ES2375867B1 - PROCEDURE FOR OBTAINING MASLINIC ACID AND OLEANOLIC ACID. - Google Patents

Abstract

Method for obtaining maslinic acid and oleanolic acid # The present invention relates to a process for obtaining maslinic acid and oleanolic acid from waste products from the milling of the olive, whereby high yields and a great yield are obtained purity of said compounds.

Description

Procedure for obtaining maslinic acid and oleanolic acid.

Object of the invention

The present invention relates to a process for obtaining maslinic acid and oleanolic acid, from waste products from the milling of the olive by means of which said compounds are obtained with high yields and high purity.

Background of the invention

The cultivation of the olive grove has great industrial importance especially in countries of the Mediterranean area. Its main use is the olive oil of which there are studies that support the benefits of olive oil in health, for its antioxidant and antithrombotic properties, among others.

Oleanolic (3-β-hydroxy-28-carboxylealeane) and maslinic (2-α-3-β-dihydroxy-28-carboxylealene) acids are triterpenic acids that are frequently found in the plant kingdom (http: //probe.nalusda .gov: 8300 / cgi-bin / browse / phytochemdb).

Among many other applications, it is known that maslinic acid inhibits serine proteins, has been proven effective against Cryptosporidium and in recent years the effects and selective capacity of maslinic acid as a compound that can inhibit apoptosis (programmed cell death) have been studied.

Where maslinic acid may be able to report greater economic benefits is in the cosmetic industry, due to its use to remove skin blemishes and wrinkles, fix collagen, inhibit melanoma formation and as an anti-cellulite (WO 2008116959).

On the other hand, oleanolic acid is a triterpenic acid that is found naturally in numerous plants (http://probe.nalusda.gov:8300/cgi-bin/browse/phytochemdb) among which the olive tree (olea is counted European) and the vine. It is one of the components of olive oil, olives and grapes.

This compound has pharmacological application in therapeutics as a vasodilator agent and a possible restorative function of endothelial function, as well as the elucidation of the mechanism of action of said effects. Oleanolic acid protects the vascular system against arteriosclerosis and its possible complications, such as heart attacks, through a mechanism similar to one that uses good cholesterol or HDL.

There are several references about its procurement processes. Among others, in 1969, Vázquez Roncero and Col., (Vázquez Roncero, A., Janerdel Valle, ML, Fats and Oils 1969, 20, 133-138) described the obtaining of triterpernic acids from the leaf and cuticle of the olive of the European wave.

The isolation of ursolic and oleanolic acids from the olive leaf has been described in patent GR1 001173.

Subsequently, Bianchi and Col (G. Bianchi, N. Pozzi, and G. Vlahov, Phytochemistry 1994, 37, 205-207; Eur. J. Lipid Sci. Technol., 2003, 105, 229-242) describe the isolation of oleanolic and maslinic acids from olive waxes by extractions with chloroform and methanol and subsequent transformation into their methyl esters and acetyl derivatives.

Already in 1996, the P9601652 patent describes the process for industrially obtaining these two acids, separately and in a high degree of purity, from solid by-products of the industrial milling of the olive.

The main drawback of this last procedure is that the raw material, "alperujo", has a high water content and certain remains of fatty materials that have to be eliminated increasing the number of stages in the extraction process.

Description of the invention

The present invention refers to a new method of obtaining oleanolic acid and maslinic acid from the skin of the olive by means of which these compounds are obtained in a simple and cheap way with high yields and purity.

The present invention has several advantages over existing procedures:

i) The raw material is very cheap and very easy to obtain by allowing the residue obtained in the oil mills that the olive bone contains.

ii) The olive skin thus obtained has a very low percentage of moisture which facilitates the process of obtaining.

iii) The process of obtaining oleanolic and maslinic acids from the skin of the olive is carried out by extractions in Soxhlet using reduced amounts of cheap solvents, hexane and ethyl acetate, which are largely recovered and can be reused.

iv) Oleanolic and maslinic acids are obtained from the skin of the olive with yields with yields greater than 10%.

v) Oleanolic and maslinic acids are obtained with a high degree of purity, 90.8% -91.5% and 82.2-84.4%, respectively.

vi) The quantities of dough used are considerably lower than the procedures described, which facilitates the scaling and lowering of the process.

A first aspect of the present invention relates to a method of maslinic acid and oleanolic acid comprising the following steps:

a) Separation of the skin from the olive from the milling residue of the olive that mostly contains the bone.

b) Making at least two consecutive extractions of the skin obtained in the previous stage.

c) Purification of the extracts obtained in the previous stage.

According to a preferred embodiment, the separation of the skin of the olive from the bone is carried out by enabling.

According to another preferred embodiment, two consecutive extractions are carried out by any type of extractor known in the state of the art. Preferably the extractions are carried out in Soxhlet.

According to another preferred embodiment, the extractions are carried out by an organic solvent.

According to another preferred embodiment, the first extraction is carried out with hexane.

According to another preferred embodiment, after the first extraction, it is allowed to cool and then concentrated in vacuo to reduce the initial volume by half and the solid that appears to be filtered, obtaining a solid product comprising maslinic acid and olenanolic acid in a proportion from 80 to 91% and from 9 to 20% respectively, even more preferably they are obtained at 90.8 and 9.2% respectively.

According to another preferred embodiment, the material remaining in the filter is distilled and hexane is recovered for subsequent cycles.

According to another preferred embodiment, the second extraction is carried out by ethyl acetate for at least 2 hours, preferably for 3 hours.

Once the extract is obtained, it is allowed to cool and a precipitate is obtained comprising a mixture of maslinic acid and oleanolic acid. Both acids are obtained in a proportion of 80 to 90% and 10 to 20% respectively. Preferably they are obtained in a proportion of 84.4% and 15.6% respectively.

According to another preferred embodiment, the mother liquor evaporates, recovering a large part of the ethyl acetate, and the concentrate is a solid comprising a mixture of maslinic acid and oleanolic acid. Said solid comprises a proportion of each of these acids of 40 to 60% and 40 to 60% respectively, preferably they are in a proportion of 54.5 and 45.5% respectively.

According to another preferred embodiment, this latter mixture can optionally be enriched in maslinic acid by crystallization of oleanolic acid using 96% ethanol as solvent. The crystallized solid once filtered comprises a mixture of oleanolic acid and maslinic acid in proportions from 90 to 95% and 5 to 10% respectively, preferably 91.5 and 8.5% respectively.

According to another preferred embodiment, the mother liquor is evaporated and the solid obtained is dissolved in hot ethyl acetate. It is allowed to cool and a solid is obtained which is filtered and comprising a mixture of maslinic acid and oleanolic acid in proportions from 80 to 90% and from 10 to 20% respectively, preferably 82.3% and 17.7% respectively. The evaporation in vacuo of the mother liquor allows to obtain a solid comprising a mixture of maslinic acid and oleanolic acid in proportions of 50 to 70% and 30 to 50% respectively, preferably 65.2% and 34.8% respectively.

According to a last preferred embodiment the purification of the obtained extracts is carried out by crystallization.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples are provided by way of illustration, and are not intended to be limiting of the present invention.

Examples

Example 1

Obtaining maslinic and oleanolic acid

The settling of 35 kg of olive bone obtained with a pulp-bone separator was carried out from the milling waste of the olive, obtaining approximately 700 g of olive skin that underwent two consecutive extractions in soxhlet.

In the first extraction the skin obtained in the previous stage was placed in a cartridge and the extraction with hexane (3 L) was carried out for two hours. It is then allowed to cool and is concentrated at approximately half volume and the solid that appears is filtered and dried, obtaining 4.8 g, which corresponds to a mixture of oleanolic acid (90.8%) and maslinic acid (9.2%).

The olive skin was subjected to a second extraction process using ethyl acetate as solvent (3 L) and three hours of extraction time. It is allowed to cool and the solid that precipitates is filtered and dried, obtaining 53 g, which corresponds to a mixture of maslinic acid (84.4%) and oleanolic acid (15.6%).

The mother liquors are then evaporated in vacuo to dryness and a solid weighing 16.8 g is obtained, and corresponding to a mixture of maslinic acid (54.5%) and oleanolic acid (45.5%). The 16.8 g of product are dissolved in hot 96% ethanol (250 mL) and when allowed to cool a solid appears that is filtered and dried. Are obtained

5.8 g corresponding to a mixture of oleanolic acid (91.5%) and maslinic acid (8.5%). The mother liquor from the crystallization in ethanol is evaporated in vacuo and the solid obtained is dissolved in hot ethyl acetate. When allowed to cool, a solid that is fi ltered and dried weighs 2g is obtained, which consists of a mixture of maslinic acid (82.3%) and oleanolic acid (17.7%). The filtrate is evaporated and a solid 9 g is obtained, which corresponds to a mixture of maslinic acid (65.2%) and oleanolic acid (34.8%).

After the extraction process, almost 2 liters of hexane and 3 liters of ethyl acetate used initially are recovered.

Claims (26)

1. Procedure for obtaining maslinic acid and oleanolic acid comprising the following steps:

to.

separate the bone and the skin from the olive from the milling residue of the olive that contains the bone;

b.

take the skin obtained in the previous stage and carry out at least two consecutive extractions with an organic solvent; Y

C.

Purify the extracts obtained in the previous stage.

2.

The method according to claim 1, wherein the separation of the bone and the skin from the olive is carried out by clamping.

3.

The method according to any of claims 1 or 2, wherein two consecutive extractions are carried out.

4. The method according to claim 3, wherein the extractions are carried out by soxhlet.

5.

The process according to any one of claims 1 to 4, wherein the first extraction is carried out with hexane.

6.

The process according to claim 5, wherein after the first extraction the product obtained is allowed to cool and then concentrated in vacuo to reduce the initial volume by half and the solid that appears to be filtered, obtaining a solid product comprising oleanolic and maslinic acid .

7.

The process according to claim 6, wherein the solid product obtained comprises oleanolic and maslinic acid in a proportion of 80 to 91% and 9 to 20% respectively.

8.

The process according to claim 7, wherein the solid product obtained consists of oleanolic and maslinic acid in proportions of 90.8 and 9.2% respectively.

9.

The process according to any of claims 1-4, wherein the second extraction is carried out in ethyl acetate.

10.

The process according to claim 9, wherein after the second extraction a precipitate comprising maslinic and oleanolic acid and mother liquors is obtained.

eleven.

The process according to claim 10, wherein the maslinic and oleanolic acids are in a proportion of 80 to 90% and 10 to 20% respectively.

12.

The process according to claim 11, wherein the maslinic and oleanic acids are obtained in 84.4% and 15.6% respectively.

13.

The process according to claim 10, wherein the mother liquor evaporates to obtain a solid comprising a mixture of maslinic acid and oleanolic acid.

14.

The process according to claim 13, wherein the maslinic acids and oleanolic acid are in a proportion of 40 to 60% and 40 to 60%, respectively.

15. The process according to claim 14, wherein the maslinic and oleanolic acid are obtained in a 54.5 and 45.5% respectively.

16.

The process according to any one of claims 13 to 15, wherein the acid mixture is enriched in maslinic acid by crystallization with ethanol.

17.

The process according to the preceding claim, wherein the crystallized solid is filtered and a mixture of oleanolic acid and maslinic acid is obtained in proportions from 90 to 95% and 5 to 10% respectively and mother liquors.

18.

The process according to the preceding claim wherein the mixture comprises oleanolic acid and maslinic acid in 91.5 and 8.5% respectively.

19.

The process according to claim 17, wherein the mother liquor evaporates, obtaining a solid that dissolves in hot ethyl acetate, is allowed to cool and a solid which is filtered and comprising a mixture is obtained.

of maslinic acid and oleanolic acid in proportions from 80 to 90% and from 10 to 20% respectively and mother liquors.

twenty.

The process according to the preceding claim, wherein the acids are 82.3% and 17.7% respectively.

twenty-one.

The process according to claim 19, wherein the mother liquor evaporates and a solid is obtained comprising a mixture of maslinic acid and oleanolic acid in proportions of 50 to 70% and 30 to 50% respectively.

22

The process according to the preceding claim wherein the acids are 65.2% and 34.8% respectively.

2. 3.

The method according to any one of the preceding claims, wherein the purification is carried out by crystallization.

24.

The process according to any of claims 5 to 8 wherein the hexane used is recovered by distilling the material that remains in the filter.

SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 201001077 SPAIN Date of submission of the application: 06.08.2010 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: See Additional Sheet RELEVANT DOCUMENTS

Category

56 Documents cited Claims Affected

X

VAZQUEZ RONCERO, A., JANER, M.L. Triterpenic acids from the olive tree. Fats and Oils, 1969. Vol 20, pages 133-138. Experimental part, sections 1 and 2. 1-24

X

EN 2111498 A1 (UNIVERSITY OF GRANADA) 01.03.1998, the whole document. 1-12.24

TO

BIANCHI, G., POZZI, N., VLAHOV, G. Pentacyclic Triterpene Acids in olives. Phytochemistry, 1994. Vol. 37, No. 1, pages 205-207. 1-24

TO

CAPUTO, R., MANGONI, L., MONACO, P. et al. New Triterpenes from the leaves of Olea Europaea. Phytochemistry 1974 Vol 13, pages 2825-2827. 1-24

TO

ES 2076899 A1 (FUENTES CARDONA, S.A) 01.11.1995, columns 2.3. 1.2

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 16.01.2012

Examiner A. Barrios de la Fuente Page 1/5

REPORT OF THE STATE OF THE TECHNIQUE CLASSIFICATION OBJECT OF THE APPLICATION C11B13 / 00 (2006.01) C07C62 / 32 (2006.01) B01D11 / 00 (2006.01) Minimum documentation sought (classification system followed by classification symbols) C11B, C07C, B01D Electronic databases consulted during the search (name of the database and, if possible, terms of search used) EPODOC, INVENES, WPI, TXTE, MEDLINE, NPL, XPESP, BIOSIS, EMBASE  WRITTEN OPINION Date of Completion of Written Opinion: 17.01.2012 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims Claims 1-24 IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims Claims 1-24 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published.  Considerations: WRITTEN OPINION 1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

VAZQUEZ RONCERO, A., JANER, M.L. Triterpenic acids from the olive tree. Fats and Oils, 1969. Vol 20, pages 133-138. Experimental part, sections 1 and 2. 1969

D02

ES 2111498 A1 (UNIVERSITY OF GRANADA) 01.03.1998

D03

BIANCHI, G., POZZI, N., VLAHOV, G. Pentacyclic Triterpene Acids in olives. Phytochemistry, 1994. Vol. 37, No. 1, pages 205-207. 1994

D04

CAPUTO, R., MANGONI, L., MONACO, P. et al. New Triterpenes from the leaves of Olea Europaea. Phytochemistry, 1974 Vol 13, pages 2825-2827. 1974

D05

ES 2076899 A1 (FUENTES CARDONA, S.A) 01.11.1995

2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The present application has as its object a procedure for obtaining maslinic acid and oleanolic acid which comprises the steps of separating the bone and the skin from the olive from the milling residue of the olive containing the bone, taking the skin obtained in the previous step for carrying out at least two consecutive extractions with an organic solvent and purifying the extracts obtained in the previous stage (Claim 1 and following). Document D01 discloses a procedure for obtaining maslinic and oleanolic acid from olives and olive leaves. Document D02 discloses a process for obtaining maslinic acid and oleanolic acid from the milling residue of the previously dried olive and containing the skin, pulp and bone. Document D03 discloses a procedure for obtaining maslinic and oleanolic acid from the waxes of the surface of the olive, in which the olives are washed with chloroform and subsequently the extraction with methanol is carried out. Document D04 discloses a process for obtaining oleanolic and maslinic acid from olive leaves. The leaves are dried and subjected to an extraction process with hexane and acetone. The extracts are purified by chromatography and crystallization with methanol. Document D05 discloses a method of treatment of pomace from the extraction of olive oil.  NEW (Art. 6 Law 11/86) Claims 1-24 are new, within the meaning of article 6 of Patent Law 11/86.  INVENTIVE ACTIVITY (Art. 8 Law 11/86) Document D01 is considered the state of the art document closest to the object of the present application. It discloses a procedure for obtaining maslinic and oleanolic acid from different extracts, such as olive leaves, seeds, pulp or olive skin. In this procedure, two extractions with organic solvents of the dry and crushed material are carried out and subsequently, said extracts are purified. The extractions, which are carried out by means of a soxhlet extractor, are first carried out with hexane and then with ethyl ether. Purification of the extracts comprises crystallization with ethanol, and subsequent treatment with ethyl acetate of the mother liquor enriched with maslinic acid as a result of crystallization with ethanol.  WRITTEN OPINION The main difference between the procedure object of the present application and the one disclosed in D01, is that the starting material used is the skin of the olive that takes advantage of the milling residue and that is separated by a process of setting or setting, while that in D01 it is indicated that an extract of the olive skin is made but it is not indicated how it is obtained. It is common in the industry to take advantage of the milling waste of the olive for different purposes, among which is the obtaining of maslinic and oleanolic acid from the dried milling residue that contains the skin, pulp and bone of the olive (see D02). Taking into account that the fact that maslinic and oleanolic acid are concentrated in the skin of the olive is well known, it would be an obvious option to use as starting material to obtain these acids, only the skin of the olive instead of the milling residue that also contains other remains, and thus achieve a simplification and greater yield of the process of obtaining. On the other hand, devices (aventadores) are known in the state of the art that, by means of air currents, allow different components to be separated according to their weight, for example, the use of aventadores to separate the pulp from the bone of the olive from the dried pomace (see D05). Taking into account the principles on which this separation process is based and even more, taking into account that it is not indicated that it has been necessary to overcome any technical difficulty to be able to separate only the skin by applying this method, it is considered that the use of the process As a method of separating the skin from the olive, it would represent an obvious option for the person skilled in the art. On the other hand, the process object of the present application also differs from that disclosed in D01 in that the second solvent used for extraction is not ethyl acetate but ethyl ether. However, the use of ethyl acetate as a solvent to carry out the extraction represents an option already known in the state of the art (see D02) Therefore, and on the basis of what is disclosed in D01, it is considered that the process for obtaining maslinic and oleanolic acid, object of claims 1-24, would not imply inventive activity for one skilled in the art. Document D02 discloses a process for obtaining maslinic acid and oleanolic acid from the milling residue of the previously dried olive and containing the skin, pulp and bone. This process comprises the extraction steps first with hexane and then with ethyl acetate and then the purification of the extracts. On the basis of the foregoing and in the light of what is disclosed in D02 it is considered that the process object of claims 1-12,24, would not imply inventive activity for a person skilled in the art. In conclusion, it is considered that the object of claims 1-24 would not imply inventive activity for a person skilled in the art within the meaning of article 8 of Patent Law 11/86.