WO2007074490A1 - Process for producing triacξtylhydroxytyrosol from olive oil mill waste waters for use as stabilized antioxidant - Google Patents
Process for producing triacξtylhydroxytyrosol from olive oil mill waste waters for use as stabilized antioxidant Download PDFInfo
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- WO2007074490A1 WO2007074490A1 PCT/IT2005/000781 IT2005000781W WO2007074490A1 WO 2007074490 A1 WO2007074490 A1 WO 2007074490A1 IT 2005000781 W IT2005000781 W IT 2005000781W WO 2007074490 A1 WO2007074490 A1 WO 2007074490A1
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F1/00—Treatment of water, waste water, or sewage
- C02F1/26—Treatment of water, waste water, or sewage by extraction
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/08—Preparation of carboxylic acid esters by reacting carboxylic acids or symmetrical anhydrides with the hydroxy or O-metal group of organic compounds
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/56—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F1/00—Treatment of water, waste water, or sewage
- C02F1/66—Treatment of water, waste water, or sewage by neutralisation; pH adjustment
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/32—Nature of the water, waste water, sewage or sludge to be treated from the food or foodstuff industry, e.g. brewery waste waters
- C02F2103/322—Nature of the water, waste water, sewage or sludge to be treated from the food or foodstuff industry, e.g. brewery waste waters from vegetable oil production, e.g. olive oil production
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/582—Recycling of unreacted starting or intermediate materials
Definitions
- the present invention concerns a process for producing triacetylhy- droxytyrosol from olive oil mill waste waters, for use as stabilized antioxidant. More specifically, the invention relates to a new method for synthesizing the triacetyl derivative of hydroxytyrosol by direct treatment of the organic extracts of olive mill waste waters, which contain hydroxytyrosol in considerable amount, with a suitable acylating mixture, thus affording the triacetyl derivative in high yield.
- the latter is biochemically convertible in situ to the active form hydroxytyrosol, and may be used as a stabilized antioxidant for the protection of human cells.
- Antioxidant compounds play a key role in human nutrition as well as in industry. Dietary intake of both vitamin and non vitamin antioxidants, indeed, is known to decrease the incidence of several pathologies, including cardio- vascular diseases.
- natural and synthetic antioxidants are widely used in cosmetic, pharmaceutical as well as in nutritional preparations.
- several studies have been devoted to explore the biological effects of non vitamin phenolic antioxidants, which occur widely in the vegetal kingdom and therefore in plant-derived food, including olive oil. The latter is the typical lipid source of the Mediterranean Diet.
- the beneficial health effects of this dietary habit have been partially attributed to the high content of antioxidant compounds, including polyphenols.
- hydroxytyrosol i.e. 3,4-dihydroxyphenylethanol or (4-(2- hydroxyethyl-1 ,2-benzenediol), having the following structural formula: which is an antioxidant compound naturally occurring in olive oil, and has been shown to have a marked chemoprotective activity (Manna, C. et. al., Biological effects of hydroxytyrosol, a polyphenol from olive oil endowed with antioxidant activity, Adv. Exp. Med. Biol. 1999, 472, 115-130).
- Hydroxytyrosol derives from the hydrolysis of oleuropein, an intensely bitter glucoside present in olives in high amounts (up to 6.5 g/kg of the fresh weight in unripe olives).
- the oleuropein concentration in olives increases during maturation.
- two different extraction systems are used in the production of extra-virgin olive oil: the traditional olive mill, which implements a discontinuous pressing process, and the continuous system, which works by cen- trifugation. Both systems envisage an initial processing stage wherein the olives are first sorted to eliminate the leaves, washed in water and then re- cuted to a paste in a mechanical crusher.
- the paste then undergoes pressing, with the production of a solid residue and a liquid product called "must".
- the “must” obtained is centrifuged to separate out an oily fraction that constitutes the oil of first pressing, while the aqueous fraction is the wastewater of this process.
- the olive paste is sent directly to separation by centrifugation, after being appropriately diluted with hot water. This operation yields three phases: the oil of first pressing, the olive residues (i.e. the pomace) and the wastewater.
- the aqueous solution obtained as residue is, with the same amount of olives processed, quantitatively greater, albeit with a lower content of dis- solved solids.
- the olive mill wastewaters generally contain, as dissolved or suspended substances, mostly sugars, polyphenol compounds, organic acids, proteins and related nitrogen com- pounds, fatty substances, mixed phenol-polysaccharide polymers, polyalco- hols, cellulose and hemicellulose, pectins and tannins.
- the inorganic substance present in lower amount, includes mainly potassium and, to a lesser degree, sodium, calcium and magnesium as cations, and chlorides, phosphates and sulphates as anions.
- one feature that makes olive mill wastewater particularly harmful if dispersed unprocessed, such as by directly pouring it into soils as a fertilizer, is its content of polyphenol compounds.
- the polyphenol compounds which act in the olives as protective agents with antioxidant and bacteriostatic functions until the olives ripen, may have, con- versely, a phytotoxic action (within certain limits) and may be harmful to the bacterial flora in the soil when they are contained in the olive processing wastes.
- hydroxytyrosol is present in considerable amounts in olive mill solid-liquid wastes (OMW) from two-phase olive oil processing (Femandez-Bolanos, J. et al., Production in large quantities of highly purified hydroxytyrosol from liquid-solid waste of two phase olive oil processing or "Alperujo", J. Agric. Food Chem. 2002, 50, 6804-681 1 ), as well as in olive mill waste waters (OMWW) from traditional and industrial three-phase plants (Al- louche, N. et al., Toward a high yield recovery of antioxidants and purified hydroxytyrosol from olive mill wastewaters, J. Agric. Food Chem.
- Hydroxytyrosol acetate is also the main hydroxytyrosol monoester concerned by the patent application EP-A-1541544 (Consejo Superior de Investigaations Cientificas and Universidad de Sevilla), disclosing a method of preparing hydroxytyrosol esters and their use as antioxidants for food products, as additives for cosmetic products and in pharmaceutical preparations.
- the monoester derivative of hydroxytyrosol is prepared by chemical synthesis (regioselective esterification) starting from either natural or synthetic hydroxytyrosol or derivatives thereof.
- Hydroxytyrosol is more active than antioxidant vitamins and more active than the usual industrial synthetic antioxidants, such as 2,6-di-te/ ⁇ f-butyl-p-hydroxytoluene and 3-tert- butyl-6-hydroxyanisole.
- hydroxytyrosol prevents passive smoking- induced oxidative stress.
- Clear epidemiological and biochemical evidence indicates that hydroxytyrosol is endowed with significant antithrombotic, antiatherogenic and anti-inflammatory activities. Mechanisms underlying these biological effects include inhibition of platelet aggregation and eicosanoid production, and inhibition of lipoxygenases and cycloxygenases activity. Further, this phenolic com- pound also prevents copper sulfate-induced lipoprotein oxidation. It should be stressed in this respect that oxidized lipoproteins have been shown to be more atherogenic than the native ones.
- oleuropein i.e. the hydroxytyrosol precursor
- Oleuropein prevents oxidative myocardial injury induced by ischemia and reperfusion, J. Nutr. Biochem. 2004, 15, 461-466. It is also able to significantly reduce myocardial injury induced by ischemia and reperfusion, using isolated rat heart as model system.
- hydroxytyrosol As far as the transport of hydroxytyrosol is concerned, it was demonstrated that it permeates cell membranes via a passive diffusion mechanism, and that it is rapidly distributed in all organs and tissues when intravenously injected in rats. Moreover, its high bioavailability in humans has been reported (Visioli, F. et al., Olive oil phenolics are dose-dependently absorbed in hu- mans, FEBS Lett. 2000, 468, 159-16032). Hydroxytyrosol is highly metabolized in vivo both in the aromatic moiety as well as in the lateral chain, yielding metabolites that still retain the antioxidant power of the native molecule (D'Angelo, S. et al., Pharmacokinetics and metabolism of hydroxytyrosol, a natural antioxidant from olive oil, Drug Metab. Dispos. 2001 , 29, 1492-1498).
- hydroxytyrosol represents a good candidate for a potential utilization as antioxidant for either nutritional or pharmaceutical and cosmetic preparations.
- ROS reactive oxygen species
- hydroxytyrosol cannot be added to lipophylic preparations because of its relative polarity (specifically, it is amphiphilic in nature, and is more soluble in the water than in the oil phase).
- hydroxytyrosol after oil extraction is mostly dissolved in the olive mill wastes (wastewater and pomace), while the oil obtained contains no more than 1-2% of the antioxidants originally contained in the olives (Panayotis, S. R. et al., Partitioning of olive oil antioxidants between oil and water phases, J. Agric. Food Chem. 2002, 50, 596-601 ).
- hydroxytyrosol should be conveniently produced as a derivative chemically more stable, which should be able to be biochemically converted in situ into its original active form, in order to exert in vivo its biological antioxidant activity.
- WO 03/082798 discloses the application of hydroxytyrosol and tyrosol esters as antioxidants for use in the treatment of cardiovascular, hepatic, renal and inflammatory diseases, to be incorporated in nutritional products (such as juice, milk, nutritional beverages and butter preparations), as well as their use as atioxidants for cosmetic preparations.
- the hydroxytyrosol esters disclosed may be mono-, di- or triesters, and are preferably obtained by binding hydroxytyrosol with fatty acid chains. These esters were found to be more resistant than hydroxytyrosol to air oxidation in an edibile matrix at 120 0 C.
- the concerned document describes the synthesis of a tristearyl derivative of hydroxytyrosol by reaction of hy- droxytyrosol as starting product with stearic acid and dicyclohexylcarbodiimide (DCC), in the presence of dimethylamminopyridine (DMAP), with a yield of 32%.
- This method obviously requires the preliminary preparation of synthetic or natural hydroxytyrosol, both preliminary steps having their own yield (about 41 % for the extraction of hydroxytyrosol from olive oil mill wastewaters according to the metod reported herein, about 80-85% by the most efficient methods reported in the literature, such as Allouche, N. et al., 2004, loc. cit.; Capasso, R. et al., 1999, loc. cit. ), so that the production of the tristearyl derivative of hydroxytyrosol can be obtained with an overall maximum yield of about 27%.
- the disclosure also reports the production of the triacetyl derivative of hydroxytyrosol, i.e. 2-(3,4-diacetoxy- phenyl)ethyl acetate, starting from hydroxytyrosol.
- the only method disclosed for synthesizing triacetylhydroxytyrosol is by reaction of hydroxytyrosol with acetic anhydride in THF (tetrahydrofuran), and requires the use of DMAP (which is considered to be highly toxic) as a catalyst. Such acylation reaction takes about 7 hours to be completed.
- the advantage of producing the triacetyl derivative of hydroxytyrosol from the OMWW extracts is due to two fundamental factors: i) the stabilization of hydroxytyrosol into its triacetyl derivative, ensuring the protection of hydroxytyrosol against oxidative and heating processes, which can occur during the chromatographic and work-up processes deriving from the direct recovery of hydroxytyrosol, and thus ensuring a higher yield in the production of the antioxidant; and ii) the preservation of the intact protective agent triacetylhydroxytyrosol in the biological matrices (alimentary, cosmetic and pharmaceutical preparations), which is only activated into hydroxytyrosol against the oxidative stress of human cells, by membrane esterases, as explained below.
- the present invention proposes to produce the pure triacetyl derivative by direct treatment of the organic extracts of OMWW, without any previous isolation and purification of hydroxytyrosol, with an acetylating system consisting of perchloric acid adsorbed on silica gel (HCIO 4 -SiO 2 ) as a catalyst and acetic anhydride (Ac 2 O), or another suitable acetyl donor, such as acetic acid (HOAc) or its salts.
- HCIO 4 -SiO 2 silica gel
- Ac 2 O acetic anhydride
- HOAc acetic acid
- the latter is a recently developed method to acetylate phenols and similar compounds having a poor nucleophilicity (Chakraborti, A. K.
- the present invention specifically provides a process for producing triacetylhydroxytyrosol from olive oil mill waste waters (OMWW) including, in sequence, the following steps: a) treating OMWW by liquid-liquid extraction with a polar organic solvent, to obtain an organic extract containing hydroxytyrosol; b) contacting the organic extract obtained from the step a) with a catalyst mixture of perchloric acid and silica and an acetyl donor compound, to obtain an acetylation reaction product containing triacetylated hydroxytyrosol; c) purifying the reaction product obtained from the step b) by means of medium-pressure liquid chromatography (MPLC), to obtain pure triacetylhydroxytyrosol.
- MPLC medium-pressure liquid chromatography
- the liquid-liquid extraction of step a) may be carried out by semi-continuous Soxhlet extraction, by continuous counter-current extraction or by discontinuous batch extraction.
- the use of the Soxhlet apparatus proved to be a very convenient liquid-liquid extraction procedure in view of its high yield, simple work-up and small volumes of solvent used, and therefore for its overall low cost. This was demonstrated in a recent paper by the same Authors (Sannino, F.
- the polar organic solvent for the liquid-liquid extraction of step a) is chosen from the group consisting of: ethyl acetate, methyl isobutyl ketone, methyl ethyl ketone, diethyl ether, methanol and n-butanol, ethyl acetate being the preferred solvent.
- the pH of the OMWW undergoing step a) can be the native pH or it can be adjusted in the range from pH 2.0 to pH 8.0. It is worth noting that the most convenient extraction procedure showed to be that performed using the native OMWW (pH 4.5) in a Soxhlet apparatus.
- the OMWW pH is the native pH (i.e. pH 4.5-5.5)
- the polar organic solvent is ethyl acetate
- the ratio solvent/OMWW is comprised between 1 :1 and 4:1. Most preferably, said ratio is 2:1.
- the catalyst mixture of the triacetylation reaction (step b) is, more specifically, perchloric acid adsorbed on silica gel, while said acetyl donor compound is prefarably acetic anhydride.
- the catalyst HCIO 4 -Si ⁇ 2 mixed with Ac 2 O is an acetylating agent developed recently (Chakraborti, A. K. et al. 2003, loc. cit.).
- this catalyst has been applied to the direct treatment of the organic extracts of OMWW in view of the high production yield it offered in the acetylation of other phenolic compounds, and in view of the fact it appears to limit the secondary reactions which occur with the mixtures of H 2 SO 4 or pyridine with Ac 2 O utilized as conventional acetylating agents.
- said acetylation reaction of step b) is carried out at a temperature comprised between room temperature and 110 0 C, and for a total reaction time of from 5 minutes to 24 hours. It is worth noting that in the specific example reported below the triacetylation of hydroxytyrosol showed to be complete after 5 minutes only and at room temperature.
- the purification step c) is carried out by MPLC on silica gel, preferably eluting with ethyl acetate/petroleum ether 1 :1 (v/v).
- the process according to the invention also provides an exhausted fraction from the OMWW liquid-liquid extraction, which is deprived of the potentially harmful phenolic compounds. This exhausted fraction can also be exploited as amendments, applying the possible procedures recently published (El Hadrami A. et al., Physico-chemical characterization and effects of olive oil mill wastewaters fertirrigation on the growth of some mediterranean crops. J. Agron. 2004, 3, 247-254).
- triacetylhydroxytyrosol appears to be much more convenient using the method which employs the treatment of the Soxhlet phenol extracts with HCIO 4 -SiO 2 and Ac 2 O than the known methods. Therefore, a possible industrial production of triacetylhydroxytyrosol, which is a stabilized form of hydroxytyrosol, is expected to find convenient applications, mainly for the exploitation of triacetylhydroxytyrosol as additive in nutritional, cosmetic and pharmaceutical preparations. In such preparations, the compound remains preserved against possible chemical oxidations and is only activated into hydroxytyrosol by the cellular contact, ensuring the highest antioxidant activity and therefore the highest cellular protecting effect, highly compatible with human health.
- polyphenols exert their biological activities in vitro at micromolar concentrations, i.e. within the concentration range expected after nutritional intake of plant-derived food.
- a complete protection against oxidative hemolysis was observed in red blood cells pretreated with as little as 5 ⁇ g total ortho-diphenols, corresponding to about 300 ⁇ l of virgin olive oil containing 100 mg/kg ortho-diphenols (Visioli, F. and GaIIi, C, Biological properties of olive oil phytochemicals, Crit. Rev. Food ScL Nutr. 2002, 42, 209-221 ).
- the daily intake of antioxidant fortified FF is useful to design dietary strategies for the prevention of cardiovascular diseases.
- triacetylhydroxytyrosol can be regarded as a non-toxic compound, being biochemically transformed into hydroxytyrosol when administrered in vivo. Previous tests on animal models have shown that this product is compatible with human health.
- Figure 1 shows a scheme of production of triacetylhydroxytyrosol from raw OMMW according to the process of the invention
- Figure 2 shows a HPLC chromatogram of the purified triacetylhy- droxytyrosol obtained by the process of Figure 1 ;
- Figure 3 shows a scheme of production of hydroxytyrosol by extraction from raw OMMW, for comparison purposes;
- Figure 4 shows a HPLC chromatogram of the purified hydroxytyrosol obtained by the process of Figure 3;
- Figure 5 shows the results of the FRAP (ferric reducing/antioxidant power) test for hydroxytyrosol(hdrx) and for triacetylhydroxytyrosol (triachdrx);
- Figures 6 A) and 6 B) show, respectively, the protective effects of hy- droxytyrosol and triacetylhydroxytyrosol on the viability of human Caco-2 cells and the protective effects of the same compounds against the thiobarbituric acid reactive substances (TBARS) formation in human Caco-2 cells; and
- Figures 7 A) and 7 B) show, respectively, the protective effects of hydroxytyrosol and triacetylhydroxytyrosol against hemolysis in human red blood cells (RBC) and the protective effects of the same compounds against the TBARS formation in human RBC.
- RBC red blood cells
- the method includes as a first step the continuous liquid-liquid extraction mode of a sample of OMWW, using a Soxhlet apparatus.
- the organic extracts were directly treated with perchloric acid supported on silica gel as acetylating cata- Iyst, and acetic anhydride as reagent, producing a mixture of compounds containing triacetylhydroxytyrosol, which appeared quantitatively acetylated.
- HCIOd-SiO? The preparation of the HCIO 4 -SiO 2 catalyst (perchloric acid adsorbed on silica gel) to perform the acetylation reaction with Ac 2 O (acetic anhydride) was carried out according to a modified proce- dure of Charkraborti and Gulhane (Chakraborti, A. K. et al. 2003, loc. cit.).
- HCIO 4 (1.25 mmol, as a 65% aqueous solution) was added to a suspension of silica gel (2.37 g, 230-400 mesh) in 7.5 mL of diethyl ether. The mixture was kept for 3 h at room temperature under magnetic stirring and then it was heated at 70 0 C for 28 h under vacuum to afford HCIO 4 -SiO 2 as a free flowing powder.
- reaction mixture was then washed with ethyl acetate, filtered under vacuum and evaporated under reduced pressure at 30 0 C, leaving a residue of 262 mg containing triacetylhydroxytyrosol.
- the mixture obtained from the acetylation step was chroma- tographed by a single MPLC step giving pure triacetylhydroxytyrosol, resulting in an overall yield of 35.6%, related to the amount of hydroxytyrosol naturally contained in the organic extracts.
- Example 1 The materials and methods employed were the same as in Example 1.
- the synthetic standard corresponding to hydroxytyrosol was prepared ac- cording to the previously reported procedure (Capasso, R. et al., 1999, loc. cit.).
- the TLC analysis procedure was the same as in Example 1 , except for the eluting solution, which was acetone/petroleum ether 50:50 (v:v).
- Spectroscopic data 1 H NMR and El MS
- the method includes as a first step the continuous liquid-liquid extraction of a sample of OMWW, using a Soxhlet apparatus, according to the same procedure described in Example 1.
- Increasing amounts of selected compounds were added to 1 ml of working solution, prepared by mixing 25 ml of 300 mM acetate buffer [pH 3.6], 2.5 ml TPTZ solution [10 mM TPTZ in 40 mM HCI] and 2.5 ml of 20 mM FeCI 3 -6H2O; after incubation for 6 min at room temperature the absorbance was read at 593 nm.
- Caco-2 cells (carcinoma colon cells, as model for epithelial intestinal cells) were used as a model system, according to the previously disclosed procedures (Manna, C. et al., The protective effect of the olive oil polyphenol (3,4-dihydroxyphenyl)ethanol counteracts reactive oxygen metabolite-induced cytotoxicity in Caco-2 cells. J. Nutr. 1997, 127, 286-292.41, 42).
- Colon carcinoma cells simulate epithelial intestinal cells.
- these cells which are able to differentiate in culture, are considered a very suitable model to mimic the food-intestinal tract interactions in vitro.
- Reactive oxygen species ROS
- ROS Reactive oxygen species
- Example 2 high glucose Dulbecco's modified Eagle's medium (DMEM), fetal calf serum, non-essential amino acids, N-2-hydroxy-ethylpiperazine-N'-2-ethane sulfonic acid (HEPES), glutamine, penicillin, streptomycin and PBS tablets were pur- chased from Gibco, Life Science Technologies (S. Giuliano Milanese, Ml, Italy).
- DMEM Dulbecco's modified Eagle's medium
- HEPES N-2-hydroxy-ethylpiperazine-N'-2-ethane sulfonic acid
- glutamine penicillin
- streptomycin and PBS tablets were pur- chased from Gibco, Life Science Technologies (S. Giuliano Milanese, Ml, Italy).
- the cells were pre-treated for 30 min, in the presence or absence of increasing concentrations of each selected antioxidant, before the induction of oxidative stress.
- the viability of the concerned cells is reduced to 62% by the treatment with 4 mM t-BHP, as assessed by MIT test.
- a remarkable protection against ROS-induced cytotoxicity was detected in the antioxidant-treated Caco-2 cells, the protective effect of triacetylhydroxytyrosol being of same order of magnitude as hydroxytyrosol in a micromolar concentration range.
- Membrane phospholipids are a major target of oxidative damage as the lipid peroxidation involves the cleavage of polyunsaturated fatty acids in their double bonds, leading to the formation of TBARS.
- a very significant protection of the Caco-2 cells by hydroxytyrosol and triacetylhydroxytyrosol is also observed against the lipoperoxidation of the membranes in the TBARS test.
- a very significant reduction of TBARS pretreated with the two tested compounds is observed with respect to the sample treated with t-BHP.
- the oxidative treatment proves to be of about three-fold increase in TBARS concentration compared with control cells; in this test also, pre-treatment of Caco-2 cells with either hydroxytyrosol or triacetylhydroxytyrosol significantly and dose-dependently decrease TBARS formation both at 50 and 100 ⁇ M, indicating that both the tested compounds equally protect against lipid peroxidation.
- triacetylhydroxytyrosol shows a biological antioxidant activity very close to that of the parent compound strongly suggests that the triacetyl derivative is metabolized by cell esterase into hydroxytyrosol, which is the effective antioxidant compound, and, very probably, also in vivo at the intestinal level.
- red blood cells were selected as a second experimental system.
- the experimental procedure is according to the previously published literature (Manna, C. et al, Olive oil hydroxytyrosol protects human erythrocytes against oxidative damages, J. Nutr. Biochem. 1999, 10, 159-165).
- RBC human red blood cells
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Abstract
The invention relates to a process for producing triacetylhydroxytyrosol as a pure compound by direct treatment of the organic extracts of olive oil mill waste waters, rich in hydroxytyrosol, with an acylating mixture consisting of perchloric acid adsorbed on silica gel and a source of acetyl radicals, such as acetic anhydride, followed by a single purification step of the reaction product by means of medium-pressure liquid chromatography (MPLC). The procedure, consisting of only three steps starting from the raw olive mill wastewaters, affords an overall yield in triacetylhydroxytyrosol of at least 35%. Triacetylhydroxytyrosol shows a biological antioxidant activity comparable with the antioxidant activity of the parent compound, i.e. hydroxytyrosol, as the two compounds have been shown to equally protect against oxidative damage, at micromolar concentrations, in two different experimental systems. Thus, tiacetylhydroxytyrosol is metabolized in vivo to yield the active deacetylated form, but is in turn chemically more stable than hydroxytyrosol, and more easily miscible with lipophilic alimentary, pharmaceutical and cosmetic preparations.
Description
PROCESS FOR PRODUCING TRIACETYLHYDROXYTYROSOL FROM OLIVE OIL MILL WASTE WATERS FOR USE AS STABILIZED
ANTIOXIDANT
SPECIFICATION
The present invention concerns a process for producing triacetylhy- droxytyrosol from olive oil mill waste waters, for use as stabilized antioxidant. More specifically, the invention relates to a new method for synthesizing the triacetyl derivative of hydroxytyrosol by direct treatment of the organic extracts of olive mill waste waters, which contain hydroxytyrosol in considerable amount, with a suitable acylating mixture, thus affording the triacetyl derivative in high yield. The latter is biochemically convertible in situ to the active form hydroxytyrosol, and may be used as a stabilized antioxidant for the protection of human cells.
Antioxidant compounds play a key role in human nutrition as well as in industry. Dietary intake of both vitamin and non vitamin antioxidants, indeed, is known to decrease the incidence of several pathologies, including cardio- vascular diseases. In addition, natural and synthetic antioxidants are widely used in cosmetic, pharmaceutical as well as in nutritional preparations. In recent years, several studies have been devoted to explore the biological effects of non vitamin phenolic antioxidants, which occur widely in the vegetal kingdom and therefore in plant-derived food, including olive oil. The latter is the typical lipid source of the Mediterranean Diet. The beneficial health effects of this dietary habit have been partially attributed to the high content of antioxidant compounds, including polyphenols.
Among the different phenolic compounds, particular attention has been focused on hydroxytyrosol, i.e. 3,4-dihydroxyphenylethanol or (4-(2- hydroxyethyl-1 ,2-benzenediol), having the following structural formula:
which is an antioxidant compound naturally occurring in olive oil, and has been shown to have a marked chemoprotective activity (Manna, C. et. al., Biological effects of hydroxytyrosol, a polyphenol from olive oil endowed with antioxidant activity, Adv. Exp. Med. Biol. 1999, 472, 115-130).
Hydroxytyrosol derives from the hydrolysis of oleuropein, an intensely bitter glucoside present in olives in high amounts (up to 6.5 g/kg of the fresh weight in unripe olives). The oleuropein concentration in olives increases during maturation. In general, two different extraction systems are used in the production of extra-virgin olive oil: the traditional olive mill, which implements a discontinuous pressing process, and the continuous system, which works by cen- trifugation. Both systems envisage an initial processing stage wherein the olives are first sorted to eliminate the leaves, washed in water and then re- duced to a paste in a mechanical crusher. In the case of traditional olive mills, the paste then undergoes pressing, with the production of a solid residue and a liquid product called "must". The "must" obtained is centrifuged to separate out an oily fraction that constitutes the oil of first pressing, while the aqueous fraction is the wastewater of this process. As regards the continuous type systems, called "three-phase systems", which are more widespread today, the olive paste is sent directly to separation by centrifugation, after being appropriately diluted with hot water. This operation yields three phases: the oil of first pressing, the olive residues (i.e. the pomace) and the wastewater. In this case, the aqueous solution obtained as residue is, with the same amount of olives processed, quantitatively greater, albeit with a lower content of dis-
solved solids.
Over the last years, the traditional olive oil production system has been a gradually shifting from the original discontinuous pressing process to the more productive continuous process based on horizontal centrifuges (de- canters). However, the need to reduce the environmental impact of the wastewater has led to developing the so-called "two-phase" centrifugation extraction systems, wherein water is not added to the decanter and the byproduct obtained consists of a single phase made up of a mixture of solid residues and olive mill wastewater. Despite the variations of concentration and composition depending on the areas of production, the climatic conditions during olive ripening, the harvesting practices and the oil extraction process, the olive mill wastewaters generally contain, as dissolved or suspended substances, mostly sugars, polyphenol compounds, organic acids, proteins and related nitrogen com- pounds, fatty substances, mixed phenol-polysaccharide polymers, polyalco- hols, cellulose and hemicellulose, pectins and tannins. The inorganic substance, present in lower amount, includes mainly potassium and, to a lesser degree, sodium, calcium and magnesium as cations, and chlorides, phosphates and sulphates as anions. As already observed, one feature that makes olive mill wastewater particularly harmful if dispersed unprocessed, such as by directly pouring it into soils as a fertilizer, is its content of polyphenol compounds. Thus, the polyphenol compounds, which act in the olives as protective agents with antioxidant and bacteriostatic functions until the olives ripen, may have, con- versely, a phytotoxic action (within certain limits) and may be harmful to the bacterial flora in the soil when they are contained in the olive processing wastes.
Actually, hydroxytyrosol is present in considerable amounts in olive mill solid-liquid wastes (OMW) from two-phase olive oil processing (Femandez-Bolanos, J. et al., Production in large quantities of highly purified hydroxytyrosol from liquid-solid waste of two phase olive oil processing or "Alperujo", J. Agric. Food Chem. 2002, 50, 6804-681 1 ), as well as in olive mill
waste waters (OMWW) from traditional and industrial three-phase plants (Al- louche, N. et al., Toward a high yield recovery of antioxidants and purified hydroxytyrosol from olive mill wastewaters, J. Agric. Food Chem. 2004, 52, 267-273; Capasso, R. et al., A highly convenient synthesis of hydroxytyrosol and its recovery from agricultural waste water; J. Agric. Food Chem, 1999, 47, 1745-1748). In view of that, it is clear that the valorisation and marketing of antioxidant molecules of the polyphenol type that can be separated out from the olive mill wastewater (OMWW) is a promising way to recover the economical efforts needed to treat this waste according to the environmental requirements.
Besides the naturally occurring hydrolysis product of oleuropein, i.e. hydroxytyrosol, also its monoacetyl derivative, i.e. 3,4-dihydroxyphenylethyl acetate, is a naturally occurring compound, and it has been shown to have an antioxidant effect very close to that of hydroxytyrosol (Gordon, M. H. et al., Antioxidant activity of hydroxytyrosol acetate compared with that of other olive oil polyphenols, J. Agric. Food Chem. 2001 , 49, 2480-2495).
Hydroxytyrosol acetate is also the main hydroxytyrosol monoester concerned by the patent application EP-A-1541544 (Consejo Superior de Investigaciones Cientificas and Universidad de Sevilla), disclosing a method of preparing hydroxytyrosol esters and their use as antioxidants for food products, as additives for cosmetic products and in pharmaceutical preparations. According to said disclosure, the monoester derivative of hydroxytyrosol is prepared by chemical synthesis (regioselective esterification) starting from either natural or synthetic hydroxytyrosol or derivatives thereof. Abundant literature shows that hydroxytyrosol is a potent scavenger of superoxide anion as well as hydroxy radicals, in vitro inhibits the oxidation of low-density lipoproteins and confers cell protection against oxidative damages (Manna, C. et al., The protective effect of olive oil polyphenol (3,4-dihydroxy- phenyl)-ethanol counteracts reactive oxygen metabolite-induced cytotoxicity in Caco-2-cells, J. Nutr 1997, 127, 286-292; Manna, C. et al., Protective effect of the phenolic fraction from virgin olive oils against oxidative stress in human cells, J. Agric. Food Chem. 2002, 50, 6521-6526). Hydroxytyrosol is more
active than antioxidant vitamins and more active than the usual industrial synthetic antioxidants, such as 2,6-di-te/τf-butyl-p-hydroxytoluene and 3-tert- butyl-6-hydroxyanisole. In addition, hydroxytyrosol prevents passive smoking- induced oxidative stress. Clear epidemiological and biochemical evidence indicates that hydroxytyrosol is endowed with significant antithrombotic, antiatherogenic and anti-inflammatory activities. Mechanisms underlying these biological effects include inhibition of platelet aggregation and eicosanoid production, and inhibition of lipoxygenases and cycloxygenases activity. Further, this phenolic com- pound also prevents copper sulfate-induced lipoprotein oxidation. It should be stressed in this respect that oxidized lipoproteins have been shown to be more atherogenic than the native ones.
Finally, a recent finding reveals new molecular mechanisms of the antiatherogenic activity of hydroxytyrosol: this phenol, indeed, is able to inhibit leukocyte adhesion to vascular endothelial cells, which represents a key step in the formation of atherosclerotic plaque (Carluccio, M.A. et al., Olive oil and red wine antioxidant polyphenols inhibit endothelial activation. Antiatherogenic properties of Mediterranean diet phytochemicals, Arterioscler. Thromb. Vase. Biol. 2003, 23, 622-629). The experimental data on the biological activities of purified hydroxytyrosol have also been confirmed using the total olive oil phenolic fraction.
Moreover, it has been recently demonstrated that oleuropein (i.e. the hydroxytyrosol precursor) exerts a strong direct cardioprotective effect (Manna, C. et al., Oleuropein prevents oxidative myocardial injury induced by ischemia and reperfusion, J. Nutr. Biochem. 2004, 15, 461-466). It is also able to significantly reduce myocardial injury induced by ischemia and reperfusion, using isolated rat heart as model system.
As far as the transport of hydroxytyrosol is concerned, it was demonstrated that it permeates cell membranes via a passive diffusion mechanism, and that it is rapidly distributed in all organs and tissues when intravenously injected in rats. Moreover, its high bioavailability in humans has been reported (Visioli, F. et al., Olive oil phenolics are dose-dependently absorbed in hu-
mans, FEBS Lett. 2000, 468, 159-16032). Hydroxytyrosol is highly metabolized in vivo both in the aromatic moiety as well as in the lateral chain, yielding metabolites that still retain the antioxidant power of the native molecule (D'Angelo, S. et al., Pharmacokinetics and metabolism of hydroxytyrosol, a natural antioxidant from olive oil, Drug Metab. Dispos. 2001 , 29, 1492-1498).
In view of the foregoing, hydroxytyrosol represents a good candidate for a potential utilization as antioxidant for either nutritional or pharmaceutical and cosmetic preparations.
The high interest for hydroxytyrosol, deriving from all considered properties and from the high compatibility with the human health (D'Angelo, S. et al., 2001 , loc. cit.), have prompted, particularly in the last years, many scientists of multidisciplinary areas to investigate on its production by direct recovery from olive mill wastes or using chemical (Capasso, R. et al., 1999, loc. cit.), biochemical (Espin, J. C. et al., Synthesis of the antioxidant hydroxytyro- sol using tyrosinase as biocatalyst, J. Agric. Food Chem. 2001 , 49, 1 187- 1 193) and biotechnological (Allouche, N. et al., Use of whole cells of Pseudo- monas aeruginosa for synthesis of the antioxidant hydroxytyrosol via conversion of tyrosol, J. Agric. Food Chem. 2004, 70, 2105-2109) methods applied on a synthetic precursor. With regard to the recovery of hydroxytyrosol from olive oil production wastes for potential industrial exploitation (i.e. nutritional, pharmaceutical and cosmetic applications), recent methods have been published. They are consistent with the direct extraction of this biomaterial with water or steam under a pressure of 42 kg/cm2 at 160-2400C in a pilot reactor (Fernandez-Bolanos, J. et al., 2002, loc. cit.) or with recovery from traditional three phase OMWW by extraction with ethyl acetate at room temperature in a polyethylene mixer settler apparatus (Allouche, N. et al., 2004, loc. cit.).
However, the production of natural hydroxytyrosol from OMWW as pure compound requires two or three chromatographic steps with a conse- quent low yield (Capasso, R. et al., 1999, loc. cit; Capasso, R. et al., Isolation, spectroscopy and selective phytotoxic effects of polyphenols from vegetable wastewaters, Phytochemistry 1992, 31, 4125-4128; Capasso, R. et al., Pro-
duction of hydroxytyrosol from olive oil vegetation waters, Agrochimica 1994, 38, 166-171 ), although recent modifications of such methods have resulted in some improvement of the yield (Fernandez-Bolafϊos, J. et al., 2002, loc. cit; Allouche, N. et al., 2004, loc. cit). More critically, hydroxytyrosol is chemically unstable, unless it is preserved dried in absence of air and in the dark, and the standard solutions have to to be prepared in situ. Therefore, the efficiency of this molecule added in its original form to alimentary, pharmaceutical and cosmetic matrices as protective agent against the action of reactive oxygen species (ROS) in hu- man cells cannot be assured.
Moreover, hydroxytyrosol cannot be added to lipophylic preparations because of its relative polarity (specifically, it is amphiphilic in nature, and is more soluble in the water than in the oil phase). By way of example, hydroxytyrosol after oil extraction is mostly dissolved in the olive mill wastes (wastewater and pomace), while the oil obtained contains no more than 1-2% of the antioxidants originally contained in the olives (Panayotis, S. R. et al., Partitioning of olive oil antioxidants between oil and water phases, J. Agric. Food Chem. 2002, 50, 596-601 ).
In view of the foregoing, it is evident that hydroxytyrosol should be conveniently produced as a derivative chemically more stable, which should be able to be biochemically converted in situ into its original active form, in order to exert in vivo its biological antioxidant activity.
With this intended object, the international patent application published with No. WO 03/082798 (Puleva Biotech S.A.) discloses the application of hydroxytyrosol and tyrosol esters as antioxidants for use in the treatment of cardiovascular, hepatic, renal and inflammatory diseases, to be incorporated in nutritional products (such as juice, milk, nutritional beverages and butter preparations), as well as their use as atioxidants for cosmetic preparations. The hydroxytyrosol esters disclosed may be mono-, di- or triesters, and are preferably obtained by binding hydroxytyrosol with fatty acid chains. These esters were found to be more resistant than hydroxytyrosol to air oxidation in an edibile matrix at 1200C. In particular, the concerned document describes
the synthesis of a tristearyl derivative of hydroxytyrosol by reaction of hy- droxytyrosol as starting product with stearic acid and dicyclohexylcarbodiimide (DCC), in the presence of dimethylamminopyridine (DMAP), with a yield of 32%. This method obviously requires the preliminary preparation of synthetic or natural hydroxytyrosol, both preliminary steps having their own yield (about 41 % for the extraction of hydroxytyrosol from olive oil mill wastewaters according to the metod reported herein, about 80-85% by the most efficient methods reported in the literature, such as Allouche, N. et al., 2004, loc. cit.; Capasso, R. et al., 1999, loc. cit. ), so that the production of the tristearyl derivative of hydroxytyrosol can be obtained with an overall maximum yield of about 27%.
Altough the fatty acid esters of hydroxytyrosol are the preferred derivatives concerned in the cited document, the disclosure also reports the production of the triacetyl derivative of hydroxytyrosol, i.e. 2-(3,4-diacetoxy- phenyl)ethyl acetate, starting from hydroxytyrosol. The only method disclosed for synthesizing triacetylhydroxytyrosol is by reaction of hydroxytyrosol with acetic anhydride in THF (tetrahydrofuran), and requires the use of DMAP (which is considered to be highly toxic) as a catalyst. Such acylation reaction takes about 7 hours to be completed.
Therefore, it is an object of the present invention to provide a further method for producing the protected triacetyl derivative of hydroxytyrosol, i.e. the compound having the following structural formula [(4-acetoxyethyl-1 ,2- diacetoxybenzene) or 2-(3,4-diacetoxyphenyI)ethyl acetate]:
CH2CH2OCOCH3 I1 21 while recovering a valuable material from the olive oil mill wastes, the method
affording a better yield than the previously known methods and not requiring costly or complex operations, while being safer and economically convenient. The achievement of said object represents a technical advance towards a safe recovery of industrially useful products from the olive mill wastes, which at the same time results in an exhausted waste fraction free from the environmental hazards due to the presence of polyphenols.
The advantage of producing the triacetyl derivative of hydroxytyrosol from the OMWW extracts is due to two fundamental factors: i) the stabilization of hydroxytyrosol into its triacetyl derivative, ensuring the protection of hydroxytyrosol against oxidative and heating processes, which can occur during the chromatographic and work-up processes deriving from the direct recovery of hydroxytyrosol, and thus ensuring a higher yield in the production of the antioxidant; and ii) the preservation of the intact protective agent triacetylhydroxytyrosol in the biological matrices (alimentary, cosmetic and pharmaceutical preparations), which is only activated into hydroxytyrosol against the oxidative stress of human cells, by membrane esterases, as explained below.
To achieve the above object, the present invention proposes to produce the pure triacetyl derivative by direct treatment of the organic extracts of OMWW, without any previous isolation and purification of hydroxytyrosol, with an acetylating system consisting of perchloric acid adsorbed on silica gel (HCIO4-SiO2) as a catalyst and acetic anhydride (Ac2O), or another suitable acetyl donor, such as acetic acid (HOAc) or its salts. The latter is a recently developed method to acetylate phenols and similar compounds having a poor nucleophilicity (Chakraborti, A. K. and Gulhane R., Perchloric acid adsorbed on silica gel as a new, highly efficient, and versatile catalyst for acetylation of phenols, thiols, alcohols, and amines. Chem. Comm. 2003, 1896-1897), which avoids toxicity problems and affords high yields in short reaction times, under mild conditions. According to the method proposed, the acetylated compound thus produced is finally purified in a single purification step by means of medium-pressure liquid chromatography (MPLC), affording a pure product in high yield.
Accordingly, the present invention specifically provides a process for producing triacetylhydroxytyrosol from olive oil mill waste waters (OMWW) including, in sequence, the following steps: a) treating OMWW by liquid-liquid extraction with a polar organic solvent, to obtain an organic extract containing hydroxytyrosol; b) contacting the organic extract obtained from the step a) with a catalyst mixture of perchloric acid and silica and an acetyl donor compound, to obtain an acetylation reaction product containing triacetylated hydroxytyrosol; c) purifying the reaction product obtained from the step b) by means of medium-pressure liquid chromatography (MPLC), to obtain pure triacetylhydroxytyrosol.
In general, the liquid-liquid extraction of step a) may be carried out by semi-continuous Soxhlet extraction, by continuous counter-current extraction or by discontinuous batch extraction. The use of the Soxhlet apparatus proved to be a very convenient liquid-liquid extraction procedure in view of its high yield, simple work-up and small volumes of solvent used, and therefore for its overall low cost. This was demonstrated in a recent paper by the same Authors (Sannino, F. et al., Recovery of hydroxytyrosol from olive mill wastewa- ter by two different continuous liquid-liquid extraction procedures, Polyphenol Communications 2004, 803-804 (XXII International Conference on Polyphenol, 25-28August 2004, Helsinki, Finland)), showing that this procedure is the most efficient as compared with two other methods: one was consistent with a continuous liquid-liquid counter-current extraction mode, and the other with a discontinuous one, in batch mode.
The polar organic solvent for the liquid-liquid extraction of step a) is chosen from the group consisting of: ethyl acetate, methyl isobutyl ketone, methyl ethyl ketone, diethyl ether, methanol and n-butanol, ethyl acetate being the preferred solvent. The pH of the OMWW undergoing step a) can be the native pH or it can be adjusted in the range from pH 2.0 to pH 8.0. It is worth noting that the most convenient extraction procedure showed to be that performed using the
native OMWW (pH 4.5) in a Soxhlet apparatus. In fact, even if the extraction process performed at pH 2.0 produces more hydroxytyrosol, it proves to be more complex and expensive (as an example, the acidification process is an over cost and also determines problems concerning the recycle of the ex- hausted OMWW, and at pH 2 much more total phenol extracts should be processed).
Thus, in a preferred embodiment of the process, the OMWW pH is the native pH (i.e. pH 4.5-5.5), the polar organic solvent is ethyl acetate and the ratio solvent/OMWW is comprised between 1 :1 and 4:1. Most preferably, said ratio is 2:1.
The catalyst mixture of the triacetylation reaction (step b) is, more specifically, perchloric acid adsorbed on silica gel, while said acetyl donor compound is prefarably acetic anhydride. As pointed out before, the catalyst HCIO4-Siθ2 mixed with Ac2O is an acetylating agent developed recently (Chakraborti, A. K. et al. 2003, loc. cit.). According to the invention, this catalyst has been applied to the direct treatment of the organic extracts of OMWW in view of the high production yield it offered in the acetylation of other phenolic compounds, and in view of the fact it appears to limit the secondary reactions which occur with the mixtures of H2SO4 or pyridine with Ac2O utilized as conventional acetylating agents.
In fact, within the frame of the studies that lead to the instant invention the production of triacetylhydroxytyrosol using the said two acetylating agents was also investigated, but the chromatographic purification of the acetyl derivative required two steps and gave a final yield of 26.1 % and 29.8%, using H2SO4 or pyridine mixed with Ac2O, respectively. In addition, the recycle of the reagents is laborious and expensive and involves safety risks. Instead, the catalyst HCIO4-SiO2 can be recycled, with obvious economical advantages in potential industrial applications and safety.
By preference, said acetylation reaction of step b) is carried out at a temperature comprised between room temperature and 1100C, and for a total reaction time of from 5 minutes to 24 hours. It is worth noting that in the specific example reported below the triacetylation of hydroxytyrosol showed to be
complete after 5 minutes only and at room temperature.
According to some specific embodiments of the invention, the purification step c) is carried out by MPLC on silica gel, preferably eluting with ethyl acetate/petroleum ether 1 :1 (v/v). Besides recovering pure triacetylhydroxytyrosol, with an overall yield of at least 35%, for use as an antioxidant in food, pharmaceutical or cosmetic preparations, the process according to the invention also provides an exhausted fraction from the OMWW liquid-liquid extraction, which is deprived of the potentially harmful phenolic compounds. This exhausted fraction can also be exploited as amendments, applying the possible procedures recently published (El Hadrami A. et al., Physico-chemical characterization and effects of olive oil mill wastewaters fertirrigation on the growth of some mediterranean crops. J. Agron. 2004, 3, 247-254).
The production of triacetylhydroxytyrosol appears to be much more convenient using the method which employs the treatment of the Soxhlet phenol extracts with HCIO4-SiO2 and Ac2O than the known methods. Therefore, a possible industrial production of triacetylhydroxytyrosol, which is a stabilized form of hydroxytyrosol, is expected to find convenient applications, mainly for the exploitation of triacetylhydroxytyrosol as additive in nutritional, cosmetic and pharmaceutical preparations. In such preparations, the compound remains preserved against possible chemical oxidations and is only activated into hydroxytyrosol by the cellular contact, ensuring the highest antioxidant activity and therefore the highest cellular protecting effect, highly compatible with human health. A further consideration is that triacetylhydroxytyrosol does not react to hydrolysis physiological conditions (370C, pH 7 and pH 2, in this last case regarding stomach conditions), confirming that its activity occurs only at the cellular level. Finally, the lipophilic properties of this compound render it particularly versatile for the addition to lipophilic alimentary, pharmaceutical and cosmetic preparations.
The finding that the chemically stable triacetylhydroxytyrosol is endowed with biological antioxidant activity is of particular interest for its poten-
tial utilization in humans. In fact, it can be successfully utilized for the preparation of functional foods (FF) for cardiovascular protection. As discussed in the introduction, the majority of the studies on the beneficial health effects of polyphenols clearly indicate that triacetylhydroxytyrosol is able to counteract the progression of atherosclerosis by interfering in different stages of the mechanism of plaque formation.
It is worth noting that polyphenols exert their biological activities in vitro at micromolar concentrations, i.e. within the concentration range expected after nutritional intake of plant-derived food. Using human cells in cul- ture, a complete protection against oxidative hemolysis was observed in red blood cells pretreated with as little as 5 μg total ortho-diphenols, corresponding to about 300 μl of virgin olive oil containing 100 mg/kg ortho-diphenols (Visioli, F. and GaIIi, C, Biological properties of olive oil phytochemicals, Crit. Rev. Food ScL Nutr. 2002, 42, 209-221 ). The daily intake of antioxidant fortified FF is useful to design dietary strategies for the prevention of cardiovascular diseases. Moreover, these high quality FF are useful to increase the endogenous defense in those countries whose dietary habits are particularly low in fruits and vegetables and therefore lacking in antioxidants. It should be pointed out that triacetylhydroxytyrosol can be regarded as a non-toxic compound, being biochemically transformed into hydroxytyrosol when administrered in vivo. Previous tests on animal models have shown that this product is compatible with human health.
Some embodiments of the process according to the present invention, as well as some experimental data concerning the performance of the product thus obtained in comparison with hydroxytyrosol, as well as its biological activity, are reported merely for exemplification purposes in the following examples. The invention and some of the said experimental results are also shown in the accompanying drawings, wherein: Figure 1 shows a scheme of production of triacetylhydroxytyrosol from raw OMMW according to the process of the invention;
Figure 2 shows a HPLC chromatogram of the purified triacetylhy- droxytyrosol obtained by the process of Figure 1 ;
Figure 3 shows a scheme of production of hydroxytyrosol by extraction from raw OMMW, for comparison purposes; Figure 4 shows a HPLC chromatogram of the purified hydroxytyrosol obtained by the process of Figure 3;
Figure 5 shows the results of the FRAP (ferric reducing/antioxidant power) test for hydroxytyrosol(hdrx) and for triacetylhydroxytyrosol (triachdrx);
Figures 6 A) and 6 B) show, respectively, the protective effects of hy- droxytyrosol and triacetylhydroxytyrosol on the viability of human Caco-2 cells and the protective effects of the same compounds against the thiobarbituric acid reactive substances (TBARS) formation in human Caco-2 cells; and
Figures 7 A) and 7 B) show, respectively, the protective effects of hydroxytyrosol and triacetylhydroxytyrosol against hemolysis in human red blood cells (RBC) and the protective effects of the same compounds against the TBARS formation in human RBC.
EXAMPLE 1 Production of triacetylhydroxytyrosol from olive oil mill waste The complete procedure for the production of pure triacetylhydroxytyrosol by the process according to the invention is shown in the scheme of Figure 1 (where 1a is hydroxytyrosol and 1 b is triacetylhydroxytyrosol). Materials and methods
Materials - Samples of OMWW (pH 4.5) were supplied by traditional mills located in Marrakech (Morocco). They were lyophilized in a Cryodos- Totelstar lyophilisator apparatus equipped with Varian vacuum technologies in the laboratory of Prof. Ismail EI Hadrami (Department of Biology, Faculty of Science Semlalia), and then redissolved in the Applicants' laboratory in high purity water to the initial volume. Solvents were of analytical and HPLC grade, purchased from Carlo
Erba (Milan, Italy). High purity water was obtained through a double filtration system, consisting of a deionized column and a MiIIiQ (Millipore) apparatus.
The synthetic standard corresponding to triacetylhydroxytyrosol was prepared according to a previously reported procedure (Capasso, R. et al., 1999, loc. cit).
Unless otherwise specified, all other compounds were purchased from Sigma-Aldrich (Milan, Italy).
Analytical TLC - TLC analysis on triacetylhydroxytyrosol obtained from the OMWW samples was performed on silica gel plates (Merck, Kiesel- gel 60 F2S4 0.25 mm) eluted with acetone/petroleum ether 40:60 (v:v). The spots were visualized by exposure to UV radiation and/or by spraying first with 10% sulphuric acid in methanol and then with 5% phosphomolybdic acid in methanol, followed by reacting at 1100C for 10 min. The qualitative monitoring of triacetylhydroxytyrosol obtained by the process of the invention was performed by comparison with the corresponding synthetic standard.
HPLC analysis - The quantitative and qualitative analysis of triacetyl- hydroxytyrosol was performed using an Agilent 1100 series liquid chromato- graph equipped with a DAD array, at 264 nm. A Nucleosil 100-5 C18 column (stainless steel 250 x 4 mm) was utilized, and elution was conducted in the isocratic mode at flow rate of 1.0 mL min"1 with acetonitrile and water (45:55, v:v) as mobile phase (tr=7.62 min). Sample volume of 20 μL was used for the injection.
The quantitative and qualitative analysis of hydroxytyrosol was performed by the same apparatus as above, at 280 nm, and with the same column, eluting at flow rate of 1.2 mL min"1 with acetonitrile (15%) and water (85%) containing 0.35% acetic acid as mobile phase (tr=3.13 min). HPLC analysis was conducted after every purification step. The quantitative analysis of hydroxytyrosol and triacetylhydroxytyrosol obtained from OMWW samples was performed elaborating their corresponding calibration curves (100-2000 μg/mL and 50-1000 μg/mL, respectively) using the corresponding synthetic standards. 1H-NMR and EI-MS analyses - Triacetylhydroxytyrosol obtained as pure compound from the OMWW samples was identified by comparison of its
1HNMR (400 MHz on a Bruker AC 400 spectrometer, using tetradeuterated methanol as solvent) and EI-MS (70 eV on a Fisons Trio 2000 spectrometer) data, and of its FAB-MS (Kratos MS 50, xenon as the bombarding atoms at 8kV acceleration potential) spectrum with those of the corresponding synthetic standards and those previously reported (Capasso, R. et al., 1999, loc. cit.). Production process
The method includes as a first step the continuous liquid-liquid extraction mode of a sample of OMWW, using a Soxhlet apparatus.
Liquid-liquid extraction of OMWW - OMWW samples of 50 ml_ at native pH (4.5) were extracted with 100 ml_ of ethyl acetate by refluxing for 8 hours in a continuous liquid-liquid Soxhlet extractor, obtaining an oil residue of 1102.0 mg. The amount of hydroxytyrosol was quantitatively monitored by HPLC analysis during the extraction process until a total recovery of 252.2 mg was obtained. Thus, the total phenol extracts showed a maximal amount of 1102.0 mg, containing 252.2 mg of hydroxytyrosol.
Production of triacetylhydroxytyrosol (1b)
As shown in the scheme of Figure 1 , the organic extracts were directly treated with perchloric acid supported on silica gel as acetylating cata- Iyst, and acetic anhydride as reagent, producing a mixture of compounds containing triacetylhydroxytyrosol, which appeared quantitatively acetylated.
Preparation of HCIOd-SiO? - The preparation of the HCIO4-SiO2 catalyst (perchloric acid adsorbed on silica gel) to perform the acetylation reaction with Ac2O (acetic anhydride) was carried out according to a modified proce- dure of Charkraborti and Gulhane (Chakraborti, A. K. et al. 2003, loc. cit.). HCIO4 (1.25 mmol, as a 65% aqueous solution) was added to a suspension of silica gel (2.37 g, 230-400 mesh) in 7.5 mL of diethyl ether. The mixture was kept for 3 h at room temperature under magnetic stirring and then it was heated at 700C for 28 h under vacuum to afford HCIO4-SiO2 as a free flowing powder.
Production of triacetylhvdroxytyrosol by treatment of OMWW extracts - A sample of 167 mg (containing 38.3 mg of hydroxytyrosol) of extracts ob-
tained by the soxhlet apparatus was acetylated using as catalyst the HCIO4- Siθ2 obtained as above (22.5 mg) and Ac2O (1 mL) in presence of diethyl ether (1mL) under magnetic stirring. The reaction, carried out at room temperature and monitored by TLC and HPLC, showed the complete formation of triacetylhydroxytyrosol after 5 min.
The reaction mixture was then washed with ethyl acetate, filtered under vacuum and evaporated under reduced pressure at 300C, leaving a residue of 262 mg containing triacetylhydroxytyrosol.
Purification of triacetylhvdroxytyrosol by MPLC - An aliquot of 260 mg of the product obtained as above was purified by medium-pressure liquid chromatography (MPLC) in order to afford the final product. The purification procedure was performed using a column of 36 mm (i.d.) x 460 mm packed with silica gel (Merck, Kieselgel 0.040 - 0.063 μm) and eluted under medium pressure (20 bar) using a Buchi 681 pump. Hexane/ethyl acetate 1 :1 (v:v) was used as eluent, and a flow rate of 2.5 mL/30 sec. (fraction volume collected was of 2.5 mL).
The homogeneous fractions with the same Rf at the TLC analysis were pooled and evaporated under reduced pressure at 30 0C. The pooled fractions monitored by TLC and quantitative HPLC showed 24.8 mg of pure triacetylhydroxytyrosol (35.6% yield), which was also identified by 1H NMR and EI-MS and FAB-MS as reported above.
Thus, the mixture obtained from the acetylation step was chroma- tographed by a single MPLC step giving pure triacetylhydroxytyrosol, resulting in an overall yield of 35.6%, related to the amount of hydroxytyrosol naturally contained in the organic extracts.
The high purity grade of the product obtained is indicated by the corresponding single and sharp peak which appears at tr=7.62 min. in the HPLC- chromatogram of the enclosed Figure 2. In addition, the 1H-NMR, EI-MS and FAB-MS data of this compound prove to be fully coincident with those re- ported previously for the corresponding synthetic sample. These data, as well as the data of the parent compound hydroxytyrosol, are summarized in detail in the following TABLE 1.
OO
* These data prove to be coincident with those reported in a previous paper for the corresponding synthetic compounds (Capasso R. et al., 1999, loc. cit.)
COMPARATIVE EXAMPLE 1 Production of hydroxytyrosol from olive oil mill waste
Pure hydroxytyrosol from the same OMWW source was also produced, according to the scheme of Figure 3, in order to homogeneously com- pare the data of its chemical antioxidant activity and protective effects with those of the triacetylated form according to the invention. Materials and methods
The materials and methods employed were the same as in Example 1. The synthetic standard corresponding to hydroxytyrosol was prepared ac- cording to the previously reported procedure (Capasso, R. et al., 1999, loc. cit.). The TLC analysis procedure was the same as in Example 1 , except for the eluting solution, which was acetone/petroleum ether 50:50 (v:v). Spectroscopic data (1H NMR and El MS) for hydroxytyrosol were also previously reported in the same literature. Production process
The method includes as a first step the continuous liquid-liquid extraction of a sample of OMWW, using a Soxhlet apparatus, according to the same procedure described in Example 1.
Purification of hydroxytyrosol from organic extracts of OMWW by MPLC - An aliquot of 155 mg (containing 35.5 mg of hydroxytyrosol) obtained by the soxhlet extraction was purified by MPLC in the same column of Example 1 , using as eluent hexane/ethyl acetate 15:85 (v:v) and a flow rate of 2.5 mL/30 sec. The homogeneous fractions with the same Rf of the synthetic standard hydroxytyrosol were pooled, leaving a residue of 14.6 mg of pure hydroxytyrosol (yield 41 %), identified by HPLC and by the spectroscopic data (1H NMR and EI-MS), as shown in Table 1.
Also in this case, the high purity of the hydroxytyrosol obtained is shown by the corresponding single and sharp peak which appears at tr=3.13 min. in the HPLC-chromatogram of the enclosed Figure 4.
EXPERIMENTAL RESULTS
Evaluation of antioxidant activity of triacetylhydroxytyrosol and
hydroxytyrosol
The chemical antioxidant ability of triacetylhydroxytyrosol was evaluated in comparison with hydroxytyrosol in the chemical ferric reducing/anti- oxidant power (FRAP) test, according to the method previously reported (Ben- zie, I. F. F., Strain, J.J., Ferric reducing/antioxidant power assay: Direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration, Methods Enzymology 1999, 299, 15-27). Such assay is a colori- metric method based on the reduction of a ferric-tripyridyltriazine complex to its ferrous form. Materials and methods involved in this test tests are mentioned under Example 1.
Increasing amounts of selected compounds were added to 1 ml of working solution, prepared by mixing 25 ml of 300 mM acetate buffer [pH 3.6], 2.5 ml TPTZ solution [10 mM TPTZ in 40 mM HCI] and 2.5 ml of 20 mM FeCI3-6H2O; after incubation for 6 min at room temperature the absorbance was read at 593 nm.
The data reported in Figure 5 clearly indicate a high antioxidant power of hydroxytyrosol (hdrx), which increases linearly, by increasing its concentration. As expected, triacetylhydroxytyrosol (triachdrx) is devoid of antioxidant power, even at high concentrations, because the orf/io-diphenol system of hydroxytyrosol is blocked by the acetyl groups and therefore totally inactive as hydrogen donor.
Biological test for the antioxidant activity of hydroxytyrosol and triacetylhydroxytyrosol in Caco-2 cells In order to assess the antioxidant effects of triacetylhydroxytyrosol in comparison with hydroxytyrosol in human cells, and, in particular, their protective effects against oxidative damages, Caco-2 cells (carcinoma colon cells, as model for epithelial intestinal cells) were used as a model system, according to the previously disclosed procedures (Manna, C. et al., The protective effect of the olive oil polyphenol (3,4-dihydroxyphenyl)ethanol counteracts reactive oxygen metabolite-induced cytotoxicity in Caco-2 cells. J. Nutr. 1997,
127, 286-292.41, 42).
Colon carcinoma cells (Caco-2 cell line) simulate epithelial intestinal cells. In particular, these cells, which are able to differentiate in culture, are considered a very suitable model to mimic the food-intestinal tract interactions in vitro. Reactive oxygen species (ROS) have been associated with several gastrointestinal injuries and may play a major role as mediators of inflammation. Therefore this cell line, amply used to examine a variety of intestinal functions, has also been used to investigate the injurious effects of ROS on the gastrointestinal tract. Materials and methods
In addition to materials and methods mentioned under Example 1 , high glucose Dulbecco's modified Eagle's medium (DMEM), fetal calf serum, non-essential amino acids, N-2-hydroxy-ethylpiperazine-N'-2-ethane sulfonic acid (HEPES), glutamine, penicillin, streptomycin and PBS tablets were pur- chased from Gibco, Life Science Technologies (S. Giuliano Milanese, Ml, Italy).
Results are reported as means ± SD; n =4. Student's West was routinely utilized. Experimental procedure Human colon carcinoma (Caco-2) cells were grown in DMEM, supplemented with 10% FCS, glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 U/ml), 1 % non-essential amino acids, in a humidified incubator with 5% CO2/95% air atmosphere. Cells were seeded at a density of 90,000 cells/cm2 in multiwell dishes and 12-14 days after confluence cells were treated with t- butyl hydroperoxide (f-BHP) to a final concentration of 4 mM. At the end of 6 hr incubation, both the cell viability and lipoperoxidation extent were evaluated as described below.
To assay the antioxidant protective effect on Caco-2 cells from oxidative injury, the cells were pre-treated for 30 min, in the presence or absence of increasing concentrations of each selected antioxidant, before the induction of oxidative stress.
Evaluation of Caco-2 cell viability (MTT assay) - The t-BHP-induced
cytotoxicity on Caco-2 cells was measured evaluating cell viability using 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. This colorimetric method is based on the reduction of the tetrazolium ring of MTT by mitochondrial dehydrogenases, yielding a blue formazan product, which can be measured spectrophotometrically; the amount of formazan produced is proportional to the number of viable cells. After the oxidative treatment , the medium was removed and MTT (5 mg/mL in DMEM without phenol red) was added to the wells (10 % final concentration). After 4 h incubation, the medium was aspired and the insoluble formazan produced was dissolved in isopropa- nol. The optical densities were measured at 570 nm.
Evaluation of Caco-2 lipoperoxidation - After the removal of the medium, cells were solubilized and lysates centrifuged at 12,00Og for 5 min; the obtained supernatants were finally assayed for lipoperoxidation products. Aliquots of supernatants were added to thiobarbituric acid (TBA) in 0.05 N NaOH (0.2% w/v final concentration) and heated in a boiling-water bath for 10 min. The absorbance of developed pink chromophore was determined at 532 nm, to evaluate the amount of thiobarbituric acid reactive substances (TBARS).
As shown in Figure 6A, the viability of the concerned cells is reduced to 62% by the treatment with 4 mM t-BHP, as assessed by MIT test. A remarkable protection against ROS-induced cytotoxicity was detected in the antioxidant-treated Caco-2 cells, the protective effect of triacetylhydroxytyrosol being of same order of magnitude as hydroxytyrosol in a micromolar concentration range. Membrane phospholipids are a major target of oxidative damage as the lipid peroxidation involves the cleavage of polyunsaturated fatty acids in their double bonds, leading to the formation of TBARS. A very significant protection of the Caco-2 cells by hydroxytyrosol and triacetylhydroxytyrosol is also observed against the lipoperoxidation of the membranes in the TBARS test. In fact, as shown in Figure 6B, a very significant reduction of TBARS pretreated with the two tested compounds is observed with respect to the sample treated with t-BHP. In particular, the oxidative treatment proves to be
of about three-fold increase in TBARS concentration compared with control cells; in this test also, pre-treatment of Caco-2 cells with either hydroxytyrosol or triacetylhydroxytyrosol significantly and dose-dependently decrease TBARS formation both at 50 and 100 μM, indicating that both the tested compounds equally protect against lipid peroxidation.
The finding that triacetylhydroxytyrosol shows a biological antioxidant activity very close to that of the parent compound strongly suggests that the triacetyl derivative is metabolized by cell esterase into hydroxytyrosol, which is the effective antioxidant compound, and, very probably, also in vivo at the intestinal level.
In order to rule out its artifact degradation, triacetylhydroxytyrosol was incubated in the same experimental conditions in the absence of cells. The results clearly indicated that such compound is not chemically degraded in the experimental conditions of the biological assays; moreover it also appeared stable in the simulated acidic and thermal conditions of the stomach.
The transport of triacetylhydroxytyrosol probably occurs, like hydroxytyrosol through the Caco-2 cells, by a passive diffusion and bidirection- ally according to the previous investigations (Manna, C. et al., Transport mechanism and metabolism of olive oil hydroxytyrosol in Caco-2 cells. FEBS Lett. 2000, 470, 341-344.), considering the acetyl derivative to be endowed with a protection power very close to hydroxytyrosol.
Biological tests for the antioxidant activity of hydroxytyrosol and triacetylhydroxytyrosol in human red blood cells
As a further test in order to elucidate the antioxidant effects of triace- tyl hydroxytyrosol in comparison with hydroxytyrosol in human cells against t- BHP-induced molecular alterations, red blood cells (RBC) were selected as a second experimental system. The experimental procedure is according to the previously published literature (Manna, C. et al, Olive oil hydroxytyrosol protects human erythrocytes against oxidative damages, J. Nutr. Biochem. 1999, 10, 159-165).
Human RBC have been amply used to study both oxidative stress- induced cytotoxicity as well as the protective effect played by antioxidants,
both in physiological and pathological conditions. These cells are characterized by a particularly high ROS production, deriving from the spontaneous autooxidation of hemoglobin. A very efficient antioxidant defense system rapidly removes these high reactive molecular species; however, if ROS are overproduced or the antioxidant defenses are impaired, severe oxidative alterations occur, eventually leading to hemolysis.
The materials and methods employed were the same as in Example 1 and in the previous biological test. Results are reported as means ± SD; n =4. Student's f-test was routinely utilized. Experimental procedure
The preparation of human red blood cells (RBC) was performed employing heparinized fresh human blood from healthy donors, as previously reported. The RBC suspensions (2% hematocrit) were treated with f-BHP (500 μM final concentration). After 2 h incubation, both the extent of hemolysis and lipoperoxidation were evaluated as described below. To assay the antioxidant protective effect on RBC from oxidative injury, the cells were pre-treated for 15 min, in the presence or absence of increasing concentrations of each selected antioxidant, before the induction of oxidative stress.
Evaluation of RBC hemolysis - The extent of hemolysis was measured spectrophotometrically, as previously described (Manna, C. et al., 1999, loc. cit). After the oxidative treatment, samples were centrifuged at 1 ,500 g for 10 min and the absorption (A) of the supernatant (S1 ) at 540 nm was measured. The precipitates (packed RBC) were then hemolyzed with 40 volumes of ice- cold distilled water and centrifuged at 1 ,500 g for 10 minutes. The supernatant (S2) was then added to S1 and absorption (B) of the combined supernatants (S1 +S2) was measured at 540 nm; percentage hemolysis was calculated from the ratio of the readings (A:B) x 100.
Evaluation of lipoperoxidation - After the oxidative treatment, RBC samples were mixed with trichloroacetic acid (10% w/v final concentration) and centrifuged at 5,000 g for 15 min. The obtained supernatants were finally assayed for lipoperoxidation products as reported above. As shown in Figure 7A, t-BHP induces a high increase of hemolysis with
respect to the control. On the contrary, a significant reduction of hemolysis is observable in all preliminary cells treated with hydroxytyrosol and triacetylhy- droxytyrosol, with the most effective concentration at 50 μM. Both these compounds are endowed with protective effects on the ROS-mediated cytotoxicity and at very close level.
The incubation of RBC with 500 μM of t-BHP significantly increases TBARS concentration, because of the oxidative stress (Figure 11B). Also in this case pretreatment of RBC with hydroxytyrosol and triacetylhydroxytyrosol, in amounts of 10 and 50 μM, causes a significant decrease of TBARS close to the control level, indicating a remarkable protection, especially at the higher concentration, of both compounds with the same order of magnitude.
These data confirm that the triacetyl form according to the invention is metabolized into the active form hydroxytyrosol by cellular esterase also in this cellular system, and suggest that it can be completely metabolized when injected intravenously.
The foregoing experimental results clearly show that triacetylhydroxytyrosol and hydroxytyrosol equally protect against oxidative damage, at micromolar concentrations. This finding is consistent with the conclusion that the acetylated derivative is metabolized to hydroxytyrosol either at the intesti- nal level or when intravenously injected, so that the chemically more stable triacetylated form is biochemically convertible in situ into its original active form, hydroxytyrosol, thus protecting more efficiently human cells against oxidative stress.
The present invention has been disclosed with particular reference to some specific embodiments thereof, but it should be understood that modifications and changes may be made by the persons skilled in the art without departing from the scope of the invention as defined in the appended claims.
Claims
1. A process for producing triacetylhydroxytyrosol from olive oil mill waste waters (OMVWV) including, in sequence, the following steps: a) treating OMVWV by liquid-liquid extraction with a polar organic solvent, to obtain an organic extract containing hydroxytyrosol; b) contacting the organic extract obtained from the step a) with a catalyst mixture of perchloric acid and silica and an acetyl donor compound, to obtain an acetylation reaction product containing triacetylated hy- droxytyrosol; c) purifying the reaction product obtained from the step b) by means of . medium-pressure liquid chromatography (MPLC), to obtain pure triacetylhydroxytyrosol.
2. A process according to claim 1 , wherein said liquid-liquid extraction of step a) is carried out by semi-continuous Soxhlet extraction, by continuous counter-current extraction or by discontinuous batch extraction.
3. A process according to claim 2, wherein said polar organic solvent of step a) is chosen from the group consisting of: ethyl acetate, methyl isobutyl ketone, methyl ethyl ketone, diethyl ether, methanol, n-butanol.
4. A process according to claim 1 , wherein the pH of the OMWW undergoing step a) is the native pH or it is adjusted in the range from pH 2.0 to pH 8.0.
5. A process according to claim 4, wherein said liquid-liquid extraction of step a) is carried out by Soxhlet extraction, the pH of the OMWW is 4.5, the polar organic solvent is ethyl acetate and the ratio solvent/OMWW is comprised between 1 :1 and 4:1.
6. A process according to claim 5, wherein said ratio solvent/OMWW is 2:1.
7. A process according to any one of claims 1-6, wherein said catalyst mixture of step b) is perchloric acid adsorbed on silica gel.
8. A process according to claim 7, wherein said acetyl donor compound of step b) is acetic anhydride.
9. A process according to claims 7 or 8, wherein said acetylation reaction of step b) is carried out at a temperature comprised between room temperature and 11O0C.
10. A process according to claim 9, wherein said acetylation reaction of step b) is carried out for a total reaction time of from 5 minutes to 24 hours.
11. A process according to any one of claims 1-10, wherein said purification step c) is carried out by MPLC on silica gel.
12. A process according to claim 11 , wherein said purification step c) is carried out eluting with ethyl acetate/petroleum ether 1 :1 (v/v).
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WO2009013596A3 (en) * | 2007-07-23 | 2009-04-09 | Probelte Pharma S A | Fortification of nutritional products with olive extracts containing hydroxytyrosol and hydroxytyrosol fortified nutritional products |
US9132104B2 (en) | 2007-07-23 | 2015-09-15 | Probelte Pharma S.A. | Fortification of nutritional products with olive extracts containing hydroxytyrosol and hydroxytyrosol fortified nutritional products |
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WO2013007850A1 (en) | 2011-07-08 | 2013-01-17 | Consejo Superior De Investigaciones Científicas (Csic) | Method for obtaining hydroxytyrosol extract, mixture of hydroxytyrosol and 3,4-dihydroxyphenylglycol extract, and hydroxytyrosyl acetate extract, from by-products of the olive tree, and the purification thereof |
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