Eutectic extract formation and purification

Abstract

The present invention relates to processes for forming eutectic extracts, processes for purifying eutectic extracts and uses of the eutectic extracts such as in food-stuffs, pharmaceuticals, nutraceuticals and supplements, such as food supplements and sports supplements.

Claims

1. A process for the purification of a eutectic extract or eutectic combination comprising: (i) mixing a eutectic extract or eutectic combination with a liquid that is immiscible in the eutectic extract or eutectic combination to form a mixture; (ii) allowing the mixture formed in step (i) to equilibrate into two phases; and (iii) separating the eutectic phase from the phase comprising the liquid that is immiscible in the eutectic extract or eutectic combination, wherein the liquid that is immiscible in the eutectic extract or eutectic combination is a lipid phase in a liquid form, a terpenic solvent, or an alcohol.

2. The purification process according to claim 1, wherein steps (i) to (iii) are repeated using the separated eutectic phase as the eutectic extract or eutectic combination in step (i) of the purification process.

3. The purification process according to claim 1, wherein the eutectic extract or eutectic combination may be prepared by (a) forming a mixture between at least one exogenous amine and biological material which is at least one of plant material, animal material, or prokaryotic biological material, (b) allowing the mixture to interact to form a liquid, gel or gel-like eutectic extract, and optionally, separating the liquid or gel/gel-like mixture from undissolved solids, or may be a eutectic extract or eutectic combination that has been previously prepared by extracting a biological material using a pre-prepared eutectic solvent.

4. The purification process according to claim 1, wherein the lipid phase in a liquid form is selected from free fatty acids, free fatty alcohol, triacylglycerols, and/or vegetable oil.

5. The purification process according to claim 1, wherein the terpene solvent is limonene or para-menthane.

6. The purification process according to claim 1, wherein the weight ratio of eutectic extract to immiscible liquid is from about 1:0.1 to about 1:10.

7. The purification process according to claim 1, wherein step (iii) separates the eutectic phase from the phase comprising the liquid that is immiscible in the eutectic extract by removing exogenous amine that has not formed a complex with the endogenous compounds in the biological material.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Visual representations of the eutectic solvents obtained using the process of the invention.

(2) FIG. 2: Content of selected antioxidants extracted from dried rosemary biomass with pure amines compared with Eutectys and hydroglycerinated extractants, measured by H PLC.

(3) FIG. 3. LC-MS chromatogram (ESI-TIC) of the Choisya eutectic extracts obtained through eutectigenesis-assisted extraction of Choisya leaves by ChCl (1:2) and BTEAC (1:2).

(4) FIG. 4. LC-MS chromatogram of the lime eutectic extract obtained through solid-state extraction of rind and juice of lime by ChCl.

(5) FIG. 5. Effect of the purification process of Cedrus bark eutectic extracts on their ORAC and CAT values (A), BTEAC content (B) and total peak area of compounds absorbing at 280 and 350 nm (C).

(6) FIG. 6. Effect of the purification process of rosemary eutectic extracts on the ORAC and CAT values (A) and the content of some phenolics (B) and BTEAC, water and oleic acid (C).

(7) FIG. 7. Effect of the purification process on the ORAC values of Ziziphus eutectic extracts.

EXAMPLES

(8) The present invention will be further described by reference to the following, non-limiting examples.

Example 1—General Procedure for the Preparation of Eutectic Extract

(9) Different parts of naturally occurring plants as well as fruits and plant barks were used to form deep eutectics with different amines.

(10) The leaves used were sourced from rosemary (Rosmarinus officinalis) and Choisya×dewitteana ‘Aztec Pearl’ (Choisya ternate); fruits used were Lime (Citrus aurantifolia), Blueberry (Cyanococcus), Capsicum (Capsicum annuum), Green chilli (Capsicum frutescens) and Aubergine (Solanum melongena). Ginger (Zingiber officinale) was used as an example of plant root and barks of the Cedrus spp. tree was used as an example of bark.

(11) The desired amount of each was ground using a mortar and pestle in the presence of liquid nitrogen to make the material brittle and easier to shatter.

(12) Juicy fruits such as lime and blueberry were separated into juice and pulp by mechanical expulsion using a kitchen juicer and treated separately while Capsicum and green chilli were physically separated to seeds and skins before treatment. The aubergine skin was removed prior to treatment. The filtered juices were freeze dried (48 h) to give respective powders and some portion of the juices were also heat dried (50° C. for 48 h) to remove excess water.

(13) The ground powders thus obtained were sieved (Fisherbrand Test Sieve, 1 mm) to form homogeneous fine powders.

(14) Different amines were used to demonstrate the differences and specificities between aliphatic and aromatic amines: (i) Three quaternary ammonium salts: choline chloride (ChCl) (Sigma-Aldrich, >99%), and benzyltriethyl ammonium chloride (BTEAC), benzyltrimethyl ammonium chloride (BTMAC) (Sigma-Aldrich, 99%), (ii) one quaternary ammonium zwitterion: betaine (Sigma-Aldrich, 98%), (iii) and one dialkylimidazolium salt: 1-butyl-3-methylimidazolium chloride (Sigma-Aldrich, ≥98%) (BmimCl).

(15) The amines listed above in a solid state were mixed with the natural product powders in different mass ratios to produce in situ (i.e. during the extraction) eutectic mixtures by specifically interacting with the hydrogen bond donors (HBDs) present in the natural products. FIG. 1 illustrates some representative examples of the eutectic extracts formed in each of Examples 3A1 to 3A19 discussed below.

Example 2—General Procedure for the Analysis of Eutectic Extracts and Combination Measurement of the Antioxidant Activity and Identification and Quantification of Compounds of Interest by High Performance Liquid Chromatography (HPLC))

(16) Test of Physico-Chemical Efficacy as Antioxidant—Oxygen Radical Absorbance Capacity (ORAC) Method

(17) The capacity of the raw or purified eutectic extracts to trap peroxyradicals was determined using the reference method published by Ou et al. (Determination of total antioxidant capacity by oxygen radical absorbance capacity (ORAC) using fluorescein as the fluorescent probe: First action 2012.23. Journal of AOAC International, 2013, 96, 1372-1376) in the AOAC Official Journal. For information, the AOAC is the Association of Official Agricultural Chemists of the United States Department of Agriculture (USDA).

(18) Phosphate buffer solutions (pH 7.2) containing the desired concentrations of eutectic extracts were prepared. Fifty millilitres of each solution were transferred by multi-channel pipette into a Fluotrac 96-well microplate (Greiner). Each well was then topped up with 100 μL of phosphate buffer solution, pH 7.2, containing 0.126 μM of fluorescein disodiium salt.

(19) To improve the repeatability, the microplate was pre-heated at 37° C. under orbital stirring at 1,200 rpm in a temperature-controlled thermoshaker (PHMT series, Grant Instruments Ltd, Shepreth, England) for 20 minutes.

(20) 50 μL of AAPH solution in freshly prepared phosphate buffer solution was then added using a multi-channel pipette.

(21) Each well contained 200 μL of the final mixture which was composed of 0.063 M of fluorescein disodiium salt, 12.7 mM of AAPH and increasing concentrations of eutectic extracts in phosphate buffer solution.

(22) A drop in fluorescence to 515 nm (λex: 490 nm) was immediately recorded. Measurements were then taken every minute for 2 hours at 37±0.1° C. with 5 seconds of stirring before each measurement using a microplate reader. The results were then calculated according to Ou et al. Development and validation of an improved oxygen radical absorbance capacity using fluorescein as the fluorescent probe. J. Agric. Food Chem. 2001, 49, 4619-4626) in μmol of Trolox equivalent per g of liquid extract (ORAC value).

(23) General Method for the Conjugated Autoxidizable Triene (CAT) Assay Used in the Following Examples

(24) Antioxidant capacity of raw or purified eutectic extracts was measured using the CAT procedure described by (Laguerre M, López-Giraldo L J, Lecomte J, Baréa B, Cambon E, Tchobo P F, Barouh N, Villeneuve P. Conjugated autoxidizable triene (CAT) assay: a novel spectrophotometric method for determination of antioxidant capacity using triacylglycerol as ultraviolet probe. Anal Biochem. 2008, 380, 282-290) with slight modifications.

(25) Antioxidant solutions were prepared as follows: a methanol solution of eutectic extract or Trolox (reference) was prepared at the desired concentration. The eutectic extract solutions were filtered (0.45 μm) to remove impurities which can interfere in the CAT value measurement.

(26) Various volumes of this solution (25, 50, 75, and 100 μL) were then added to 24.9 mL of phosphate buffer solution (PBS), pH 7.2, and then filled to 25.0 mL with pure methanol (75, 50, 25, and 0 μL, respectively).

(27) In this way, all buffered solutions of eutectic extract contained the same methanol volume (100 μL), which minimizes any possible bias among samples.

(28) Samples of these solutions (50 μL/well) were transferred using a multichannel micropipet into UV-Star 96-well microplate (Greiner, Frickenhausen, Germany) (absorbance at 273 nm=0.03). The microplate was then prewarmed and stirred in a thermostated shaker (PHMT Grant Instruments, Shepreth, England) at 37° C. for 5 min at 1,200 rpm.

(29) Twenty-five milliliters of a buffered solution (pH 7.2) containing 34 μM Brij 35 (neutral emulsifier, estimated MW=1,198 g/mol) was added to 5 mg tung oil (non-stripped) in a brown glass flask.

(30) For the next step, this mixture was stirred for 10 s using a Vortex apparatus, before its homogenization in an Ultra Turrax homogenizer (Janke & Kunkel, Staufen, Germany) at approximately 2,400 rpm for 90 s.

(31) Each well was then filled with 100 μL of this tung oil-in-PBS emulsion. To improve repeatability, the microplate was then immediately pre-warmed and shaken, sheltered from light, in a thermostated shaker (PHMT Grant Instruments) at 37° C. for 1 min at 1,200 rpm.

(32) Fifty microliters of a freshly prepared AAPH solution in PBS (4 mM) was added with a multi-channel micropipet.

(33) In the end, each well contained 200 μL of the final mixture consisting of 115 μM stripped tung oil, 17 μm Brij 35, 1 mM AAPH, and various concentrations of Trolox (from 0 to 2 μM) and eutectic extracts in PBS. The progress of reactions was immediately monitored by recording the decrease in absorbance at 273 nm. Measurements were performed each minute for 5 h at 37±0.1° C., with 5 seconds of stirring before each measurement, using a Saffire 2 microplate reader (Tecan, Groedig, Austria) equipped with Magellan software.

(34) Each experiment was performed in triplicate (three wells), and results were expressed as the mean of CAT value (see below) ±SD.

(35) Similarly, to the ORAC assay, the antioxidant value of a sample was calculated through the difference between the area under the curve of this sample and that of the blank (without eutectic extract). The result of this operation gave the net area under the curve (AUC) which was then plotted on a graph as a function of the concentration. Only the linear part of the curve was taken into account to calculate the slope which was then divided by the slope of the Trolox (standard) calculated in the same conditions and analyzed on the same microplate. As such, CAT values were expressed as μmol Trolox equivalent/g eutectic extract.

(36) Example of HPLC-ESI/MS Analysis—Identification of the Main Compounds Contained in the Chemical Profile of the Liquid Extracts of Choisya and Lime

(37) The chromatographic conditions hereinbelow were applied with a view to identifying and quantifying the compounds present in the various extracts produced.

(38) Column: Waters BEH C18 50 mm×2.1 mm 1.7 μm

(39) Temperature: 45° C.

(40) Flow: 0.3 mL/min

(41) Injection volume: 2 μL

(42) Negative mode

(43) Mobile phase: A: 0.1% formic acid in acetonitrile B: 0.1% formic acid in Water

(44) TABLE-US-00001 Time (min) A % B % 0 10 90 20 10 90 30 90 10

(45) Note: Re-Equilibrate at Starting Mobile Phase Conditions for at Least 10 Minutes Between Each Run

(46) Sample preparation: 100 mg of DES were diluted into 5 ml of methanol/water (50:50), filtered and injected into LC system

(47) Example of HPLC-DAD Analysis—the Concentration of Rosmarinic Acid in the Liquid Extracts of Rosemary

(48) The liquid extracts obtained using the method described in Example 1 were analyzed directly by HPLC without preliminary concentration or drying.

(49) Quantification and identification of rosmarinic acid are performed using an analytical standard (Extrasynthese—reference: 4957S) and by plotting a calibration curve. The Agilent 1100 HPLC apparatus is equipped with a UV-Visible DAD detector or equivalent. An elution gradient is used via a mixture of HPLC grade acetonitrile and HPLC grade water with an addition of 99% trifluoroacetic acid (TFA). The following chromatography conditions are used:

(50) Zorbax Eclipse XDB C18 column, 1.8μη.sub.I, 4.6 mm×50 mm or equivalent.

(51) Mobile phase:

(52) TABLE-US-00002 Time (min) % acetonitrile 0.1% TFA % water 0.1% TFA 0 15 85 2 15 85 2.5 18 82 2.7 100 0 3.5 100 0

(53) Flowrate: 2 mL/min.

(54) Detection: 328 nm

(55) Temperature: 60° C.

(56) Injection volume: 2 pL

(57) Pressure: 210 bars±5 bars

(58) The following retention times are observed:

(59) Rosmarinic acid: 2.0 min; Luteolin 3-glucuronide: 2.3 min.

(60) Example of Gas Chromatography (GC) Analysis Coupled to a Flame Ionization Detector (FID)—the Concentration of Oleic Acid in the Upper and Lower Phases after the Purification Process Applied to a Raw Eutectic Extract

(61) The liquid phases (upper and lower) obtained using the purification method described in Examples 3B1 to 3B3 were prepared for the analysis as follows: 50 mg of each liquid phase were dispensed into a 20 mL glass autosampler vial, then added with 1 mL of the internal standard (methyl pentadecanoate) and 4 mL of 0.5 N NaOH solution in methanol. Before being hermetically closed with a septum, vials were flushed with nitrogen then stirred and heated at 100° C. for 5 min. The samples were then allowed to cool down to room temperature then opened carefully and 5 mL of a 14% BF3 solution in methanol was added. The samples were then flushed with nitrogen before being closed, stirred and heated at 100° C. for 30 min with stirring starting at 15 min. The samples were then allowed to cool down to room temperature then opened and 4 mL of a 10% NaCl aqueous solution and 4 mL of pure n-hexane was added. The samples were then vortexed and centrifuged at 3000 rpm for 3 min. The resulting upper phase was transferred into 2 mL-vials, then placed on a autosampler for GC-FID analysis.

(62) The samples were analyzed using a 7890 Agilent gas chromatograph equipped with autosampler and a RTX®-capillary column (2330 60 m×0.25 mm with 0.20 μm film thickness). Injector temperature was set at 250° C. in a split mode (1:50) and with a liner (ref 092003, SGE Analytical Sciences). The GC oven temperature was set at 120° C. for 5 min, ramped to 200° C. at a rate of 5° C./min, held at 200° C. for 10 min, then ramped to 230° C. at a rate of 2° C./min and finally held at 230° C. for 15 min. Helium at 2 mL/min was used as the carrier gas (constant pressure at 30 psi). FID temperature was set at 300° C.

Example 3—Specific Examples of Eutectic Extracts

(63) Examples of methods used to produce eutectic extracts, and more preferably to produce highly active and concentrated liquid eutectic extracts, are set out below as Examples 3A and 3B.

(64) Examples 3A are a set of 19 generic examples demonstrating the feasibility of the eutectigenesis-assisted extraction process on a diverse range of biological raw materials such as seeds, peels, juice, whole fruits, veggies, roots, leaves, and barks.

(65) Examples 3B are a set of results exemplifying the enhancement of the extract activity and profile using the purification process.

Examples 3A

Example 3A1: Fresh Rosemary Leaves (Rosmarinus officinalis)+Amines

(66) Fresh rosemary leaves were ground using liquid N.sub.2 then mixed with choline chloride and left at 20° C. in an open crystallising dish for five days. At the end of this period, a liquid had formed in the dish. Optimal weight ratios of amine to fresh rosemary were found to 1 to 10 times.

(67) Example 3A1 is the result of combining 0.5 g of fresh rosemary powder and 2.0 g of choline chloride (ChCl) (weight ratio: 1:4, resp.).

(68) Other eutectic extracts were obtained from fresh rosemary. Some with a different weight ratio with ChCl (1:2, 1:6, and 1:8) and another with benzyltriethylammonium chloride (BTEAC) (1:2).

(69) In all cases, the liquid was separated from the biomass components via centrifugation and analysed using the oxygen radical absorbance capacity (ORAC) and the conjugated autoxidizable triene (CAT) methods. Results show that they exert antioxidant activity, which suggests that phytoactive compounds were extracted from fresh rosemary.

(70) The antioxidant activities of the five extracts are shown in Table 1. As it can be seen, the more the amine (ChCl), the lower the antioxidant activity, which is probably due to a dilution effect of the antioxidant compounds by the amine.

(71) TABLE-US-00003 TABLE 1 Compositions and antioxidant activities of different eutectic extracts. ORAC CAT Natural Nat. value value Product Added product:amine Eutectic (μmol (μmol Material amine weight ratio formation TE/g) TE/g) Fresh ChCl 1:2 Melt 10.8 3.90 Rosemary Fresh ChCl 1:2 (repetition) Melt 12.7 — Rosemary Fresh ChCl 1:4 Melt 7.4 2.2 Rosemary Fresh ChCl 1:6 Melt 6.0 1.5 Rosemary Fresh ChCl 1:8 Melt 4.6 1.60 Rosemary Fresh ChCl 1:8 (repetition) Melt 5.7 — Rosemary Fresh BTEAC 1:2 Melt 24.3 7.70 Rosemary Fresh BTEAC 1:2 (repetition) Melt 27.5 — Rosemary ChCl alone — Powder 0.3 0 — BTEAC — Powder 2.8 0.7 alone

Example 3A2: Dried Rosemary Leaves (Rosmarinus officinalis L.)+Amines

(72) Dry rosemary leave powder was produced by freezing the rosemary leaves with liquid N.sub.2 and grinding with a pestle and mortar. The resulting powder was mixed with different amines in different ratios to make deep eutectic mixtures and extract components which have ability to form hydrogen bonds with the tested amines.

(73) The dried biomass was mixed with amines and left at room temperature in an open vessel for 10 days to extract as much natural product as possible.

(74) Optimal weight ratios of amines to dried rosemary were found to 1 to 10 times. Example 3A2 is the result of combining 0.5 g of dried rosemary powder and 2.0 g of ChCl (weight ratio: 1:4, resp.).

(75) At the end of this period (10 days) a large volume of coloured liquid had formed due to the hygroscopic nature of the amines. The liquid was analysed using HPLC and found to contain a variety of natural products extracted from the plant. The colour of the extract is noticeably darker than for the fresh rosemary which is due to the higher relative concentration of natural products in the dried biomass.

(76) Other eutectic extracts were obtained from dried rosemary: some with a different weight ratio with ChCl (1:6), others with BTEAC (1:8) or 1-Butyl-3-methylimidazolium chloride (BmimCl) (1:4).

(77) TABLE-US-00004 TABLE 2 Compositions and antioxidant activities of different eutectic extracts. ORAC CAT Nat. value value Natural Product Added product:amine Eutectic (μmol (μmol Material amine weight ratio formation TE/g) TE/g) Dried rosemary ChCl 1:4 Melt 84.3 22.9 powder Dried rosemary ChCl 1:6 Melt 84.8 25.4 powder Dried rosemary ChCl 1:8 Melt 106.9 21.0 powder Dried rosemary BTEAC 1:8 Melt 81.2 23.9 powder Dried rosemary BTEAC 1:8 (repetition) Melt 70.2 19.4 powder Dried rosemary BmimCl 1:4 Melt 207.4 53.4 powder Dried BmimCl 1:6 Melt 158.2 39.6 rosemary powder Dried rosemary BmimCl 1:8 Melt 153.5 34.7 powder — ChCl alone — Powder 0.3 0 — BTEAC — Powder 2.8 0.7 alone — BmimCl — Powder 1.1 0.24 alone

(78) FIG. 2 shows the content in some important phytoactives extracted from dried rosemary leaves such as luteoline-3-glucuronide, rosmarinic acid and carnosic acid. The data presented here also contain the values corresponding to rosemary extracts obtained with (i) already formed DES used as extraction solvent (Rosemary Eutectys™) and (ii) already formed hydroglycerinated solvent (Rosemary hydroglycerinated).

Example 3A3: Dried Rosemary Leaves (Rosmarinus officinalis L.)+55% Weight H.SUB.2.O+Amines

(79) Example 3A3 shows a typical mixture of dried rosemary powder first added with 55% weight of H.sub.2O then added with amine in various stoichiometric ratios.

(80) The value of 55% water was chosen as it typically corresponds to the difference observed between fresh and dried rosemary leaves. The mixture was left in an open vessel for three days after vigorous mixing via mechanic stirring. The addition of H.sub.2O is to increase the rate of extraction by decreasing the viscosity of the amine, hence improving the mass transport. This would naturally occur in an open system due to the hygroscopic nature of the amines used, however, this would reduce the time taken to reach equilibrium.

(81) The optimum weight ratio range of dried rosemary leaves:amine was found to be between 1:1 and 1:10. Example 3A3 is the result of combining 0.5 g of dried rosemary leave powder, 2.0 g of ChCl and 0.275 g of H.sub.2O added prior to the amine addition (weight ratio: 1:4:0.55, resp.).

(82) Other eutectic extracts were obtained from dried rosemary powder and ChCl (1:6) or BTEAC (1:6). These latter two systems have been assessed for their antioxidant activity (Table 3) and their content in rosmarinic acid, carnosic acid and luteoline-3-glucuronide (FIG. 2).

(83) TABLE-US-00005 TABLE 3 Compositions and antioxidant activities of different eutectic extracts. Nat. Nat. product Added Added prod.:amine:water Eutectic ORAC value CAT value Material amine water weight ratio formation (μmol TE/g) (μmol TE/g) Dried ChCl 55% 1:6:0.55 Melt 43.7 4.5 rosemary (w) Dried BTEAC 55% 1:6:0.55 Melt 100.4 26.8 rosemary (w)

Example 3A4: Fresh Choisya Leaves (Choisya ternata)+Amines

(84) The extraction of phenolic compounds and other active compounds such as alkaloids can also take place from other leaves such as Choisya (Choisya ternata) belonging to the Rutaceae family.

(85) The same trends are observed in extract efficiency with amine amount and type. Fresh Choisya leaves were mixed together with amines in a pestle and mortar and left in an open vessel for seven days. The optimum ratio range of fresh Choisya:amine was found to be between 1:1 and 1:10. Example A4 (a) is the result of mixing 0.75 g of fresh Choisya leaves with 4.5 g of ChCl (weight ratio: 1:6, resp.) and example A4 (b) is the result of mixing 0.75 g of fresh Choisya leaves with 4.5 g of BTEAC (weight ratio: 1:4:2, resp.). In both cases the resulting liquid was centrifuged and filtered. When ChCl was used as amine, a brown liquid was obtained, while a green liquid was produced with BTEAC.

(86) ORAC and CAT value measurements of these mixture showed good content of antioxidant material within the extract.

(87) In this example (FIG. 3), no significant difference can be observed in the LC-MS profiles of eutectic extracts obtained using an aliphatic (ChCl) or an aromatic (BTEAC) amine salt.

(88) Moreover, LC-MS data show that the extracted compounds from Choisya leaves are mainly phenolics such as flavonoids (e.g. apigenin-diglucoside, rutin, luteolin-7-rutinoside), phenolic acid derivatives (rosmarinic acid glucoside), and alkaloids (quinoline alkaloid and its numerous isomers, scopolamine, balfourodine, evoxine, methylevoxine, and choisyne to only cite a few examples) (FIG. 4). They mostly contain HBD moieties that can give a H-bond to the tested amine salt, either ChCl or BTEAC (Table 4).

(89) TABLE-US-00006 TABLE 4 MS identification of compounds from choisya eutectic extracts. Formula Fragment Δ HBD HBA Aromatics Bp Peak Identification found [M + H].sup.+ ion +/− moiety moiety (homo/hetero) (° C.).sup.h LogP.sup.i  1 Apigenin 6,8-di-C-glucoside C.sub.27H.sub.30O.sub.15 595.1662 — 0.95 11 15 2/0 974 −2.621  2 Quinoline alkaloid 1ª C.sub.16H.sub.19NO.sub.3 274.1438 — −0.22  3 Rutin C.sub.27H.sub.30O.sub.16 611.1607 465.1036/ −0.07 10 16 2/0 983 −0.903 303.0507 Scopolamine C.sub.17H.sub.21NO.sub.4 304.1548 — −1.39 1 5 1/0 460 0.693  5 Rosmarinic acid β-D-glucoside C.sub.24H.sub.26O.sub.13 523.1448 361.0929 1.05 8 13 2/0 876 −1.411  5′ Luteolin 7-O-rutinoside C.sub.27H.sub.30O.sub.15 595.1662 287.0558 −1.21 9 15 2/0 954 0.180  6 Balfourodine C.sub.16H.sub.19NO.sub.4 290.1388 — −0.62 1 5 1/0 436 −0.174  7 Evoxine C.sub.18H.sub.21NO.sub.6 348.1448 — −1.92 2 7 1/1 546 1.718  8 Quinoline alkaloid 2.sup.b C.sub.18H.sub.21NO.sub.5 332.1496 — −1.68  9 Methylevoxine C.sub.19H.sub.23NO.sub.6 362.1595 — 0.45 1 7 1/1 525 2.151 10 Skimmianine C.sub.14H.sub.13NO.sub.4 260.0921 — −1.65 0 5 1/1 402 2.178 10′ Choisyine C.sub.18H.sub.19NO.sub.5 330.1338 — −0.72 1 6 1/1 508 2.036 11 Kokusaginine C.sub.14H.sub.13NO.sub.4 260.0924 — −3.7 0 5 1/1 402 2.178 11′ Quinoline alkaloid 3.sup.c C.sub.17H.sub.17NO.sub.4 300.1244 — −5.79 nd nd nd nd nd 12 Quinoline alkaloid 4.sup.d C.sub.18H.sub.19NO.sub.4 314.1390 — −1.33 nd nd nd nd nd 13 Quinoline alkaloid 5.sup.c C.sub.18H.sub.20NO.sub.5 330.1337 — −0.62 nd nd nd nd nd 14 Quinoline alkaloid 6.sup.f C.sub.18H.sub.19NO.sub.4 314.1393 — −2.11 nd nd nd nd nd 15 Quinoline alkaloid 7.sup.g C.sub.19H.sub.21NO.sub.5 344.1510 — −4.92 nd nd nd nd nd 16 Choisyine isomer C.sub.18H.sub.19NO.sub.5 330.1337 — −2.16 1 6 1/1 508 2.036 ªDaurine (or forthucine) .sup.bDihydroepimacronine (or Tazettine or Unsevine) .sup.c2-[(2R)-10-Methoxy-1,2-dihydrodifuro[2,3-b:3′,2′-f]quinolin-2-yl]-2-propanol .sup.dand .sup.f (3E)-4-(4,6-Dimethoxyfuro[2,3-b]quinolin-5-yl)-2-methyl-3-buten-2-ol .sup.e(2R)-3-Chloro-1-[(4,8-dimethoxyfuro[2,3-b]quinolin-7-yl)oxy]-3-methyl-2-butanol .sup.g3-Methyl-1-(4,6,7-trimethoxyfuro[2,3-b]quinolin-5-yl)-3-buten-2-ol

(90) Furthermore, ORAC and CAT value measurements of these eutectic mixtures showed significant antioxidant activities for crude liquid extracts (Table 5), which is corroborated by the LC-MS identification of four potent antioxidant molecules: rutin, luteolin 7-O-rutinoside, apigenin 6,8-di-C-glucoside, and rosmarinic acid β-D-glucoside (Table 4).

(91) TABLE-US-00007 TABLE 5 Compositions and antioxidant activities of different eutectic extracts. ORAC CAT Natural Nat. value value Product Added product:amine Eutectic (μmol (μmol Material amine weight ratio formation TE/g) TE/g) Fresh choisya ChCl 1:4 Melt 31.7 4.9 Fresh choisya ChCl 1:6 Melt 23.9 5.1 Fresh choisya ChCl 1:8 Melt 18.1 3.1 Fresh choisya ChCl 1:8 (repetition) Melt 21.2 5.2 Fresh choisya BTEAC 1.4 Melt 30.0 4.5 Fresh choisya BTEAC 1:4 (repetition) Melt 37.3 8.0

(92) None of the identified alkaloids bear reducing group, such as a phenolic moiety, capable of antioxidant potential. However, they are endowed with a broad range of biological activities. One can cite scopolamine which has as a number of uses in medicine, where it is used to treat postoperative nausea and vomiting and sea sickness, leading to its use by scuba divers. It is also used in the treatment of motion sickness, gastrointestinal spasms, renal or biliary spasms, bowel colic, irritable bowel syndrome, clozapine-induced hypersalivation (drooling), and eye inflammation.

Examples 3A5 and 3A6: Dried Blueberry Juice (Cyanococcus)+Amines

(93) Examples 3A5 and 3A6 show ChCl mixed with dried blueberry juice powder using both thermal evaporation (example A5) and freeze drying (example A6) and to produce a white powder. Upon addition of the components (amines+dried blueberry juice), mixing occurred by using a pestle and mortar.

(94) The mixture was left in an open vessel at room temperature for seven days. A very pale yellow/brown liquid was formed. This was the same for all amines tried (ChCl/BTEAC/BmimCl).

(95) The weight ratio of amines to natural product did not alter the colour of the liquid. The optimum natural product:amine ratio for this system was found to be between 1:2 and 1:8. Example A5 is the result of mixing 0.5 g of thermally evaporated (heat-dried) blueberry juice with 2.0 g of ChCl (weight ratio: 1:4, resp.) and example A6 is the result of mixing 0.5 g of freeze dried blueberry juice with 2.0 g of ChCl (weight ratio: 1:4, resp.).

(96) Other eutectic extracts were obtained from dried blueberry juice either in its heat-dried or freeze-dried form (Table 6). Some melt were obtained with a different weight ratio with ChCl (1:2, 1:6), while others were found with BTEAC (1:4, 1:8) and BmimCl (1:4).

(97) TABLE-US-00008 TABLE 6 Compositions and antioxidant activities of different eutectic extracts. ORAC CAT Natural Juice Nat. value value Product drying Added prod.:amine Eutectic (umol (μmol Material technique amine weight ratio formation TE/g) TE/g) Blueberry Heat-dried ChCl 1:4 Melt 9.6 3.7 juice Blueberry Heat-dried ChCl 1:4 Melt 9.0 2.9 juice (repetition) Blueberry Heat-dried BTEAC 1:4 Melt 6.6 2.2 juice Blueberry Freeze- ChCl 1:2 Melt 17.5 6.4 juice dried Blueberry Freeze- ChCl 1:4 Melt 12 2.8 juice dried Blueberry Freeze- ChCl 1:6 Melt 10.7 4.0 juice dried Blueberry Freeze- BTEAC 1:4 Melt 11.9 4.6 juice dried Blueberry Freeze- BTEAC 1:8 Melt 7.9 2.8 juice dried Blueberry Freeze- BmimCl 1:4 Melt 13.8 5.7 juice dried

Example 3A7: Dried Blueberry Pulp (Cyanococcus)+Amines

(98) Example 3A7 is the result of mixing 0.5 g of dried blueberry pulp with 4.0 g of ChCl (weight ratio: 1:4, resp.) with a pestle and mortar and left in an open vessel for seven days.

(99) The optimum natural product:amine weight ratio for this system was found to be between 1:2 and 1:8 as other eutectic melts were obtained using BTEAC at a weight ratio of 1:4 and 1:8.

(100) The resulting liquid extract is darker in colour than the resulting extracts from the dried blueberry juice tests. This was the same for all amines tried with the pulp and the ratio of natural product:amine did not alter the colour of the liquid. In terms of antioxidant activity, we can observe a slight decrease of the activity when the amine proportion is increased which is probably due to a sort of dilution effect of the antioxidant molecules in the amine (Table 7).

(101) TABLE-US-00009 TABLE 7 Compositions and antioxidant activities of different eutectic extracts. ORAC CAT Natural Natural value value Product Added Product:amine Eutectic (μmol (μmol Material amine weight ratio formation TE/g) TE/g) Blueberry skin ChCl 1:8 Melt 10.9 4.2 Blueberry skin BTEAC 1:2 Melt 16.5 6.2 Blueberry skin BTEAC 1:8 Melt 13.9 6.1

(102) Examples 3A8, 3A9, 3A10, 3A11: Capsicum (Capsicum annuum)/Green Chilli (Capsicum frutescens)+Amines

(103) The hulls and seeds of Capsicum and green chilli fruits were separately mixed with amines to recover the natural substances (able to donate hydrogen bonds) contained in these parts of the plants.

(104) Example 3A8 (a) shows the result of 0.5 g of fresh Capsicum hull mixed with 3.0 g of ChCl (ratio: 1:6) and example 3A10 (a) shows the result of combining 0.5 g of fresh green chilli hull with 4.0 g of ChCl (ratio: 1:8). Example 3A8 (b) shows the result of 0.5 g of fresh Capsicum hull mixed with 3.0 g of BTEAC (ratio: 1:6) and example 3A10 (b) shows the result of 0.5 g of fresh green chilli hull mixed with 4.0 g of BTEAC (ratio: 1:8).

(105) The colour of the eutectic extract varies depending on the amine used. When ChCl is used (examples 3A8 (a) and 3A10 (a)) a very lightly yellow coloured solution is formed, however when BTEAC (examples 3A8 (b) and 3A10 (b)) is used, a pink solution is formed.

(106) Example 3A9 shows the result of 0.5 g of ground Capsicum seeds with 4.0 g ChCl (ratio: 1:8) and example 3A11 shows the result of 0.5 g of ground green chilli seeds with 4.0 g of ChCl (ratio: 1:8).

(107) Other ratios were also found to lead to the formation of eutectic melts (Table 8). In all examples the pure amine was mixed with the plant material using a pestle and mortar and left in an open vessel for seven days at room temperature. The resulting liquid was centrifuged and filtered. The optimum weight ratio range of Capsicum:amine or green chilli:amine was found to be between 1:2 and 1:10.

(108) TABLE-US-00010 TABLE 8 Compositions and antioxidant activities of different eutectic extracts. ORAC CAT Natural Nat value value Product Added product:amine Eutectic (μmol (μmol Material amine weight ratio formation TE/g) TE/g) Capsicum skin ChCl 1:4 Melt 3.9 2.2 Capsicum skin ChCl 1:6 Melt 10.5 3.4 Capsicum skin BTEAC 1:2 Melt 2.6 0.5 Capsicum skin BTEAC 1:2 (repetition) Melt 3.5 0.6 Capsicum skin BTEAC 1:4 Melt 2.1 0.5 Capsicum seed ChCl 1:4 Melt 3.8 1.3 Capsicum seed ChCl 1:8 Melt 4.4 1.1 Green chilli skin ChCl 1:2 Melt 13.9 3.9 Green chilli skin ChCl 1:8 Melt 7.2 1.3 Green chilli skin ChCl 1:8 (repetition) Melt 5.9 1.7 Green chilli skin BTEAC 1:4 Melt 11 1.8 Green chilli skin BTEAC 1:8 Melt 12.6 2.4 Green chilli skin BTEAC 1:8 (repetition) Melt 10.8 2.7 Green chilli ChCl 1:8 Melt 3.9 0.7 seed Green chilli ChCl 1:8 (repetition) Melt 3.1 0.9 seed

Example 3A12: Dried Aubergine (Solanum melongena)+Amines

(109) Example 3A12 shows the results of mixing 0.5 g of dried aubergine juice with 2.0 g of ChCl and had been left in an open vessel for seven days (weight ratio: 1:4, resp.). The liquid was centrifuged and filtered. A light yellow coloured liquid resulted and did not vary in colour when alternative amines were used. The flesh of an aubergine was juiced and freeze-dried. The resulting dried pulp was then ground into a powder. The optimum weight ratio range of aubergine:amine was found to be between 1:2 and 1:8. Example A12 shows the resulting liquid eutectic extract using ChCl as the amine. Table 9 also exemplifies the possibility to use BTEAC to form a eutectic melt.

(110) TABLE-US-00011 TABLE 9 Compositions and antioxidant activities of different eutectic extracts. ORAC CAT Natural value value Natural Product Added Product:amine Eutectic (μmol (μmol Material amine weight ratio formation TE/g) TE/g) Freeze Dried ChCl 1:4 Melt 4.7 0.2 Aubergine flesh Freeze Dried BTEAC 1:4 Melt 15.9 3.1 Aubergine flesh

Example 3A13: Ginger (Zingiber officinale)+Amines

(111) Example 3A13 shows how a root, in this case whole ginger root could be extracted with an amine. The ginger was juiced, freeze dried and ground into a powder before addition of the amine. Example 3A13 demonstrates the end result of mixing 0.5 g of dried ginger with 2.0 g of ChCl (weight ratio: 1:4, resp.) using a pestle and mortar and leaving in an open vessel for seven days. This eutectic extract was centrifuged and filtered in the same manner as other samples.

(112) The optimum ratio range in which dried ginger could be extracted from was found to be 1:2 to 1:8 ginger:amine as other ratios and amines (BTEAC) also yield eutectic melts.

(113) TABLE-US-00012 TABLE 10 Compositions and antioxidant activities of different eutectic extracts. Natural Natural Product Added Product:amine Eutectic ORAC value CAT value Material amine weight ratio formation (μmol TE/g) (μmol TE/g) Ginger ChCl 1:2 Melt 4.6 1.9 Ginger ChCl 1:4 Melt 6.1 1.7 Ginger BTEAC 1:2 Melt 5.2 2.5 Ginger BTEAC 1:4 Melt 7.4 1.6

Examples 3A14 and 3A15: Lime (Citrus aurantifolia)+Amines

(114) The rind and juice of a lime, once separated from the flesh of the lime, have been mixed with various amines independently and have been shown to yield a eutectic extract with a high concentration of malic, citric and ascorbic acids. The rind was frozen with liquid N.sub.2 and then ground into a powder prior to addition of amines and the juice was freeze dried.

(115) To remove any insoluble solid material from the liquid phase, the mixture was centrifuged and then decanted. Example A14 is the result of mixing 0.5 g of dried and powdered lime rind with 3.0 g of ChCl (weight ratio: 1:6, resp.) and example A15 is the result of mixing 0.5 g of freeze dried lime juice with 3.0 g of ChCl (weight ratio: 1:6, resp.). Both samples were mixed using a pestle and mortar and left in an open vessel for ten days. The optimum lime:amine weight ratio to obtain a eutectic melt was found to range from 1:2 to 1:10 (Table 11).

(116) TABLE-US-00013 TABLE 11 Compositions of two eutectic extracts from Lime. Natural Product Added Natural Product:amine Eutectic Material amine weight ratio formation Dried lime rind ChCl 1:6 Melt Freeze-dried lime ChCl 1:6 Melt juice

(117) TABLE-US-00014 TABLE 12 MS identification of compounds from lime eutectic extracts. Formula Fragment Δ ppm HBD HBA Aromatics Mp Bp Peak Possible identification found [M + H].sup.+ ion +/− moiety moiety (homo/hetero) (° C.).sup.h (° C.).sup.i LogP.sup.j  1 Cinamic acid C.sub.9H.sub.8O.sub.2 149.0598 — −2.97 1 2 1/0 300 1.212  3 Dimethyl citrate C.sub.8H.sub.12O.sub.7 221.0665 203.0551/ 2.85 2 7 0 — 405 0.244 185.0445  4 Dimethyl citrate isomer C.sub.8H.sub.12O.sub.7 221.0665 — 2 7 0 — 405 0.244  8 Apigenin 6,8-di-C-glucoside C.sub.27H.sub.30O.sub.15 595.1659 — −0.39 11 15 2/0 — 974 −2.621  9 Diosmetin 6,8-di-C-glucoside C.sub.28H.sub.32O.sub.16 625.1765 — −0.73 11 16 2/0 — 986 −1.871 11 Eriocitrin (eriodictyol C.sub.27H.sub.32O.sub.15 597.1815 435.1301/ −0.51 9 15 2/0 160 957 −1.813 7-O-rutinoside) 289.0715 12 Rutin C.sub.27H.sub.30O.sub.16 611.1628 465.1058/ −4.01 10 16 2/0 — 983 −0.903 303.0513 12 Limonin C.sub.26H.sub.30O.sub.8 471.2021 — −1.69 0 8 0/1 298 668 0.474 13 Narirutin (Naringenin C.sub.27H.sub.32O.sub.14 581.1871 435.1305/ −2.21 8 14 2/0 180 924 −0.522 7-O-rutinoside) 273.0766 13′ Diosmetin 8-C-glucoside C.sub.22H.sub.22O.sub.11 463.0124 — −1.2 7 11 2/0 — 784 0.736 13″ Natsudaidain C.sub.21H.sub.22O.sub.9 419.1345 — −4.08 1 9 2/0 156 608 2.124 14 Eriodictyol C-glucoside C.sub.21H.sub.22O.sub.11 451.1217 — 4.76 8 11 2/0 — — — 15 Rhoifolin (or sorhoifolin)ª C.sub.27H.sub.30O.sub.14 579.1715 — −1.24 8 14 2/0 260 916 −0.062 16 Hesperidin C.sub.28H.sub.34O.sub.15 611.1972 449.1448/ −0.31 8 15 2/0 262 930 −1.212 303.0870 17 Scutellarein 7,4-dimethylether C.sub.29H.sub.32O.sub.17 653.1712 — −0.47 8 17 2/0 — — — sophoroside 18 Nomilin glucoside C.sub.34H.sub.46O.sub.15 694.7274 — −0.42 5 15 0/1 — 852 0.524 19 Poncirin.sup.b (or its isomers: C.sub.34H.sub.28O.sub.14 595.2035 — −1.29 7 14 2/0 210 900 0.537 neoponcirin or didymin) 19′ Limonexin C.sub.26H.sub.30O.sub.10 503.1918 — −2.60 1 10 0 — 784 −2.915 21 Citrobilin C.sub.28H.sub.34O.sub.11 564.2469 — −1.57 1 11 0 — 725 −1.550 21′ Hesperetin glucoside C.sub.22H.sub.24O.sub.11 464.2645 — −5.34 6 11 2/0 — — — 22 Citropten C.sub.11H.sub.10O.sub.4 207.0651 — 0.26 0 4 1/1 149 388 1.891 23 Bergapten C.sub.12H.sub.8O.sub.4 217.0499 — −2.07 0 4 1/2 188 412 2.035 23′ Isopimpinellin C.sub.13H.sub.10O.sub.5 247.0604 — −1.74 0 5 1/2 151 449 1.407 24 Limonin isomer C.sub.26H.sub.30O.sub.8 471.2023 — −2.06 0 8 0/1 — — — 24′ Geranyl propionate C.sub.13H.sub.22O.sub.2 211.1692 — −1.04 0 2 0

(118) Table 12 gives the chemical compounds founds and identified by LC-MS in ESI+ for the two eutectic extracts. First, it appears that the chemical profile of lime juice eutectic extract is different from that obtained from the rind part. Among other differences, one can note the higher concentration of hesperidin in lime peel compared to the juice, as well as the higher contents of citrobilin (triterpenoid), citropten (a coumarin currently found in lime essential oils), and bergapten (a furocoumarin also currently found in citrus essential oils). Table 13 provides quantitation of the compounds found in eutectic extracts of lime peel and juice.

(119) TABLE-US-00015 TABLE 13 Quantitation of compounds from lime peel and juice eutectic extracts Lime juice Lime peel Compounds ppm Apigenin 6,8-di-glucoside .sup.(1) 88.9 157.1 Diosmetin 6,8-di-glucoside .sup.(1) 84.8 35.95 Eriocitrin .sup.(2) 144.3 202.15 Rutin .sup.(1) 25.45 0 Narirutin .sup.(2) 37.77 66.93 Eriodyctiol C-glucoside .sup.(1) 7.4 35.9 Rhoifolin .sup.(1) 21.15 166.25 Hesperidin .sup.(2) 171.8 1442.5 Scutellarein dimethylether sophoroside.sup.(1) 0 41.9 Nomilin glucoside .sup.(3) 0 246.43 Poncirin .sup.(4) 3.75 2.31 Furanocoumarin .sup.(4) 0 3.67 Citropten .sup.(4) 0.57 21.06 Bergapten & isopimpinellin .sup.(4) 0.56 9.16 .sup.(1) Expressed as rutin .sup.(2) Expressed as hesperidin .sup.(3) Expressed as nomilin .sup.(4) Expressed as coumarin

Example 3A16: Cedrus Bark (Cedrus Spp.)+Amines

(120) Example 3A16 shows that a bark can be extracted using the same eutectigenesis-assisted extraction process to yield an extract high in phytoactives. The sample shown in example 3A16 was the result of combining 0.5 g of Cedrus bark powder with 3.0 g of BmimCl (weight ratio: 1:6, resp.) using a pestle and mortar and leaving in an open vessel for eight days. Cedrus bark was frozen using liquid N.sub.2 and then ground into a fine powder using a pestle and mortar.

(121) Noteworthy, other weight ratios (1:8) and amines (BTEAC, BmimCl) allows obtaining eutectic melts (Table 14). The optimum weight ratio range of Cedrus bark to amine was found to be 1:2 to 1:10. The resulting extract was a deep brown colour and ORAC and CAT values showed relatively strong antioxidant properties demonstrating that antioxidant components had been recovered by the amine. Cedrus bark:BTEAC (1:8) followed by Cedrus bark:BmimCl (1:6) exhibited the highest activities we have found using the eutectigenesis-assisted extraction technique (without further purification): 302.9 and 285.2 μmol TE/g of liquid extract (ORAC values), respectively (Table 14). The highest CAT value (79.1 μmol TE/g) was also observed for the eutectic extract from Cedrus bark:BTEAC (1:8), though it was not from the same repetition.

(122) TABLE-US-00016 TABLE 14 Compositions and antioxidant activities of different eutectic extracts from Cedrus bark. ORAC CAT Natural value value Natural Product Added Product:amine Eutectic (μmol (μmol Material amine weight ratio formation TE/g) TE/g) Powdered Cedrus ChCl 1:8 Melt 152.3 45.5 bark Powdered Cedrus BTEAC 1:8 Melt 302.9 42.7 bark Powdered Cedrus BTEAC 1:8 (repetition) Melt 258.2 79.1 bark Powdered Cedrus BmimCl 1:6 Melt 285.2 37.0 bark Powdered Cedrus BmimCl 1:8 Melt 170.5 35.2 bark

Example 3A17: Java Tea, Orthosiphon aristatus (Blume) Miq. (Syn. Orthosiphon stamineus Benth.), aerial part (dry & milled)+amines

(123) Example 3A17 shows a typical extract of the herb Orthosiphon using pure amines. In this example, 0.5 g of dried Orthosiphon was mixed with 3.0 g ChCl (weight ratio: 1:6, resp.) and mixed together using a pestle and mortar. The mixture was then left for in an open vessel for eight days. The resulting liquid was a light brown/orange colour. Other ratios (1:4, 1:8) and another amine (BmimCl) were also found to yield eutectic melts (Table 15). The optimum weight ratio range of Orthosiphon:amine was found to be between 1:2 to 1:8. The ORAC and CAT values shows that antioxidant compounds are present in this liquid extract, with relatively high antioxidant activities for the eutectic extract coming from Orthosiphon:BmimCl (1:4): 133.1 (ORAC) and 40.9 (CAT) μmol TE/g.

(124) TABLE-US-00017 TABLE 15 Compositions and antioxidant activities of different eutectic extracts from Orthosiphon. ORAC CAT Natural value value Natural Product Added Product:amine Eutectic (μmol (μmol Material amine weight ratio formation TE/g) TE/g) Orthosiphon herb ChCl 1:6 Melt 44.5 12.6 Orthosiphon herb ChCl 1:8 Melt 28.6 7.3 Orthosiphon herb BmimCl 1:4 Melt 133.1 40.9 Orthosiphon herb BmimCl 1:8 Melt 51.4 15.1

Example 3A18: Ziziphus (Ziziphus jujuba Mill., Seed (Dry & Milled))+Amines

(125) Example 3A18 shows a typical extract of the seeds from the Ziziphus plant (jujube). In this example, 0.5 g of dried Ziziphus seed was mixed with 3.0 g ChCl (weight ratio: 1:6, resp.) and mixed using a pestle and mortar and left in an open vessel for seven days. Other ratios (1:4, 1:8) and amines (BTEAC, BmimCl) were also found to yield eutectic melts (Table 16). The optimum weight ratio range of Ziziphus:amine was found to be 1:2 to 1:8. The resulting liquid was a light brown/orange colour. Noteworthy, only BTEAC extract (ratio 1:6) gives a significant antioxidant activity in the ORAC method with a value of 41.4 μmol TE/g.

(126) TABLE-US-00018 TABLE 16 Compositions and antioxidant activities of different eutectic extracts from Ziziphus. ORAC CAT Natural value value Natural Product Added Product:amine Eutectic (μmol (μmol Material amine weight ratio formation TE/g) TE/g) Ziziphus seed ChCl 1:6 Melt 4.3 2.7 Ziziphus seed ChCl 1:8 Melt 3.7 1.7 Ziziphus seed BmimCl 1:4 Melt 9.7 3.1 Ziziphus seed BmimCl 1:8 Melt 4.5 1.7 Ziziphus seed BTEAC 1:6 Melt 41.4 3.6

Example 3A19: Cilantro (Coriandrum sativum L., Leaf (Dry & Grinded))+Amines

(127) Example 3A19 shows the resulting extract when 0.5 g of the herb cilantro (or coriander) is mixed with 2.0 g of ChCl (weight ratio: 1:4, resp.). The plant material was dried and powdered when mixed with different amines and left in an open vessel for ten days. Other ratios (1:6, 1:8) and another amine (BmimCl) were also found to yield eutectic melts (Table 17). The optimum weight ratio range of cilantro:amine was found to be 1:2 to 1:8. The resulting liquid was a light brown/orange colour.

(128) TABLE-US-00019 TABLE 17 Compositions and antioxidant activities of different eutectic extracts from Cilantro. ORAC CAT Natural value value Natural Product Added Product:amine Eutectic (μmol (μmol Material amine weight ratio formation TE/g) TE/g) Cilantro leaves ChCl 1:6 Melt 9.7 3.6 Cilantro leaves ChCl 1:8 Melt 5.6 2.1 Cilantro leaves BmimCl 1:8 Melt 10.5 4.0

Examples 3B

(129) Here are presented a set of results using various plant materials as bark, leaves, or seeds exemplifying the enhancement of the eutectic extract activity and profile using the purification process.

Example 3B1: Cedrus Bark Eutectic Extract Purification

(130) Purification of the eutectic extract was carried out by liquid-liquid extraction using a long chain monounsaturated fatty acid as the immiscible phase. To 1.0 mL of the Cedrus bark eutectic extract, 0.75 mL of pure oleic acid was added and mixed via magnetic stirrer at 50° C. for 30 minutes. The resulting mixture was then centrifuged to maximise separation of the two layers. The upper layer was then isolated from the lower layer with a Pasteur pipette and both phases were evaluated in terms of antioxidant activity, BTEAC content and profiles of compounds absorbing at 280 nm (phenolics) and 350 nm (mostly flavonoids).

(131) Both ORAC and CAT values increased by a factor >2 which represents a strong enhancement of the antioxidant activity (FIG. 4). This activity improvement following the purification step can be explained by a rise of compounds absorbing at 280 nm (mostly phenolics having antioxidant potential) and 350 nm (mostly flavonoid phenolics having antioxidant potential). Concomitantly, the content in BTEAC (amine devoid of any antioxidant activity) slightly decreases from 71 to 63% when the purification process is applied to the extract.

Example 3B2: Rosemary Leaf Eutectic Extract Purification

(132) To 1.0 mL of the rosemary leaf eutectic extract, 0.5 or 0.75 mL of pure oleic acid was added and mixed via magnetic stirrer at 50° C. for 30 minutes. The resulting mixture was then centrifuged at 5000 rpm for 15 minutes to maximise separation of the two layers. The upper layer was then isolated from the lower layer.

(133) FIG. 5A shows an increase of the antioxidant activity of the rosemary eutectic extracts in both ORAC and CAT measurements following the application of the purification step. When 0.5 mL of pure oleic acid was used to wash 1 mL of liquid extract, the ORAC value of the resulting extract (lower layer) increased by 37% and the CAT value by 25%. Similarly, when using 0.75 mL oleic acid (for 1 mL extract), the ORAC value increased by 17.5% and CAT value by 19%.

(134) This significant improvement of the activity is concomitant with an increase of two relatively hydrophilic phenolic antioxidants, namely rosmarinic acid and luteoline-3-O-glucuronide (FIG. 5B). Concurrently, the BTEAC and water are partially depleted from the extracts when the purification procedure is applied, although, as for Cedrus bark eutectic extract, the depletion remains modest (<10% for BTEAC; <20% for water). The BTEAC and the water removed from the extract are thus present in the upper layer of which they constitute a significant part (38-42%) almost equalling the proportion of oleic acid (43-46%) (FIG. 5C). Surprisingly, despite the immiscibility of water and oleic acid, the upper layers contain around 10% of water (FIG. 5C), which probably migrates as water H-bonded to BTEAC molecules (water-BTEAC complex).

(135) Altogether, these results demonstrate that the purification step enables to raise the level of active molecules through a partial migration of non-antioxidant molecules into the phase formed by the long chain fatty acid, which consequently increases the activity that is considered. Noteworthy, this activity improvement can be obtained with other properties than just antioxidant properties.

(136) In our experiments, no oleic acid migrates to the extracts during the purification step. Indeed, only low amounts of oleic acid (<1%) are found in the lower layer, whereas the non-purified rosemary eutectic extract already contain 1.7% oleic acid (FIG. 5C). It is also worth noting the slight antioxidant activity of the upper phase as measured by the ORAC method (FIG. 5A). First, compositional data shows that carnosic acid partially migrate from the extract into the washing phase (FIG. 5B). Second, it has been found that pure oleic acid can induce a positive value, although a modest one, for the ORAC method, unlike the CAT method.

Example 3B3: Ziziphus Seed Eutectic Extract Purification

(137) To 1.0 mL of the Ziziphus seed eutectic extract, 0.75 mL of oleic acid was added and mixed via magnetic stirrer at 50° C. for 30 minutes. The resulting mixture was then centrifuged (5000 rpm for 15 mins) to maximise separation of the two layers. The upper layer was then isolated from the lower layer with a Pasteur pipette.

(138) FIG. 6 shows that the purification technique leads to an increase of the ORAC activity of the eutectic extract of 61%. The parent extract contains around 67.5% of BTEAC before purification and 59.7% after purification, showing a partial depletion of the amine (13%). Consequently, the active molecules which are partitioned in the lower phase are concentrated, hence, probably, the increase of the antioxidant activity.