Patent Description:
Polyhydroxylated Fatty Alcohols have known cosmetic and pharmaceutical benefits, for example, a significant increase in the inhibition of T-cell proliferation which may be highly helpful when treating inflammations and various pathogenic conditions, such as atopic dermatitis, contact dermatitis, rheumatoid arthritis etc. Polyhydroxylated fatty alcohols (PFA) are crystalline lipophilic molecules. PFA molecules have a long aliphatic (mostly C17) chain with one end unsaturated with a double or triple (acetylenic) bond, and the other end having three hydroxyl groups. Some of the PFA molecules are polar and the other are non-polar molecules. The polarity of the different types of PFA molecules depends on the presence of hydroxyl groups, double or triple bonds or other polar groups in the molecule. Therefore, since all known solvents are either polar (e.g., suitable for extracting polar extracts) or non-polar (e.g., suitable for extracting non-polar extracts) it is almost impossible to extract all PFA molecules using the same solvent.

Some types of PFA can be extracted from Avocado seeds, using organic solvents, for example, methanol, alkyl acetates (e.g., butyl acetate) and the like. These organic solvents are toxic and environmentally unfriendly. Therefore, any cosmetic and pharmaceutical product that includes PFA extracted using an organic solvent must be cleaned from any residual solvent. An example of extracting PFA from Avoacdo seeds using an organic solvent is given in <CIT>. The patent discloses: crushing raw undried avocado seeds that were not subject to any type of drying treatment, to provide crushed avocado seeds at a bulk density between about <NUM> and <NUM>/ml; incubating the crushed avocado seeds at a temperature between <NUM> and <NUM>° C. for between <NUM> minutes to <NUM> hours; and extracting PFAs from the crushed seeds with an organic solvent selected from the group consisting of methanol and alkyl acetates or any combination thereof.

There are three solvents that are considered toxin-free (e. g, non-toxic), water, ethanol and supercritical CO<NUM>. Water and ethanol are polar solvents not suitable for extractions of oils and the non-polar PFA molecules. Supercritical CO<NUM> is intrinsically non-polar, and it is suitable solvent for extraction of non-polar compounds, such as, oil and some low molecular weight, volatile, polar compounds. However, it is less effective in the extraction of polar phytochemicals embedded in the cell wall, such as polar PFA molecules.

Furthermore, even for non-polar extracts such as oils from cursed seeds, the known practice in the field includes the addition of <NUM>-<NUM> wt. % of organic cosolvent, to the supercritical CO<NUM>. Such an addition is known to be essential for extraction of oils from crushed seeds. Therefore, in all known commercial lines a cosolvent tank is included in the line for adding cosolvent to the supercritical CO<NUM>. The added cosolvents may enhance the solvation power of supercritical CO<NUM> and improve the recovery of bioactive compounds (e.g., the diffusion of the bioactive compounds via the cells wall). This process is used for extraction of oils from crushed seeds, such as, lemon, mandarin, orange, watermelon, Cucurbita pepo, tomato and the like. The organic cosolvent, although in smaller amounts than in methods based solely on the organic cosolvent, still needs to be removed and recycled and can make the extracted oil contaminated with residual cosolvent. Any process that uses such cosolvents cannot be regarded as <NUM>% environmentally friendly or non-toxic. An example for extracting non-polar molecules such as terpenes from cumin seeds is given in the article "<NPL>). The article discloses extracting terpenes from cumin seeds using a solvent comprising <NUM>% supercritical CO<NUM>.

Accordingly, there is a great benefit in having a non-toxic process for extracting all molecules of PFA from avocado seeds using a single solvent.

Aspects of the invention are directed to a method of extracting Polyhydroxylated Fatty Alcohols according to claim <NUM>. The method includes: drying the crushed avocado seeds to a humidity level of at most <NUM> wt. %; inserting crushed avocado seeds to one or more extraction chambers; flowing supercritical CO<NUM> fluid, at a pressure range of <NUM> bar to <NUM> bar, through the crushed avocado seeds in one or more extraction chambers; and separating from the supercritical CO<NUM> flown through the crushed avocado seeds the Polyhydroxylated Fatty Alcohols extracted and dissolved in the supercritical CO<NUM>. The supercritical CO<NUM> fluid is the only solvent used for dissolving and extracting the Polyhydroxylated Fatty Alcohols.

In some embodiments, the crushed avocado seeds may have a particle size of at most <NUM>. In some embodiments, the supercritical CO<NUM> fluid may have a temperature range of <NUM> to <NUM>.

In some embodiments, flowing the supercritical CO<NUM> fluid may include: obtaining liquidized CO<NUM>; increasing the pressure of the liquidized CO<NUM> to reach a required supercritical pressure; heating the pressurized liquid CO<NUM> to reach a required supercritical temperature; and inserting the supercritical CO<NUM> fluid to the one or more extraction chambers.

In some embodiments, separating the Polyhydroxylated Fatty Alcohols may include: introducing the supercritical CO<NUM> flown through the crushed avocado seeds into a separator having two or more separation stages. In some embodiments, for every kilogram (kg) of crushed avocado seeds at least <NUM> gram (gr) of Polyhydroxylated Fatty Alcohols is extracted and collected. In some embodiments, a time of production of at least 1gr of the Polyhydroxylated Fatty Alcohols from <NUM> of crushed avocado seeds is at most <NUM> hours.

In some embodiments, the method may further include collecting the CO<NUM> separated from the Polyhydroxylated Fatty Alcohols; and recycling the collected CO<NUM>.

<FIG> is an illustration of an extraction system according to some embodiments of the invention; and.

<FIG> is a flowchart of a method of extracting PFA using supercritical CO<NUM> according to some embodiments of the invention.

In other instances, well-known methods, procedures, and components, modules, units and/or circuits have not been described in detail so as not to obscure the invention. Some features or elements described with respect to one embodiment may be combined with features or elements described with respect to other embodiments. For the sake of clarity, discussion of same or similar features or elements may not be repeated.

Although embodiments of the invention are not limited in this regard, discussions utilizing terms such as, for example, "processing", "computing", "calculating", "determining", "establishing", "analyzing", "checking", or the like, may refer to operation(s) and/or process(es) of a computer, a computing platform, a computing system, or other electronic computing device, that manipulates and/or transforms data represented as physical (e.g., electronic) quantities within the computer's registers and/or memories into other data similarly represented as physical quantities within the computer's registers and/or memories or other information non-transitory storage medium that may store instructions to perform operations and/or processes. Although embodiments of the invention are not limited in this regard, the terms "plurality" and "a plurality" as used herein may include, for example, "multiple" or "two or more". The terms "plurality" or "a plurality" may be used throughout the specification to describe two or more components, devices, elements, units, parameters, or the like. The term set when used herein may include one or more items. Unless explicitly stated, the method embodiments described herein are not constrained to a particular order or sequence. Additionally, some of the described method embodiments or elements thereof can occur or be performed simultaneously, at the same point in time, or concurrently.

Aspects of the invention may be directed to a method of extracting all molecules of PFA, both the polar and non-polar molecules, using supercritical CO<NUM> without any additional cosolvent. Although, prior art extraction method of oils from seeds using supercritical CO<NUM> requires the addition of <NUM>-15wt. % of cosolvent, the applicant has surprisingly found that such an addition is not required when extracting PFA using supercritical CO<NUM> from avocado seeds, while maintaining or even increasing the required production yield. Furthermore, although supercritical CO<NUM> is known to be non-polar solvent, a method of extraction according to embodiments of the invention resulted in the extraction of both the polar and non-polar PFA molecules. Therefore, unlike the prior art methods, a method and an extract according to some embodiments of the invention may be completely non-toxic, or cosolvent free. A method according to embodiments of the invention may be executed by any known supercritical CO<NUM> extraction system, for example, the system illustrated in <FIG>.

Referring now to <FIG> which is an illustration of an extraction system <NUM> according to some embodiments of the invention. System <NUM> may include an extraction unit <NUM> and a separation unit <NUM>. Extraction unit <NUM> may include: a CO<NUM> reservoir <NUM> configured to hold CO<NUM>, a condenser <NUM> configured to condense and liquidize the CO<NUM>, a pump <NUM> (e.g., a dual piston pump) for increasing the extraction pressure of the CO<NUM> and a heater <NUM> for heating the pressurized CO<NUM> to supercritical conditions. Accordingly, the CO<NUM> leaving heater <NUM> is a supercritical CO<NUM> fluid. As used herein, supercritical CO<NUM> fluid may refer to a CO<NUM> fluid that may have a density of liquid CO<NUM> (e.g., <NUM>-<NUM>/m<NUM>) and a dissolving power (solubility) of CO<NUM> gas. In some embodiments, supercritical CO<NUM> fluid may have a temperature range of <NUM> to <NUM>. The supercritical CO<NUM> fluid has a pressure range of <NUM> bar to <NUM> bar.

In some embodiments, the supercritical CO<NUM> fluid is introduced into one or more extraction chambers <NUM> included in extraction unit <NUM>. One or more extraction chambers <NUM> may be preloaded with crushed seeds, for example, crushed avocado seeds. The supercritical CO<NUM> fluid is flown through the crushed seeds in at least one of one or more extraction chambers <NUM>. In some embodiments, PFA may be dissolved from the crushed seeds into the supercritical CO<NUM> fluid to form a supercritical CO<NUM> solution. In some embodiments, other materials may also be extracted from the crushed avocado seeds in the process, for example, tocopherols, carotenoids, unsaturated fatty acids, etc. It should be appreciated by those skilled in the art that a cosolvent reservoir is not required in systems according to embodiments of the present invention.

In some embodiments, the supercritical CO<NUM> solution that includes the extracted PFA may be introduced into separation unit <NUM> to be separated. Separation unit <NUM> may include one or more separators <NUM> each being in fluid connection to one or more tanks <NUM>. In some embodiments, separation unit <NUM> may further include one or more filters and one or more containers (not illustrated) for collecting the separated PFA. In some embodiments, separators <NUM> may each include a tank at which the supercritical CO<NUM> solution may be depressurized using aback-pressure regulator and maintained in a desired temperature (e.g., <NUM>-<NUM>) until the CO<NUM> may gradually be converted into gas. In some embodiments, in order to avoid pressure and temperature changes the processes may be carried out is several stages in two or more separators <NUM>. The evaporated CO<NUM> may be recycled and condensed back into a liquid CO<NUM>.

It should be understood by one skilled in the art that the invention is not limited to system <NUM> disclosed herein and illustrated in <FIG>, which is given as an example only.

Reference is made to <FIG> which is a flowchart of a method of extracting PFA according to some embodiments of the invention. In step <NUM>, crushed avocado seeds are introduced to one or more extraction chambers (e.g., one or more chambers <NUM>). The avocado seeds are crushed using any known method, for example, grinding, milling, etc. In some embodiments, the crushed avocado seeds may have a particle size (e.g., average diameter) of at most, <NUM>, <NUM>, <NUM>, <NUM> or less. In some embodiments, the smaller the particle size the higher is the specific surface area exposed to the supercritical CO<NUM> fluid, therefore the higher is the extraction rate of the PFA from each particle. Therefore, the smaller the particle size the higher is the yield of the extraction process.

Undried raw avocado seeds may be crushed at room temperature. The crushed avocado seeds are dried to a humidity level of at most <NUM> wt. PFA are insoluble in water therefore the presence of water is an impediment to the diffusion of the PFA into the supercritical CO<NUM> fluid. Furthermore, it is easier to recycle the CO<NUM> if it does not contain water.

In some embodiments, the crushed seeds may be incubated for at least <NUM> hours, for example, about <NUM> hours, at a temperature range of <NUM>-<NUM>.

In step <NUM>, supercritical CO<NUM> fluid is flown through the crushed avocado seeds in at least one of the one or more extraction chambers (e.g., chambers <NUM>). The supercritical CO<NUM> fluid is obtained by pressurizing and heating CO<NUM> into supercritical conditions as disclosed hereinabove with respect to <FIG>. Some embodiments of the invention may include: obtaining liquidized CO<NUM>, increasing the pressure of the liquidized CO<NUM> to reach a required supercritical pressure and heating the pressurized liquid CO<NUM> to reach a required supercritical temperature; and inserting the supercritical CO<NUM> fluid to at least one extraction chambers.

In some embodiments, the supercritical CO<NUM> fluid may have a density of liquid CO<NUM> and a dissolving power (solubility) of the PFA in the supercritical CO<NUM> gas. In some embodiments, the supercritical CO<NUM> fluid may have a temperature range of <NUM> to <NUM>. The supercritical CO<NUM> fluid has pressure range of <NUM> bar to <NUM> bar.

In some embodiments, as the supercritical CO<NUM> fluid is flowing and wetting the crushed avocado seeds, PFA may be extracted from the surface area of the crushed avocado seed particles. The PFA may be dissolved into the supercritical CO<NUM> fluid. In some embodiments, the PFA dissolved into the supercritical CO<NUM> fluid may include both polar and non-polar PFA molecules.

In step <NUM>, PFA extracted and dissolved in the supercritical CO<NUM> may be separated from the supercritical CO<NUM> flown through the crushed avocado seeds. In some embodiments, PFA from within the avocado seed cells may diffuse from the cells and dissolve in the supercritical CO<NUM>. The supercritical CO<NUM> fluid is the only solvent used for dissolving and extracting the PFA.

In some embodiments, the extracted PFA may be non-toxic and/or cosolvent free extracted PFA including both polar and non-polar PFA molecules. As used herein, the term non-toxic refers to cosolvent amounts which are lower than the required standard (e.g., lower than <NUM> ppm) or the lowest detection limit, for example, lower than <NUM> ppm, lower than <NUM> ppm or lower than <NUM> ppm measured, for example, using Headspace Gas Chromatograph (HSGC). In some embodiments, the lowest detection limit may depend form the type of product material (oil, paste, solid, solution, etc.), the type of the toxic cosolvent and the like.

In some embodiments, for every kg of crushed avocado seeds at least <NUM>-gram, for example, at least <NUM>-gram, <NUM>-gram, <NUM>-gram, <NUM>-gram or more. of the PFA may be extracted and collected. In some embodiments, a time of production of at least <NUM> gram of the PFA from <NUM> of crushed avocado seeds may be at most <NUM> hours.

Three batches of <NUM>-<NUM> of crushed avocado seeds were inserted into extraction chambers, such as chambers <NUM>. Each batch was exposed to <NUM> of flowing supercritical CO<NUM> fluid having pressure of: <NUM>, <NUM> and <NUM> bar at <NUM>. No additional cosolvent was used in the process. The flowing rate of the supercritical CO<NUM> fluid was <NUM>-<NUM>/hour. Table <NUM> summarized the results of obtained PFA.

As known in the art the typical production yield of solvents (e.g., ethyl acetate, hexane, etc.) based extraction process is <NUM>-<NUM> %. As clearly shown in Table <NUM> above, a process according to embodiments of the invention may result in a higher production yield without the use of any cosolvent.

Using the same raw material as in the previous experiments, additional five batches of <NUM>-<NUM> and <NUM> batches of <NUM>-<NUM> of crushed avocado seeds were inserted into extraction chambers, such as chambers <NUM>. Small scale batches were exposed to <NUM>-<NUM> of flowing supercritical CO<NUM> fluid and Large-scale batches were exposed to <NUM>,<NUM> of flowing supercritical CO<NUM> fluid, having pressure of: <NUM>, <NUM>, <NUM> and optionally also <NUM> bar at <NUM>. No additional cosolvent was used in the process. The flowing rate of the supercritical CO<NUM> fluid was <NUM>-<NUM>/hour for small scale batches and <NUM>/hour for large-scale batches. Table <NUM> summarized the results of obtained PFA.

As shows in table <NUM>, as pressure increases the yield increases and the selectivity decreases, accordingly, more compounds are extracted which lead to a change in the appearance and odor of the whole extract. Batches that were extracted above <NUM> bar appear as dark green extract with an unpleasant odor and larger number of undesirable compounds (such as pigments). Higher pressure during extraction enable producing in higher yields.

Claim 1:
A method of extracting Polyhydroxylated Fatty Alcohols (PFA) comprising:
drying the crushed avocado seeds to a humidity level of at most <NUM> wt.%;
inserting crushed avocado seeds to one or more extraction chambers (<NUM>);
flowing supercritical CO<NUM> fluid, at a pressure range of <NUM> bar to <NUM> bar through the crushed avocado seeds in the one or more extraction chambers (<NUM>); and separating from the supercritical CO<NUM> flown through the crushed avocado seeds the PFA extracted and dissolved in the supercritical CO<NUM>,
wherein the supercritical CO<NUM> fluid being the only solvent used for dissolving and extracting the PFA from the seeds.