti-Solvent, Recovery of the Crude Extract by Centrifugation, and Removal of Residual Deep Eutectics by a Washing Procedure with Water

[0342] A homogenous solution of 1.2 betaine:pyruvic acid mixture containing 25% of water was prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 160 mL (plant:solvent: 1:8), 200 mL (plant:solvent 1:10), or 300 mL (plant:solvent: 1:15) of this deep eutectic solution for 0.5 hour, and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be 34, 44, and 56%, respectively (FIG. 21a).

[0343] Then, the filtrate was diluted by 4 volumes of water at room temperature. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the deep eutectics from the extract. The resulting precipitate was magnetically stirred for 15 minutes and left to settle for another 15 minutes.

[0344] Then, an additional washing procedure using the same volume of water as the volume of solvent used for the extraction (160, 200, or 300 mL) was applied to remove the residual deep eutectics from the extract and further increase the carnosic acid concentration. The solvent-free eutectic extract that has been freeze-dried overnight finally contained 39% (plant:solvent: 1:8), 44% (plant:solvent: 1:10), and 42% (plant:solvent: 1:15) of carnosic acid (HPLC quantification) (FG. 21b). The final mass yield was found to be 1.6, 2.1, and 2.3%, respectively (FIG. 18c).

Example 24

Extraction of Rosemary Using Urea:Choline chloride DES, Cake Removal by Centrifugation then Filtration, Separation of the Extract from the Deep Eutectics Using Water as an Anti-Solvent, and Recovery of the Crude Extract by Centrifugation

[0345] A homogeneous solution of a 2:1 urea:choline chloride neat mixture (UChCl2/1_0% w) was prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 200 mL (plant:solvent: 1:10) of this deep eutectic solutions for one hour, and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be 13% (FIG. 22a, sample ‘UChCl2/1_0% w_10M_RT_1h_9vol_crude’). Then, the filtrate was diluted by 9 volumes of water. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the deep eutectics from the extract. The resulting precipitate was magnetically stirred for 15 minutes and left to settle for another 15 minutes. The precipitate was recovered by centrifugation and freeze-dried overnight. Finally, dried pellets were collected. They contained 6% of carnosic acid (HPLC quantification) (FIG. 22b). The final mass yield was 2.1% (FIG. 22c).

Example 25

Extraction of Rosemary Using Glycerol:Lactic Acid DES, Cake Removal by Filtration, Separation of the Extract from the Deep Eutectics Using Water as an Anti-Solvent, Recovery of the Crude Extract by Centrifugation, and Removal of Residual Deep Eutectics by a Washing Procedure with Water

[0346] A homogeneous solution of a 1:1 glycerol:lactic acid mixture containing 20% of water (GlyLa1/1_20% w) was prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 200 mL (plant:solvent:1:10) of this deep eutectic solution for 0.5 hour, and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be 24% (FIG. 22a). Then, the filtrate was diluted by 4 volumes of water at room temperature. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the deep eutectics from the extract. The resulting precipitate was magnetically stirred for 15 minutes and left to settle for another 15 minutes.

[0347] Then, an additional washing procedure using 200 ml of water was applied to remove the residual deep eutectics from the extract and further increase the carnosic acid concentration. The solvent-free eutectic extract that has been freeze-dried overnight finally contained 19% of carnosic acid (HPLC quantification) (FIG. 22b). The final mass yield was found to be 1.6% (FIG. 22c).

Example 26

Extraction of Rosemary Using Glycerol:Levulinic Acid DES, Cake Removal by Filtration, Separation of the Extract from the Deep Eutectics Using Water as an Anti-Solvent, Recovery of the Crude Extract by Centrifugation, and Removal of Residual Deep Eutectics by a Washing Procedure with Water

[0348] A homogeneous solution of a 1:1 glycerol:levulinic acid mixture containing 20% of water (GlyLe1/1_20% w) was prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 200 mL (plant:solvent 1.10) of this deep eutectic solution for 0.5 hour, and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be 79% (FIG. 22a). Then, the filtrate was diluted by 4 volumes of water at room temperature. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the deep eutectics from the extract. The resulting precipitate was magnetically stirred for 15 minutes and left to settle for another 15 minutes.

[0349] Then, an additional washing procedure using 200 mL of water was applied to remove the residual deep eutectics from the extract and further increase the carnosic acid concentration. The solvent-free eutectic extract that has been freeze-dried overnight finally contained 33% of carnosic acid (HPLC quantification) (FIG. 22b). The final mass yield was found to be 0.2% (FIG. 22c).

Example 21

Extraction of Rosemary Using Sorbitol:Lactic Acid DES, Cake Removal by Filtration, Separation of the Extract from the Deep Eutectics Using Water as an Anti-Solvent, Recovery of the Crude Extract by Centrifugation, and Removal of Residual Deep Eutectics by a Washing Procedure with Water

[0350] A homogeneous solution of a 1:1 sorbitol:lactic add mixture containing 20% of water (SLa1/1_20% w) was prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 200 mL (plant:solvent:1:10) of this deep eutectic solution for 0.5 hour, and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be 7% (FIG. 22a). Then, the filtrate was diluted by 4 volumes of water at room temperature. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the deep eutectics from the extract. The resulting precipitate was magnetically stirred for 15 minutes and left to settle for another 15 minutes.

[0351] Then, an additional washing procedure using 200 mL of water was applied to remove the residual deep eutectics from the extract and further increase the carnosic acid concentration. The solvent-free eutectic extract that has been freeze-dried overnight finally contained 8% of carnosic acid (HPLC quantification) (FIG. 22b). The final mass yield was found to be 0.1% (FIG. 22c).

Example 28

Extraction of Rosemary Using Xylitol:Levulinic Acid DES, Cake Removal by Filtration, Separation of the Extract from the Deep Eutectics Using Water as an Anti-Solvent Recovery of the Crude Extract by Centrifugation, and Removal of Residual Deep Eutectics by a Washing Procedure with Water

[0352] A homogeneous solution of a 1:1 xylitol:levulinic acid mixture containing 20% of water (XLe1/1_20% w) was prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 200 mL (plant solvent:1:10) of this deep eutectic solution for 0.5 hour, and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be 67% (FIG. 22a). Then, the filtrate was diluted by 4 volumes of water at room temperature. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the deep eutectics from the extract. The resulting precipitate was magnetically stirred for 15 minutes and left to settle for another 15 minutes.

[0353] Then, an additional washing procedure using 200 mL of water was applied to remove the residual deep eutectics from the extract and further increase the carnosic acid concentration. The solvent-free eutectic extract that has been freeze-dried overnight finally contained 33% of carnosic acid (HPLC quantification) (FIG. 22b). The final mass yield was found to be 0.4% (FIG. 22c).

Example 29

Extraction of Rosemary Using Glucose:Levulinic Acid DES, Cake Removal by Filtration, Separation of the Extract from the Deep Eutectics Using Water as an Anti-Solvent Recovery of the Crude Extract by Centrifugation, and Removal of Residual Deep Eutectics by a Washing Procedure with Water

[0354] A homogeneous solution of a 1:2 glucose:levulinic acid mixture containing 30% of water (GluLe1/2_30% w) was prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 200 mL (plant:solvent:1:10) of this deep eutectic solution for 0.5 hour, and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be 77% (FIG. 22a). Then, the filtrate was diluted by 4 volumes of water at room temperature. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the deep eutectics from the extract. The resulting precipitate was magnetically stirred for 15 minutes and left to settle for another 15 minutes.

[0355] Then, an additional washing procedure using 200 mL of water was applied to remove the residual deep eutectics from the extract and further increase the carnosic acid concentration. The solvent-free eutectic extract that has been freeze-dried overnight finally contained 28% of carnosic acid (HPLC quantification) (FIG. 22b). The final mass yield was found to be 0.2% (FIG. 22c).

Example 30

Extraction of Rosemary Using Proline:Levulinic Acid DES, Cake Removal by Filtration, Separation of the Extract from the Deep Eutectics Using Water as an Anti-Solvent, Recovery of the Crude Extract by Centrifugation, and Removal of Residual Deep Eutectics by a Washing Procedure with Water

[0356] A homogeneous solution of a 1:1 proline:levulinic acid mixture containing 20% of water (PLe1/1_20% w) was prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 200 mL (plant:solvent:1.10) of this deep eutectic solution for 0.5 hour, and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be 95% (FIG. 22a). Then, the filtrate was diluted by 4 volumes of water at room temperature. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the deep eutectics from the extract. The resulting precipitate was magnetically stirred for 15 minutes and left to settle for another 15 minutes.

[0357] Then, an additional washing procedure using 200 mL of water was applied to remove the residual deep eutectics from the extract and further increase the carnosic acid concentration. The solvent-free eutectic extract that has been freeze-dried overnight finally contained 38% of carnosic acid (HPLC quantification) (FIG. 22b). The final mass yield was found to be 4.1% (FIG. 22c).

Example 31

Extraction of Rosemary Using Lysine:Levulinic Acid DES, Cake Removal by Filtration, Separation of the Extract from the Deep Eutectics Using Water as an Anti-Solvent, Recovery of the Crude Extract by Centrifugation, and Removal of Residual Deep Eutectics by a Washing Procedure with Water

[0358] A homogeneous solution of a 1:1 lysine:levulinic acid mixture containing 20% of water (PLe1/1_20% w) was prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at 60° C. with 200 mL (plant:solvent: 1:10) of this deep eutectic solution for 0.5 hour, and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be 53% (FIG. 22a). Then, the filtrate was diluted by 4 volumes of water at room temperature. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the deep eutectics from the extract. The resulting precipitate was magnetically stirred for 15 minutes and left to settle for another 15 minutes.

[0359] Then, an additional washing procedure using 200 mL of water was applied to remove the residual deep eutectics from the extract and further increase the carnosic acid concentration. The solvent-free eutectic extract that has been freeze-dried overnight finally contained 32% of carnosic acid (HPLC quantification) (FIG. 22b). The final mass yield was found to be 2.9% (FIG. 22c).

Example 32

Extraction of Rosemary Using Urea:Betaine HCI DES, and Cake Removal by Centrifugation then Filtration

[0360] A homogeneous solution of a 2:1 urea:betaine HCl neat mixture (UBHCl2/1) was prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 200 mL (plant:solvent: 1:10) of this deep eutectic solution for one hour, and the enriched solution was separated from the plant by centrifugation, then filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be 10% (FIG. 23).

Example 33

Extraction of Rosemary Using Betaine:Glycerol DES, and Cake Removal by Filtration

[0361] Homogeneous solutions of 1:2 betaine:glycerol mixtures containing 10% (BGly1/2_10% w) and 30% (BGly1/2_30% w) of water was prepared as well as 1:1 betaine:glycerol mixtures containing 20% of water (BGly1/1_20% w), and 2:1 betaine:glycerol mixtures containing 30% of water (BGly2/1_30% w). Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 200 mL (plant:solvent: 1:10) of this deep eutectic solution for one hour, and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be comprised between 19 and 33% (FIG. 23).

Example 34

Extraction of Rosemary Using Malic Acid:Choline Chloride DBS, and Cake Removal by Filtration

[0362] Homogeneous solutions of 1:1 malic acid:choline chloride mixtures containing 20% (MChCl1/1_20% w) and 30% (MChCl1/1_30% w) of water was prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 200 mL (plant:solvent: 1.10) of this deep eutectic solution for one hour, and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be comprised between 8 and 18% (FIG. 23).

Example 35

Extraction of Rosemary Using Glycerol:Choline Chloride DES, and Cake Removal by Filtration

[0363] Homogeneous solutions of a 2:1 glycerol:choline chloride neat mixture (GlyChCl2/1_0% w), a 1:1 glycerol:choline chloride mixture containing 10% of water (GlyChCl1/1_10% w), and a 1:2 glycerol:choline chloride mixture containing 20% of water (GlyChCl1/2_20% w)_were prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 200 mL (plant:solvent: 1:10) of this deep eutectic solution for one hour, and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be composed between 9 and 13% (FIG. 23).

Example 36

Extraction of Rosemary Using Glycerol:Sorbitol DES, and Cake Removal by Filtration

[0364] A homogeneous solution of a 1:1 gycerol:sorbitol mixture containing 20% of water (GlyS1/1_20% w) was prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 200 mL (plant.solvent:1:10) of this deep eutectic solution for 0.5 hour, and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be 4% (FIG. 23).

Example 37

Extraction of Rosemary Using Lysine:Levulinic Acid DES, and Cake Removal by Filtration

[0365] A homogeneous solution of a 1:2 lysine:levulinic acid mixture containing 20% of water (LLe1/2_20% w) was prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 200 mL (plant:solvent:1:10) of this deep eutectic solution for 0.5 hour, and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be 87% (FIG. 23).

Example :38

Extraction of Rosemary Using Betaine:Proline DES, and Cake Removal by Filtration

[0366] A homogeneous solution of a 1:1 betaine:proline mixture containing 30% of water (BPr1/1_30% w) was prepared. Twenty grams of ground rosemary leaves (containing 2.77% carnosic acid) were incubated at room temperature with 200 mL (plant:solvent:1:10) of this deep eutectic solution for 0.5 hour, and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was found to be 14 (FIG. 23).

Example 39

Negative Controls Showing the Importance of the DES Combination for the Precipitation/Flocculation Step

[0367] To further verify that the process of the invention necessitates the combination of two molecules (hence the formation of a DES), such as betaine:levulinic acid far example, we tested some negative control for the precipitation step. Indeed, we reproduced the exact same process as for 1:2 betaine:levulinic acid containing 10% or 25% of water on the sole levulinic acid without any betaine added in the mixture (Table 2). Ground rosemary leaves were incubated in these mixtures at a plant:solvent weight ratio of 1:10 for one hour at room temperature. While the first step of extraction was successfully performed with carnosic acid recovery rates ranging from 84 to 93%, then it was not possible to recover any crude extract by filtration or centrifugation after we applied the standard precipitation step with the addition of water.

[0368] Then, we directly used pure levulinic acid without any water addition (nor betaine) and performed the extraction on ground rosemary leaves at a plant:solvent weight ratio of 1:10 for one hour at room temperature. Again, we were unable to collect the precipitate formed (if any) with the same procedure that the one currently applied on DES.

[0369] A last verification was done by using betaine alone containing 40% of water (near the saturation level in water) using the same extraction and precipitation procedure as for levulinic acid alone except that the extraction duration was 0.5 h. Although 9.2% of carnosic acid was extracted, again, the collect of the precipitate (if any) was unsuccessful.

TABLE-US-00002 TABLE 2 Negative controls used for the precipitation/flocculation step CA Vol. of Quantity of extract Studied Water Plant:solvent recovery water to collected after molecule content ratio Time rate (%) precipitate precipitation (mg) Levulinic 10% 1:10 1 h 84.5 4 0 acid Levulinic 10% 1:10 1 h 87.0 4 0 acid Levulinic 25% 1:10 1 h 92.7 4 0 acid Levulinic  0% 1:10 1 h 97.9 4 0 acid Betaine 40% 1:10 0.5 h 9.2 4 0

[0370] These negative controls demonstrate in the example of levulinic acid:betaine DES that the combination of the two compounds is absolutely required to obtain a crude or a purified rosemary extract, because no extract can be collected when either levulinic acid alone or betaine alone are used instead of the corresponding DES.

Example 40

Antioxidant Activity (Rancimat Method) of the Crude and Purified Eutectic Extracts of Rosemary Obtained with Urea:Betaine Deep Eutectics and Formulated in a Sunflower Oil

[0371] The crude (non-washed) and purified (washed) rosemary extracts obtain using the process of the invention described in Examples 7 and 8 with urea betaine deep eutectics were assessed for their antioxidant activity by the Rancimat method. Extracts were solubilized in sunflower oil at a final concentration of 1000 mg/kg, and were introduced in glass tubes in the Rancimat apparatus. The samples were heated at 110° C. and flushed with air at a flux of 10 L/h to accelerate the oxidation kinetics. The conductivity of the collected headspace allows to precisely evaluate the lag phase that passes until oxidation significantly increases (induction time). By dividing the induction time of the sample treated with a rosemary extract by that of an untreated sunflower oil control, we can determine the protection factor (PF). A PF of 1 shows that the extract is non-antioxidant, while a PF with a higher value demonstrates an antioxidant activity. Here we show that three different crude rosemary extracts obtained using 2:1 urea:betaine or 1:1 urea:betaine mixtures near their saturation levels (950 and 957 g/L. respectively) exhibit a significant antioxidant activity in oil formulation with PFs between 1.1 and 1.2 (FIG. 24). When these crude extracts are washed to yield purified rosemary extracts, the PF drastically increases to reach values ranging from 1.8 to 2.0. This means that by applying the process of the invention with the additional washing procedure, we doubled the oxidative stability of an oily formulation (here a sunflower oil).

Example 41

Antioxidant Activity (Rancimat Method) of the Crude and Purified Eutectic Extracts of Rosemary Obtained with Betaine:Levulinic Acid DES and Formulated in a Sunflower Oil

[0372] With the same Rancimat protocol as described in Example 40, we show that seven different purified rosemary extracts obtained using 1:2 betaine:levulinic acid DES exhibit a significant antioxidant activity in oil formulation with PFs between 2.7 and 3 (FIG. 25). This means that by applying the process of the invention with the additional washing procedure, we can tripled the oxidative stability of an oily formulation (here a sunflower oil). Interestingly, we shows a standard rosemary extract obtained using a traditional extraction with acetone followed by a complex purification process. The extracts of the invention are all-comparable or even better that this rosemary extract reference.

Example 42

Correlation between the Protection Factor Measured using the Rancimat Assay and the Carnosic Acid Content in the Final Extract (%)

[0373] Here we plotted the the protection factor measured using the Rancimat assay and exemplified in Example 40 and 41 as a function of the carnosic acid content in the final extract (%). A linear positive correlation that can be mathematically expressed as y=0.0523x+0.5173 was found with a R.sup.2 of 0.96 (FIG. 26). This means that the higher the carnosic acid content in the final extract, the higher the corresponding antioxidant activity.

Example 43

Effect of the Content of Salt (NaCl) in Water on the Carnosic Acid (CA) Hydrosolubility

[0374] To further improve the washing procedure that leads to a high carnosic acid content in the final extract and, concomitantly, a strong antioxidant activity, here we studied the effect of the addition of sodium chloride on the CA hydrosolubility. Results shown in FIG. 27 clearly demonstrate that salt can help to reduce the amounts of CA lost during the washing procedure. Indeed, the water solubility of CA is strongly reduced by adding 3 g/L of salt both in distilled water and in acidified distilled water. Thus, the amount of CA retrieved after centrifugation following the washing procedure could be strongly improved by adding salt.

[0375] This salt addition at the washing step has been performed on an extraction of camosic acid from ground rosemary leaves for 0.5 h at room temperature and at a plant:solvent weight ratio of 10 using a 1:2 betaine:levulinic acid mixture containing 25% of water. After filtration, the carnosic acid recovery rate was found to be 79%. We then added 9 volumes of water at room temperature under magnetic stirring for 15 min and we let for settling for other 15 minutes. At the end of the procedure, we used water added with sodium chloride for the washing step (3.5%). The final carnosic acid content of the purified extract was found to be 38%, while the final mass yield was 4.5%. With this verification, we demonstrated that the addition of salt is compatible with the obtention of an extract.

[0376] Besides the washing procedure, the addition of salt can also help during the precipitation/flocculation step by using water added with 3 g/L or more of salt as an antisolvent. This could minimize the CA amounts lost during the precipitation.

Example 44

Extraction of Ground Sage Leaves (1 h), Cake Removal by Centrifugation then Filtration, Separation of the Extract from the Deep Eutectics Using Water as an Anti-Solvent, Recovery of the Crude Extract by Centrifugation, and Removal of Residual Deep Eutectics by a Washing Procedure with Water

[0377] A homogeneous aqueous solution of a 2:1 urea:betaine mixture was prepared near the saturation level (950 g/L). Twenty grams of sage (Salvia officinalis) leaves (containing 1.75% carnosic acid) were incubated at ambient temperature with 200 mL of this deep eutectic solution for 1 h and the enriched solution was separated from the plant by centrifugation followed by a filtration. The filtrate was analyzed by HPLC and the concentration of carnosic acid was found to be 0.67 g/L, which corresponds to a recovery rate of 28.6%. Then, the filtrate was diluted by water by a factor 10. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the deep eutectics from the extract. The resulting precipitate was magnetically stirred for 15 minutes and let for settling for other 15 minutes. The precipitate was recovered by centrifugation and dried overnight at 45° C. in a vacuum oven. Finally, 573 mg of dried pellets was collected (mass yield: 2.86%), containing 7.30% of carnosic acid (HPLC quantification), which corresponds to a final recovery rate of 11.92% relative to the sage starting material.

[0378] This process was then reproduced in a separate experiment. A recovery rate of carnosic acid of 31.6% was observed in the filtrate after removal of sage residues, which is comparable to what was obtained previously. Then, an additional washing procedure using 200 mL of water was applied to remove the residual deep eutectics from the extract and further increase the carnosic acid concentration. The solvent-free eutectic extract that has been dried overnight at 45° C. in a vacuum oven final contained 14.3% of carnosic acid (HPLC quantification). The final mass yield was 1.22%, with a global recovery rate of carnosic acid of 9.95%. The extract is easily recoverable and forms a fluid powder that can be further ground.

Example 45

Extraction of Ground Turmeric Roots (1 h), Cake Removal by Filtration, Separation of the Extract from the Deep Eutectic Solvent Using Water as an Anti-Solvent Recovery of the Crude Extract by Centrifugation, and Removal of Residual Deep Eutectic Solvent by a Washing Procedure with Water

[0379] Homogeneous aqueous solutions of 1:1 sorbitol:levulinic acid mixture containing 20% water (SLe1/1_20% w), 1:1 proline:levulinic acid mixture containing 20% water (PLe1/1_20% w), 1:2 betaine:glycerol mixture containing 30% water (BGly1/2_30% w), 1.2 betaine:levulinic acid mixture containing 25% water (BLe1/2_25% w), and 1:2 betaine:lactic acid mixture containing 10% water (BLa1/2_10% w) were prepared. Twenty grams of ground turmenc roots (Curcuma longa) leaves (containing 2.93% curcuminoids and 1.74% curcumin) were incubated at ambient temperature or 60° C. with 200 mL of each DES (plant-solvent ratio: 1:10) for one hour and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was comprised between 9 and 117% for curcuminoids and between 7 and 112% for curcumin (FIG. 27a). Then, the filtrate was diluted with four volumes of water. This step of using water as an anti-solvent to precipitate the active components that will then constitute the crude extract is essential to separate most of the DES from the extract. The resulting precipitate was magnetically stirred for 15 min and let for settling for other 15 min. The precipitate was recovered by centrifugation and then washed using 200 mL of acidic water to remove the residual DES from the extract and further increase the carnosic acid concentration. Again a centrifugation step enables to recover the pellets which constitute the solvent-free eutectic extracts. These latters have been freeze-dried overnight and finally contained between 21 and 46% of curcuminoids and between 12 and 26% for curcumin (HPLC quantification) (FIG. 27b). The final mass yield was between 0.2 and 3.9% (FIG. 27c).

Example 46

Chemical Composition of the Purified (Washed) Turmeric Extract Obtained in Example 45 Using a 1:2 Betaine:Lactic Acid DES

[0380] Table 3 shows some of the identified compounds found in a eutectic turmeric extract obtained using a 1:2 betaine:lactic acid containing 10% of water and described in Example 45 and FIG. 27 (sample ‘BLa1/2_10% w_10M_1h_4vol_1 washed’).

TABLE-US-00003 TABLE 3 Identified compounds in a eutectic turmeric purified extract Identified compounds in the eutectic extract Formula Chemical family Ar-turmerone C.sub.15H.sub.21O Sesquiterpene Curcumin C.sub.21H.sub.20O.sub.6 Curcuminoid Dihydrocurcumin C.sub.21H.sub.22O.sub.6 Curcuminoid Demethoxycurcumin C.sub.20H.sub.18O.sub.5 Curcuminoid Dehydrodemethoxycurcumin C.sub.20H.sub.20O.sub.5 Curcuminoid Bisdemethoxycurcumin C.sub.19H.sub.18O.sub.4 Curcuminoid Dehydro bisdemethoxycurcumin C.sub.19H.sub.18O.sub.4 Curcuminoid Curcumadione C.sub.15H.sub.23O.sub.2 Sesquiterpene Procurcumadiol C.sub.15H.sub.23O.sub.3 Sesquiterpene Dehydrocurdione C.sub.15H.sub.23O.sub.2 Sesquiterpene Deoxy bisdemethoxycurcumin C.sub.19H.sub.16O.sub.3 Curcuminoid Deoxy dehydrobisdemethoxycurcumin C.sub.19H.sub.18O.sub.3 Curcuminoid O-Demethyldemethoxycurcumin C.sub.19H.sub.16O.sub.5 Curcuminoid

Example 47

Extraction of Ground Orange (Citrus sinensis) peel (1 h) and Cake Removal by Filtration

[0381] Homogeneous aqueous solutions of 1:1 sorbitol:levulinic acid mixture containing 20% water (SLe1/1_20% w), 1:1 proline:levulinic acid mixture containing 20% water (PLe1/1_20% w), 1:2 betaine:glycerol mixture containing 30% water (BGly1/2_30% w), 1:2 betaine:levulinic add mixture containing 25% water (BLe1/2_25% w), and 1:2 betaine:lactic acid mixture containing 10% water (BLa1/2_10% w) were prepared. Twenty grams of ground orange peel (containing 2.67% hesperidin) were incubated at ambient temperature with 200 mL of each DES (plant:solvent ratio: 1:10) for one hour and the enriched solution was separated from the plant by filtration. The filtrate was analyzed by HPLC and the recovery rate was comprised between 49 and 93% for hesperidin (FIG. 29).