Method of obtaining a hydroxytyrosol-rich composition from vegetation water

ES 2 392 706 T3

DESCRIPTION

Procedure for obtaining a composition rich in hydroxytyrosol from vegetation water (0001) The present invention refers to a phenolic fraction of a group of components, present in olive plants, known as hydroxytyrosol (3,4-dihydroxy- phenylethanol). More specifically, the invention provides both an olive extract - which contains hydroxytyrosol, with reduced amounts of oleuropein and tyrosol, or which is substantially free of these - and also provides a process for obtaining this extract.

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Deiana, M. and others, Journal of Free Radical Biology Medicine No. 26, 762-769 (1999); de la Puerta, R. and others, Revista Bioquímica Farmacológica No. 57, 445-449 (1999);

Ficarra, P. and others, Revista Fármacos No. 46, 803-815 (1991);

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Owen, RW et al., European Cancer Review No. 36, 1235-1247 (2000b);

Owen, RW et al., Food, Chemistry, Toxicology Magazine No.38, 647-659 (2000c);

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Visioli, F. and Galli, C., Nutrition Magazine No. 56, 142-147 (1998).

(0002) High amounts of dietary fat have been found to be involved in the development of various diseases (Owen et al., 2000c). Atherosclerosis (Kuller, 1997), coronary heart disease (Gerber, 1994), breast cancer (La Vecchia et al., 1998), prostate cancer (Chan et al., 1999), ovarian cancer (Risch et al. , 1994) and colon cancer (Armstrong and Doll, 1975) have all been associated with high amounts of dietary fat. The evidence shows, however, that it is not only the amount, but also the type of dietary fat that is important in the etiology of some cancers (Bartsch et al., 1999).

(0003) Olive oil, the main component of fat in the Mediterranean diet, has been associated with a lower incidence in coronary heart disease (Owen et al., 2000b; Parthasarathy et al., 1990; Mattson and Grundy, 1985) and in certain types of cancer (d'Amicis and Farchi, 1999; Braga and others, 1998; Martín-Moreno and others, 1994). Several laboratories have reported that the hydrolysis of phenolic oleuropein in olive oil, and other elements of this family, leads to small phenolic components with marked chemical protective activity (Owen et al., 2000a; Manna et al., 2000). Especially the phenolic hydroxytyrosol in olive oil prevents low-density lipoprotein (LDL) oxidation (Visioli and Galli, 1998), platelet aggregation (Petroni et al., 1995) and inhibits 5- and 12-lipoxygenesis (from Puerta and others, 1999; Kohyama and others, 1997). It has also been discovered that hydroxytyrosol exerts an inhibitory effect on tyrosine nitration and on peroxynitrite-dependent basic modification of DNA (Deiana et al., 1999) as well as acting against cytotoxicity, induced by reactive oxygen species in different systems. human cell phones (Manna et al., 2000). Finally, studies have shown that hydroxytyrosol is absorbed - depending on the dosage - in people after its ingestion, thus indicating its biological availability (Visioli et al., 2000).

(0004) The production of olive oil generally involves crushing the olives, including the pits, to produce a viscous paste. During this process, the crushed olives are continuously washed with water, a process that is known as "malaxation". The paste is then mechanically pressed to squeeze out the oil content. Apart from providing the olive oil, this pressing also squeezes the content of the pasta into water. These phases of washing and pressing produce a considerable amount of water which is known as “vegetation water”.

(0005) Both the pit and the pulp of olives are rich in phenolic combinations, which are soluble in water. These combinations are extracted from the olives during the malaxation, depending on their coefficients

ES 2 392 706 T3 of its proportional parts, and they end up in the vegetation water. This explains why different phenolic combinations - such as, for example, oleuropein and its derivatives, produced in the pulp of olives - can be found abundantly in vegetation waters. Similarly, monophenolic combinations such as tyrosol and its derivatives, produced by the hollows of olives, are also found abundantly in vegetation waters.

(0006) Due to the marked chemical protective activity of hydroxytyrosol, it is desirable to develop a process that produces an aqueous extract with a high percentage of hydroxytyrosol from the olive.

(0007) On the one hand, the present invention encompasses a process for producing a composition rich in hydroxytyrosol. This procedure comprises the phases of: (a) Producing the vegetation water from the olives, preferably from the meat of pitted olives; (b) Adding an acid to the vegetation water, in an amount sufficient to obtain a pH value between 1 and 5, preferably between 2 and 4; and (c) Incubate the acidified vegetation water for a period of at least two months, normally 6 to 12 months, until at least 75%, preferably at least 90% of the oleuropein, initially present in vegetation water, it has been converted to hydroxytyrosol.

(0008) According to one embodiment, the incubation is carried out until the vegetation water has a ratio between the weight of hydroxytyrosol and the weight of oleuropein of 5: 1 to 200: 1, preferably between 10: 1 to 100: 1. According to a similar embodiment, this incubation is carried out until the vegetation water has a weight ratio between hydroxytyrosol and tyrosol of 3: 1 to 50: 1, preferably 5: 1 up to 30: 1.

(0009) The procedure may also include the fractionation of the incubated vegetation water to separate the hydroxytyrosol from the other components and / or to dry the vegetation water from the isolated hydroxytyrosol, this in order to produce a dry extract; This procedure in which the aforementioned fractionation comprises, after the aforementioned incubation, putting the acidified vegetation water in contact with a supercritical fluid as well as recovering the composition, rich in hydroxytyrosol, from the contacted vegetation water, whereby the composition, rich in hydroxytyrosol, it comprises at least 95% by weight of hydroxytyrosol. According to another embodiment, it appears that the composition, rich in hydroxytyrosol, comprises at least about 97% by weight of hydroxytyrosol. According to yet another embodiment, the composition, rich in hydroxytyrosol, comprises at least about 99 percent by weight of hydroxytyrosol.

(0010) According to one embodiment it appears that the recovery phase, described above, comprises the steps (a) of recovering the supercritical fluid, this supercritical fluid containing hydroxytyrosol, and (b) of evaporating this supercritical fluid in order to extract the composition rich in hydroxytyrosol. According to another embodiment, the phase of putting into contact, described above, comprises the steps (a) of providing a porous membrane, the opposite sides of which are inside a modular element under pressure, the membrane serving as barrier boundary surface between a fluid and a dense gas, and not acting as a selector for said hydroxytyrosol; (b) putting the supercritical fluid inside the module on one side of the membrane and the vegetation water on the opposite side of the membrane; and (c) of extracting the hydroxytyrosol through the membrane, driven by a concentration gradient of the hydroxytyrosol between the vegetation water and the supercritical fluid. According to another embodiment, it appears that the porous membrane is a hollow fiber membrane. According to yet another embodiment, the supercritical fluid is carbon dioxide.

(0011) According to another aspect, it appears that the present invention comprises a dietary supplement consisting of an aqueous extract of olives, with a ratio between the weight of hydroxytyrosol and the weight of oleuropein of 5: 1 to 200: 1, preferably from 10: 1 to 100: 1.

(0012) According to another aspect, related to this, the present invention comprises a dietary supplement consisting of an aqueous extract of olives, with a ratio between the weight of hydroxytyrosol and the weight of tyrosol of 3: 1 to 50: 1, preferably 5: 1 to 30: 1.

(0013) The above mentioned supplement can be dried to provide a powdery extract which can then be shaped into a tablet, capsule, pill or as a sweet food additive.

(0014) These as well as other objects and aspects of the present invention may be fully appreciated in the detailed description, set forth below, in conjunction with the corresponding Figures and Tables.

Brief description of the Figures (0015)

Figure 1 shows the structures of the phenolic combinations and their precursors, detected in olive oil: Ligstrósidos (I); oleuropein glycosides (II); ligstroside aglycones (III); oleuropein glycoside aglycones (IV); a dialdehyde form of ligstroside aglycones in the absence of a carboxymethyl group (V); a dialdehyde aglycone form of oleuropein glycosides lacking a carboxymethyl group (VI); tyrosol (VII); hydroxytyrosol (VIII).

Figure 2 indicates the HPLC analysis (High Pressure Liquid Chromatography or high pressure liquid chromatography)

ES 2 392 706 T3 of a composition, rich in hydroxytyrosol, according to the invention after the supercritical carbon dioxide has been extracted from the vegetation water.

Figure 3 shows the HPLC analysis of a composition, rich in hydroxytyrosol, of the invention after extraction of the supercritical carbon dioxide with synthetic hydroxytyrosol.

Figure 4 indicates the mass spectrum of a composition, rich in hydroxytyrosol, according to the invention.

Figure 5 shows the fragmentation path of hydroxytyrosol.

I. Definitions (0016) If nothing to the contrary is stated, all terms used herein have the same meaning for a person familiar with the technical field of the present invention. It should be borne in mind that this invention is not limited to the methodology, protocols, or the particular reagents described herein, since they may vary.

(0017) By "oleuropein" is to be understood an oleuropein of secoiridoid glycosides (structure II in Figure 1).

(0018) By "tyrosol" is to be understood a 4-hydroxyphenethylalcohol (structure VII in Figure 1).

(0019) By "hydroxytyrosol" is to be understood a 3,4-dihydroxyphenethyl alcohol (structure VIII in Figure 1).

II. Process of the invention (0020) The present invention provides, on the one hand, a composition, rich in hydroxytyrosol, produced from vegetation water that is derived from olives. It has been discovered that, under specific conditions as described below, hydroxytyrosol can be obtained from the water of olive vegetation. The steps to be taken in order to carry out the present invention are considered below.

A. Producing the vegetation water (0021) The process of the present invention uses olives that can be obtained from conventional and commercially available sources, such as, for example, olive growers. The vegetation water is preferably obtained from pitted olives. The olives, processed according to the procedure described here, can be pitted with any appropriate means. The pits within olives contain tyrosol which represents an undesirable component for vegetation water, and they cannot be appreciably destroyed by the acid treatment, described below. The bones can be separated from the pulp manually or automatically, as described below. Preferably, these means should be capable of removing the bones without breaking them; otherwise, higher concentrations of tyrosol would be produced within the vegetation water. According to another embodiment, the hydroxytyrosol is extracted from vegetation water that is obtained from olives that have not been pitted.

(0022) To produce the vegetation water, the pulp from the olives is first pressed to obtain a liquid phase mixture comprising olive oil, vegetation water and solid by-products. Next, the vegetation water is separated from the rest of the liquid phase mixture to be collected. Examples of procedures for obtaining water from vegetation are described in Patent Nos. 6 165,475 and 6,197,308 of the United States, both from the same Holder of the present Patent Application and granted to R. Crea.

(0023) For the purposes of commercial scale production, it may be desirable to automate various aspects of the present invention. In this connection, one embodiment contemplates the use of an apparatus, such as that disclosed in Patent Nos. 4,452,744, 4,522,119 and 4,370,274 to the United States, all issued to Finch et al. Briefly, Finch et al. Present an apparatus for recovering olive oil from olives. Initially, the olives are fed to a grinder that separates the hollows of the olives to obtain a boneless olive meat. This olive meat is then collected by an extraction screw that subjects the meat to an extraction pressure, high enough to be able to remove a liquid phase comprising oil, water and a smaller proportional part of olive pulp. The liquid phase is collected in a hopper and then sent to a clarification centrifuge that separates the polish from the liquid phase to obtain a mixture comprising olive oil and vegetation water. Next, a purification centrifuge separates the vegetation water and a small proportional part of solid substances from the mixture to obtain an olive oil that is mainly free from vegetation water and which is collected in a tank. According to Finch and others, water is drawn into a means of leaving it, such as a sewer. In stark contrast, the present invention envisages the collection of vegetation water to save it and use it for the purpose of extracting hydroxytyrosol.

(0024) Other additional devices, which can be used to carry out the present invention, are disclosed in Italian Patent Nos. 1 276 576 and 1 287 025. As previously mentioned, these devices can also be used to separate the pulp from the bones, before processing the

ES 2 392 706 T3 crushed olive pulp to get oil, water and solid waste.

B. Conversion of oleuropein to hydroxytyrosol (0025) On the other hand, the present invention provides that the oleuropein contained in the vegetation water is converted to hydroxytyrosol. The pH value of the vegetation water can be reduced by the addition of acid, which allows the vegetation water to be incubated under conditions which, according to the discovery of the present invention, promote the hydrolysis of the oleuropein acid to hydroxytyrosol. The sample can then be fractionated in order to separate the hydroxytyrosol from other combinations.

(0026) According to a preferred embodiment, the added acid is citric acid. This acid is added to the vegetation water to adjust the pH value to 1 to 5, preferably 2 to 4. A solid citric acid can be added during continuous stirring and with an amount of preferably 11.34 to 22.68 kgs. (25 to 50 pounds) of citric acid for approximately 3,785 liters (1,000 gallons) of vegetation water. The pH value of the resulting solution can be controlled, and another acid addition may, where appropriate, be necessary to achieve the desired pH value. Some process examples, which relate to the conversion of oleuropein into hydroxytyrosol after the addition of citric acid, are represented in Examples Nos. 1 and 2.

(0027) Instead of citric acid, another organic or inorganic acid can also be used as acid. The acids, which by way of example can be used for the present invention, comprise inorganic substances, known as mineral acids - sulfuric acid, nitric acid, hydrochloric acid and phosphoric acid, as well as organic combinations, belonging to carboxylic acid, to acid sulfonic and phenolic groups. The addition of the acid to the vegetation water serves several purposes, that is, to: (i) Stabilize the vegetation water; (ii) prevent fermentation of vegetation water, and (iii) hydrolyze oleuropein slowly, converting it to hydroxytyrosol, as indicated in Examples Nos. 1 and 2. Tables 1 and 2 in these Examples Nos. 1 and 2, respectively, contain data from two vegetation water samples and the respective percentage of the composition of different components, formed in the samples during a certain time after the addition of citric acid. According to one embodiment, the mixture is allowed to incubate until the hydroxytyrosol is 75 to 90% of the total combination of tyrosol and hydroxytyrosol, and that substantially none of the oleuropein is found in the starting mixture.

C. Purification of hydroxytyrosol (0028) After the conversion of oleuropein to hydroxytyrosol, the incubated vegetation water can be fractionated by a series of procedures, already known in this technical field. As an alternative, the vegetation water can also be fractionated prior to its treatment with the acid. By way of example, some fractionation procedures include separation using an organic solvent, high pressure liquid chromatography (HPLC) or supercritical fluids.

(0029) Vegetation water, obtained in the manner described above, provides a solution that is rich in low molecular weight polyphenols, particularly hydroxytyrosol, as well as a small amount of tyrosol. The concentration of hydroxytyrosol within the processed water can vary between 4-5 grams per liter up to 10-15 grams per liter, depending on the degree of dilution during the extraction of olive oil. According to one embodiment, it appears that the present invention provides a process for extraction or purification, which selectively enriches the hydroxytyrosol content and without the addition of contaminants. In this way, the largest polyphenolic component, that is, hydroxytyrosol, is isolated from other substances of the polyphenol family, from impurities, from solid matter in suspension, from tannins, as well as isolated from other molecules, contained in the water of vegetation. Consequently, hydroxytyrosol can be produced in a purity and in an amount that cannot be easily achieved by current extraction procedures, be they synthetic or natural.

(0030) A supercritical fluid is a gas that becomes very dense from its critical temperature and pressure. Its properties are between the properties of a gas and a liquid, for which there is an increased capacity to dissolve the combinations. This fluid is of a relatively high density; high diffusivity and low viscosity allow you to extract blends faster than conventional liquid solvents. Carbon dioxide is the gas most widely used for food and food ingredient processing with supercritical fluid, since it is natural, it is not toxic or flammable, apart from being relatively inert and leaving no residue. in the extracted product. As a general rule, liquid extraction with supercritical carbon dioxide is carried out by dispersing one phase within another phase within large columns or contact towers; in this case, fluid containing dissolved substances - such as juices - flows downward by gravity, while supercritical carbon dioxide flows upward. At the intersection of the two phases, specifically, a mass transfer takes place.

(0031) As an alternative, the continuous extraction of liquids and suspensions can also be achieved by the use of supercritical fluids - such as, for example, carbon dioxide - and porous membranes, instead of columns or towers of Contact; the liquid is passed continuously through a few

ES 2 392 706 T3 porous polypropylene membranes that are configured as bundles of hollow fibers or as spirally bent sheets. The liquid passes through the porous membranes, arranged within a module, while the supercritical carbon dioxide flows countercurrently on the other side of the membrane. The pressure within the module is mostly the same, such that extraction is being driven by the concentration gradient between the fluid and the supercritical carbon dioxide. The extract can be recovered by evaporation of carbon dioxide for recycling purposes. A process for extraction, using supercritical carbon dioxide and porous membranes, is described, for example, in US Patent No. 5,490,884.

(0032) Use other supercritical fluids, instead of carbon dioxide or in combination with it. These fluids include methane, ethane, propane, butane, isobutane, ethene, propene, hydrofluorocarbons, tetrafluoromethane, chlorodifluoromethane, carbon dioxide, dinitrogen monoxide, sulfur hexafluorides, ammonia, and methyl chloride.

(0033) Example No. 3 describes, on a smaller scale, an experiment in support of the present invention; in this case, hydroxytyrosol has been isolated from vegetation water, using supercritical carbon dioxide and porous membranes. HPLC analysis and mass spectrometry of the extracted hydroxytyrosol indicate that the sample contains 97 to 99% pure hydroxytyrosol. Accordingly, the present invention provides a hydroxytyrosol-rich composition which contains at least about 80% hydroxytyrosol, preferably at least about 90% hydroxytyrosol, and especially preferably by at least about 95% hydroxytyrosol and, even more preferably, it contains at least about 99% hydroxytyrosol.

(0034) Prior to extraction with a supercritical fluid, the vegetation water may contain carrier agents, which are known to those familiar with this technical field and such as, for example, maltodextrin or polypropylene beads, added to the solution, and / or the solution can be dried. The drying phase removes from the vegetation water preferably at least 90%, approximately, of water; more preferably at least about 95% water; and, even more preferably, it removes at least about 98% water.

(0035) An important aspect of these membrane reactors consists in the fact that the area of ​​the contact surface between the faces can be bridged regardless of the speed of the flows. Accordingly, the present invention contemplates a large-scale unit in which the surface area of ​​the membrane, used for extraction, is at least about 83.61 m² (100 square yards); preferably it is at least about 250.83 m² (300 square yards); and especially preferably it is at least about 501.66 m² (600 square yards), in order to allow the separation of hydroxytyrosol from large volumes of vegetation water. Thus, the membrane system of the present invention could, in one aspect, be capable of accommodating a flow rate of between 1 and 20 liters per minute, preferably between 5 and 10 liters per minute.

(0036) In accordance with the present invention, and as indicated above, additional purification procedures may also be employed. The isolation of hydroxytyrosine by HPLC chromatography is described in the publications of Ficarra et al., 1991; de Romani et al., 1999; and from Tsimidou, 1992.

III. Dietary supplement rich in hydroxytyrosol (0037) It should be taken into account that hydroxytyrosol, produced by the procedure described above, can be used for a whole series of applications. By way of example, hydroxytyrosol, obtained according to the process of the present invention, can be used: (i) As a natural antibacterial, antiviral and / or fungicidal product for applications in agriculture and / or in the control of pests and (ii) as a therapeutic product and / or as an anti-oxidant for a wide variety of health-related purposes. According to an exemplary embodiment, hydroxytyrosol is administered to a mammalian subject, such as, for example, a person wishing to experience one or more of the beneficial effects related to hydroxytyrosol.

(0038) Hydroxytyrosol, obtained by the process of the present invention, can be administered orally or parenterally. Oral dosage forms can be of the solid or liquid type. These dosage forms can be formulated from a purified hydroxytyrosol or they can be established on the basis of aqueous or aqueous / alcoholic extracts. With respect to the latter, aqueous or aqueous / alcoholic extracts (such as, for example, water / methanol or water / ethanol) can be spray-dried in order to provide a dry powder that can be formulated into the forms oral dosage in conjunction with other pharmacologically acceptable carrier substances. Aqueous or aqueous / alcoholic extracts can be formulated for compositions containing different ratios between the weight of hydroxytyrosol and the weight of oleuropein, that is, between 5: 1 and 200: 1, preferably between 10: 1 and 100: 1. These extracts can also be formulated to contain different ratios between the weight of hydroxytyrosol and the weight of tyrosol, that is, between 3: 1 and 50: 1, preferably between 5: 1 and 30: 1.

(0039) Solid compositions, in oral dosage form according to the present invention, are

ES 2 392 706 T3 prepared in a manner well known in the pharmaceutical arts, and comprises hydroxytyrosol in combination with at least one carrier substance that is pharmacologically acceptable. When making these dosages, a composition rich in hydroxytyrosol - in a mainly pure form or as the component of a distillate or of a crude extract - is normally mixed, diluted or it is wrapped in a carrier substance. This carrier substance can be in solid form or it can be of the semi-solid or liquid type that acts as a vehicle, as a carrier or as an auxiliary medium for the active ingredient. Alternatively, the carrier substance may also be in the form of a capsule or a receptacle to facilitate oral administration. Thus, the solid dosage forms for oral administration according to the present invention can be in the form of tablets, pills, powders or gelatin capsules, soft or hard.

(0040) Alternatively, the hydroxytyrosol - obtained in accordance with the present invention and intended for oral administration - can also be in liquid form; in this case, the pharmacologically acceptable carrier substance can be water or an aqueous / alcoholic medium.

(0041) Compositions for oral administration according to the present invention can also be formulated with other common and pharmacologically acceptable excipients, including lactose, dextrose, sucrose, sorbitol, mannitol, starch, rubbers, silicate calcium, a microcrystalline cellulose, polyvinyl pyrrolidones, methyl cellulose, water, alcohol and other similar substances. The formulas can additionally include lubricating agents such as talc, magnesium stearate and a mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl and propyl hydroxybenzoates; as well as sweetening and taste agents. Furthermore, the compositions of the present invention may be formulated in order to provide rapid, sustained or delayed release of the active ingredient, after administration to a subject.

(0042) Parenteral formulas, for their application according to the present invention, are prepared by applying the normal techniques of the field. They are generally prepared in the form of sterile injectable solutions - using parenterally acceptable carrier substances such as, for example, an isotonic saline solution - or in the form of a powder packaged sterile and prepared for reconstitution with a sterile buffer or with an isotonic saline solution, prior to its administration to a subject. (0043) The examples, listed below, describe procedures for producing hydroxytyrosol-rich compositions according to the present invention. The examples are intended to describe the scope of the present invention, but in no way to limit it.

Examples

Example No. 1

Conversion of oleuropein to hydroxytyrosol after addition of approximately 11.34 kgs (25 pounds) of citric acid to 3.785 liters (1,000 gallons) of vegetation water (0044) Table 1 represents the conversion of oleuropein to hydroxytyrosol for some time after from the addition of approximately 11.34 kgs (25 pounds) of citric acid to 3.785 liters (1,000 gallons) of vegetation water. The percentages in Table 1 are indicated as percentages of the weight of all phenolic combinations within the solution. As Table 1 shows, hydroxytyrosol comprises, after 12 months, more than 80% of the phenolic combinations within the solution.

Table 1

<td colspan="5">Conversion of oleuropein to hydroxytyrosol after addition of approximately 11.34 kqs (25</td>

<td colspan="5">pounds) of citric acid to 3,785 liters (1,000 qalonas) of vegetation water</td>

<td colspan="5"></td>

<td>Component</td><td>Composition a T = 2 months</td><td>Composition a T = 3 months</td><td>Composition at T = 4.5 months</td><td>Composition a T = 12 months</td>

<td>Hydroxytyrosol</td><td> 30.4%</td><td> 32%</td><td> 48.4%</td><td> 80.2%</td>

<td>Tyrosol</td><td> 2.5%</td><td> 5%</td><td> 2.2%</td><td> 3.6%</td>

<td>Oleuropein</td><td> 41%</td><td> 36.6%</td><td> 25.1%</td><td> 1.2%</td>

<td>Oleuropein aglycone</td><td> 4.2%</td><td> 4.6%</td><td> 2.7%</td><td> 3.7%</td>

Example No. 2

Conversion of oleuropein to hydroxytyrosol after addition of approximately 22.68 kgs (50 pounds) of citric acid to 3.785 liters (1,000 gallons) of vegetation water (0045) Table 2 represents the conversion of oleuropein to hydroxytyrosol for some time after from adding approximately 50 pounds (22.68 kgs) of citric acid to 1,000 gallons (3,785 liters) of

ES 2 392 706 T3 vegetation. The percentages in Table 2 are indicated as percentages of the weight of all phenolic combinations within the solution. Significantly, and as shown in Table 2, hydroxytyrosol comprises, after two months, more than 45% of the phenolic combinations within the solution.

Table 2

<td colspan="3">Conversion of oleuropein to hydroxytyrosol after addition of approximately 22.68 kqs (50</td>

<td colspan="3">pounds) of citric acid to 3,785 liters (1,000 qalonas) of vegetation water</td>

<td colspan="3"></td>

<td>Component</td><td>Composition at T = 2 months</td><td>Composition at T = 12 months</td>

<td>Hydroxytyrosol</td><td> 45.7%</td><td> 78.5%</td>

<td>Tyrosol</td><td> 2.9%</td><td> 3.3%</td>

<td>Oleuropein</td><td> 28.7%</td><td> 1.5%</td>

<td>Oleuropein aglycone</td><td> 4.1%</td><td> 3.5%</td>

Example No. 3

Extraction of hydroxytyrosol from vegetation water (0046) An aliquot (0.5 milliliter) of vegetation water, containing approximately 40 mg. of a dry solid substance (maltodextrin), it has been mixed with some porous polypropylene beads and it is dried. The dry mix has been used for supercritical carbon dioxide extraction (PoroCrit Company, LLC, Berkeley, California). The collected sample (approximately 2.0 mg) has been analyzed by HPLC. The profile of the sample is indicated in Figure 2, while Table 3 indicates the area below the highest peak, which is 97%. When a synthetic hydroxytyrosol was added to the sample, which is analyzed by HPLC, a greater peak appeared, indicating, as represented in Figure 3, that most of the product is hydroxytyrosol (Table 4).

(0047) Analysis of the sample by mass spectrometry confirms, as indicated in Figure 4, that most of the product consists of hydroxytyrosol. The sample has been diluted with methanol to a final concentration of 26 micrograms per milliliter and has been analyzed as negative ionization with a Finnigan LCQ device, fitted with ESI cylinders. The infusion was 3 microliters per minute, using a pump with a built-in syringe. The temperature has been 27 ° C .; the needle voltage of + 4.2 V .; 45-unit sheath gas; and the auxiliary gas of 10 units. The hydroxytyrosol fragmentation trajectory is indicated in Figure 5. As can be seen in Figure 4, hydroxytyrosol (mass / charge 153.1) and its fragmentation products (123.1 and 105.1 mass / charge) represent the majority of the totality of products within the multiphase spectra.

Table 3

<td colspan="5">Peak Analysis of Fiqura 2 HPLC Results</td>

<td>Crest No.</td><td>Weather</td><td>Height (pV)</td><td>Zone (pV-seq.)</td><td>Zone (%)</td>

<td> 1</td><td> 5.935</td><td> 215542</td><td> 6687705</td><td> 97.476</td>

<td> 2</td><td> 11.433</td><td> 5686</td><td> 173104</td><td> 2.523</td>

Table 4

<td colspan="5">Peak Analysis of HPLC Results from Figure 3</td>

<td>Crest No.</td><td>Weather</td><td>Height (pV)</td><td>Zone (pV-seq.)</td><td>Area (%)</td>

<td> 1</td><td> 2.875</td><td> 1345</td><td> 13895</td><td> 0.26</td>

<td> 2</td><td> 3.278</td><td> 1076</td><td> 14140</td><td> 0.265</td>

<td> 3</td><td> 6.641</td><td> 211204</td><td> 5241105</td><td> 98.240</td>

<td> 4</td><td> 11.961</td><td> 2587</td><td> 65811</td><td> 1.233</td>

ES 2 392 706 T3

TRANSLATION OF THE LEGENDS OF THE DRAWINGS

FIGURE 2:

Height Time

FIGURE 3:

Height Time

FIGURE 4:

Relative absorption