Method for extraction of an agent from a plant source

Abstract

Provided is a process for extraction of a lipophilic agent from a plant source, the process including: mixing a first quantity of a plant source, the plant source being containing the lipophilic agent and a first quantity of an extraction medium to obtain a first mixture, the extraction medium being in the form of a microemulsion and comprising at least one oil, at least one hydrophilic surfactant, at least one co-surfactant and optionally at least one co-solvent; homogenizing the first mixture under conditions maintaining the microemulsion structure; and separating the homogenized mixture into a biomass slurry and an agent-loaded medium to obtain the agent-loaded medium in a microemulsion form.

Claims

1. A process for extraction of a lipophilic agent from a plant source, the process comprising: (a) mixing a first quantity of a plant source, the plant source containing the lipophilic agent and a first quantity of an extraction medium to obtain a first mixture, the extraction medium being in the form of a microemulsion and comprising at least one oil, at least one hydrophilic surfactant, at least one co-surfactant and optionally at least one co-solvent; (b) homogenizing the first mixture under conditions maintaining the microemulsion structure; and (c) separating the homogenized mixture into a biomass slurry and an agent-loaded medium to obtain the agent-loaded medium in a microemulsion form.

2. The process of claim 1, wherein the agent-loaded medium comprises between about 0.02 and 20 wt % of the lipophilic agent.

3. The process of claim 1, wherein the homogenization of step (b) is carried out in at least one condition selected from (i) a period of time of between about 1 minute and about 120 minutes, (ii) at a pressure of between about 500 and 6,000 psi, and (iii) at a temperature of between about 5 and about 70° C.

4. The process of claim 1, wherein the weight ratio (wt/wt) of the first quantity of plant source to the first quantity of extraction medium is between 1:5 and 1:80.

5. The process of claim 1, further comprising: (d) mixing the biomass slurry with a second quantity of the extraction medium being in the form of a microemulsion to obtain a second mixture; (e) homogenizing the second mixture; and (f) separating the second mixture into biomass slurry and agent-loaded medium to obtain the agent-loaded medium in a microemulsion form.

6. The process of claim 1, further comprising: (d′) mixing the agent-loaded medium in a microemulsion form with a second quantity of the plant source to obtain a second mixture; (e′) homogenizing the second mixture; and (f′) separating the second mixture into biomass slurry and highly agent-loaded medium in a microemulsion form.

7. The process of claim 1, wherein said at least one oil is selected from the group consisting of essential oils, D-limonene, mineral oil, paraffinic oils, phospholipids, polar lipids, squalenes, sphingomyelins, waxes, vegetable oils, glycerides, triglycerides, fatty acids and esters of fatty acids, and liquid hydrocarbons, wherein said at least one oil being present in the extraction medium at an amount of between about 0.5 and 20 wt %.

8. The process of claim 1, wherein said at least one hydrophilic surfactant is selected from the group consisting of polyethylene glycol (15)-hydroxystearate (Solutol HS15), polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monooleate, and polyoxyethylene esters of saturated and unsaturated castor oil, ethoxylated monoglycerol esters, ethoxylated fatty acids, ethoxylated fatty acids of short, medium and long chain fatty acids, wherein said at least one hydrophilic surfactant being present in the extraction medium at an amount of between about 30 and 85 wt %.

9. The process of claim 1, wherein said at least one co-surfactant is selected from the group consisting of polypropylene glycol, polyethylene glycol, sorbitol, xylitol, PEG 200, PEG 400 and PEG 600, wherein said at least one co-surfactant being present in the extraction medium at an amount of between about 1 and 50 wt %.

10. The process of claim 1, wherein the extraction medium further comprises at least one phospholipid, in an amount of between about 1 and 10 wt % of phospholipids; and/or at least one solvent, wherein the extraction medium comprises between about 0.1 and 25 wt % of said solvent.

11. The process of claim 1, wherein the extraction medium is essentially free of water.

12. The process of claim 1, wherein the lipophilic agent to be extracted is selected from the group consisting of astaxanthin, lycopene, beta-carotene, lutein, eugenol, piperine, anthocyanins, betain, oleuropein, trimyristin, curcumin, capsaicin, gossipol, rosmanol, chlorogenic acid, cynamaldehyde, flavones, caffeine, isoflavone, tocopherol, omega fatty acids (including DHA and EPA), caffeic acid, niacin, nicotinamide, flavonoids, cineole, borneol, thujone, carnosol, carnosic acid, fumaric acid, behenic acid; and any triglycerides, or esters of long chain fatty acids of the lipophilic agent.

13. The process of claim 1, wherein the plant source is an algae or a microalgae.

14. The process of claim 13, wherein the microalgae is Haematococcus pluvialis and the lipophilic agent is astaxanthin.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

(2) FIGS. 1A-1B show astaxanthin (AX) concentration (FIG. 1A) and yield of AX extraction (FIG. 1B) in the AX-1 extraction medium as a function of plant-to-medium ratio (w/w).

(3) FIGS. 2A-2B show AX concentration (FIG. 2A) and yield of AX extraction (FIG. 2B) in the AX-1 extraction medium as a function of extraction duration.

(4) FIGS. 3A-3B show AX concentration (FIG. 3A) and yield of AX extraction (FIG. 3B) in the AX-1 extraction medium as a function of the number of extraction cycles.

(5) FIGS. 4A-4B show AX concentration (FIG. 4A) and yield of AX extraction (FIG. 4B) in the AX-1 extraction medium as a function of the number of extraction cycles from the same biomass.

(6) FIG. 5 shows the effect of water dilution on unloaded and AX-loaded extraction medium, as measured by electrical conductivity tests.

(7) FIG. 6 shows the effect of water dilution on the viscosity of unloaded and AX-loaded extraction medium.

(8) FIG. 7 shows AX-loaded extraction medium, diluted by water (0-90 wt % water).

(9) FIGS. 8A-8B show the diffusion coefficients of the components of the extraction medium (as measured by SD-NMR) for unloaded and AX-loaded AX-1 medium: full composition (FIG. 8A), AX-limonene-tween in isolation (FIG. 8B).

(10) FIGS. 9A-9B show various agent-loaded mediums, concentrates (FIG. 9A) and diluted by 90 wt % water (FIG. 9B), from left to right: unloaded AX-1, carrots, turmeric, marigold, beetroot, nutmeg, tomato, black pepper, allspice and olive leaves.

(11) FIGS. 10A-10B show the concentration (FIG. 10A) and yield of extraction (FIG. 10B) of astaxanthin, curcumin and piperine as a function of the number of extraction cycles.

DETAILED DESCRIPTION OF EMBODIMENTS

(12) Astaxanthin as a Model Lipophilic Agent

(13) In the following examples, astaxanthin (AX) was used as a model lipophilic agent. Astaxanthin is a water-insoluble antioxidant from the carotenoids group, having the following structure (Formula I):

(14) ##STR00001##

(15) It is to be understood that various other lipophilic agents may be extracted from various plant sources by the process and extraction media disclosed herein, and the extraction of astaxanthin is brought for exemplifying purposes only. Some additional exemplary lipophilic agents will be provided below.

(16) Extraction Medium Composition and Preparation

(17) As noted above, the extraction media used for the extraction process are self-assembled systems which are formed in a spontaneous manner. Therefore, the compositions of the extraction medium were prepared by simple mixing of ingredients at 15-70° C. An exemplary process for preparing the extraction medium involves mixing together the oil, the surfactant and the co-surfactant (and where applicable also a solvent, a co-solvent and/or a phospholipid) until a homogenous, clear (transparent) mixture is obtained. In case the surfactants or oil are solid at room temperature, heating can be applied while mixing to allow full dissolution and formation of the empty extraction medium.

(18) The extraction medium is then slowly added to the plant to allow appropriate wetting and then mixed and homogenized. Another variation of the process includes adding solid plant parts (for example leaves or buds) stepwise to the empty (un-loaded) extraction medium until a homogeneous slurry is obtained.

(19) Extraction was carried out under heating and inert atmosphere, thereby solubilizing the desired lipophilic agent into the extraction medium. The mixture was allowed to settle to the bottom of the mixing vessel before filtration and/or centrifugation.

(20) Table 1 provides details of exemplary formulations used in the process of the present disclosure.

(21) TABLE-US-00001 TABLE 1 Formulations of extraction medium Formulation AX-1 Formulation AX-2 Component wt % Component wt % Oil R-(+)-Limonene 5 MCT 3.5 Hydrophilic Polysorbate 80 (Tween 45 Tween 80 35 surfactant 80) Chremophore 42 Co-surfactant Propylene glycol (PG) 45 PG 13 Solvent Ethanol 5 Ethanol 1.5

(22) Extraction Process Parameters

(23) Effect of Plant-to-Medium Ratio

(24) Haematococcus pluvialis microalgae samples provided by Algatech (batch number 219) were mixed with AX-1 extraction medium at a weight ratio of between 1:3 and 1:15 (plant:medium). The mixture was then homogenized at room temperature using lab Silverson homogenizer L5M-A for 30 minutes. After homogenization, each sample was centrifuged at 4000 rpm for 20 minutes or filtered through cotton wool. Samples were prepared in triplicates.

(25) Analysis of astaxanthin content in the extracts was carried out by UV-vis spectrophotometer (max value at 472 nm) or HPLC vis-à-vis a calibration curve (using the following conditions: C18 column, mobile phase gradient of methanol/water (69/31 v/v %) to 100% methanol, flow rate 0.3 ml/min).

(26) FIGS. 1A-1B show the concentration of astaxanthin (AX) in the extracts and the extraction yield, respectively, as a function of the plant-to-medium ratio. Although the nominal concentration decreases upon increasing the plant:medium ratio (i.e. due to the dilution effect), the higher the weight ratio between the medium and the microalgae a higher extraction yield is obtained, reaching app. 80% extraction yield in a single extraction cycle.

(27) Effect of Extraction Duration

(28) Haematococcus pluvialis microalgae samples were mixed with AX-1 extraction medium at a weight ratio of 1:40. The mixture was then homogenized at room temperature using Silverson homogenizer for various periods of time. After homogenization, each sample was centrifuged at 4000 rpm for 20 minutes or filtered through cotton wool. Samples were prepared in triplicates.

(29) Analysis of astaxanthin content in the extracts was carried out by UV-vis spectrophotometer (max value at 472 nm) or HPLC vis-à-vis calibration curves. FIGS. 2A-2B show the concentration of AX in the extracts and the extraction yield, respectively, as a function of the extraction time.

(30) As can clearly see from the results, extraction yields of AX in a single extraction round ranged between 60-80% and up to ˜0.6 wt % AX-loading in to the medium.

(31) Multiple-Extractions Processes

(32) Multiple Cycles Using the Same Quota of Extraction Medium

(33) Increasing the concentration of the lipophilic agent, in this case AX, in the medium was carried out by a multi-extraction process. For the multi-extraction process a number of extraction cycles are carried out by using the same quota of medium for several consecutive extraction cycles, in each cycle a fresh sample of microalgae is extracted according to the following procedure.

(34) A Haematococcus pluvialis microalgae biomass sample was mixed with AX-1 extraction medium at a weight ratio of 1:10. The mixture was then homogenized at room temperature using Silverson homogenizer for 10 minutes. After homogenization, each sample was centrifuged at 4000 rpm for 20 minutes or filtered through cotton wool. After separating the AX-loaded medium from the spent biomass, the AX-loaded medium was weighed and a new sample of microalgae was added at a weight ratio of 1:15 (plant:medium). Homogenization and separation were carried out for the new mixture. One additional cycle of extraction was carried out, amounting to a total of 3 extraction cycles.

(35) Samples of the medium were taken in between cycles to assess the effect of the number of cycles on the AX-loading of the medium.

(36) Analysis of AX content was done according to the description hereinabove. FIGS. 3A and 3B show the concentration of AX in the extracts and the extraction yield, respectively, as a function of the number of extraction cycles.

(37) As evident from the results, the AX content in the medium increases by at least 2.5-folds as a result of the multi-extraction process. However, as the extraction medium becomes loaded with AX, the extraction efficiency (i.e. yield) of the medium decreases compared to the extraction efficiency at the first cycle of extraction, due to the proximity of the AX content to the maximum loading capacity of the medium.

(38) Multiple Cycles Using the Same Biomass of Microalgae

(39) Similarly, the efficiency of extraction from the plant source by using multiple cycles of extraction from the same biomass was tested; namely, the capability to utilized spent biomass for extraction of additional AX from the same sample.

(40) A Haematococcus pluvialis microalgae biomass sample was mixed with AX-1 extraction medium at a weight ratio of 1:10. The mixture was then homogenized at room temperature using Silverson homogenizer for 10 minutes. After homogenization, each sample was centrifuged at 4000 rpm for 20 minutes or filtered through cotton wool. After separating the AX-loaded medium from the spent biomass, the spent biomass was weighed and a fresh quota of extraction medium was added at a weight ratio of 1:15 (plant:medium). Homogenization and separation were carried out for the new mixture. One additional cycle of extraction was carried out, amounting to a total of 3 extraction cycles.

(41) Samples of the medium were taken in between cycles to assess the effect of the number of cycles on the AX-loading of the medium.

(42) Analysis of AX content was done according to the description hereinabove. FIGS. 4A and 4B show the concentration of AX in the extracts and the extraction yield, respectively, as a function of the number of extraction cycles.

(43) As can clearly be seen, the extraction of AX from the microalgae in the first extraction cycle is shown to have relatively high extraction yield. Namely, in the second and third cycles of extraction from the same sample of microalgae (i.e. extraction of the spent biomass by fresh extraction medium quota), significantly less AX was extracted from the spent biomass. This attests to the relatively high extraction yield obtained in the first extraction round (namely when extracting the fresh microalgae).

(44) However, as also evident, it is possible to reach about 80% extraction yield cumulatively by employing multiple extraction cycles on the same spent biomass.

(45) Tables 2-3 below provide a comparative summary of the extraction yields by using the various extraction processes.

(46) TABLE-US-00002 TABLE 2 extraction yields of a single extraction cycle Extraction duration AX conc. in ME (wt %) Extraction yield (%) 10 min 0.56 ± 0.01 79.5 ± 0.8 60 min 0.55 ± 0.02 79.5 ± 0.8

(47) TABLE-US-00003 TABLE 3 extraction yields of multiple extraction cycles AX conc. in medium (wt %) Extraction yield (%) Accumulated 1.sup.st cycle 2.sup.nd cycle 3.sup.rd cycle 1.sup.st cycle 2.sup.nd cycle 3.sup.rd cycle yield (%) (1) 0.59 ± 0.01 1.10 ± 0.08 1.63 ± 0.01 76.4 ± 5.1 72.4 ± 21.4 72.9 ± 12.1 73.9 ± 12.9 (2) 0.45 ± 0.05 0.08 ± 0.01 0.06 ± 0.04 63.8 ± 6.6 30.7 ± 03.7 31.8 ± 14.8 75.9 ± 3.2  (1) same medium, new biomass (2) same biomass, fresh extraction medium

(48) Characterization and Dilutability of the AX-Loaded Medium

(49) Effect of Dilution on the Medium's Structure

(50) As noted above, the media described herein are substantially devoid of water (i.e. in the form of a concentrate), constituted by self-assembled oil-solvated clusters or short domains of surfactants, which differ from the classical reverse micelles. The concentrates are dilutable by any suitable diluent, for example by water, to form a diluted delivery system.

(51) The effect of water dilution on the medium's structure was investigated by using electrical conductivity tests. Electrical conductivity measurements were performed at 25±2° C. using a conductivity meter, type CDM 730 (Mettler Toledo GmbH, Greifensee, Switzerland). Measurements were made on empty and AX-loaded samples upon dilution with water up to 95 wt %. No electrolytes were added to the samples. The conductivity allows to distinguish between the continuous phase and the inner phase. The results are shown in FIG. 5.

(52) As can be seen, the microemulsions undergo 2 phase transitions upon increasing the amount of diluent. When in concentrate form, the system is in the form of reverse micelles, solubilizing the AX within the core of the micelle. When mixed with small amounts of water, a bicontinuous structure of solvated (oil-rich) and hydrated (water-rich) domains are formed; upon further dilution with water, the bicontinuous structure progressively and continuously transforms into oil-in-water (O/W) nanodroplets entrapping AX molecules within their oily core (or within the surfactants tails). As also noted above, the transformation to O/W microemulsions is spontaneous, i.e. without the need to employ shear stresses or excessive heating conditions. Thus, throughout the phase transformations occurring upon dilution, AX is stabilized and solubilized within the oily phase (as will be further explained below in connection with SD-NMR analysis).

(53) In addition, when comparing the unloaded system with the AX-loaded system, it seems that the presence of AX has no significant effect on the system's structure and its ability to undergo the phase transitions.

(54) Similar results were obtained in viscosity measurements of unloaded and AX-loaded medium, as shown in FIG. 6. Viscosity measurements were performed at 25±1° C. on empty and AX-loaded MEs (Thermo Electron GmbH, Karlsruhe, Germany) using a cone (60 mm diameter) and glass plate. Shear rates were 0-1000 in the water.

(55) Droplet Size

(56) One of the advantages of the concentrates described herein is the ability to be diluted at various dilution ratios, without significantly affecting the formulation's properties.

(57) The hydrodynamic radius of the oil droplets was measured at room temperature by small-angle x-ray scattering (SAXS). Scattering experiments were performed using CuKα radiation (λ=0.154 nm) from Rigaku RA-MicroMax 007 HF X-ray generator operated at a power rating up to 1.2 kW and generating a 70×70 μm.sup.2 focal spot. The osmic CMF12-100CU8 unit produced a beam size at the sample position of 0.7×0.7 mm.sup.2. The scatter radiation passed through a He-filled flight path and was detected by a Mar345 imagine plate detector from Marresearch (Nordestedt, Germany). Samples were inserted into 1.5 mm quartz capillaries and scanned for 15 min at T=25±1° C. The sample to detector distance was calibrated using silver behenate. Curve fitting of the SAXS profiles was performed using Origin (MicroCal, MA).

(58) Average droplet sizes for unloaded and AX-loaded media at different dilutions (i.e. different water concentrations) are provided in Table 4.

(59) TABLE-US-00004 TABLE 4 Average droplet size values at different dilutions Water content (wt %) Unloaded system (nm) AX-loaded (nm) 0 12.57 15.65 10 7.34 8.89 30 8.51 9.16 50 9.59 10.60 90 5.93 6.95

(60) As clearly evident from Table 4, the droplet sizes of the empty system are smaller than those measured for the loaded systems indicating that AX is located within the core/interface of the drop increasing its size (see also SD-NMR analysis below).

(61) Further, as seen in FIG. 7, the very small droplet size that characterizes the media described herein (typically have an average droplet diameter of less than 20 nm) enables obtaining clear and transparent microemulsions for prolonged periods of time, without phase separation. The transparency also enables detection of undesired contaminants, thereby allowing the user to easily identify in a visual manner contaminated formulations that are unsuitable for use.

(62) Self-Diffusion NMR (SD-NMR)

(63) In order to determine the structure of the oil droplets (or micelles) of the medium, self-diffusion NMR analysis was carried out. SD-NMR is able to locate each component within the medium via measurements of its diffusion coefficient. Rapid diffusion (>100×10.sup.−12 m.sup.2s.sup.−1) is characteristic of small molecules, free in solution, while slow diffusion coefficients (<0.1×10.sup.−12 m.sup.2s.sup.−1) suggest low mobility of macromolecules or bound/aggregated molecules.

(64) NMR measurements were performed with a Bruker AVII 500 spectrometer equipped with GREAT 1/10 gradients, a 5 mm BBO and a 5 mm BBI probe, both with a z-gradient coil and with a maximum gradient strength of 0.509 and 0.544 Tm.sup.−1, respectively. Diffusion was measured using an asymmetric bipolar longitudinal eddy-current delay (bpLED) experiment, or an asymmetric bipolar stimulated echo (known as one-shot) experiment with convection compensation and an asymmetry factor of 20%, ramping the strongest gradient from 2% to 95% of maximum strength in 32 steps. The spectrum was processed with the Bruker TOPSPIN software. NMR spectra were recorded at 25±0.2° C. The components were identified by their chemical shift in 1H NMR.

(65) Tables 5-1 and 5-2 shows the diffusion coefficients (Dx, m.sup.2/sec) of the unloaded and AX-loaded medium (with 0.5 wt % of AX), respectively, at various water dilutions. The results are also graphically presented in FIGS. 8A-8B.

(66) TABLE-US-00005 TABLE 5-1 Diffusion coefficients (m.sup.2/sec), as measured by SD-NMR, unloaded extraction medium Water content R-(+)- (wt %) Tween 80 PG limonene EtOH D.sub.2O 0 4.6 × 10.sup.−12 4.53 × 10.sup.−11 5.66 × 10.sup.−11 4.58 × 10.sup.−11 — 10 3.3 × 10.sup.−12 4.74 × 10.sup.−11 3.33 × 10.sup.−11 4.85 × 10.sup.−11  3.3 × 10.sup.−11 20 1.5 × 10.sup.−12 6.15 × 10.sup.−11 1.36 × 10.sup.−11 6.17 × 10.sup.−11 1.35 × 10.sup.−11 30   6 × 10.sup.−13 8.55 × 10.sup.−11  4.3 × 10.sup.−12 8.67 × 10.sup.−11 2.64 × 10.sup.−10 40   5 × 10.sup.−13 1.24 × 10.sup.−10  1.5 × 10.sup.−12 1.25 × 10.sup.−10 3.88 × 10.sup.−10 50   9 × 10.sup.−13 1.68 × 10.sup.−10  1.5 × 10.sup.−12 1.65 × 10.sup.−10  5.0 × 10.sup.−10 60 2.2 × 10.sup.−12 2.34 × 10.sup.−10  3.3 × 10.sup.−12 2.37 × 10.sup.−10 6.34 × 10.sup.−10 70 5.7 × 10.sup.−12 3.19 × 10.sup.−10  5.9 × 10.sup.−12  3.3 × 10.sup.−10  9.1 × 10.sup.−10 80 1.09 × 10.sup.−11  4.42 × 10.sup.−10 1.15 × 10.sup.−11 4.82 × 10.sup.−10 1.13 × 10.sup.−9  90 2.06 × 10.sup.−11  6.03 × 10.sup.−10 2.14 × 10.sup.−11 5.82 × 10.sup.−10 1.45 × 10.sup.−09

(67) TABLE-US-00006 TABLE 5-2 Diffusion coefficients (m.sup.2/sec), as measured by SD-NMR, 0.5 wt % AX-loaded medium Water content R-(+)- (wt %) Tween 80 PG limonene EtOH D.sub.2O AX  0  4.5 × 10.sup.−12 4.43 × 10.sup.−11 5.46 × 10.sup.−11 4.13 × 10.sup.−11 —  4.7 × 10.sup.−12 10  3.5 × 10.sup.−12 4.78 × 10.sup.−11 3.09 × 10.sup.−11 4.66 × 10.sup.−11  3.1 × 10.sup.−11   3 × 10.sup.−12 20  2.9 × 10.sup.−12 4.86 × 10.sup.−11 2.36 × 10.sup.−11 4.78 × 10.sup.−11 2.84 × 10.sup.−11  1.4 × 10.sup.−12 30   6 × 10.sup.−13 8.69 × 10.sup.−11  3.2 × 10.sup.−12 8.67 × 10.sup.−11  3.2 × 10.sup.−12   3 × 10.sup.−13 40   4 × 10.sup.−13 1.23 × 10.sup.−10  1.4 × 10.sup.−12  1.2 × 10.sup.−10  1.3 × 10.sup.−12   3 × 10.sup.−13 50   9 × 10.sup.−13 1.75 × 10.sup.−10  1.4 × 10.sup.−12  1.7 × 10.sup.−10 2.69 × 10.sup.−11   7 × 10.sup.−13 60  2.2 × 10.sup.−12 2.41 × 10.sup.−10  3.1 × 10.sup.−12 2.33 × 10.sup.−10 7.96 × 10.sup.−10  1.4 × 10.sup.−12 70  5.3 × 10.sup.−12 3.35 × 10.sup.−10  5.7 × 10.sup.−12 3.32 × 10.sup.−10  9.1 × 10.sup.−10  4.8 × 10.sup.−12 80 1.05 × 10.sup.−11 4.67 × 10.sup.−10 1.10 × 10.sup.−11 4.67 × 10.sup.−10 1.16 × 10.sup.−9  1.12 × 10.sup.−11 90 1.91 × 10.sup.−11 6.03 × 10.sup.−10 1.91 × 10.sup.−11 5.89 × 10.sup.−10 1.48 × 10.sup.−09

(68) As can be seen from Tables 5-1 and 5-2, the diffusion coefficient of AX is similar to that of the surfactants (Tween 80 and R-(+)-limonene). These results indicate that the AX is located within the core and at the interface of the swollen micelle. As also evident from the results, AX solubilization causes the binding of water molecules through interaction with the surfactant. This suggests that all of the AX in the media is contained within the oil droplet, and it is likely that no free AX is within the aqueous continuous phase, attesting to the ability of the medium to contain and stabilize AX.

(69) Further, at dilutions of ˜30-50% water, AX binds water through interaction with the surfactant, as evident by the low diffusion coefficients of D.sub.2O in the AX-loaded medium compared to the unladed system. This means that astaxanthin has no direct interaction with the water, however, it affects the water mobility that is induced by the surfactant.

(70) Other Lipophilic Agents

(71) Astaxanthin was demonstrated above as a model lipophilic agent for extraction processes described herein. However, the process may be used to extract a variety of lipophilic agents from a variety from natural sources.

(72) A number of plant sources containing active molecules were chosen to show the high effectiveness of the extraction medium for the extraction of various active molecules: piperine (extracted from black pepper corns), oleuropein (extracted from olive leaves), trimyristin (extracted from nutmeg), eugenol (extracted from allspice corns), curcumin (extracted from turmeric root), carotene (extracted from baby carrots), lycopene (extracted from dried tomatoes and lycopene rich cherry tomatoes), lutein (extracted from marigold flowers), and betain and anthocyanins (extracted from beetroot).

(73) According to the raw plant, pretreatment was optionally carried out. Olive leaves, nutmeg (grounded), and turmeric (grounded) were used as received. Black pepper and allspice were pounded with pestle and mortar and sieved. Beetroot, dried tomatoes and baby carrots were finely chopped and heated at vacuum oven for 1.5 hours at 60° C. Marigold flowers were washed and dried, and petals were separated and heated under vacuum for 1.5 hours at 60° C. Lycopene rich cherry tomatoes were mashed in a blender; half of the mashed tomatoes were centrifuged twice to remove most of the water. The other half was lyophilized for 20 hours.

(74) Extraction was carried out as follow: plants samples were weighted, and then AX-1 extraction medium was weighted into the same flask. The ratios between the plant and the extraction medium were determined based on the plant and were within a range of 1:1 and 1:10. A Silverson homogenizer was used for homogenization for 30 min at room temperature (25-28° C.). At the end of the extraction, the slurry was centrifuged for 20 min for separating the loaded medium from the spent biomass. The loaded medium was filtered and kept in the fridge until been analyzed.

(75) For the extraction of piperine and curcumin another series of 3 cycles of extractions was accomplished, using new plant source and loaded-ME at each cycle.

(76) Droplet Size Measurements

(77) The loaded-medium samples were diluted by 90 wt % water and droplet size was measured with DLS.

(78) Table 6 presents the droplet size of the extracts from different plant sources as well as the polydispersity index. FIGS. 9A and 9B show the concentrates and diluted mediums, respectively.

(79) TABLE-US-00007 TABLE 6 Droplet size and polydispersity index of agent-loaded mediums, 90 wt % water Plant source Drop size [nm] PDI Allspice NA NA Black pepper 12.05 ± 0.45 0.15 ± 0.01 Nutmeg 14.92 ± 0.32 0.27 ± 0.32 Olive leaves NA NA Turmeric 11.84 ± 0.19 0.21 ± 0.01 Tomatoes 12.03 ± 0.26 0.14 ± 0.03 Carrots 11.55 ± 0.21 0.18 ± 0.02 Marigold flower 11.57 ± 0.01 0.17 ± 0.01 Beetroot 18.33 ± 0.37 0.36 ± 0.01 Empty system 11.24 ± 0.26 0.115 ± 0.024

(80) Concentration of Active Molecules in the Medium and Yield of the Extraction

(81) One Step Extraction

(82) Extraction products from black pepper, turmeric, tomato paste, and lyophilized tomato were diluted with distilled water or organic solvents (ethanol or acetone) at 10.sup.2-10.sup.3 order of magnitude. UV-visible spectrum of the dilutions were recorded against suitable blank and absorption at the chosen wavelength was taken for the determination of the component in the extract. A spectrum of standards was recorded for each agent, from which the wavelength at the maximum was chosen as the wavelength at which calibration curve was constructed based on Beer-Lambert law. Further calculation based on reported data about plant capacity of each nutraceutical were carried out to determine the yield of the extraction. Table 7 details the concentration of the active molecules in the agent-loaded mediums and the yield of the extraction.

(83) TABLE-US-00008 TABLE 7 The concentration of the active molecules and yield of the extraction after one cycle Concentration Yield of Active Concentration in medium extraction molecule Source λmax [nm] in plant source [mg/g] [%] Piperine Black pepper 310 5-9% [2] 12.48 69.3-89.2 (grounded) Curcumin Turmeric 424 0.58-3.14% [3] 2.02 32.2-50.5 (grounded) Lycopene Tomato paste 480 340 ppm [4] 0.04 57.9 Lyophilized 480 900-1200 ppm 0.05 40.6-58.1 tomatoes [5]
Yield determined according to range of concentrations of component in the plant

(84) Multi-Step Extraction

(85) Extraction products from black pepper and turmeric were diluted with ethanol or acetone at 10.sup.3-10.sup.4 order of magnitude. Analysis was the same as in case of one step extraction. FIG. 10A shows the concentrations of curcumin and piperine, as compared to astaxanthin, in the medium after one, two and three cycles. Linear fit was plotted for each component. FIG. 10B shows the yield of the extraction at each of the three cycles for astaxanthin, curcumin and piperine.

(86) As clearly evident, the extraction mediums and process described herein demonstrate the ability to extract various molecules from their natural source, which appears in a variety of forms. As also evident, for the same extraction medium formulation, different concentrations and yields were achieved for each extracted molecule, suggesting that selectivity of extraction may be controlled by tailoring the composition of the microemulsion to the desired molecule to be extracted.

(87) Further exemplary target molecules which were extracted by AX-1 extraction medium formulation were rosmanol acid (extracted from rosemary leaves), cinnamaldehyde (extracted from cinnamon bark), chlorogenic acid (extracted from green coffee beans), and omega 3 fatty acid (extracted from chia seeds).

(88) Cinnamon and chia seeds were used as received. Green coffee beans were ground and pounded by pestle and mortar. Rosemary was either chopped or heated for 13 min before the extraction.

(89) Extraction was carried out as detailed above. Initial characterization results are provided in Table 8.

(90) TABLE-US-00009 TABLE 8 Characterization of mediums loaded with the active molecules Viscosity of concentrate Active molecule Drop size [nm] [μS/cm] Rosmarinic acid 21.41 (±0.36) 0.129 (±0.013) Cinamaldehyde 17.87 (±0.97) 0.141 (±0.002) Chlorogenic acid 11.45 (±0.77) 0.093 (±0.009) Linolenic acid 26.05 (±0.97) NA