WO2005075614A1

WO2005075614A1 - Method of obtaining high-value-added compounds from olive leaves

Info

Publication number: WO2005075614A1
Application number: PCT/ES2005/070013
Authority: WO
Inventors: J. Javier TABERA GALVÁN; Alejandro RUIZ RODRÍGUEZ; Francisco Javier SEÑORÁNS RODRÍGUEZ; Elena IBÁÑEZ EZEQUIEL; Guillermo J. Reglero Rada; Tomás ALBI VIRELLA; Augusto LANZÓN REY; Mª Carmen PÉREZ CAMINO; Mª Ángeles GUINDA GARÍN; Mirella Rada Rada
Original assignee: Consejo Superior De Investigaciones Científicas; Universidad Autonoma De Madrid
Priority date: 2004-02-09
Filing date: 2005-02-08
Publication date: 2005-08-18
Also published as: ES2238183A1; ES2238183B1

Abstract

The invention relates to a method of obtaining high-value-added compounds from olive leaves. The inventive method comprises the following steps, namely: a first step in which the leaves are subjected to solid-liquid extraction with organic solvents, preferably hexane or ethanol, and the crude extract thus obtained is vacuum concentrated; and a second step in which the crude extract is fractionated by means of supercritical CO2 countercurrent column extraction and separation into two cells with different fixed pressure and/or temperature conditions which alter the dissolving power of the CO2, thereby precipitating different compounds. The invention can be used to extract natural products of interest from olive leaves for the food, pharmaceutical and cosmetic industries, such as waxes, squalene, β-carotene, α-tocopherol, oleuropein, hydroxytyrosol and other phenolic compounds, β-sitosterol, α- and β-amyrin, erythrodiol, uvaol and other terpenic alcohols, oleanolic acid, ursolic acid and maslinic acid, among others.

Description

TITLE

PROCEDURE FOR OBTAINING HIGH VALUE COMPOUNDS ADDED FROM OLIVE LEAF.

OBJECT OF THE INVENTION

The object of the present invention is a process for obtaining compounds of high added value from olive leaf by solid-liquid extraction with solvents and subsequent fractionation with CO ₂ in supercritical state. In a first stage, the sheets are subjected to solid-liquid extraction with organic solvents, preferably hexane or ethanol, and the crude extract obtained is concentrated in vacuo. In a second stage, a fractioning of this crude extract is carried out by countercurrent extraction in a column with supercritical CO ₂ and separation in two cells in which different pressure and / or temperature conditions that change the solvent power of CO _{2 are set} , precipitating different compounds. The process of the invention allows natural products of interest to the food, pharmaceutical and cosmetic industries to be extracted from the olive leaf, such as waxes, squalene, β-carotene, α-tocopherol, oleuropein, hydroxytyrosol and other phenolic compounds, β-sitosterol, α and β-amirin, erythrodiol, uvaol and other terpenic alcohols, oleanolic acid, ursolic acid and maslinic acid, among others.

STATE OF THE TECHNIQUE

The present invention is related to obtaining natural compounds of high added value from olive leaf, by-product of the olive grove that enters the oil mills in considerable quantities (about 10% on the weight of the olive) and of which currently poor profitability is obtained. The compounds obtained are, among others: waxes, squalene, β-carotene, α-tocopherol, oleuropein, hydroxytyrosol and other phenolic compounds, β-sitosterol, α and β-amirin, erythrodiol, uvaol and other terpenic alcohols, oleanolic acid, acid Ursolic and maslinic acid. Some of them (squalene, β-carotene, α-tocopherol, hydroxytyrosol and other phenolic compounds) have antioxidant activity, so they find application both in prolonging the shelf life of food and in reducing oxidative stress in the human organism . For this second purpose they can be used as functional food ingredients or drugs. They are also used in pharmacy, parapharmacy and cosmetics in topical preparations protective and regenerating the skin. As a consequence of the decrease in oxidative stress in the organism, these compounds have been described as anti-thromatous (the oxidation of LDL starts the formation of atheroma plaques), anti-cancer (some free radicals can be mutagenic) and protective against cataracts (aggregation of proteins in the lens is closely related to oxidative phenomena). Oleanolic acid and β-sitosterol also have antitumor effects, which, in turn, reduces the level of LDL cholesterol in the blood. Other pharmacological activities described are anti-inflammatory (oleanolic acid, ursolic acid, maslinic acid, erythrodiol and uvaol) and bactericide (squalene, oleuropein and oleanolic acid). Ursolic acid is also used in cosmetic preparations against skin wrinkles. Currently, maslinic acid is studied as an inhibitor of proteases responsible for the infective power of HIV (ES2140329) and intestinal parasites of the genus Cryptosporidium (ES2131467). Infection with these protozoa is high risk in immunosuppressed patients and for her there is no specific therapy.

The interest in the compounds of high added value present in the olive leaf has given rise to numerous technological developments for their extraction and purification, many of them aimed at the use of their properties in the food and health field. Some of these developments (US6338865, EP1157701, FR2588159, ES2056745) are proposed to improve food preservation, mainly through the antioxidant activity of the extracted compounds, as well as some functional properties that are beneficial to health after ingestion of those food or ingredients Others (US2003185921, US5714150) address the isolation and characterization of the extracted compounds and the in vitro evaluation of their various pharmacological activities. Finally, most (DE10213032, DE10213019, DE10213031, US2003152656, US2002054927, FR2810887, FR2830195, EP1213025, FR2815852, DE3901286) use the compounds extracted from the leaf as active principles of cosmetic or dermatological formulations for skin and hair care , ranging from its use as deodorant or anti-dandruff products to photoprotection or acne treatment.

In the extraction procedures, the sheet is usually dried and / or ground (US2003152656, US2002054927, FR2810887, EP1157701), extracted with an organic solvent (US2003185921, US2003152656, FR2810887, EP1157701), with water (US2002054927, ES2056745), or with ethanol-water mixture (US5714150); In some cases, the Soxhlet extractor (DE10213032, DE10213019, DE10213031, EP1213025) is used. The extract obtained is usually concentrated or dried by evaporation or lyophilization (US2003152656, US2002054927, FR2810887, EP1157701, US5714150, ES2056745). The extract obtained by any of the above or similar procedures can be concentrated and fractionated by extraction with CO ₂ in supercritical state (P> 7.3 MPa and T> 31.1 ° C), in which it has solvation properties similar to those of liquids and viscosities and diffusivities similar to those of gases, making it an ideal extractant. The extraction capacity of supercritical carbon dioxide can be made selective towards a certain group of compounds by modifying their density, which can be achieved in the same installation with slight adjustments of pressure and temperature. In addition, supercritical CO ₂ is not toxic, it is released from products extracted by simple passage to the gaseous state, and produces effective extractions at mild temperatures and, obviously, in a non-oxidizing atmosphere. No application of supercritical fluid extraction has been found to concentrate, fractionate and / or purify the compounds extracted from the olive leaf. There is a precedent (GB644917) in which CO ₂ is used to create an inert protective atmosphere against oxidation of compounds extracted by solvents of different vegetable and animal oils; but, unlike the present invention, it is used as a gaseous state and at low pressure, so it has no extractive effect.

BRIEF DESCRIPTION OF THE INVENTION

The present invention involves a process for the extraction and fractionation of high added value compounds present in the olive leaf, applicable in the food, pharmaceutical and cosmetic industries.

The process comprises a solid-liquid extraction with organic solvents, preferably hexane or ethanol, from the olive leaf, an extraction with supercritical CC ^ of the concentrated crude extract above and a fractionation of the latter extract. Washed olive leaf is used if it comes from pruning the olive or unwashed if it comes from the separation of olives from the mill. When hexane is to be used, the olive leaf is dried by forced air at a temperature between 30 and 40 ° C for at least 24 hours and chopped in a knife chopper.

When ethanol is to be used, the olive leaf is extracted whole and without drying. For solid-liquid extraction the leaves are extracted by prolonged contact (maceration) with the organic solvent, hexane or ethanol, in a leaf / solvent ratio between 1/4 - 1/20 weight / volume and at a temperature between 20 ° C and 40 ° C. If the solvent is hexane, the maceration takes place during a period between 30 and 60 hours. If the solvent is ethanol, the maceration takes place during a period between 100 and 160 hours.

Then the clarification is carried out, by filtration or centrifugation, of the extract and then at the concentration of the clarified by evaporation under vacuum of the solvent (T <40 ° C), until a volume of crude extract between 20% and 50% of the initial maceration volume. The crude extract thus obtained is extracted in countercurrent with supercritical CO2 in a filled column in a temperature range between 35 and 50 ° C and in a pressure range between 15 and 35 Mpa obtaining a supercritical extract in which there is lower concentration of organic solvent than in the crude extract. The effluent of this column, supercritical extract, is taken to a first separation cell where, by lowering the pressure to values below 20 MPa, preferably between 10 and 15 Mpa, and / or changing the temperature, the solvent power of CO ₂ , precipitating the non-soluble compounds in these new conditions. The effluent from this first cell is taken to a second separation cell, where the pressure is again reduced to values lower than 2 MPa, preferably between 1 and 2 MPa, bringing the carbon dioxide to the gaseous state and producing precipitation of all the compounds that were dissolved in it. At the end of the process the two fractions, of different composition, obtained in the two cells are collected. A third fraction (refined), consisting mainly of the organic solvent and compounds not soluble in supercritical CO ₂ , is collected from the extraction column. DETAILED DESCRIPTION OF THE INVENTION

The process object of the present invention consists in the extraction of the olive leaf with organic solvents, preferably hexane or ethanol. The leaves, obtained from the pruning of the olive tree or separated from the olives in the oil mills, are washed or not, depending on their origin, they are dried with forced air at 30-40 ° C and chopped with a knife chopper if they are to be extracted with solvents such as hexane or extracted whole and without drying with solvents such as ethanol. The extraction is carried out by prolonged contact (maceration) for a time between 30 and 60 hours if hexane is used or between 100 and 160 hours if ethanol is used, in a leaf / solvent ratio between 1 / 4-1 / 20 ( weight / volume) and at a temperature between 20 and 40 ° C. The extract is then concentrated under vacuum (10-20 mm pressure), keeping the temperature below 40 ° C, until a volume between 20 and 20 is achieved. % and 50% of the initial. Finally it is clarified by filtration with filter plate or centrifugation. The crude extract obtained as described above is fed to a column filled with extraction, capable of withstanding pressures greater than 40 MPa, traveled in the opposite direction by carbon dioxide in a supercritical state, preferably at a pressure between 15 and 35 MPa and temperature between 35 and 50 ° C, in an amount (measured in atmospheric conditions) between 5 and 40 times the raw extract feed flow rate, preferably 12 times. When interested, you can be promoted the solubility of the polar compounds by adding to the CO ₂ stream a small proportion of a polar solvent (modifier or cosolvent). When the extraction is carried out with hexane, a suitable modifier is ethanol that would be added in a proportion of up to 10% over the flow of CO ₂ , while when the extraction is carried out with ethanol, the appropriate modifier is water that is added also in proportion of up to 10% over the flow of CO ₂ . The supercritical CO ₂ containing the soluble compounds under the conditions of the extraction column passes to a first separating cell in which, by means of reductions in pressure and / or variations in temperature, the solvent power of CO ₂ is reduced, so that they precipitate preferably a first group of compounds, insoluble in the new conditions. The color of this first fraction is usually yellowish-green. In this first separating cell the pressure of carbon dioxide is between 10 and 15 MPa. Next, the carbon dioxide, containing the compounds still soluble under the pressure and temperature conditions of the first separating cell, is taken to a second cell in which, by means of a new pressure decrease, it enters the gaseous state, all precipitating the compounds it brought dissolved. In this second separating cell the pressure of carbon dioxide is between 1 and 2 MPa. This second fraction is usually colorless or pale yellow. The third fraction consists of refining, the crude extract already extracted with supercritical CO ₂ that remains in the extraction column. It is collected by gravity, by its base, once the system is depressurized. It is enriched in compounds less soluble in carbon dioxide at the density of the latter in the extraction column and usually has a brown-greenish color, darker than the crude extract.

EXAMPLES OF EMBODIMENT OF THE INVENTION Example 1: Extraction with hexane

It starts from 1000 g of olive leaves, which are washed with water and dried with air between 30-40 ° C for 6 hours, chopped and extracted for 48 hours with 5 liters of hexane. The maceration is filtered on a vacuum filter plate and concentrated in vacuo to 1 liter at a temperature below 40 ° C. The crude hexane extract thus obtained, containing 962.4 μg / g of α-tocopherol, 123.5 μg / g of β-sitosterol and 429.0 μg / g of terpenic alcohols, is fed at a rate of 200 mL / ha the column (stainless steel AISI 316, cylindrical, 17.6 mm internal diameter and 180 cm high), filled with Fenske rings, and extracted in countercurrent with 2500 mL / h of CO ₂ at 20 MPa and 35 ° C for 35 minutes, with the addition of ethanol as a modifier (10% over the flow rate of CO ₂ ). The extract obtained is partially separated in the first separating cell (stainless steel AISI 316, cylindrical, 270 mL capacity) reducing the pressure to 12 MPa, obtaining in it a greenish yellow product containing 105.6 μg / g of α- tocopherol, 57.6 μg / g of β-sitosterol and 103.3 μg / g of terpenic alcohols. The effluent of this first is taken to the second separating cell (stainless steel AISI 316, cylindrical, 270 mL capacity) where, by lowering the pressure up to 1 MPa, the compounds dissolved in the carbon dioxide are recovered, now in a gaseous state, obtaining a pale yellow product containing 80.4 μg / g of α- tocopherol, 474.7 μg / g of β-sitosterol and 1661.6 μg / g of triterpenic dialcohols. Both β-sitosterol and terpenic alcohols are concentrated in the second separator cell, while α-tocopherol is, for the most part, refined.

Example 2: Extraction with ethanol

It starts from 1000 g of olive leaves and is extracted for 140 hours with 20 liters of 96% ethanol. The macerate is filtered on a vacuum filter plate and decolorized with 20 g of powdered active carbon (CLARIMEX type 061C), filtered through MN 616 filter paper (Machery-Nagel). The solution is concentrated in vacuo to 20% of the original volume, at a temperature below 40 ° C, producing a precipitate (mostly oleanolic acid) that is separated by centrifugation or filtration The crude extract in ethanol thus obtained, containing among other compounds 78.6 μg / g of hydroxytyrosol, 140.2 μg / g of erythrodiol and 70.0 μg / g uvaol are fed at a rate of 150 mL / ha column (stainless steel AISI 316, cylindrical, 17.6 mm internal diameter and 180 cm high), filled with Fenske rings, and extracted in countercurrent with 2500 mL / h of CO ₂ at 25 MPa and 35 ° C for 120 minutes, with the addition of water as a modifier (5% on CO ₂ flow). The extract obtained is partially separated in the first separating cell (stainless steel AISI 316, cylindrical, 270 mL capacity) reducing the pressure to 10 MPa, obtaining a yellowish product containing 34.8 μg / g of hydroxytyrosol, 320 , 2 μg / g of erythrodiol and 240.0 μg / g uvaol. The effluent of this first is taken to the second separating cell (stainless steel AISI 316, cylindrical, 270 mL capacity) where, by lowering the pressure up to 2 MPa, the compounds dissolved in the carbon dioxide are recovered, now in a gaseous state, obtaining a pale yellow product containing 29.0 μg / g of hydroxytyrosol, 70.2 μg / g of erythrodiol and 50.6 μg / g uvaol. Both erythrodiol and uvaol are concentrated in the first separating cell, while hydroxytyrosol is, for the most part, refined (284.2 μg / g).

Claims

1.- Procedure for obtaining high added value compounds from olive leaf by solid-liquid extraction with organic solvents and subsequent fractionation with CO ₂ in a supercritical state characterized in that it comprises the following steps: a) obtaining a solid-liquid extract of fresh olive leaves by mashing in organic solvents, preferably hexane or ethanol; b) filtration or centrifugation of the macerate obtained in the previous stage to clarify it; c) concentration of the clarified from the previous stage to obtain a crude olive leaf extract; d) passage of carbon dioxide under supercritical conditions through a column filled with supercritical extraction in countercurrent with the raw extract fed thereto; e) conduction of the effluent formed, consisting mostly of some of the compounds of interest dissolved in carbon dioxide, to a first separation cell where the less soluble compounds precipitate in greater quantity; f) conduction of the effluent from the first separation cell to a second one in which the carbon dioxide is transformed into gas, precipitating the rest of the compounds dissolved in it; g) collection of the precipitated compounds in the two previous stages and the refining that remains in the column, whereby three fractions of the crude extract are achieved.

2. Method for obtaining compounds of high added value from olive leaf according to claim 1, characterized in that washed olive leaf is used if it comes from pruning the olive or unwashed if it comes from the separation of olives from the oil press.

3. Method for obtaining compounds of high added value from olive leaf according to claims 1 and 2, characterized in that when hexane is used, the olive leaf is dried by forced air at a temperature between 30 and 40 ° C for at least 24 hours and chopped in a knife chopper.

4. Process for obtaining compounds of high added value from olive leaf according to claims 1 and 2, characterized in that when ethanol is used, the olive leaf is extracted whole and without drying.

5. Process for obtaining compounds of high added value from olive leaf according to claims 1-4, characterized in that a ratio of sheet / solvent comprised between 1/4 -1/20 weight / volume is used.

6. Process for obtaining compounds of high added value from olive leaf according to claims 1, 2, 3 and 5 characterized in that if the solvent is hexane the maceration takes place during a period between 30 and 60 hours.

7. Method for obtaining compounds of high added value from olive leaf according to claims 1, 2, 4 and 5 characterized in that if the solvent is ethanol the maceration takes place during a period between 100 and 160 hours.

8. Method for obtaining compounds of high added value from olive leaf according to claims 1-7, characterized in that a maceration temperature between 20 ° C and 40 ° C is used.

9. Process for obtaining compounds of high added value from olive leaf according to claims 1-8, characterized in that the filtrate is concentrated, by evaporation under vacuum of the solvent (T <40 ° C), until a volume of Gross extract comprised between 20% and 50% of the initial maceration volume.

10. Process for obtaining compounds of high added value from olive leaf according to claims 1-9, characterized in that the extraction operation Supercritical countercurrent of the crude extract is performed in a temperature range between 35 and 50 ° C.

11. Method for obtaining compounds of high added value from olive leaf according to claims 1-10, characterized in that the operation of supercritical extraction in countercurrent of the crude extract is carried out in a pressure range between 15 and 35 MPa.

12. Method for obtaining compounds of high added value from olive leaf according to claims 1-11, characterized in that the fractionation operation in the first separating cell of the compounds of interest present in the CO ₂ stream is carried out by pressure decrease to values below 20 MPa, preferably between 10 and 15 MPa.

13. Method for obtaining compounds of high added value from olive leaf according to claims 1-12, characterized in that the fractionation operation in the second separating cell of the compounds of interest present in the CO ₂ stream is carried out by pressure decrease to values below 2 MPa, preferably between 1 and 2 MPa.