USOO896.8811 B2

(12) United States Patent (10) Patent No.: US 8,968,811 B2 Lopez Mas et al. (45) Date of Patent: *Mar. 3, 2015

(54) HYDROXYTROSOL CONTAINING EXTRACT (56) References Cited OBTANED FROM OLIVES AND SOLIDS CONTAINING RESIDUES OF U.S. PATENT DOCUMENTS EXTRACTION 6,361,803 B1 3/2002 Cuomo et al. 6,849,770 B2 2/2005 Guzmann et al. (75) Inventors: Jose A. Lopez Mas, Murcia (ES); Sergio 6,942,890 B1 9, 2005 van Buuren et al. A. Streitenberger, Murcia (ES); Marcos 2004.0102657 A1 5/2004 Fernandez-Bolanos Guzman Peñalver Mellado, Murcia (ES); Pedro 2004/0176647 A1 9, 2004 Perdices et al. Martinez Ortiz, Murcia (ES) FOREIGN PATENT DOCUMENTS

(73) Assignee: Probelte Pharma, S.A., Espinardo, EP 1582 512 A1 10/2005 Murcia (ES) EP 1623 96.0 A1 2, 2006 WO WO 2004/OO5228 A1 1, 2004 (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 OTHER PUBLICATIONS U.S.C. 154(b) by 56 days. Frank et al., “Characterization of an Intense Bitter-Tasting 1 H, 4 This patent is Subject to a terminal dis H-Quinolizinium-7-olate by Appliation of the Taste Dilution Analy claimer. sis, a Novel Bioassay for the Screening and Identification of Taste Active Compounds in Foods,” J. Agric. Food Chem. 2001, 49, 231 (21) Appl. No.: 13/543,086 238. Filed: Jul. 6, 2012 Mateos et al., “Evaluation of Virgin Olive Oil Bitterness by Quanti (22) fication of Secoridoid Derivatives.” JAOCS, vol. 81, No. 1 (2004), 71-75. (65) Prior Publication Data Andrewes et al., “Sensory Properties of Virgin Olive Oil US 2013/0178537 A1 Jul. 11, 2013 : Identification of Deacetoxy-ligstroside Aglycon as a Key Contributor to Pungency.” J. Agric. Food Chem. 2003, 51, 1415 Related U.S. Application Data 1420. Beauchamp et al., “Ibuprofen-like activity in extra-virgin olive oil.” (63) Continuation-in-part of application No. 12/524,603, Nature, vol. 437, Sep. 2005, 45-46. filed as application No. PCT/IB2008/000173 on Jan. Gutierrez-Rosales et al., “Main Polyphenols in the Bitter Taste of 25, 2008, now Pat. No. 8,236,993. Virgin Olive Oil. Structural Confirmation by On-Line High-Perfor mance Liquid Chromatography Electrospray Ionization Mass Spec (30) Foreign Application Priority Data trometry.” J. Agric. Food Chem. 2003, 51, 6021-6025. Database CAPLUS on STN. Acc. No. 2004: 143424, Fernandez Jan. 26, 2007 (EP) ...... O7001791 Bolanos Guzmann, ES 2172429 A1 (Sep. 16, 2002) (abstract). Jul. 23, 2007 (EP) ...... O7014390 International Search Report for PCT International Application No. PCT/IB2008/000173 mailed May 23, 2008. (51) Int. C. A2.3L. I./00 (2006.01) Primary Examiner — Brian J Davis C07C37/68 (2006.01) (74) Attorney, Agent, or Firm — RatnerPrestia A6 IK3I/05 (2006.01) A6 IK 8/34 (2006.01) A61O 19/00 (2006.01) (57) ABSTRACT A 6LX8/97 (2006.01) A2.3L I/30 Hydroxytyrosol extracted from olives and/or from the solid (2006.01) residues of olives after the extraction of olive oil, by acid A6 IK 36/63 (2006.01) hydrolysis and purification on resin columns eluted with (52) U.S. C. water contains hydroxytyrosol and , is free from Sug CPC ...... A61 K3I/05 (2013.01); A61 K8/347 ars, has a residual content of BenZoapyrene that is less than (2013.01); A61O 19/00 (2013.01); A61K 8/97 2 microg/Kg (weight BaF/weight of extract as dry matter), (2013.01); A23L I/3002 (2013.01); A61K 36/63 containing a weight ratio of hydroxytyrosol to hydroxymeth (2013.01); A61K 2800/10 (2013.01); A23V ylfurfural of between 45:1 and 10000:1, and the content of 2002/00 (2013.01); A6 IK 2236/39 (2013.01) hydroxytyrosol in the extract is at least 0.5% (w/w) with USPC ...... 426/431; 426/429;568/750; 568/753 purity of at least 40% (by HPLC 280 nm). (58) Field of Classification Search None See application file for complete search history. 26 Claims, 8 Drawing Sheets U.S. Patent US 8,968,811 B2

U.S. Patent Mar. 3, 2015 Sheet 2 of 8 US 8,968,811 B2

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U.S. Patent Mar. 3, 2015 Sheet 3 of 8 US 8,968,811 B2

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US 8,968,811 B2 4 a column with non activated ion exchange resin and Subse quently loaded on a XAD non-ionic column, from where the OH hydroxytyrosol is eluted with methanol or ethanol. The pro cess results in a poor yield in hydroxytyrosol, considering the process in its entirety. OH EP 1623960, which is incorporated by reference, 0, dis closes a process of recovery of hydroxytyrosol, and tyrosol, from alpechin by means of filtration in a complicated plant O CH consisting of three units (EX. 1) and Subsequent separation. ALDEHYDIC FORM OF OLEUROPEIN AGLYCON 10 The tyrosol is then oxidated to hydroxytyrosol in a protic solvent ( or water) to obtain a final product that is OH semi-synthetic. Additionally, EP 1623960 discloses that con centration by nanofiltration and reverse osmosis of vegetation waters (alpechin) are carried out at neutral or alkaline pH OH 15 (claim 4). This process has two important problems. A first drawback is that hydroxytyrosol degradation increases at neutral and alkaline conditions giving unwanted products that are difficult to remove, and, in addition, due to the fact that no acid hydrolysis is carried out the oleuropein content should be still high. Secondly, the obtainable concentration factor is It is known to carry out acid hydrolysis of the pomaces (or poor, thus the maximum concentration allowed for hydroxy of vegetation water) to have the cleavage of the ester bond in tyrosol according EX-1 (see table 1) is in the range from 1.2 to the oleuropein molecule and obtain hydroxytyrosol. 1.6 g/L. U.S. Pat. No. 6,361,803, which is incorporated by refer U.S. Patent Application Publication 2004/0176647, which ence, discloses extraction of hydroxytyrosol (and other com 25 is incorporated by reference, discloses an extraction process pounds) by neutral or acid hydrolysis of olive pulp residues at of phenols from alperujo under stirring at 180-240° C. in reflux for one hour (ex. 12). The extracted water solution is autoclave, in water. No acid is added, but the thermal treat loaded on an absorption XAD-7 column that is eluted with ment results in a “liberation of acetyl groups and in an methanol to recover the extracted hydroxytyrosol. U.S. Pat. alleged consequent reduction of the pH (page 3, first para No. 6,361,803 requires the use of organic polar solvents, to 30 graph). Nevertheless, at the pHs produced in the described recover hydroxytyrosol with a minimum purity grade, in conditions hydrolysis of oleuropein is far to be complete, addition (ex.12) freeze precipitation of some impurities from meaning two things, lower hydroxytyrosolyields and remain the methanol Solution is necessary. Polaraqueous solvents are ing hydrolysed oleuropein, that as we discuss above is caus selected among methanol, ethanol, acetonitrile or acetone, ing non-pleasant taste in food products containing Such while polar organic solvents are selected, for example, among 35 extracts, that are thus not suitable for food applications. The esters, amides, dimethyl sulfoxide, dioxane, DMF and their products are tyrosol and hydroxytyrosol, that are separated by mixtures. Most of these solvents are toxic and very difficult to HPLC with sulphuric acid/acetonitrile eluent. completely eliminate from the desired hydroxytyrosol prod Summarizing, the above mentioned techniques are either uct. Accordingly, traces of the utilized solvents will be found too long or too complex, or too harsh, or all of the above; this in the final product even after several purification steps, thus 40 results in that the amount of remaining oleuropein, i.e. the rendering the hydroxytyrosol obtained according to this pro amount that is not hydrolyzed, and/or the amount of the cess not suitable for a safe application in the alimentary, hydrolysis by-products such as hydroxymethylfurfural is cosmetic and pharmaceutical field. Moreover, the final prod high enough to make difficult the Subsequent purification uct is not suitable for use in fortifying foods, particularly steps. edible oils, without use of ethanol and acetic acid as additives 45 Obtaining a good source of hydroxytyrosol is very impor to the extract to obtain a stable food product: the resulting tant for the fortification of foods, especially of edible oils. food product (oil plus hydroxytyrosol containing extract, Extracts should not transfer to oils any bitter polyphenolic ethanol and acetic acid) is not acceptable in the food industry. Substances, however, the health beneficial compounds, WO 2004/005228, which is incorporated by reference, namely hydroxytyrosol, are always associated to bitter discloses hydrolysis at room temperature of vegetation water 50 polyphenols in the known extracts. obtained from olive oil extraction by incubation of the acidi Frank and co-authors in J. Agric. Food Chem. 2001, 49. fied vegetation water for at least two months and preferably 231-238 disclose a procedure called “taste dilution analysis” 6-12 months until at least 50% (preferably 90%) of the oleu in order to point out the sensory threshold of bitter for oleu ropein originally present in the vegetation water has been ropein derivatives. Bitterness was assessed by preparing converted to hydroxytyrosol. The incubated vegetation water 55 serial dilutions of samples in water and then tasting them is extracted with an organic solvent, for example ethyl according to increasing concentration, until the concentration acetate, or it is contacted with a Supercritical fluid (CO), to at which the diluted sample can be differentiated from water produce a fraction rich in hydroxytyrosol. The main problems as judged in a triangle test is found. of this process are the very long time required for the incu It was also shown that at least for these compounds there is bation of the acidified vegetation water and the use of organic 60 no direct correlation of bitterness with the absorbance at 225 solvents, that should be avoided, particularly when the final (the K225 value): Mateo et al. J. Am. Oil Chem. Soc. 2004, 81, product obtained is to be used in the alimentary, cosmetic and 71-75, verified the correlation between the aldehydicic form pharmaceutical field. ofoleuropeinaglycone (obtained by hydrolysis of oleuropein U.S. Patent Application Publication 2004/0102657, which with B-glucosidase from almonds purchased from Sigma) is incorporated by reference, discloses acidic hydrolysis with 65 and bitterness. a steam explosion process at high temperatures (about 190 Andrewes et al., J. Agric. Food Chem. 2003, 51, 1415 220°C.). The obtained solution is firstly partially purified on 1420, assessed the relationship between polyphenols and US 8,968,811 B2 5 6 olive oil pungency; p-HPEA-EDA was the key source of the polyphenols (standardized by their content of hydroxytyrosol burning sensation found in many olive oils. and its derivatives) and protection of blood lipids from oxi In 2005, Beauchamp and co-authors, Nature 2005, 437, dative stress. 45-46, measured the pungent intensity of p-HPEA-EDA iso According to Regulation (EC) No 1924/2006 of the Euro lated from different extra virgin and virgin olive oils confirm pean Parliament and of the Council of 20 Dec. 2006 on ing this molecule is the principal agent in extra virgin and nutrition and health claims made on foods, a claim that a food virgin olive oils responsible for throat irritation. is Sugars-free, and any claim likely to have the same meaning Additionally, the bitter and pungent polyphenols present in for the consumer, may only be made where the product con olive oil, are also presents in olives and are also responsible tains no more than 0.5g of sugars per 100 g or 100 ml. for their bitterness and pungency, those being the ones that 10 best correlate with the bitterness attribute the oleuropein and SUMMARY OF THE INVENTION the aldehydic form of oleuropein aglycone. The crushing of olives to prepare olive oil or olive extracts produces the It is an aim of the present invention to solve the above hydrolysis of oleuropein by endogenous B-glucosidase ren mentioned problems and to provide olive extracts to be used dering oleuropeinaglycone as transient specie that forms the 15 as a source of hydroxytyrosol to prepare fortified food oils aldehydic form of oleuropeinaglycone. Thus, the use of olive and products in general and dietary Supplements for protec extracts containing oleuropein and/or the aldehydic form of tion of blood lipids from oxidative stress, thus preventing or oleuropein aglycone can modify the taste of foods in which treating cardiovascular diseases, plaque build-up in the arter the aforementioned extract is incorporated, resulting in a ies, arterial hypertension, and metabolic syndrome, but lower acceptance by consumers. avoiding non-pleasant alterations of organoleptic properties Summarizing, any way set up in order to increase antioxi of the fortified foods. dant capacity using techniques based on fortification of foods In fact, the applicant found that generation of by-products with olive polyphenols, should not alter food natural organo is a much bigger problem in the production of hydroxytyrosol leptic characteristics, nor increase the amount of the bitter 25 extracts than it was previously thought, as it appears from the tasting olive polyphenols, otherwise incurring with the Sub above discussed prior art documents: the starting materials, sequent alteration of organoleptic properties of the food, thus especially olives and pomaces, contain inter alia a high causing non-pleasant taste due to excessive bitterness, pun amount of phenolic compounds in the form of glucoside gency and/or astringency, and Subsequently causing rejection and/or its esters including oleuropein, ligustroside, Verbasco of the fortified food from many consumers. 30 side and several . Several other natural compounds It is known to add polyphenols and hydroxytyrosol to food are present. It is very easy to degrade the starting compounds products. into a plethora of by-products. U.S. Pat. No. 6,942,890 which is incorporated by refer As by-products must be eliminated to purify hydroxytyro ence, discloses a method offortifying food products adding to Sol, in the known art the use of organic solvents is required in such products solid matter derived from olive fruits, resulting 35 order to purify the hydroxytyrosol during the Subsequent in an increase of the level of , particularly of olive purification steps on chromatographic columns. The use of polyphenols. organic solvents, for example methanol, when eluting from a It has also to be noted that all the previously mentioned resin column, is inconvenient, particularly when the final techniques allow to obtain a fortified food with an increased product obtained is to be used in the alimentary, cosmetic and content of total olive polyphenols but with a low content in 40 pharmaceutical field. hydroxytyrosol with respect to the total olive polyphenols Moreover, the known hydroxytyrosol-rich extracts are not content, due to the fact that most of the polyphenols incorpo suitable to be used as additives for foods, especially edible rated to the fortified food are secoiridoids, oleuropein related oils. compounds as oleuropein aglycone (AOA), 3,4-DHPEA Therefore, there is the need for a hydroxytyrosol contain EDA and p-HPEA-EDA, at a higher content than hydroxyty 45 ing extract that is rich in hydroxytyrosol and has low content rosol, and that those olive polyphenols are mainly responsible of starting products and of bitter products such as oleuropein for the bitter taste and pungency of the fortified oil. The and , and of by-products, particularly incorporation of these secoiridoids, able to produce undesir hydroxymethylfurfural and in general has a very low content able changes in the organoleptic properties of the fortified in Sugars and in salts. food, is in fact directly related with the source of olive 50 It is an aim of the present invention to solve the above polyphenols used in all the previously mentioned techniques, mentioned problems and to provide a hydroxytyrosol extract which in fact present a low content in hydroxytyrosol and/or from olives and/or olive oil extraction by-products that is a low purity degree. simple to obtain, not expensive and that is a high purity A few studies addressed the bioavailability of polyphenolic product free from organic solvents and Suitable for use as compounds in olive showing that the absorption of olive oil 55 additive for food, cosmetics etcetera. phenolics is probably larger than 55-66 mol %, and that the Such aim is achieved by means of the present invention that absorption of hydroxytyrosol is dose-dependent, suggesting provides an extract, as obtainable by extraction from a start that olive oil phenolics are absorbed from the intestine, that ing material selected from olives and/or pomaces, residues of tyrosol and hydroxytyrosol are incorporated in lipoprotein olives after the extraction of olive oil, characterized in that fractions, and that hydroxytyrosol is excreted in urine as 60 said extract contains hydroxytyrosol and tyrosol, the amount glucuronide conjugate. The incorporation of phenolic com of sugars per 100 g or 100 mL of extract is 0.5g or less, the pounds from olive oil in LDL particles has been proposed as residual content of BenZoapyrene in said extract is less than the mechanism by which olive phenolics may protect LDL 2 microg/Kg (weight BaF/weight of extract as dry matter), particles from peroxidation. containing a weight ratio of hydroxytyrosol to hydroxymeth The EFSA Panel on Dietetic Products, Nutrition and Aller 65 ylfurfural of between 45:1 and 10000:1 and in that the content gies (NDA) concludes that a cause and effect relationship has of hydroxytyrosol in said extract is at least 0.5% (w/w) and been established between the consumption of olive oil the hydroxytyrosol purity is at least 40% (by HPLC 280 nm). US 8,968,811 B2 7 8 More particularly, a preferred olive extract is that one In vitro tests and studies on rats suggest potential toxicity obtained according to the process hereinafter disclosed, hav and carcinogenicity of HMF. In humans, no correlation ing a content in hydroxytyrosol of at least 0.5% by weight between intakes of HMF and disease has yet been demon (w/w), and a purity of hydroxytyrosol of at least 40% (by strated. HPLC at 280 nm). This extract is free of organic solvents (the 5 Polycyclic aromatic hydrocarbons (PAHs), are potent residual content of organic Solvents is less that 1 ppb and atmospheric pollutants that consist of fused aromatic rings preferably 0 ppm) and Substantially free of Sugars, and the and do not contain heteroatoms or carry substituents. PAHs content in hydroxytyrosol of that extract is at least 0.5% occur in oil, coal and tar deposits, and are produced as (w/w) and the purity of hydroxytyrosol is at least 40% (by byproducts of fuel burning (whether fossil fuel or biomass). HPLC at 280 nm). A hydroxytyrosol content of at least 10% 10 As a pollutant, they are of concern because some compounds (w/w) is preferred, and more preferably at least 35% (w/w); have been identified as carcinogenic, mutagenic and terato the purity of hydroxytyrosol at 80% is preferred, and more genic. PAHs are also found in cooked foods. Studies have preferably at 90% (by HPLC at 280 nm). shown that high levels of PAHs are found, for example, in The Substantial absence of even minimal traces of organic meat cooked at high temperatures such as grilling or barbe Solvents such as methanol, ethanol, isopropanol that are 15 cuing, and in Smoked fish. As olive oil extraction residues are employed in the purification steps of the prior art, is of great complex materials it is possible that harsh conditions in tem importance for the intended use of the aforementioned perature (i.e. steam explosion extraction) during olive eXtractS. extraction could produce such PAHs at residual Additionally, Some of the polyphenols present in olives are levels, being then desirable that a process to extract olive responsible for their bitterness, the ones that best correlate polyphenols avoid such PAHs residual levels formation. with this attribute being the oleuropein and the aldehydic Benzoapyrene (BaP) is commonly used as an indicator form of oleuropein aglycone. Thus, the use of olive extracts species for PAHs contamination and most of the available containing oleuropein and/or the aldehydic form of oleu data refer to this compound. ropein aglycone can modify the taste of foods in which the AIIs, HMF, PAHs and above mentioned have aforementioned extract is incorporated, resulting in a lower 25 molecular structures (e.g. the core of triazines in most of the acceptance by consumers. pesticides) capable of interacting with the absorption resins Olive extract rich in hydroxytyrosol obtained according to and then are eluted from the resin together with other com the invention process (as per co-pending U.S. application Ser. ponents of the extract when using organic solvents. Thus the No. 12/524,603) is virtually free of oleuropein and of the methods of preparing extracts from olives based on the use of aldehydic form of oleuropein aglycone, the total amount of 30 an absorbent resin and Subsequent elution with organic Sol each compound being less than 500 ppm (their detection vents must exercise extreme quality control on the raw mate limit), because no organic solvent is used, where these mol rial residues, but also in the hydrolysis and extrac ecules, much more hydrophobic than hydroxytyrosol, are tion process of olive polyphenols in acidic conditions from more soluble, for obtaining the extract, together with the olive materials containing Sugar able to degradate to HMF. combination of purifying steps used, chromatographic col 35 since the absorption resin can act as a trap for PAHs, HMF and umns, for obtaining the extract. these pesticides, that will concentrate on the resin and then Also the olive extract rich in hydroxytyrosol obtained elute when an organic solvent is used to recover the hydroxy according to the invention process has a very low content of tyrosol present in the olive. hydroxymethylfurfural, being the ratio hydroxytyrosol/hy The above problem was solved by the invention with the droxymethylfurfural (w/w) higher than 45:1 before column 40 preparation of an olive extract rich in hydroxytyrosol, due to purification or higher than 9000:1 after purification steps, the fact that the non-ionic absorption resins were specifically because the controlled extraction conditions pH time and selected to elute the active compounds of the said extract by temperature, together with the combination of purifying steps use of water only, or of an aqueous solution basified and not used, chromatographic columns, for obtaining the extract. by the use of organic solvents. An additional advantage of the above mentioned olive 45 The process of preparing the extract of the inventionentails extracts is the purification from the residues of pesticides the acidic hydrolysis in water of the starting materials at a present in raw materials, i.e. in the olives and olive pomaces. temperature not exceeding 140° C., preferably within the Thus, potentially toxic chemicals such as insecticides, herbi range from 70° C. to 130° C. and most preferably above the cides, fungicides, rodenticides, etc. . . . . Such as: diuron, reflux temperature, in the range of 110°C. to 130° C. and in terbuthylazine, simazine, C-, and B-endosulfan 50 a pressurized condition, e.g. in an continuous sterilization may be present in the starting material. system, at a pressure within the range of atmospheric pressure Hydroxymethylfurfural (HMF), also 5-(Hydroxymethyl) to 10-20 psi above the atmospheric pressure. The pH of the furfural, is an organic compound derived from dehydration of acidified mixture that undergoes the hydrolysis is within the certain Sugar, especially in acidic conditions. This yellow range of 1.0 to 6.0. The mixture after the hydrolysis is clari low-melting solid is highly water-soluble. The molecule con 55 fied by physical methods known in the art, e.g. by filtering sists of afuran ring, containing bothaldehyde and alcohol and and/or centrifuging, to remove the Suspended Solids from the functional groups. HMF has been identified in a wide variety hydrolysis water solution, and to obtain a clarified solution of heat-processed foods including milk, fruit juices, spirits, Substantially free of Solids in Suspension. honey, etc. . . . The starting materials are olives or pomaces, i.e. the above HMF is a natural component in heated food but is usually 60 defined residues from olive oil extraction. Preferred olives are present in low concentrations. The daily intake of HMF may green olives, pomaces are preferably free from oil. Oil is underlie high variations due to individual consumption-pat removed either before or after the hydrolysis step by means terns. It has been estimated that in a western diet, about 5-10 known in the art (e.g. those used to prepare defatted orujo or mg of HMF are ingested per day from food. orujillo) or by use of diatomaceous earth or other filter means. In former times, HMF was used in food for flavoring pur 65 According to the invention the above steps (acid hydrolysis poses, but in Europe this practice now is suspended. HMF is and clarification) are followed by the steps of loading the also found in cigarette Smoke. product thus obtained in at least one chromatographic column US 8,968,811 B2 10 of an acid activated anion exchange resin, and of eluting the require additional process steps that, at the end of the full products retained in said chromatographic column with process, result in a lower yield in the desired final product. Water. According to the present invention, the hydrolysis step con In a preferred aspect, the invention provides for a further temporaneously allows to carry out the hydrolysis reaction purification step carried out by loading the water-eluted Solu and provides for the necessary sterilization of the products tion from the first column (anion exchange) in at least one involved, thus avoiding the necessity to carry out any steril chromatographic column of adsorbent non-ionic resins and of ization step on the starting material as well as providing for a eluting the products retained in said second chromatographic hydrolysed material that is ready for further uses, without any column with water. additional sterilization treatment. According to a further aspect of the invention the liquid 10 product is concentrated e.g. by reverse osmosis concentra In addition, the hydrolysis step is followed by at least a tion. According to a further step, after chromatographic puri purification step, which is carried out by loading the product fication and reverse osmosis concentration, the resulting liq obtained from the hydrolysis step in at least a single chroma uid product is brought to a solid form, e.g. by freeze-drying, tography column system or in a two chromatography col vacuum rotaevaporation or spray drying, with or without 15 umns system and eluting the hydroxytyrosol retained in said carriers such as maltodextrines. chromatographic columns with water. In this case, as there are The extract of the invention has a ratio by weight of very low amounts of by-products coming from the hydrolysis hydroxytyrosol:oleuropein that is greater than 200:1, prefer step, it is possible to make use of a chromatographic column ably also a ratio (by weight, i.e. w/w) of hydroxytyrosol: that can be eluted with water in the absence of any organic aldehydic form of oleuropein aglycone that is between 5:1 solvent, either alone or mixed with water. This means that no and 850:1. organic solvents are contacted with the hydroxytyrosol, thus Additionally, in a preferred embodiment the extract obtaining a purified hydroxytyrosol that is particularly Suit according to the invention is free from oleuropein and alde able to be used in the alimentary, cosmetic and pharmaceuti hydic form of oleuropein aglycone (by HPLC 280 nm). cal field. According to another aspect, the invention extract has a 25 Another advantage of the present invention is due to the ratio hydroxytyrosol: tyrosol that is greater than 55:1 by concentration step, that allows to enrich the hydroxytyrosol weight, preferably within the range of 55:1 to 618:1. content after the purification steps. These purification steps Moreover, the extract is free from organic polar solvents. produce a liquid extract, Substantially free from Sugars and A further object of the invention is the use of an extract as salts, resulting in a very important reduction of the osmotic above disclosed as a source of hydroxytyrosol in the food 30 pressure during the concentration step of the purified liquid industry as additive or as functional ingredient; as a source of extract permitting thus a very high concentration factor (until hydroxytyrosol in the cosmetic industry or as a source of 350 times). This concentration step provides a “concentrate' hydroxytyrosol in the pharmaceutical industry. To put it in characterized by a high content of hydroxytyrosol, which can other words, it is an object of the invention a process of be directly used for further treatments. According to the prior preparing an enriched food, cosmetic or pharmaceutical 35 art, where no “concentration' steps are usually provided or product, wherein an extract according to the invention is when provided they have poor concentration factors (about 2 added to said product. to 5 times) due to the lack of a previous purification that The invention provides several advantages over the prior removes Sugars and salts, in the described processes for art techniques. hydroxytyrosol preparation, the hydroxytyrosol concentra First of all, the hydrolysis step is carried out, according to 40 tion in the processed solutions/dispersions is always very low, the present invention, using just water and mineral acid, keep thus resulting in the necessity to operate with great Volumes ing the temperature in the claimed range preferably above the with consequent lower yields in the final product. reflux temperature, using a combination of heating and pres Always according to the present invention, the possibility sure. When pressure is applied together with the claimed to obtain a solid final product that is not mixed with any heating temperature, the resulting hydrolysis reaction is 45 carrier, for example maltodextrines, gives the opportunity to almost completed in about half an hour, without significant formulate purified hydroxytyrosol according to its final formation of by-products and with a very good conversion of intended use and according to any formal requirement, pos the starting material (oleuropein) into the final desired prod sibly required. uct (hydroxytyrosol). The hydrolysis step according to the As above mentioned, an additional advantage of the inven present invention allows to obtain, in a short time, a very good 50 tion’s olive extracts is the purification from the residues of yield in hydroxytyrosol together with a quite complete pesticides present in raw materials, i.e. in the olives and absence of those by-products, that are difficult to eliminate pomaces. and which are instead formed, according to the prior art, in significant amounts. BRIEF DESCRIPTION OF THE DRAWINGS The hydrolysis step, carried out according to the invention, 55 provides for another advantage. The combined use of acids The invention will now be further disclosed in greater and temperature, beside its main objective, that is to carry out detail with reference to the enclosed non-limiting drawings the hydrolysis with a good conversion rate and avoiding the wherein: formation of detrimental by-products, results in a sterilization FIG. 1 is a schematic view of an apparatus according to an of the mixture. In fact, the hydrolysis step provides a steril 60 exemplary embodiment of the invention; and ization of the water solution, i.e. of the products involved, that FIGS. 2-4 are chromatograms of three hydroxytyrosol con is very useful, as the starting material (for example pomaces) taining products according to three embodiments of the used in the processes for the preparation of hydroxytyrosol, invention process. usually comes from already treated materials, and generally FIG. 5 is a chromatographic profile showing the separation requires some depuration-sterilization pre-treatments, to give 65 of hydroxytyrosol and tyrosol peaks in adsorption column a safe hydroxytyrosol final product. Said depuration-steril eluted with water. Also 2 marks are signaling the bed Volumes ization pre-treatments are often complex treatments and at which sample spot 2a and sample spot 2b were taken. US 8,968,811 B2 11 12 FIG. 6a is a HPLC chromatogram of the sample spot 2a supply of demineralized water 4 and with means 6 to stir the (punctual sample at 2 BV) taken during collection of eluate water mixture of starting product. Usually, the ratio water to fraction showing a complete separation of hydroxytyrosol solids is within the range of 1:1 to 4:1. and tyrosol in adsorption column eluted with water. The hydrolysis process is carried out at a pH of 1.0 to 6.0, FIG. 6b is a HPLC chromatogram of the sample spot 2b preferably of 1.0 to 3.0. The required amount of acid, prefer taken (punctual sample at 11.8 BV), showing impurification ably sulphuric acid, is obtained from acid tank 5. of hydroxytyrosol with tyrosol that is eluted later than The combination of acidic conditions and oftemperature in hydroxytyrosol in adsorption column eluted with water. the claimed ranges results in a hydrolysis process that is fast Collection of eluate fraction is completed after 9.8 BV and efficient and can last for 30 minutes only, at 120-123°C. avoiding ratios of hydroxyrtyrosol/Tyrosol lower than 55. 10 and a pressure of 15 psi above the atmospheric pressure. FIG. 7 is a chromatogram of hydrolysis reaction of oleu Moreover, the process also results in the sterilization of the ropein. hydrolysis products. FIG. 8a is a graphical representation of ratio hydroxytyro The outlet of continuous sterilization system, i.e. reactor, 1 sol/tyrosol (w/w) in 17 different production lots of olive is connected with a filter 7 for removing solids from the extracts production from orujillo. 15 reaction mixture containing hydroxytyrosol and other phe FIG. 8b is a graphical representation of the ratio hydroxy nols. The filtered portion is then sent to a further separation tyrosol/hydroxymethylfurfural (w/w), at the crude aqueous means, preferably a centrifuge 8, where further solids are extract after centrifugation in 17 different production lots of removed from the reaction mixture to obtain a liquid Substan olive extracts production from orujillo. tially free from suspended solids, i.e. a solution that is suitable FIG. 9 shows the ion species detected in purified olive for the following purification. The clarified liquid after cen extract in liquid form having a hydroxytyrosol concentration trifugation has a brownish color and is preferably stored in of 41.5% (w/w) with an HPLC purity of 94.8% and a ratio reservoir 9. The clarified liquid A is now containing hydroxy hydroxytyrosol?tyrosol (w/w) of 110/1 obtained according to tyrosol, a residue of oleuropein and minor amounts of phenols Example 15 on the HPLC-DAD-MS systems. and other products. FIG. 2 chromatogram shows the compo FIG. 10 is a Chromatogram DAD (280 nm): BPC (Base 25 sition and the percent of the detected compounds in this liquid Peak Chromatogram) in negative (-) and positive (+) ioniza and is obtained by HPLC at 280 nm, the major peak at 9.317 tion mode, for purified olive extract in liquid form having a minutes is hydroxytyrosol and the peak at 13.150 minutes is hydroxytyrosol concentration of 41.5% (w/w) with an HPLC tyrosol. purity of 94.8% and a ratio hydroxytyrosol?tyrosol (w/w) of According to a preferred embodiment of the invention, 110/1 obtained according to Example 15 dissolved in MeOH 30 liquid A is concentrated by evaporation or tangential flow 0.1% HCOOH 100 mg/ml. filtration (TFF), (e.g. by using a reverse osmosis system). The FIG. 11 is a graphical representation of a number of cap concentration step can be carried out on liquid A directly or, Sules VS. net weight of olive extract inside the dietary Supple preferably, after a purification step by means of at least one ment. chromatographic column. To this end, Storage tank, or reser FIG. 12 is a graphical representation of the plasmatic level 35 Voir, 9 is connected with means of concentrating 10, e.g. by of oxidized LDL in the participants of the groups 1 and 2 of TFF or preferably by reverse osmosis, and to purification the intervention study. The difference at the level of oxidized CaS. LDL was determined as statistically significantive when com Purification means comprises in one embodiment a single pared Group 2 with the control Group 1, p<0.01 (Tukey's column system comprising a chromatographic column of a Test). 40 resin selected from acid activated anion exchange resins. In another preferred embodiment purification means comprises, DETAILED DESCRIPTION OF THE INVENTION in addition to the above mentioned first column of acid acti vated anion exchange resins at least a further, second, chro With reference to FIG. 1, the invention apparatus com matographic column of a resin selected from adsorbent non prises a means, or reactor, 1, preferably a continuous steril 45 ionic resins. ization system, in which the hydrolysis according to the In another more preferred embodiment, the acid activated invention process is carried out. anion exchange resin is a weakly basic anion exchange resin, As previously mentioned, the hydrolysis is carried out at a and the adsorbent non-ionic resin is a macroreticular cross temperature that does not exceed 140°C., in a reactor. Pref linked aromatic polymer. erably the temperature range is 70 to 130° C. and most pref 50 The anion exchange resins used as a chromatographic resin erably it is above reflux temperature, in a range 110 to 130° for the purification of hydroxytyrosol of the present invention C., at a pressure of 10 to 20 psi above the atmospheric pres are not particularly limited so long as they can be acid acti sure. and for a time length within the range of 15 to 45 vated. Examples of preferred weakly basic anion exchange minutes. Preferably, the hydrolysis temperature is within the resins include polyamine-type resins (including polyamine range of 118 to 126° C. and in the most preferred embodiment 55 type chelating resins) such as reaction products of a styrene? the hydrolysis is carried out at 120-121°C., at 15 psi (over the divinylbenzene copolymer and diethylenetriamine or other, atmospheric pressure) for 30 minutes. A preferred reactor is a and resins as polymerization products of compounds mainly continuous sterilization system. comprising allylamine, vinylamine or other, and acrylic res The continuous sterilization system 1 is provided with ins. Such as copolymers of divinylbenzene and amide com feeding means 2 for feeding to it the starting materials, 60 pounds which comprise acrylic acid or methacrylic acid and namely the residues, or by-products, of olive oil extraction, dimethylaminopropylamine or other. Other resins may also namely pomaces (i.e. the Solid residues of the pressed olives). be used in which the aforementioned weakly basic anion Continuous sterilization system 1 is suitable to treat the exchange resin is partly Substituted with strongly basic starting materials in a continuous process rather than batch exchange groups such as trimethylamine, dimethylethanola wise. Heating means 3 for heating the reactor 1 to the above 65 mine or other. More specifically, known in the art resins mentioned temperatures are provided in a known way, e.g. as which have been used so far can be used, for example, Diaion a jacket around the reactor. Reactor 1 is also provided with a WA10, WA20, WA21 and WA30 (Mitsubishi Chemical), US 8,968,811 B2 13 14 Amberlite IRA-35, IRA-67 (IRA-68), IRA-93ZU, IRA-94S, the adsorbed hydroxytyrosol is recovered by elution with IRA-478 (Rhom & Haas), WGR-2 (Dow Chemical) and demineralized water from supply 13 and is collected in res other. ervoir 17. According to the invention process, these resins are acid Liquid C, i.e. the liquid collected in reservoir 17 has a 5 purity in hydroxytyrosol of at least 90%, and generally of at activated before being used, preferably with acetic acid. least 95%, the purity being determined as the percent of the These resins are particularly suitable for the invention process peak areas in a chromatogram by HPLC at 280 nm. The in view of their low cost and of the fact that they can be recovery of retained hydroxytyrosol from the resin is at least regenerated in mild conditions. Additionally, the weakly 90% and is generally at least 95% and substantially quantita basic nature of the resin allows the partial separation of tive. FIG. 4 shows the relevant HPLC chromatogram of the hydroxytyrosol and tyrosol molecules. From the comparison 10 obtained purified liquid product C. of the peak ratiohydroxytyrosol/tyrosol and the HPLC purity FIG. 1 also shows a source of acetic acid 18 that is con of the hydroxytyrosol peak of FIGS. 2 and 3, it can be appre nected to column 11 for acid activation of resin 12 and a ciated the efficiency of the chromatographic separation source of NaOH 19 or other suitable base for regeneration of according to the invention. the same. Additionally, a source of NaOH 19 for regeneration 15 of resin 16 is connected to column 15 and a source of sulphu In fact, the use of a weakly anionic exchange resin allows ric acid 21 for resin Surface activation is also shown. to separate very similar compounds during the elution, this is As for liquid A, also liquid B and liquid C can be concen not possible by the prior art techniques using strong anionic trated in concentration means 10, e.g. by evaporation or TFF, exchange resins, because the elution of Such resins is gener preferably by reverse osmosis; concentration is preferably ally an all-or-nothing process. As previously mentioned, a carried out on liquids B and C, preferably liquid C, to a further advantage is that the retained products can be eluted hydroxytyrosol content that is easily up to 10%, and that can with water, without using any polar solvent Such as methanol. reach 20%, 35% and even 40% by weight of hydroxytyrosol Suitable adsorption resins are based on non-ionic, hydro in the concentrated liquid product (i.e. extract). phobic, macroreticular cross-linked aromatic polymer. Such In a further step of the invention process, the liquid prod ucts obtained by the previously discussed steps are dried in resins are typically aromatic polymers, such as styrene and 25 dryer means 20, e.g. freeze-dryer, vacuum rotoevaporator or divinylbenzene copolymers, which may be cross-linked. preferably by spray-dryer, to produce a solid final product. Such resins are known and are generally prepared by poly The final product characteristics will be different according to merization of the appropriate monomers. The adsorption res the starting product (A, B or C) that is dried, the purity of the ins used as a chromatographic resin for the purification of dry product being within the range of 45% to 99% (HPLC at hydroxytyrosol of the present invention are not particularly 30 280 nm). limited so long as they can be water eluted. Examples of This drying step is preferably carried out on liquid products preferred adsorption resins include: Amberlite R. X.AD-4, B and C, after concentrating them as above disclosed. The XAD-7, XAD-1180, XAD-16 and XAD-1600 (available drying step can advantageously make use of carriers Suitable for the final use of the dry product; suitable carriers are e.g. from Rohm & Haas): XUS-40323.00, XUS-40285.00 and 35 maltodextrines, lactose, lecithins, caseinates etcetera. XUS-40283.00 (available from Dow Chemical Co.); and Suitable drying techniques are known in the art and com SP-700, SP-825, SP850, Diaion HP 10, HP 20, HP 30, HP 40 prise spray drying (usually with the use of carriers), freeze and HP 50 (available from Mitsubishi Chemical). drying and water evaporation under vacuum. The resulting These type of resins are particularly suitable for the inven products will have a hydroxytyrosol content of 0.5% to 10% tion process in view of their very high adsorption capacity for 40 and up to 20% (w/w) if a carrier is used, the hydroxytyrosol hydroxytyrosol and of the fact that, thanks to the low content content can reach up to about 95% (w/w) if no carrier is used. of by-products in the solution obtained after hydrolysis, the It should be noticed that the invention process provides a adsorbed hydroxytyrosol can be recovered by elution with purified liquid product (Band especially C) that is so pure that water, only, without any polar solvent Such as methanol or it can be evaporated to a dry powdereven without carriers, this ethanol, as was instead required by the prior art techniques. 45 being not possible with known techniques. Adsorbed hydroxytyrosol is recovered substantially quanti The extracts of the invention are suitable to prepare oral tatively. composition comprising olive polyphenols in an amount effective to provide in a daily serving at least 5 mg of In the shown preferred embodiment, the process of the hydroxytyrosol and its derivatives (e.g. oleuropein complex invention provides for a two-step purification on chromato and tyrosol) wherein said olive polyphenols comprises a graphic columns. 50 weight ratio of hydroxytyrosol to aldehydic form of oleu Liquid A, as obtained from the initial steps a) and b), i.e. ropein aglycone of between 5:1 and 850:1. hydrolysis and solid separation, is charged into column 11, The oral composition of the invention is comprising olive containing the anion exchange resin 12 as above detailed. The polyphenols in an amount effective to provide in a daily permeate is sent to waste treatment (not shown). Demineral serving at least 5 mg of hydroxytyrosol and its derivatives ized water from water supply 13 is then fed to column 11 to 55 (e.g. oleuropein complex and tyrosol) wherein said olive elute the retained products and the eluted liquid is collected in polyphenols comprises a weight ratio of hydroxytyrosol to reservoir 14. The thus obtained liquid product (liquid B) has oleuropein greater than 200:1. a purity in hydroxytyrosol of at least 75%, and generally of at The invention will now be further disclosed with reference least 80%, the purity being determined as the '% of the peak to the following non-limiting examples. areas in a chromatogram by HPLC at 280 nm. The recovery of 60 retained hydroxytyrosol from the resin is at least 85% and is Example 1 generally at least 90%. FIG. 3 shows the relevant HPLC chromatogram of the Hydroxytyrosol Extraction from Olive Waste obtained purified liquid product B. (Orujillo), Purification of the Water Phase In the second purification step, liquid product B is charged 65 into column 15, containing a non-ionic adsorption resin 16, as 250 g of a sample of dry olive waste, orujillo, are mixed above detailed. The permeate is sent to waste treatment and with 838 ml of demineralized water and 16.7 g of sulphuric US 8,968,811 B2 15 16 acid (98%). The obtained mixture is kept in autoclave for 30 Example 5 minutes at 121°C. After that, the aqueous phase is separated from the solid residue, by filtering on a filter. The solid phase, Concentration of Hydroxytyrosol Ion Exchange retained on the filter, is washed with 310 ml of demineralized Purified Extract Enriched by Reverse Osmosis water, and the water coming from this washing operation is 5 collected with the aqueous phase previously recovered. The A sample of 801 of crude aqueous extract containing 150g aqueous phase, approximately 860 ml, is then centrifuge of hydroxytyrosol obtained in a pilot plant according to Example 2, is concentrated using a reverse osmosis pilot refined to eliminate solid particles passed through the filter. plant, equipped with a 2.5 m polymeric membrane, in order After solid elimination, 835 ml of crude aqueous extract, to reduce the volume to 101 of concentrate product. A 0.3 m containing 1.41 g of hydroxytyrosol, with a HPLC purity of 10 membrane made of the same material is then used in order to 47.5%, are obtained. obtain a hydroxytyrosol concentrate containing 10.8% of hydroxytyrosol with an HPLC purity of 80.53%. Example 2 Example 6 15 Ion Exchange Hydroxytyrosol Purification Concentration of Hydroxytyrosol Ion Exchange and Adsorption Purified Extract by Reverse Osmosis A sample of 835 ml of crude aqueous extract containing 1.41 g of hydroxytyrosol obtained according to Example 1, is A sample of 5461 of crude aqueous extract containing 135 loaded on a column containing a ion exchange resin of the g of hydroxytyrosol obtained in a pilot plant according to anionic type, previously activated by means of acetate cycle. Example 3, is concentrated using a reverse osmosis pilot For example, Diaion WA10 may be used. The liquid phase plant, equipped with a 2.5 m polymeric membrane, in order recovered at the end of the column, does not contain any to reduce the volume to 101 of concentrate product. A 0.3 m hydroxytyrosol, which is instead continuously eluted with membrane made of the same material is then used, in order to demineralized water until at least 90% of the initially charged 25 obtain a hydroxytyrosol concentrate containing 12.20% of hydroxytyrosol is recovered. The eluted phase contains hydroxytyrosol with an HPLC purity of 95.27%. approximately 1.27g of hydroxytyrosol with an HPLC purity of about 80.85%. Example 7 Spray-Drying of the Crude Extract Enriched in Example 3 30 Hydroxytyrosol without any Purification Ion Exchange and Adsorption Hydroxytyrosol A sample of 442 ml of crude aqueous extract containing Purification 1.02 g of hydroxytyrosol obtained according to Example 1, is mixed with 100 g of maltodextrin, until maltodextrin was 35 completely dissolved. For example, equivalent 10 dextrose A sample of 835 ml of crude aqueous extract containing potato maltodextrin may be used. A peristaltic pump is used 1.41 g of hydroxytyrosol obtained according to Example 1, is to feed the spray-dryer, which is previously equilibrated with loaded on a column containing an ion exchange resin of the an inlet air temperature of 150° C. The feeding speed is anionic type, previously activated by means of acetate cycle. adjusted in order to obtain an outlet air temperature which is For example, IRA-67 may be used. The liquid phase recov 40 ered at the end of the column, does not contain any hydroxy less than 100° C.95g of a brown powder, with a moisture of tyrosol, which is instead continuously eluted with deminer 6.85% (Karl Fischer) and a hydroxytyrosol richness of alized water until at least 90% of the initially charged 0.98%, are obtained. hydroxytyrosol is recovered. Example 8 The eluted phase coming from the first column, is charged 45 on a column containing an adsorption resin. For example, Spray-Drying of the Partially Purified Aqueous resin XAD-1180 may be used. The liquid phase recovered at Extract Enriched in Hydroxytyrosol the end of the column, does not contain any hydroxytyrosol. Then, hydroxytyrosol is eluted from the resin with deminer A sample of 290 ml of aqueous extract containing 0.38g of alized water until at least 90% of the initially charged 50 hydroxytyrosol obtained according to Example 2 and Subse hydroxytyrosol is recovered. quently concentrated by reverse osmosis is mixed with 50g of The eluted phase contains approximately 1.14 g of maltodextrin until maltodextrin was completely dissolved. hydroxytyrosol with an HPLC purity of about 95.72%. For example, equivalent 10 dextrose potato maltodextrin may be used. A peristaltic pump is used to feed the spray-dryer, Example 4 55 which is previously equilibrated with an inlet air temperature of 150°C. The feeding speed is adjusted in order to obtain an outlet air temperature which is less than 100° C. 48.25 g of a Concentration of Hydroxytyrosol Crude Extract greyish powder, with a moisture of 6.72% (Karl Fischer) and Enriched by Evaporation a hydroxytyrosol richness of 0.71%, are obtained. 60 The aqueous phase obtained in Example 1 before the cen Example 9 trifugation step, approximately 860 ml, is concentrated by evaporation in order to reach a final volume of about 193 ml. Spray-Drying of the Purified Aqueous Extract The aqueous phase is then centrifuge refined to eliminate Enriched in Hydroxytyrosol solid particles passed through the filter. After solid elimina 65 tion, 160 ml of crude aqueous extract, containing 1.41 g of A sample of 188 ml of purified aqueous extract containing hydroxytyrosol, with a HPLC purity of 47.5%, are obtained. 0.29 g of hydroxytyrosol obtained according to Example 3, US 8,968,811 B2 17 18 and Subsequently concentrated by reverse osmosis, is slowly using a reverse osmosis pilot plant, equipped with a 2.5 m stirred with 28.5g of maltodextrin. For example, equivalent polymeric membrane, in order to reduce the volume to 101 of 10 dextrose potato maltodextrin may be used. A peristaltic concentrate product. A 0.35 m membrane made of the same pump is used to feed the spray-dryer, which is previously material is then used, in order to obtain a hydroxytyrosol equilibrated with an inlet air temperature of 175° C. The 5 concentrate containing 3.5% of hydroxytyrosol. Finally the feeding speed is adjusted in order to obtain an outlet air RO concentrate is rotaevaporated at 78° C. under a vacuum temperature which is less than 100° C. 27.1 g of a white pressure of 245 mbar to allow about 10 times concentration of powder, with a moisture of 5.45% (Karl Fischer) and a the olive fruit extract in liquid form reaching a final concen hydroxytyrosol richness of 0.97%, are obtained. 10 tration of 37.2% (w/w) with an HPLC purity of 93.3%. Example 10 Example 12 Preparation of a Highly Rich Hydroxytyrosol Preparation of Olive Fruit Extract Powder by Powder Spray-Drying 15 A sample of 1750 1 of aqueous extract containing 432 g of hydroxytyrosol obtained, according to Example 3, in a pilot A sample of 260 ml of purified olive extract in liquid form plant is concentrated according to Example 6, to obtain a containing 19.5 g of hydroxytyrosol obtained according to hydroxytyrosol concentrate containing 39.04% of hydroxy Example 11, is slowly stirred with 58 g of maltodextrin pre tyrosol with an HPLC purity of 95.60%. The concentrated 20 viously dissolved in 260 ml of demineralized water. For solution is used to feed the spray-dryer, which is previously example, potato maltodextrin may be used. A peristaltic equilibrated with an inlet air temperature of 150° C. The pump is used to feed the spray-dryer, which is previously feeding speed is adjusted in order to obtain an outlet air equilibrated with an inlet air temperature of 150° C. The temperature less than 100° C. 375.84 g of a light brown feeding speed is adjusted in order to obtain an outlet air powder, with a moisture of 4.35% (Karl Fischer) and a 25 temperature which is less than 100° C. 76 g of a white powder, hydroxytyrosol richness of 94.74%, are obtained. with a moisture of 5.4% (Karl Fischer) and a hydroxytyrosol richness of 21.9% (w/w), are obtained. Example 11 Example 13 Olive Extracts Production from Olives Fruits 30 Hydroxytyrosol Extraction from Three Phases Pomaces 25 Kg of a sample of olives fruits are mixed with 50 L of (Defatted Orujo), Purification of the Aqueous Phase demineralized water. The obtained mixture is blended for a 475.5g of a sample of three phases pomace with a humidity few minutes, and then 636 g of sulphuric acid (98%) were of 60.55%, are mixed with 800 ml of demineralized water and added. The obtained mixture is kept in autoclave for 30 min 35 26.36 g of sulphuric acid (98%). The obtained mixture is kept utes at 121°C. After that, the aqueous phase is separated from in autoclave for 30 minutes at 121°C. After that, the aqueous the solid olive residue, by filtering on a filter. The solid phase, phase is separated from the solid residue, by filtering on a 600 retained on the filter, is washed with 12.5 L of demineralized micron polypropylene filter. The filtered aqueous phase, water, and the water coming from this washing operation is approximately 795 ml, is concentrated by evaporation in collected with the aqueous phase previously recovered. The 40 order to reach a final volume of about 343.8 ml. The aqueous aqueous phase, approximately 63 L, is then filtered trough a phase is then centrifuge refined to eliminate solid particles Kieselguhr filter pre-coated with a CeliteTM 500 diatoma passed through the filter. After solids elimination, 275 ml of ceous earth to eliminate the extracted oil. The oil-free aque crude aqueous extract, containing 0.97g of hydroxytyrosol, ous phase, about 56 L. is then centrifuge refined to eliminate with a HPLC purity of 54%, are obtained. solid particles passed through the Kieselguhr filter After solid 45 elimination, 52 L of crude aqueous extract, containing 141 g Example 14 of hydroxytyrosol, with a HPLC purity of 50.5%, are obtained. Ion Exchange and Adsorption Purification of Then, crude aqueous extract, is loaded on a column con Hydroxytyrosol Deriving from Three Phases Pomace taining an ion exchange resin of the anionic type, previously 50 (Defatted Orujo) activated by means of acetate cycle. For example, IRA-67 may be used. The liquid phase recovered at the end of the A sample of 275 ml of crude aqueous extract containing column, does not contain any hydroxytyrosol, which is 0.97g of hydroxytyrosol obtained according to Example 13, instead continuously eluted with demineralized water until at is loaded on a column containing anion exchange resin of the least 90% of the initially charged hydroxytyrosol is recov 55 anionic kind, previously activated by means of acetate cycle. ered. For example, Diaion WA10 resin may be used. The liquid The eluted phase coming from the first column is charged phase recovered at the end of the column, does not contain on a column containing an adsorption resin. For example, any hydroxytyrosol, which is instead continuously eluted resin XAD-1180 may be used. The liquid phase recovered at with demineralized water until at least 90% of the initially the end of the column, does not contain any hydroxytyrosol. 60 charged hydroxytyrosol is recovered. Then, hydroxytyrosol is eluted from the resin with deminer The eluted phase coming from the first column is loaded on alized water until at least 90% of the initially charged a column containing an adsorption resin. For example, resin hydroxytyrosol is recovered. Diaion HP20 may be used. The liquid phase recovered at the The eluted phase contains approximately 114 g of end of the column, does not contain any hydroxytyrosol. hydroxytyrosol with an HPLC purity of about 96.7%. 65 Then, hydroxytyrosol is eluted from the resin with deminer Then, a 461 L fraction of purified extract containing 114g alized water until at least 90% of the initially charged of hydroxytyrosol obtained in a pilot plant is concentrated hydroxytyrosol is recovered. US 8,968,811 B2 19 20 The eluted phase contains approximately 0.80 g of indene(1.2.3-cd)pyrenes-10 microg/Kg (quantification limit: hydroxytyrosol with an HPLC purity which is higher than 10 microg/Kg) 95%. Then, the crude aqueous extract is loaded on a column As previously mentioned, the process of the invention pro containing an ion exchange resin of the anionic type, previ vides for preparing an extract containing hydroxytyrosol 5 ously activated by means of citrate cycle. For example, IRA starting from olives and/or pomaces without making use of 67 may be used. The liquid phase recovered at the end of the organic polar solvents. The products obtained according to column, does not contain any hydroxytyrosol, which is this process, both the final products and the intermediate instead continuously eluted with demineralized water until at products, are therefore free from organic polar solvents. The least 90% of the initially charged hydroxytyrosol is recov products are also substantially free from Sugars and salts. This 10 ered. is possible by the combined use of the claimed hydrolysis The eluted phase coming from the first column is concen conditions and purification procedure. trated by reverse osmosis till a final volume equivalent to 1.8 The final product, because of its high purity and high bed Volumes of the adsorption resin contained at the second hydroxytyrosol content, in particular a solid hydroxytyrosol 15 column. The reverse osmosis concentrate is charged at the extract free from carriers and organic Solvents is highly Suit second column containing an adsorption resin. For example, able for use in the food industry, and in cosmetic and phar 1000 L resin XAD-1180 may be used, then the loaded reverse maceutical industries. osmosis concentrate is 1800 L (1.8 bed volumes). The liquid FIG. 5 is a chromatographic profile showing the separation phase recovered at the end of the column after complete the of hydroxytyrosol and tyrosol peaks in adsorption column loading phase of reverse osmosis concentrate (column per eluted with water. Also 2 marks are signaling the bed Volumes meate fraction), does not contain any hydroxytyrosol. After at which sample spot 2a and sample spot 2b were taken. FIG. that, resin is washed with 1.33 bed volumes of water. Then the 6a is a HPLC chromatogram of the sample spot 2a (punctual water elution phase starts, and the column eluate fraction is sample at 2 BV) taken during collection of eluate fraction collected into the purified product tank. Hydroxytyrosol is showing a complete separation of hydroxytyrosol and tyrosol 25 eluted from the resin with 8.45 bed volumes of demineralized in adsorption column eluted with water. FIG. 6b is a HPLC water (same quality that the one used for the washing step) chromatogram of the sample spot 2b taken (punctual sample until the HPLC analysis of a spot sample of the eluate take at at 11.8 BV), showing contamination of hydroxytyrosol with outlet of the column presents a ratio hydroxytyrosol?tyrosol tyrosol that is eluted later than hydroxytyrosol in adsorption (w/w) lower than 15/1. The chromatographic profile showing column eluted with water. 30 the separation of hydroxytyrosol and tyrosol peaks is pre Collection of eluate fraction is preferably completed after sented in FIG. 5. Presence of oleuropein and aldehydic form 9.8 BV avoiding ratios of hydroxyrtyrosol/Tyrosol lower than of oleuropein aglycone is checked by HPLC but these com 55. pounds are not detectable at any moment during the elution Example 15 35 step, being the detection limits for both compounds 500 ppm. The eluted phase contains approximately 7400 g of Olive Extracts Production from Orujillo hydroxytyrosol (at least 90% of the initially loaded hydroxy tyrosol) with an HPLC purity of about 95.7% and a ratio 2367 Kg of orujillo are loaded with 10250 L of deminer hydroxytyrosol/tyrosol (w/w) of 113/1. alized water into a stirred glass lined reactor at atmospheric 40 Then, the eluted fraction of purified extract containing pressure. The obtained mixture is stirred for a few minutes, 7400 g of hydroxytyrosol is concentrated by reverse osmosis, and then sulphuric acid (98%) was added to adjust to pH1.50. in order to reduce the volume to 230 L of concentrate product. The obtained mixture is kept stirred into a glass lined reactor Finally the RO concentrate is rotaevaporated at 78°C. under 4 hours at 70° C. After that, NaOH (50%) was added to the a vacuum pressure of 245 mbar to allow about 14 times reactor to adjust to pH 2.5-0.5, and then, the aqueous phase is 45 concentration of the olive fruit extract in liquid form reaching separated from the Solid olive residue, by using a centrifugal a final hydroxytyrosol concentration of 41.5% (w/w) with an decanter. The Solid phase was sent to waste handling. The HPLC purity of 94.8% and a ratio hydroxytyrosol?tyrosol aqueous phase, approximately 7900 L, is then centrifuge (w/w) of 110/1 (see FIG. 5) and a ratio hydroxytyrosol/hy refined to eliminate the Small solid particles passed through droxymethylfurfural (w/w), of 9050 was obtained. Presence the centrifugal decanter step. After solid elimination, 7500 L 50 of oleuropein and aldehydic form of oleuropein aglycone is of crude aqueous extract, containing 9.55 Kg of hydroxyty checked by HPLC at olive fruit extract in liquid form (final rosol, with a HPLC purity of 52.7%, were obtained; the product) but these compounds are not detectable, being the weight ratios hydroxytyrosol/hydroxymethylfurfural (w/w), detection limits for both compounds 500 ppm (see FIG. 6a, hydroxytyrosol/oleuropein and hydroxytyrosol/aldehydic FIG. 6b and FIG. 7 for identification of hydroxytyrosol, tyro form of oleuropein aglycone were found to be the values of 55 Sol, oleuropein and aldehydic form of oleuropein aglycone, 82,289 and 8 respectively. A sample of crude aqueous extract comparison of peak profile and retention time of every com was dried till moisture content lower than 10%, and then the pound). As the hydroxytyrosol content (w/w) was 41.5% and content in PAHs was analyzed. The following data are the the detection limits for oleuropein and aldehydic form of results: oleuropein aglycone, 500 ppm a theoretical weight ratio benzoapyrene-2 microg/Kg (quantification limit: 2 microg/ 60 hydroxytyrosol/oleuropein and hydroxytyrosol/aldehydic Kg) form of oleuropein aglycone could be calculated, being the benzo(b)fluorantenes 10 microg/Kg (quantification limit: 10 theoretical value higher than 830. microg/Kg) The oleuropein of FIG. 7 is initially present in a sample of benzo(k)fluorantenes 10 microg/Kg (quantification limit: 10 extract purchased from Naturex; it was hydrolysed microg/Kg) 65 with beta-glucosidase from almond (purchased at Sigma) BenZogh.iperylene<10 microg/Kg (quantification limit: 10 during 1 h at 25°C. at pH 5.0 in 100 mM sodium phosphate microg/Kg) buffer. It is shown for comparative reasons with FIGS. 6a and US 8,968,811 B2 21 22 6b, especially to identify the retention times of oleuropeinand an HPLC purity of 94.8% and a ratio hydroxytyrosol?tyrosol aldehydic form of oleuropein aglycone formed during the (w/w) of 110/1 obtained according to Example 15 was used as reaction test item with the aim of identifying described phenolic com The ion species detected in purified olive extract in liquid pounds (hydroxytyrosol, tyrosol, etc.) and the presence of form having a hydroxytyrosol concentration of 41.5% (w/w) with an HPLC purity of 94.8% and a ratio hydroxytyrosol/ impurities. HPLC-DAD-MS systems with two types of mass tyrosol (w/w) of 110/1 obtained according to Example 15 on analyzers: TOF (Time of Flight) and IT (Ion Trap). The the HPLC-DAD-MS systems are shown in FIG.9. former provides the exact mass of a given molecule and with FIG.10 shows a chromatogram DAD (280 nm); BPC (Base the aid of the external laboratory of analysis database a pos Peak Chromatogram) in negative (-) and positive (+) ioniza sible identity can be established. In addition, the identity of tion mode, for purified olive extract in liquid form having a 10 the target compound could be confirmed by MS' experiments hydroxytyrosol concentration of 41.5% (w/w) with an HPLC provided by the Ion Trap. A specific search of oleuropein and purity of 94.8% and a ratio hydroxytyrosol?tyrosol (w/w) of aldehydic form of oleuropeinaglycone has been carried out. 110/1 obtained according to Example 15 dissolved in MeOH HPLC-DAD-TOF System 0.1% HCOOH 100 mg/ml. 15 The chromatographic separation is performed on a rapid Example 16 resolution C18 column 50x3.0 mm, 1.8 Lum under gradient conditions. The mobile phase consists of a mixture of metha Analysis of Ratios nol and water, both containing 0.1% of formic acid, with the Hydroxytyrosol/Hydroxymethylfurfural (w/w), aim of promoting the ionization of the compounds in samples. Hydroxytyrosol/Tyrosol (w/w) in 17 Different The signals are registered in a diode array detector (at 280 Production Lots of Olive Extracts Production from nm) and in a TOF mass spectrometer detector with an elec Orujillo, and Analysis of Oleuropein and Aldehydic trospray ion source (ESI). Form of Oleuropein Aglycone HPLC-DAD-IT System In addition, an ion trap mass analyzer has been used with The process of production of olive extracts from orujillo 25 described at example 15, was carried out for 17 different the aim of elucidating and confirming the molecules struc production lots. The extension of the washing step was main tures, which has been previously identified in the RRLC-TOF tained fix (1.3 to 1.5 bed volumes), but the extension of the system. For this purpose, a C18 column 150x2.1 mm, 3.5 um elution step was tested in the range 7 to 10 bed volumes, as is used undergradient conditions, becaused the system can shorter the bed volumes used for elution as higher the ratio not work at pressures above 400 bars. The rest of chromato hydroxytyrosol?tyrosol (w/w) obtaining values for such ratio 30 graphic conditions are the same as those described in the from 55/1 to 618/1 (FIG. 8a). For all the analyzed lots the previous section. recovery of initially charged hydroxytyrosol at the eluted The identified chromatographic peaks are compiled in fraction is at least 80%. Presence of oleuropein and aldehydic FIG. 9 and the chromatographic profile and composition is form of oleuropein aglycone is checked by HPLC for every presented in FIG. 10. There are only selected the ones that batch but these compounds are not detectable at any moment 35 have a certain absorbance at 280 nm, because this is the during the elution step nor in the final product. specific wavelength for phenolic compounds. The ratio hydroxytyrosol/hydroxymethylfurfural (w/w) FIG. 9 shows the results obtained with IT and TOF mass was calculated for the crude aqueous extract after centrifuga analyzers. In both cases, the ionization has been carried out in tion, but before column purification the values were in the negative and positive polarity. Therefore, the ions obtained in range 45:1 to 136:1 (see FIG. 8b). 40 each ionization mode are shown in the table attached to the ionic species detected. Example 17 Concerning to the phenolic compounds, the presence of hydroxytyrosol (peak 2) and tyrosol (peak 4) has been con Preparation of Olive Fruit Extract Powder by firmed. The tyrosol molecule is not ionized in the operating Spray-Drying 45 conditions, and it has been identified by comparison of the retention time with a commercial standard. Oleuropein and A sample of 16 Kg of purified olive extract in liquid form aldehydic form of oleuropeinaglycone, which are other phe containing 6640 g of hydroxytyrosol obtained according to nolic compounds of interest, have not been detected in the Example 15, is slowly stirred with 19 Kg of maltodextrin analyzed olive extract. previously dissolved in 220 L of demineralized water. For 50 example, potato maltodextrin may be used. The Solution is Example 19 pumped to feed the spray-dryer, which is previously equili brated with an inlet air temperature of 150° C. The feeding Preparation of a Dietary Supplement Having as speed is adjusted in order to obtain an outlet air temperature Active Ingredient an Olive Fruit Extract which is less than 78° C. A production lot of 26.2 Kg of a 55 white powder, with a moisture of 3.4% (Karl Fischer) and a A sample of 518 g of purified olive extract inform of white hydroxytyrosol content of 22.8% (w/w), and a ratiohydroxy powder, with a moisture of 3.4% (Karl Fischer) and a tyrosol?tyrosol (w/w) of 115/1 and a ratio hydroxytyrosol/ hydroxytyrosol content of 22.8% (w/w), and a ratiohydroxy hydroxymethylfurfural (w/w), of 9136 were obtained. tyrosol?tyrosol (w/w) of 115/1 obtained according to 60 Example 16, was filled into a biconic mixer and after that Example 18 1978 g of potato maltodextrin, 52 g of magnesium esterate and 52 g of tri-calcium phosphate, total batch size 2600 g. HPLC-DAD-MS Characterization of Olive Fruit After 15 minutes of mixing the powder has an improved Extract flowability in comparison with the initial olive extract, allow 65 ing a very good filling of hard gelatine capsules of size 00, A sample of 500 g of purified olive extract in liquid form with a very accurate net weight of 0.294+0.012 g (see FIG. having a hydroxytyrosol concentration of 41.5% (w/w) with 11). US 8,968,811 B2 23 24 The capsules were automatically cleaned externally and invention. Accordingly, it is intended that the appended filled into ablistering machine that produces the blisters with claims cover all such variations as fall within the spirit and 30 capsules. Finally blisters were manually introduced into Scope of the invention. the final packing. The invention claimed is: Example 20 1. A process for producing a food in the food industry, comprising adding an extract obtainable by extraction from a Preparation of a Olive Oil Having as Active starting material selected from olives and/or pomaces, resi Ingredient an Olive Fruit Extract and Use of Such dues of olives after the extraction of olive oil, wherein said Oral Compositions for Contribute to the Protection 10 extract contains hydroxytyrosol and tyrosol, the amount of of Blood Lipids from Oxidative Stress sugars per 100 g or 100 mL of extract is 0.5g or less, the residual content of BenZoapyrene in said extract is less than The objective of the study was to analyze the protection of 2 microg/Kg (weight BaF/weight of extract as dry matter), blood lipids from oxidative stress due to the intake of the oral 15 containing a weight ratio of hydroxytyrosol to hydroxymeth compositions of olive oil added with a hydroxytyrosol-rich ylfurfural of between 45:1 and 10000:1 and the content of olive extract in a human intervention study by measuring the hydroxytyrosol in said extract is at least 0.5% (w/w) and the serum levels of oxidized LDL. hydroxytyrosol purity is at least 40% (by HPLC 280 nm) to The selection of participants in the pilot study has followed said food. a series of criterialisted below: 2. A process for producing a cosmetic composition, com Inclusion Criteria: prising adding an extract obtainable by extraction from a 1. All participants must give their written informed consent starting material selected from olives and/or pomaces, resi after receiving oral and written information about the study dues of olives after the extraction of olive oil, wherein said 2. Aged between 18 and 65 extract contains hydroxytyrosol and tyrosol, the amount of 3. Body mass index (BMI) between 18.5 and 30 kg/m 25 sugars per 100 g or 100 mL of extract is 0.5g or less, the 4. Healthy men (without known diseases, including hyperten residual content of BenZoapyrene in said extract is less than Sion, hypercholesterolemia, diabetes and psoriasis) 2 microg/Kg (weight BaF/weight of extract as dry matter), 5. Not having used dietary Supplements or having donated containing a weight ratio of hydroxytyrosol to hydroxymeth blood two months before the study and during its develop ylfurfural of between 45:1 and 10000:1 and the content of ment 30 hydroxytyrosol in said extract is at least 0.5% (w/w) and the Exclusion Criteria: hydroxytyrosol purity is at least 40% (by HPLC 280 nm) to 1. Cardiovascular disease at the time of the study or before said cosmetic composition. that 3. A process for producing a pharmaceutical composition, 2. Diabetes mellitus or other serious chronic disease, includ comprising adding an extract obtainable by extraction from a ing allergies and severe psoriasis at the time of the study or 35 starting material selected from olives and/or pomaces, resi before that dues of olives after the extraction of olive oil, wherein said 3. Hypertension at the time of the study or before that extract contains hydroxytyrosol and tyrosol, the amount of 4. Knowledge or belief of alcohol, drugs or medication abuse sugars per 100 g or 100 mL of extract is 0.5g or less, the All participants have the right to withdraw at any time residual content of BenZoapyrene in said extract is less than without explanation. In addition, participants may be 40 2 microg/Kg (weight BaF/weight of extract as dry matter), excluded from the intervention if failure to follow the study containing a weight ratio of hydroxytyrosol to hydroxymeth guidelines occurs. ylfurfural of between 45:1 and 10000:1 and the content of There are no side effects expected in this study. hydroxytyrosol in said extract is at least 0.5% (w/w) and the The number of participants in this study was that of 20, hydroxytyrosol purity is at least 40% (by HPLC 280 nm) to divided in two groups of 10 participants who consumed: 45 said pharmaceutical composition. Group 1: 25 ml extra virgin olive oil (without addition of 4. A process for producing a food according to claim 1, olive extract, used as control) daily at a single dose. wherein said extract is Suitable to provide in a daily serving at Group 2: 25 ml fortified extra virgin olive oil (with addition least 5 mg of hydroxytyrosol and its derivatives. of an olive extract prepared according ex. 15, providing 5. A process according to claim 2, wherein said extract is a a dose of 5 mg of hydroxytyrosol and its derivatives (e.g. 50 liquid extract and has a total phenol content of at least 35% oleuropein complex and tyrosol) daily at a single dose. (w/w), anhydroxytyrosol content of at least 35% (w/w) and a The duration of the study was that of 4 weeks. Blood purity of at least 90% (by HPLC 280 nm). samples were taken in fasting at days 0, 14 and 28. Blood 6. A process according to claim 5, wherein the content of sampling was performed by Laboratorios Munuera S.L. hydroxytyrosol of said extract is at least 45% (w/w). With the blood samples the measurement of oxidized LDL 55 7. A process according to claim 2, wherein said extract is a was performed: solid, has a total phenols content of at least 20% (w/w), the The analysis of results showed a 35% decrease at the plas content of hydroxytyrosol in said extract is at least 20% (w/w) matic level of oxidized LDL in the group 2 participants. Such and the hydroxytyrosol purity is at least 90% (by HPLC 280 difference at the level of oxidized LDL was determined as nm). statistically significantive when compared with the control 60 8. A process according to claim 7, wherein said extract has group, p<0.01 (Tukey's Test). The results of the intervention a hydroxytyrosol content of at least 90% (w/w) a purity of at study are shown in the FIG. 12. least 90% and a total phenols content of at least 92% (w/w). While preferred embodiments of the invention have been 9. A process according to claim 8, wherein said extract is shown and described herein, it will be understood that such free from carriers. embodiments are provided by way of example only. Numer 65 10. A process according to claim 2, wherein the weight ous variations, changes and Substitutions will occur to those ratio of hydroxytyrosol:oleuropein in the extract is greater skilled in the art without departing from the spirit of the than 200:1. US 8,968,811 B2 25 26 11. A process according to claim 2, wherein the weight 19. A process according to claim 18, wherein said extract ratio of hydroxytyrosol:aldehydic form of oleuropein agly has a hydroxytyrosol content of at least 90% (w/w) a purity of cone in the extract is between 5:1 and 850:1. at least 90% and a total phenols content of at least 92% (w/w). 12. A process according to claim 2, wherein said extract is 20. A process according to claim 19, wherein said extract is free from oleuropein and aldehydic form of oleuropeinagly free from carriers. cone (by HPLC 280 nm). 13. A process according to claim 2, wherein the weight 21. A process according to claim 3, wherein the weight ratio of hydroxytyrosol:tyrosol in the extract is greater than ratio of hydroxytyrosol:oleuropein in the extract is greater 55:1. than 200:1. 14. A process according to claim 2, wherein the weight 22. A process according to claim 3, wherein the weight ratio of hydroxytyrosol:tyrosol in the extract is within the 10 ratio of hydroxytyrosol:aldehydic form of oleuropein agly range of 55:1 to 618:1. cone in the extract is between 5:1 and 850:1. 15. A process according to claim 2, wherein said extract is 23. A process according to claim3, wherein said extract is free from organic polar solvents. free from oleuropein and aldehydic form of oleuropeinagly 16. A process according to claim3, wherein said extract is 15 cone (by HPLC 280 nm). a liquid extract and has a total phenol content of at least 35% 24. A process according to claim 3, wherein the weight (w/w), anhydroxytyrosol content of at least 35% (w/w) and a ratio of hydroxytyrosol:tyrosol in the extract is greater than purity of at least 90% (by HPLC 280 nm). 55:1. 17. A process according to claim 16, wherein the content of hydroxytyrosol of said extract is at least 45% (w/w). 25. A process according to claim 3, wherein the weight 18. A process according to claim 2, wherein said extract is ratio of hydroxytyrosol: tyrosol in the extract is within the a solid, has a total phenols content of at least 20% (w/w), the range of 55:1 to 618:1. content of hydroxytyrosol in said extract is at least 20% (w/w) 26. A process according to claim3, wherein said extract is and the hydroxytyrosol purity is at least 90% (by HPLC 280 free from organic polar solvents. nm). k k k k k