PROCESS FOR EXTRACTING SUBSTANCES OF INTEREST

Abstract

The present description relates to a method for extracting at least one substance of interest, the method comprising the following steps: —providing an admixture comprising water, a source comprising the at least one substance of interest or at least one precursor thereof, at least one surfactant and at least one lipophilic compound; —the emulsification of the admixture, leading to the formation of an emulsion; wherein the emulsion comprises: —a continuous aqueous phase; and a discontinuous lipidic phase comprising the at least one lipophilic compound, the at least one surfactant and at least one substance of interest extracted from the source during the emulsification; wherein the method further comprises the addition of at least one cryoprotectant to the admixture and the lyophilisation of the emulsion.

Claims

1. A process for extracting at least one substance of interest from a source, the process comprising the following steps of: providing a mixture comprising water, a source comprising the at least one substance of interest or at least one precursor thereof, at least one surfactant, and at least one lipophilic compound; and emulsifying the mixture, thereby forming an emulsion; wherein the emulsion comprises: a continuous aqueous phase; and a discontinuous lipid phase comprising the at least one lipophilic compound, the at least one surfactant, and the at least one substance of interest extracted from the source during emulsifying; wherein the source is a plant, an animal, a mineral, a prokaryote, and/or a unicellular eukaryote source; and wherein the process further comprises adding at least one cryoprotectant to the mixture and freeze-drying the emulsion; and wherein adding at least one cryoprotectant to the mixture is performed after emulsifying the mixture, in a weight-to-volume ratio of at least 5% w/v based on the total volume of the mixture excluding the source.

2. The process of claim 1, wherein emulsifying the mixture comprises sonicating the mixture.

3. (canceled)

4. The process of claim 1, wherein the source is a plant source.

5. The process of claim 1, wherein the at least one surfactant is selected from the group comprising Dendri-TAC type dendrimers, F.sub.iTAC.sub.n or H.sub.iTAC.sub.n type oligomers, TPGS 1000, TPGS 750M, sugar- and/or amino acid-derived surfactants, and combinations thereof.

6. The process of claim 1, wherein emulsifying is performed by maintaining the temperature of the mixture between about 4° C. and about 60° C.

7. The process of claim 1, wherein the duration of emulsifying is comprised between about 0.5 seconds and about 5 hours.

8. The process of claim 1, wherein said discontinuous lipid phase is in the form of droplets of a predetermined mean diameter D.

9. (canceled)

10. The process of claim 1, wherein the at least one cryoprotectant is selected from the group comprising polymers, amino acids, saccharide compounds such as mono-, di- and polysaccharides, and combinations thereof.

11. The process of claim 1, wherein the at least one lipophilic compound, the at least one surfactant, and the at least one cryoprotectant are biocompatible.

12. The process of claim 1, further comprising a step of pretreating prior to emulsifying the mixture, wherein the step of pretreating comprises macerating the mixture at a temperature comprised between about 20° C. and about 60° C.

13. A dry formulation obtained by the process of claim 1.

14. A process of manufacture of a product containing at least one substance of interest extracted from a source comprising the at least one substance of interest or at least one precursor thereof, comprising adding a compound selected from the group comprising water or an oil to the dry formulation of claim 13.

15. (canceled)

16. The process of claim 14, wherein the product is a cosmetic, a food supplement or additive, a pharmaceutical, a nutraceutical and/or a probiotic.

17. The process of claim 2, wherein the source is a plant source.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0068] FIG. 1, a schematic diagram illustrating an example of a process according to an embodiment of the invention.

DETAILED DESCRIPTION

[0069] In the following detailed description of embodiments of the present invention, a number of specific details are set forth to provide a more thorough understanding of the present description. However, it will be apparent to the skilled person that the present description can be implemented without these specific details. In other instances, well-known features have not been described in detail to avoid unnecessarily complicating the description.

[0070] FIG. 1 illustrates an embodiment of the extraction process according to the present description, further comprising an additional step of freeze-drying an emulsion obtained by sonication. The mixture to be emulsified comprises water (1), a plant source (2), i.e., Curcuma longa root, a surfactant (3), a lipophilic compound (4). The mixture is contained in a container (5), and emulsifying the mixture comprises a step of sonicating performed by means of an ultrasonic rod (6), capable of emitting ultrasonic waves (7). Sonicating the mixture leads to the formation of an emulsion comprising a continuous aqueous phase (10) and a discontinuous lipid phase in the form of droplets (40) having a certain content of substance of interest (21), i.e., in this case, curcumin. A cryoprotectant is then added to the mixture, and the freeze-drying step leads to the formation of a dry formulation (400). Adding water to this dry formulation, i.e., rehydration, can allow the dry formulation to rehydrate and leads to an emulsion with the same characteristics as before drying. However, in embodiments of the extraction process according to the present description other than that illustrated in FIG. 1, rehydration of the emulsion may lead to an emulsion having different characteristics than the emulsion before drying, e.g., in terms of mean diameter D and polydispersity index.

[0071] Example embodiments of an extraction process according to the present description are detailed below, in which the chosen source is a Curcuma longa root in order to extract curcumin, the substance of interest. Curcumin is a polyphenol whose varied pharmacological properties (anti-inflammatory, antioxidant, anticancer, treatment of certain neurodegenerative diseases, etc.) are only partially explored to date. Moreover, curcumin shows a low solubility in water (11 ng/ml in a buffer solution pH=5), which makes the process according to the present description particularly attractive for extracting curcumin from Curcuma longa. Prior to their attempts to extract curcumin, the inventors tested the solubility of this molecule in various lipophilic compounds, by HPLC assay. Among the lipophilic compounds tested, tributyrin, in particular, proved to be a good candidate. Interestingly, this lipophilic compound is a biocompatible oil, which may make it attractive for certain applications of the process of the invention in the fields of cosmetics, food processing, pharmaceuticals, nutraceuticals and/or probiotics.

[0072] The emulsions numbered 12 to 18, for which certain characteristic conditions of the process for obtaining them, which are otherwise identical, have been collected in Table 1, are emulsions obtained by the process of the invention according to as many different example embodiments of the process of the invention. These embodiments of the process resulting in emulsions 12 to 18 all involve the use of the surfactant TPGS 1000 (α-tocopheryl polypropylene glycol 1000 succinate), namely a commercial surfactant derived from vitamin E (tocopherol), as well as the use of tributyrin as at least one lipophilic compound in the mixture. In emulsions 13 to 18, a second lipophilic compound was added to the mixture provided by the process, in the form of a wax, i.e., jasmine wax for emulsion 13 and Suppocire NB for emulsion 14, or in the form of another oil, i.e., C8-C10 triglycerides for emulsion 18. As apparent for emulsion 14, the inventors succeeded in obtaining, via the process of the invention, an emulsion containing a curcumin content corresponding to an extraction rate of 100%. This extraction rate, fixed at 100% in acetone for a Soxhlet extraction set-up for 8 hours, was obtained under the inventors' conditions in only 2 minutes of sonicating the mixture leading to the formation of emulsion 14.

TABLE-US-00001 TABLE 1 Emulsion 12 13 14 18 m.sub.tributyrin (mg) 206 181 175 85 m.sub.C8-C10 triglycerides (mg) 0 0 0 15 m.sub.SuppocireNB (mg) 0 0 25 0 m.sub.jasmine wax (mg) 0 25 0 0 m.sub.Curcuma longa (mg) 100 100 100 100 m.sub.TPGS 1000 (mg) 60.8 60.8 60.8 60.8 m.sub.H2O (mg) 2027.6 2027.6 2027.6 2027.6 D (nm) 286.5 168.3 194.7 113 PdI 0.296 0.232 0.254 0.257 Extraction rate 67 88 100 93 (%)

[0073] Moreover, it appears that replacing an amount of the first compound, i.e., tributyrin, with an amount approximately equal to its mass equivalent in jasmine wax (emulsion 13) or SuppocireNB (emulsion 14) improves the extraction rate and reduces the polydispersity index PdI, compared with emulsion 12 obtained only from tributyrin. Some protocol details for the production and analysis of emulsions 12 to 18 are provided in the paragraphs below.

[0074] Protocol for Preparing Emulsions 12 to 18:

[0075] The lipophilic compound alone (tributyrin) or the two lipophilic compounds (tributyrin/C8-C10 triglycerides or tributyrin/jasmine wax or tributyrin/Suppocire NB) was/are weighed, with a certain ratio in the case of the mixture of two lipophilic compounds. Furthermore, in the case of an oil/wax mixture, the oil/wax mixture was placed in a 40° C. bath until a homogeneous mixture was obtained.

[0076] The aqueous phase was prepared by dispersing a surfactant (TPGS 1000) in Milli-Q® water. The aqueous phase was then added to the at least one lipophilic compound and the whole was stirred for 20 seconds using a vortex. This solution was then poured into a 50 mL conical centrifuge tube in which a certain mass of Curcuma longa was previously weighed (100 mg).

[0077] A coarse emulsion was prepared by emulsifying the at least one lipophilic compound, the aqueous phase and the plant source first by vortexing for 10 seconds and then placing the centrifuge tube in the ultrasonic bath for 5 minutes at room temperature.

[0078] From this coarse emulsion, a finer emulsion was then prepared using an ultrasonic probe (BIOBLOCK SCIENTIFIC, Vibracell 7504). The ultrasonic probe (3=13 mm) was placed in the centrifuge tube for 16.75 minutes (in pulsed mode, corresponding to 2 minutes of sonicating in total) in an ice bath. The temperature of the mixture during sonicating the mixture was measured and was in the range of about [4° C.-20° C.]. The duty cycle applied was 11.94% and the sonication intensity was 60% (450 W).

[0079] After removing the ultrasound probe from the emulsion, the same probe was dipped into a tube containing 2 mL of Milli-Q® water. The rinse water was then added to the emulsion and the whole was centrifuged 1 minute at 1100 G.

[0080] The resulting emulsion can then be diluted and aliquoted in 2 mL batches. 300 μL of an aqueous cryoprotectant solution (concentration 500 mg/mL), for example trehalose or maltose, can then be added. These aliquots can then be placed in the freezer overnight and freeze-dried for one day.

[0081] Analysis Protocol for Emulsions 12 to 18:

[0082] The supernatant obtained after centrifugation was recovered and the droplet size distribution was analyzed by dynamic light scattering (DLS) using a Nano-S Nanosizer (Malvern Instrument). The supernatant, diluted 1:10 in Milli-Q® water, was placed in a 45 μL quartz cell and underwent 10 measurements of 10 seconds each. The hydrodynamic diameter or mean diameter D was obtained by averaging the results of the 10 measurements. The measurements were performed at an angle of 173° using a laser with a wavelength of 633 nm. The DLS data are calculated on an intensity basis.

[0083] The curcumin concentration of the supernatant was determined by high-performance liquid chromatography after diluting the emulsion in acetonitrile and then filtered through 0.2 μm nylon syringe filter.

[0084] A Shimadzu system with a diode array detector (SPD-M20A) was equipped with a pumping system (LC-20 AD), a degasser (DGU-20A3), a communication module (CBM-20A) and a column oven (Waters). Separation was performed on a Phenomenex Kinetex Biphenyl column (100 Å, 4.6*100 mm, 2.6 μm) with a column temperature set at 30° C. Data analysis was performed with the LabSolution software. A linear gradient of A (water containing 0.1% trifluoroacetic acid) and B (acetonitrile containing 0.1% trifluoroacetic acid) was used with an elution gradient as follows (v/v): 0 min, B 37%; 10 min, B 50%; 15 min, B 100% maintained for 10 min. The flow rate was 1.3 mL/min and the injection volume was 5 Detection was obtained at 420 nm.

[0085] The following Tables 2 to 9 collect certain characteristics of emulsions, i.e., emulsions 19 to 65, prepared according to one or more embodiments of the process of the invention. These emulsions were prepared according to non-detailed embodiment protocols but similar to those described above for the preparation of emulsions 12 to 18. The analysis protocol for emulsions 19 to 65, on the other hand, is identical to that used for emulsions 12 to 18.

[0086] Each Table 2 to 9 aims to present comparable results as obtained under otherwise identical conditions.

TABLE-US-00002 TABLE 2 Emulsion 19 18 20 22 m.sub.tributyrin (mg) 100 85 85 42.5 m.sub.C8-C10 triglycerides (mg) 0 15 0 0 m.sub.SuppocireNB (mg) 0 0 15 7.5 m.sub.Curcuma longa (mg) 100 100 100 100 m.sub.TPGS 1000 (mg) 60.8 60.8 60.8 60.8 m.sub.H2O (mg) 2027.6 2027.6 2027.6 2027.6 D (nm) — 113 109 — PdI >0.3 0.257 0.251 >0.3 Extraction rate 74 93 87 82 (%)

[0087] Table 2 shows emulsions 19 and 20 prepared by weighing the same total mass of lipophilic compound(s) as emulsion 18 of Table 1, namely 100 mg. The results show in particular the benefit of adding a second lipophilic compound in order to reduce the PdI. Emulsion 22 corresponds to emulsion 20 for which the mass of lipophilic compound has been reduced by half, i.e., 50 mg, to the detriment of the homogeneity of the emulsion, whose PdI is higher than 0.3.

[0088] The inventors also showed that emulsion 18, when freeze-dried in the presence of 6.5% (w/v) trehalose (used as cryoprotectant) and then rehydrated, has a D of 147 nm and a PdI of 0.26 (result not shown in Table 2).

TABLE-US-00003 TABLE 3 Emulsion 23 24 25 26 27 m.sub.tributyrin 100 100 100 100 100 (mg) m.sub.Curcuma longa 100 150 200 250 300 (mg) m.sub.TPGS 1000 60.8 60.8 60.8 60.8 60.8 (mg) V.sub.H2O (mL) 4000 4000 4000 4000 4000 D (nm) — 134 112 111 119 PdI >0.3 0.278 0.234 0.209 0.182 Extraction rate 87 90 87 94 97 (%)

[0089] Table 3 indicates that the extraction rate increases with increasing amount of source (Curcuma longa) included in the mixture, as does the homogeneity of the emulsions, which for the latter results in a decrease in PdI.

TABLE-US-00004 TABLE 4 Emulsion 27 28 29 30 31 m.sub.tributyrin (mg) 100 100 100 100 100 m.sub.Curcuma longa (mg) 300 300 300 300 300 m.sub.TPGS 1000 (mg) 60.8 60.8 60.8 60.8 60.8 V.sub.H2O (mL) 4000 4000 4000 4000 4000 Post-extraction 1100 G 1100 G 1100 G 1100 G 1100 G (centrifugation) 1 min 1 min + 1 min + 1 min + 1 min + 5000 G 10000 G 15000 G 20000 G 5 min 5 min 5 min 5 min D (nm) 119 116 116 115 113 PdI 0.182 0.127 0.116 0.112 0.114 Extraction rate 97 81 77 83 81 (%)

[0090] Unlike the opaque emulsion 27, emulsions 28 to 31 presented in Table 4 are translucent, due to the inclusion in the extraction process from which they are derived of a “post-extraction” treatment step comprising several ultra-centrifugation steps.

[0091] The addition of a second ultra-centrifugation step in the post-treatment step results in a slight decrease in the extraction rate, but also in an increase in the homogeneity of the emulsions reflected by the decrease in PdI.

TABLE-US-00005 TABLE 5 Emulsion 32 33 34 35 36 F1 Tributyrin Capryol90 Squalene Tocopherol Triglycerides (C8-C10) m.sub.F1 (mg) 100 100 100 100 100 m.sub.Curcuma longa 100 100 100 100 100 (mg) m.sub.TPGS 1000 60.8 60.8 60.8 60.8 60.8 (mg) m.sub.H2O 2027.6 2027.6 2027.6 2027.6 2027.6 (mg) Pretreating UltraTurrax ® UltraTurrax ® UltraTurrax ® UltraTurrax ® UltraTurrax ® D (nm) — — 138 199 — PdI >0.3 >0.3 0.230 0.277 >0.3 Extraction rate 76 80 79 83 81 (%)

[0092] Table 5 shows the influence of the nature of the lipophilic compound in the context of emulsions comprising a single lipophilic compound (or Fat 1, F1).

[0093] The series of emulsions 32 to 36 presented in Table 5 shows in particular the good homogeneity of emulsions containing tocopherol (also called vitamin E) or squalene as lipophilic compounds.

TABLE-US-00006 TABLE 6 Emulsion 35 37 38 39 F1 Tocopherol Tocopherol Tocopherol Tocopherol m.sub.F1 (mg) 100 100 85 85 m.sub.squalene (mg) 0 0 15 0 m.sub.tributyrin (mg) 0 0 0 15 m.sub.Curcuma longa (mg) 100 100 100 100 m.sub.TPGS 1000 (mg) 60.8 60.8 60.8 60.8 m.sub.H2O (mg) 2027.6 2027.6 2027.6 2027.6 Pretreating UltraTurrax ® — — UltraTurrax ® D (nm) 199 — 136 133 PdI 0.277 >0.3 0.226 0.245 Extraction rate 83 76 79 71 (%)

[0094] Table 6 shows the influence of a specific step of pretreating, as well as the influence of the addition of a second lipophilic compound in the context of emulsions comprising tocopherol as lipophilic compound (or Fat 1, F1).

[0095] The results show that the pre-treatment step including a passage of the mixture to the disperser-homogenizer (UltraTurrax® type) lowers the PdI of the emulsions. The same effect is observed when a second lipophilic compound is used, compared with the case of a single lipophilic compound.

TABLE-US-00007 TABLE 7 Emulsion 40 41 42 Emulsifying Ultrasound UltraTurrax ® UltraTurrax ® 2 min 16 min 2 min m.sub.tributyrin (mg) 255 255 255 m.sub.C8-C10 triglycerides (mg) 45 45 45 m.sub.Curcuma longa (mg) 300 300 300 m.sub.TPGS 1000 (mg) 180 180 180 V.sub.H2O (mL) 6000 6000 6000 D (nm) 88 134 121 PdI 0.260 0.230 0.240 Extraction rate 77 83 83 (%)

[0096] Table 7 compares an emulsion obtained by a process comprising a step of emulsifying the mixture by sonicating (emulsion 40) with two emulsions obtained by a process comprising a step of emulsifying by passage to the disperser-homogenizer (of the UltraTurrax® type).

[0097] The total sonicating time of 2 min for emulsion 40, shown in Table 7, corresponds to the total time that ultrasound is applied to the mixture. Similarly, for emulsions 41 and 42, the time shown is the total time the disperser-homogenizer is applied to the mixture.

[0098] The extraction rate and homogeneity of emulsions 41 and 42 (obtained with the disperser-homogenizer) are similar and slightly higher than the corresponding values characterizing emulsion 40 (obtained by sonicating).

[0099] The inventors have also shown that when the emulsions undergo a “post-extraction” treatment step consisting of ultracentrifugation at 5000 G for 5 min, the values of (D; PdI) for emulsions 40 and 42 are, respectively, (81; 0.200) and (111; 0.222) (result not shown in Table 7).

[0100] Tables 8 and 9 below present the characteristics of other emulsions obtained by extraction processes according to the present description, applying to sources other than Curcuma longa root, such as lavender flower (Table 8) or orange peel (Table 9).

[0101] In both cases, the source is a plant source and contains an essential oil representing the substance of interest.

[0102] Two different surfactants were used to extract the lavender essential oil, namely TPGS 1000 for emulsions 43 to 52 and H.sub.12TAC.sub.5 for emulsions 53 to 55. Emulsions 43 to 47, which do not contain sunflower oil, also illustrate the possibility that the at least one lipophilic compound is exclusively contained in the source, as contemplated in the present description according to one or more embodiments of the process. Table 8 further illustrates the use of a cryoprotectant (trehalose) in different amounts in order to lower the polydispersity index of the emulsions after freeze-drying/rehydration.

[0103] Emulsions 56-60, which do not contain sunflower oil, illustrate the possibility that the at least one lipophilic compound is exclusively contained in the source, as contemplated by the present description according to one or more embodiments of the process. Table 9 further illustrates the use of a cryoprotectant (trehalose) in different amounts to lower the polydispersity index of the emulsions after freeze-drying/rehydration.

TABLE-US-00008 TABLE 8 TPGS Analysis m.sub.sunflower oil m.sub.lavender 1000 H.sub.12TAC.sub.5 V.sub.H2O Trehalose Emulsion conditions (mg) (g) (g/L) (g/L) (mL) (% w/v) D (nm) PdI 43 Before 0 2 0.66 0 15 0 120 0.226 44 After 0 2 0.66 0 15 0 252 0.399 45 After 0 2 0.66 0 15 6.5 173 0.247 46 After 0 2 0.66 0 15 10 167 0.243 47 After 0 2 0.66 0 15 15 152 0.216 48 Before 10 1.25 1 0 10 0 106 0.206 49 After 10 1.25 1 0 10 0 338 0.551 50 After 10 1.25 1 0 10 6.5 172 0.177 51 After 10 1.25 1 0 10 10 177 0.163 52 After 10 1.25 1 0 10 15 152 0.173 53 Before 10 1.25 0 1 10 0 132 0.209 54 After 10 1.25 0 1 10 0 460 0.545 55 After 10 1.25 0 1 10 6.5 137 0.218

TABLE-US-00009 TABLE 9 TPGS Analysis m.sub.sunflower oil m.sub.orange peel 1000 V.sub.H2O Trehalose Emulsion conditions (mg) (g) (g/L) (mL) (% w/v) D (nm) PdI 56 Before 0 2 1 10 0 137 0.160 57 After 0 2 1 10 0 160 0.462 58 After 0 2 1 10 6.5 146 0.362 59 After 0 2 1 10 10 159 0.426 60 After 0 2 1 10 15 152 0.343 61 Before 10 2 1 10 0 137 0.201 62 After 10 2 1 10 0 140 0.914 63 After 10 2 1 10 6.5 126 0.970 64 After 10 2 1 10 10 233 0.734 65 After 10 2 1 10 15 131 0.967