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Title:
PROCESS FOR THE EXTRACTION AND PURIFICATION OF LONG-CHAIN BI-FUNCTIONAL SUBERIN ACIDS FROM CORK
Document Type and Number:
WIPO Patent Application WO/2014/092591
Kind Code:
A1
Abstract:
The present application describes a multi-step process for the isolation and purification of some of the main suberin acids from cork, based in solvent-fractionation and chromatographic techniques, affording these compounds in very-high purity, in excess of 99.5 %. Cork is first extracted with solvents and suberin is then depolymerised. The ensuing suberin acids mixtures are then partitioned using differential temperature and solvent-solubility precipitation. The fractions enriched in the different suberin acids thus obtained, are sequentially treated by medium-pressure normal phase chromatography (MPLC) and high-performance reversed-phase chromatography (HPLC), leading to individual suberin acid purities approaching 100 % purity. The process of the present invention is useful for several industrial applications to provide compounds which may be applied to pharmaceuticals, cosmetics, surfactants, engineering polymers, and bio-mimetic membranes.

Inventors:
SANTOS, Sara C. P. G. R. (Av. do Uruguai, 36 2B, -615 Lisboa, P-1500, PT)
GRAÇA, José A. R. (Rua do Borja, 103 3F, -046 Lisboa, P-1350, PT)
Application Number:
PT2012/000049
Publication Date:
June 19, 2014
Filing Date:
December 12, 2012
Export Citation:
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Assignee:
INSTITUTO SUPERIOR DE AGRONOMIA (Tapada da Ajuda, -017 Lisboa, P-1349, PT)
International Classes:
B27J5/00; C07C51/42; C08L97/00; C11B3/06; C11C1/00
Domestic Patent References:
WO2010093320A12010-08-19
WO2010093320A12010-08-19
Foreign References:
US20020043577A12002-04-18
RU2175326C12001-10-27
US20090182158A12009-07-16
EP0711746A11996-05-15
US2617814A1952-11-11
PT101683A1996-11-29
RU2119503C11998-09-27
US20090182158A12009-07-16
US20050158414A12005-07-21
US20030109727A12003-06-12
Other References:
CORDEIRO N ET AL: "CORK SUBERIN AS A NEW SOURCE OF CHEMICALS", INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, ELSEVIER BV, NL, vol. 22, 1 January 1998 (1998-01-01), pages 71 - 80, XP002479577, ISSN: 0141-8130, DOI: 10.1016/S0141-8130(97)00090-1
P. HÄRMÄLÄ, 1 January 1992 (1992-01-01), XP055052852, Retrieved from the Internet [retrieved on 20130208]
GRAGA, J.; SANTOS, S.: "Suberin: A Biopolyester of Plants' Skin", MACROMOLECULAR BIOSCIENCE, vol. 7, 2007, pages 128 - 135, XP055231045, DOI: doi:10.1126/science.208.4447.990
ABE, A.; SUGIYAMA, K.: "Growth inhibition and apoptosis induction of human melanoma cells by omega-hydroxy fatty acids", ANTICANCER DRUGS, vol. 16, 2005, pages 543 - 549
METZGER, J.: "Fats and oils as renewable feedstocks for chemistry", EUR. J. LIPID SCI. TECHNOL., vol. 111, 2009, pages 865 - 876
HUF, S. ET AL.: "Biotechnological synthesis of long-chain dicarboxylic acids as building blocks for polymers", EUR. J. LIPID SCI. TECHNOL., vol. 113, 2011, pages 548 - 561
Attorney, Agent or Firm:
FERREIRA PINTO, Francisca (Av. da República, 25 - 1º, -186 Lisboa, P-1050, PT)
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Claims:
CLAIMS

1. Process for the extraction and purification of long-chain bi- functional suberin acids from cork comprising: a) an extraction step by sequential extraction with solvents having increasing polarity, and filtration and concentration of the resulting extractives; b) a depolymerisation step by methanolysis or by alkaline hydrolysis; c) an isolation step by solvent fractionation with at least one non-polar solvent and at least one polar solvent; and d) a purification step by sequential treatment of the extracts obtained in c) with medium-pressure normal phase chromatography (MPLC) and high-performance reversed-phase chromatography (HPLC) techniques.

2. A process, according to claim 1, wherein the extraction of step a) is performed with at least 3 solvents with increasing polarity, with dielectric constants respectively from 1.5 to 5.0 relative to a non-polar solvent, 5.0 to 50.0 relative to a polar solvent and above 50 relative to a highly polar solvent .

3. A process, according to claim 1, wherein in step b) the depolymerization by methanolysis comprises contacting the extractive-free cork obtained in a) with a solution of a strong base in methanol, at pH > 10 followed by acidification to pH < 6.

1

4. A process, according to claim 3, wherein the strong base comprises a methoxide of an alkali metal.

5. A process, according to claim 1, wherein in step b) the depolymerization by alkaline hydrolysis comprises contacting the extractive-free cork obtained in a) with a solution of a strong, base in ethanol/water 0.1' to 3.0 M at pH > 10 and a temperature close to the solvent mixture boiling point followed by acidification to pH < 6.

6. A process, according to any of the claims 3 - 5, wherein the temperature of the depolymerization step is between 50-80°C.

7. A process, according to claim 1, wherein the isolation step c) of the depolymerised extract obtained in b) is contacted with a non-polar solvent and heated at a temperature close to the solvent boiling point followed by a cooling step at a temperature of approximately 0°C.

8. A process, according to claim 1, wherein the purification step d) is performed by dual chromatography, by first using a medium pressure normal-phase chromatography (NP-MPLC) and afterwards a high-performance reversed-phase chromatography (RP-HPLC) .

9. A process, according to claim 8, wherein the purification step d) is performed by NP-MPLC with n-hexane/ethyl acetate or other solvent mixtures of similar polarity for the elution of suberin acids methyl esters.

10. A process, according to claim 8, wherein the purification step d) is performed by NP-MPLC with n-hexane/2-propanol slightly acidified or other solvent mixtures of similar polarity for the elution of free suberin acids.

2

11. A process, according to claim 8, wherein the purification step d) is performed by RP-HPLC with methanol or acetonitrile/water mixtures as eluent, for the purification of suberin acid methyl esters and free suberin acids.

Lisbon, December 12, 2012.

3

Description:
DESCRIPTION

PROCESS FOR THE EXTRACTION AND PURIFICATION OF LONG- CHAIN BI-FUNCTIONAL SUBERIN ACIDS FROM CORK

Field of the invention

The present invention relates to a process for extraction and purification of long-chain bi-functional suberin acids from cork. Said compounds are useful in a large number of applications, such as cosmetics and pharmaceuticals and also in polymer synthesis. Thus, the process of the present invention is useful for several industrial applications to provide compounds which may be applied to pharmaceuticals, cosmetics, and engineering polymers.

Background of the invention

Suberin is the specific chemical constituent of cork, accounting for about 40% of its dry weight. Cork is found in all tree barks, although in highly variable amounts depending on the tree species. Cork is particularly abundant in the cork-oak (Quercus suber) outer bark, where it reaches a thickness of several centimetres in a few years growth. This is the commercial cork used for the manufacture of cork-stoppers and a myriad of other artefacts. Cork is harvested from the cork-oak trees in a renewable and sustainable manner. Cork suberin is a heteropolymer made of long-chain omega-hydroxy fatty acids (LC-OHFAs) and long-chain alpha, omega-dicarboxylic acids (LC-DCAs) , together with glycerol, all assembled as a complex biopolyester [1] . The polyester structure of suberin can be broken by any reaction that cleaves ester bonds, like hydrolysis, either acidic or basic, alcoholysis, hydrogenolysis, etc., releasing the LC-OHFAs and LC-DCAs as individual compounds. These suberin acids have even chain-lengths, from Ci 6 to C 2 6 - Some of them have saturated hydrocarbon chains or, particularly the Cie, have mid-chain functional groups, like an unsaturation, an epoxide ring or a vicinal diol.

The high potential of the long-chain bi-f nctional suberin acids for a large number of uses, from cosmetics [2] and pharmaceuticals [3] to polymer synthesis [4], has long been recognized and expressed in abundant scientific literature.

Because of this, processes for the preparation of these bi- functional acids by synthetic [2] or biotechnological [5] routes have been disclosed and developed. However, the extraction of these compounds from a natural source such as cork will have the highly prized advantages of renewability and sustainability, together with the certifiable status of "plant natural products" and "green chemicals".

A long known process for the extraction of suberin acids from cork dates back to 1952 [6]. The process was based in the differential solubility of suberin acid salts, and this same principle was retaken in another process for the extraction of some of these acids from a cork by-product, the "smoked washed solids" [7] . However, these processes give low yields and they are not capable to separate the several individual compounds of the suberin acids in significant purity.

More recently, a number of processes have been disclosed for the extraction of suberin acids from the cork of birch bark (Betula sp.) [8-12]. These methods are however only able to provide said compounds in the form of crude "enriched" fractions, through more or less complex fractionation methods.

Document [8] WO 2010/093320 discloses a method for converting plant parts into chemicals, in particular, a method for converting biopolyesters, by hydrolysis with an alkali in water to extract the correspondent hydroxyl-fatty acid monomers with the aim of eliminating the use of organic solvents. The dissolved extracts obtained in the alkali hydrolysis step are then precipitated by acidification. However, the disclosed method only provides an extract enriched in cis-9, 10-epoxy-18- hydroxyoctadecanoic acid.

Document [9] RU 2175326 also discloses a method for recovering some compounds from birch bark fibres, namely betulin and derivatives thereof, by solvent extraction and separation using for that purpose toluene and further filtration of the extracts. However, with this method is not possible to extract other compounds from suberin, such as long-chain omega-hydroxy fatty acids (LC-OHFAs) and long-chain alpha, omega-dicarboxylic acids (LC-DCAs) .

Document [10] US2009/0182158 discloses a method for extraction of compounds from birch bark comprising a depolymerisation extraction with an alkaline solution and a water-soluble organic solvent. The resulting triterpene alcohols, namely betulin and lupeol, and suberin acid salts, are then recovered to a water- insoluble organic solvent, after removal of the water-soluble organic solvent, water and insoluble materials from the extracts. Betulin may be further purified by crystallization, filtration and drying to yield approximately 16-18% of betulin of about 93-96% purity.

However, with said process is not possible to obtain the several suberin acids present in the suberin extracts in isolated and purified form, but only mixtures or fractionated extracts of them. In fact, document [10] only discloses crude extracts, depolymerised extracts and (i) fractionated, extracts comprising lupeol, betulin and other non identified compounds, (ii) fractionated extracts comprising suberin acid salts and betulinic acid salts, and (iii) fractionated extracts comprising suberin free acids and betulinic free acids.

Therefore, it would be desirable to develop a process that allows obtaining improved yields of some, of the most valuable compounds of suberin in high purity rates in a straightforward way.

It has now been found that said drawbacks may be overcome by a multi step process comprising an extraction step followed by a depolymerisation step, a solvent fractionation step and a sequential treatment of the isolated fractions with medium- pressure normal phase chromatography (MPLC) and high-performance reversed-phase chromatography (HPLC) techniques, leading to individual suberin acids approaching 100% purity. Summary of the invention

The present invention relates to an integrated and comprehensive process for the extraction, isolation, and purification of all the main suberin acid monomers that can be found in suberin-rich plant tissues like cork. The process starts from the very complex mixture that arises from the depolymerisation of the suberin polyester, and ends with all the most valuable individual compounds of cork in high purity rates in a simple, accurate and convenient way for their further use or chemical processing: either with the carboxylic acid as a free group or protected in the form of methyl ester.

The higher purity degree of the compounds extracted according to the process of the present invention is required for the use of said compounds in performance demanding polymer, cosmetic, and pharmaceutical industries.

Therefore, the objective of the present invention is to provide a process for extraction and purification of long-chain bi- functional suberin acids, the most valuable individual compounds of cork, in improved yields and in high purity rates in a simple and accurate way, according to claim 1.

General description of the invention

The present application describes a multi-step process for the isolation of some of the main suberin acids from cork, based in solvent-fractionation and chromatographic techniques, affording these different suberin compounds in very-high purity, in excess of 99.5%. Cork is first extracted with solvents and suberin is then depolymerised by conventional methods, preferably by methanolysis and alkaline hydrolysis, and the ensuing suberin acids mixtures are first partitioned using differential temperature and solvent-solubility precipitation. The fractions enriched in the different suberin acids thus obtained, are sequentially treated by medium-pressure normal phase chromatography (MPLC) and high-performance reversed-phase chromatography (HPLC) , leading to individual suberin acid purities approaching 100% purity.

Two alternative routes are presented: (i) one for the isolation and purification of suberin acids in the form of methyl esters; and (ii) the other for the isolation and purification of suberin acids as free carboxylic acids.

Definitions :

The structure, systematic and common names, and acronyms used herein of the main long-chain bi-functional suberin acids of the present invention are presented in the formulae below,

wherein

R is OH, for free carboxylic acids;

R is OCH3, for methyl esters Hid22 R

22-hydroxydocosanoic acid

[phellonic acid; ω-hydroxy C22:0 fatty acid]

Formula I

Formula II

f/?reo-9,10,18-trihydroxyoctadecanoic acid

[phloionolic acid; 9,10,18-trihydroxystearic acid]

Formula I I I

[C18:1 dicarboxylic acid]

Formula IV

tftreo-9,10-dihydroxyoctadecanedioic acid

[phioionic acid]

Formula V Compounds Hid22 (C22) with formula I, are representative of the saturated, Ci 6 -C 2 6, long-chain omega-hydroxy fatty acids (LC- OHFAs) . Compounds Hidl8:l with formula II and compounds Hidl8diol with formula III are C i8 , mid-chain substituted, long- chain omega-hydroxy fatty acids (LC-OHFAs) .

Compounds Dil8:l with formula IV and compounds Dil8diol with formula V ' are C i8 mid-chain substituted long-chain alpha, omega- dicarboxylic acids (LC-DCAs) .

In the scope of the present invention, the term "long-chain" means an organic compound comprising a hydrocarbon chain length of C 8 -C 30 ; the long-chain preferably comprises C 8 -C 30 , more preferable Ci5-C 25 , even more preferably Ci 8 -C 2 2- The term "mid- chain substituted", in the scope of the present invention, means a Ci8 suberin acid with the substituent group linked to the carbon atoms at positions 9 and 10; and "short-chain" means any aliphatic acid compound with a chain of less than 8 carbons.

Cork is defined as the outer bark of the cork-oak tree {Quercus suber L.). Cork definition is extended to the cork, periderm or suberized tissue, present in the bark of trees and shrubs or other plant organs.

Cork powder is defined as cork particles of size < 0.25 mm. Cork powder can be obtained: (i) by grinding raw or boiled cork, by means of a mill, followed by sieving; (ii) as a by-product of the cork industry, in the form of (a) under-sized granulated cork; (b) powder recovered from cleaning operations from

granulated cork production; (c) powder recovered from milling or polishing operations of cork artefacts (cork stoppers, etc.). 1. Cork extractives removal

Cork is mostly built by polymeric structural components, namely suberin, polysaccharides and polyaromatics, but also includes in its chemical composition a significant proportion of non- polymeric compounds, which represent typically 10 to 25% of its dry matter. These non-polymeric compounds are collectively known as "extractives", since they can be removed by solvent extraction. Cork extractives are complex mixtures including acyclic lipids, triterpenoids, flavonoids and oligosaccharides, with a wide array of polarities. Extractives have to be removed from cork before suberin depolymerisation, since some of them have a chemical behaviour, namely in terms of polarity, similar to the suberin acids intended to be recovered and isolated. Cork extractives are removed in the present process by conventional solid-liquid extraction techniques, such as immersion, percolation or Soxhlet-type extraction by appropriate solvents. To ensure that all types of extractives are removed from cork, solvents with increasing polarity are applied in sequence.

Organic compounds used as solvents, in the present invention, include aromatic compounds and other hydrocarbons, alcohols, esters, ethers, ketones, amines, and nitrated and halogenated hydrocarbons. Water is also included in the definition of organic compounds according to the present invention.

The extraction step begins with the most non-polar organic solvent, typically a halogenated hydrocarbon such as chloroform, or short-chain alkane such as pentane or hexane, then followed by a polar solvent, typically a short-chain alcohol such as methanol or ethanol or a ketone such as acetone and ending with a high-polarity solvent like water. In a preferred embodiment of the present invention, up to three solvents with increased polarity are used for extractives removal .

In a more preferred embodiment the three solvents used in the scope of the invention are first chloroform or hexane, the following solvents are methanol or ethanol and the last solvent used is water.

The polarity of the solvents is defined according to their dielectric constant and therefore, non-polar solvents are, in the scope of the present invention, those which have a dielectric constant between 1.5 and 5.0, a polar solvent has dielectric constant between 5.0 and 50.0 and a highly polar solvent has a dielectric constant above 50.0.

According to the present invention, examples of non-polar solvents are n-hexane, ether, chloroform, polar solvents are ethanol, methanol, acetone, and a high-polarity solvent is water .

After the extraction with the highest polarity solvent is completed the extractives are separated from the solvent and optionally filtered, concentrated and/or dried into a powder.

After washing out the extractives, an "extractive-free cork powder" material is obtained, and can be used as the starting point for suberin depolymerisation and recovery of the suberin acids .

The solvents used in this extraction step may be recycled by distillation or evaporation under reduced pressure in a rotary evaporator or other distillation apparatus and reused. 2. Cork suberin depolymerisation

Suberin acids are solubilised and recovered from the extractive- free cork powder after suberin depolymerisation. The polyester structure of suberin is depolymerised either by (i) a methanolysis reaction, which solubilises its long-chain suberin acids in the form of methyl esters, or (ii) by an alkaline hydrolysis reaction followed by acidification to obtain the suberin acids as free carboxylic acids.

Suberin depolymerisation is determinant to release the suberin acids as individual compounds, by cleaving the ester bonds that kept them together in the original polyester structure.

2.1 The methanolysis reaction

This reaction is performed by contacting the extractive-free cork powder with a solution of a strong base, such as NaOH or NaOCH 3 in methanol, at a pH > 10 under reflux or at a temperature of 50-80°C for a few hours, for example between 1 and 5 hours, till the depolymerisation reaction is completed.

In a preferred embodiment, the strong base comprises a methoxide group. The use of a base comprising methoxide as the catalyst, such as sodium methoxide or potassium methoxide, for the methanolysis reaction is advantageous compared to other strong bases like alkali hydroxides, such as KOH or NaOH, due to the fact that the depolymerisation reaction is more selective towards the suberin polyester. This means that the methanolysate mixture recovered has less contaminant materials from other cork cell wall constituents, like lignin or other polyaromatics . In general, the base methoxide-catalysed reaction simplifies the ensuing isolation and purification of suberin acids methyl esters. Methanolysis is also preferred over other alcoholysis reactions, like ethanolysis, due to kinetic reasons: the methanolysis reaction proceeds at a higher rate and the total depolymerisation of the suberin polyester can be achieved more quickly.

The methanolysate solution is filtered to remove the non reacted cork powder residue and further acidified with a strong acid in an alcohol solution (ex. methanol), down to a pH value ≤6. Completeness of the suberin polyester depolymerisation can be assessed by infrared analysis of the residue, in which case the absorption band at ca. 1740 cm -1 resulting from the ester carbonyl group, is absent.

2.2. The alkaline hydrolysis reaction

This reaction is performed by contacting the extractive-free cork powder with a solution of a strong base, like sodium or potassium hydroxide, dissolved in ethanol/water, in molar concentrations of 0.1 to 3.0 M. The mixture is reacted at the solution boiling point temperature in reflux for up to 3 hours, till completeness of the depolymerisation reaction, which can be assessed by the IR analysis of the un-reacted residue as described above. The hydrolysate is separated from the non- reacted residue by filtration and afterwards acidified to pH ≤6 with a solution of a strong acid; after acidification, most of the ethanol/water is removed by evaporation and the precipitated material partitioned between a water immiscible organic solvent and water. The organic phase is recovered and the solvent is removed by evaporation, giving rise to a solid extract made up of a mixture of free suberin acids. 3. Suberin acids isolation by solvent fractionation

The first separation of the target suberin acids from the suberin mixtures produced in the depolymerisation process is achieved by solvent fractionation, taking advantage of both solvent polarity and temperature-dependent solubility (Figure 1) . Two solvents with contrasting polarity, a non-polar and a polar solvent are sequentially used, to dissolve in hot and precipitate in cold temperature conditions, fractions of the desired suberin monomers. In the case of the suberin acids in the free carboxylic acid form, due to its comparatively higher polarity, the solvent-based isolation steps use only a low- polarity solvent (Figure 2) .

Thus, by using this solvent fractionation process it is possible to obtain the different compounds present in the depolymerised extracts grouped in function of their polarity and solubility at different temperatures. In this way, fractions enriched in the desired compounds, in concentrations from 40 to more than 80%, are obtained.

The first isolation step is therefore performed with a low- polarity solvent, such as n-hexane or other solvent of similar dissolving characteristics, like other low-dielectric constant hydrocarbons .

The mixtures resulting from suberin depolymerisation, obtained by methanolysis or alkaline hydrolysis, are extracted with such a solvent, at a temperature close to its boiling point or in reflux conditions. This solvent-extraction is carried out either directly on the actual depolymerisation extract, or on its organic phase, the later obtained by partition of the acidified depolymerised materials between water and an organic solvent, typically a short-chain chlorinated hydrocarbon like dichloromethane . This first extraction in the hot low-polarity solvent separates most of the target suberin acids, in which they are soluble, from other suberin-associated compounds present in the depolymerisation extracts, which remain in the solid residue separated by filtration.

The hot solvent solution with the solubilised suberin acids is allowed to cool to ambient temperature or forced to cool down to 0°C, using a chilling fluid. The resulting precipitated material is isolated by filtration and/or decantation, affording the first separation of two groups of suberin acids: (1) the insoluble fraction, enriched in the higher-polarity suberin acids, namely the long-chain saturated ω-hydroxyalkanoic acids (including Hid22) and the C18 diol-substituted acids (Hidl8diol and Dil8diol) ; and (2) the soluble fraction, enriched in the lower-polarity suberin acids, including the saturated chain mono- and dicarboxylic acids and the C18 monoene acids, namely Hidl8:l and Dil8:l.

A second isolation step follows involving the extraction of the above fractions with a polar solvent, typically methanol, or other solvent with similar polarity properties, including other short-chain alcohols. In this second isolation step fractions enriched in the suberin acids, are obtained in purities reaching 85%.

In this second isolation step the two fractions obtained in the first isolation step are extracted with the hot polar solvent, and the ensuing solutions are afterwards cooled to 0°C. The precipitated materials are again separated by filtration and/or decantation, each of the initial fractions giving rise to two new fractions, either insoluble (lower-polarity) or soluble (higher-polarity) in the cold polar solvent. In this way, the initial fraction insoluble in the cold low- polarity solvent obtained in the first isolation step (1), which included the Hid22 and C18-diol acids, is here divided in two new fractions: (1.1) the insoluble fraction, enriched in Hid22 and other long-chain ω-hydroxyalcanoic acids; and (1.2) the soluble fraction, enriched in the C18-diol acids. Furthermore, the initial fraction soluble in the cold low-polarity solvent in the first isolation step (2), which included the C18 monoene acids, namely Hidl8:l and Dil8:l, is now divided in two other fractions : (2.1 ) the insoluble fraction, which includes the saturated chain mono- and dicarboxylic acids; and (2.2) the soluble fraction, which includes the targeted Hidl8:l and Dil8:l suberin acids .

This second polar solvent fractionation is particularly adequate for the isolation of the suberin acids in the form of methyl esters. In the case of the suberin acids in the free carboxylic acid form, which are more polar compared to the corresponding methyl esters, this step can be substituted by a re- crystallization with the initial low-polarity solvent, affording isolate fractions with levels of purity of the same order of those obtained with the second polar solvent solubility partition .

4. Suberin acids purification by dual chromatography

With the aim of obtaining substantially pure long-chain bi- functional suberin acids from cork, the fractionated extracts described above in point 3 are further purified by chromatographic techniques. For this purpose a two-fold sequential contrasting normal-phase and reversed-phase chromatography is used. A first level of purification is obtained by normal-phase medium-pressure liquid chromatography (NP-MPLC) , and a second and final level of purity is achieved with preparative reversed-phase high-performance liquid chromatography (RP-HPLC) . Some of these chromatographic steps can be repeated whenever higher purities are desired.

The first purification step by NP-MPLC is carried out in columns filled with a polar stationary phase like silica, to be eluted by a relatively non-polar mobile phase, typically composed of mixtures of a low-polarity solvent, such as n-hexane, and a silica-compatible polar solvent, like ethyl acetate or 2- propanol. The proportion of the polar solvent in the eluent mixture will follow the polarity of the extract fraction or compound to be isolated. Isocratic elution is preferred both for the simplicity of the process and for easier recovery of the used solvent. This first step of purification by NP-MPLC will be applied to each of the suberin acid-enriched fractions described in point 3, attaining after this stage purity levels for the individual target compounds from 70 up to 90%.

The second and final purification step by RP-HPLC is carried out in high-pressure columns filled with a non-polar stationary phase like octadecyl-bonded silica (Cie) , or the relatively more polar octyl-bonded silica (Cs) , preferably of small particle size (≤ 10 μπι) , for better resolution. Elution is made in gradient mode, with the initial mobile phases composed of binary mixtures of a suitable polar strong solvent, such as methanol or acetonitrile, and 10 to 30% of water, as the weak solvent. Each of the relatively purified target compounds obtained in the previous NP-MPLC step, will be subject to this final purification stage, affording final very high purity levels, above 99% for most of them, and in some cases technically "100% pure" . 5. Solvent-recycling and side-products usage

The cork residue that remains after suberin depolymerisation and extraction of suberin acids is mainly composed by lignin-like aromatics and polysaccharides, thus resembling wood in its composition. Therefore, it can be used as a highly convenient biomass source for energy production through burning in steam generators. In the end, this can make the process of suberin acids extraction and purification form cork eventually self- sufficient in terms of energy consumption.

The suberin by-products that will not be used in the next solvent-fractionation or chromatographic purification step are conveniently looped back into the process and are not to be discarded. In most cases they are mixtures that still contain significant quantities of the target suberin acids, which can be further extracted by re-entering the process in the appropriate previous step.

The fractions that are mostly composed of the non-targeted suberin acids, namely those with mixtures of saturated long- chain mono and dicarboxylic acids, can also be a useful material for technical polymer synthesis and be commercialized for such purpose.

The solvents used in all the steps described in the isolation and purification steps of the suberin acids, are recycled by evaporation and/or distillation with a recovery rate above 90%. This means that only comparatively small quantities of solvents will be used for make-up, and will be effectively consumed in each batch of the extraction and purification processes. Description of the Figures

Figure 1 represents a flow-chart for the isolation of suberin acid methyl esters by solvent fractionation of the process of the present invention. The methanolysate that results from the depolymerisation of cork is extracted in succession by a non- polar and a polar solvent, in each step separating fractions of contrasting polarity. These isolates will be subject to purification steps by chromatographic techniques, affording pure suberin acid methyl esters.

Figure 2 represents a flow-chart for the extraction and isolation steps of free suberin acids of the process of the present invention. The hydrolysate that results from the alkaline hydrolysis of cork is subject to a non-polar solvent isolation step resulting in two fractions of contrasting polarity which are further submitted to purification steps by chromatographic techniques, affording pure free suberin acids.

Detailed description of the invention

1. Cork extractives removal

Cork is extracted with solvents to remove its non-polymeric constituents, also called "extractives", giving rise to "extractive-free cork powder". The process of the present invention comprises first the removal of the extractives of the cork by using solvents of different polarity.

Extractives removal by solvent extraction is performed by a conventional extraction procedure such as the ones explained below carried out by means of:

(i) in a reaction vessel coupled to a condenser, the cork powder is mixed with the solvent, the mixture is heated up to the boiling point of the solvent, and the extraction carried out under reflux conditions; after the extraction is completed, the solvent with the solubilised extract is washed away by filtering;

(ii) in a Soxhlet-type apparatus, where cork powder is confined in a porous bag, and is washed with fresh solvent cyclically regenerated by evaporation/condensation.

In both cases, up to three solvents of increasing polarity - non-polar, polar, and highly-polar, are used sequentially until complete extractives removal.

Also in both cases the used solvents may be recycled by distillation or evaporation under reduced pressure in a rotary evaporated, and the corresponding extracts recovered as dry matter.

2. Cork suberin depolymerisation

2.1 Methanolysis reaction

The polyester structure of suberin is depolymerised by a methanolysis reaction, which solubilises its long-chain suberin acids in the form of methyl esters.

The extractive-free cork powder, prepared as described previously, is reacted under reflux in a solution of a strong base, preferably with a solution of sodium methoxide, in a concentration of 0.1-3.0 M in methanol, for at least, 1 hour.

After methanolysis, the reaction mixture is filtered to remove the non reacted cork powder residue, and the methanolysis filtrate acidified to a pH value 6 with a strong acid in methanol solution. The solvent is removed by evaporation providing a viscous solid of depolymerised suberin materials, including the suberin acid methyl esters.

2.2 Alkaline hydrolysis

Suberin is depolymerised by an alkaline hydrolysis reaction ("saponification") , and the suberin acids are recovered as free carboxylic acids after acidification. The starting material is extractive-free cork powder prepared as previously mentioned. The cork powder is mixed with an alkaline solution made of a metallic strong base, like sodium or potassium hydroxide, dissolved in ethanol/water , typically in a 9:1 proportion, in molar concentrations 0.1 to 3.0 M. The mixture is reacted in reflux for at least 1 hour, the non-reacted residue filtered away, and the hydrolysate acidified to pH 6 with a solution of a strong acid. After acidification, most of the ethanol/water is removed by evaporation and the residual material partitioned between a water immiscible organic solvent and water. The organic phase is recovered and the solvent is removed by evaporation, giving rise to a sticky solid made up of a mixture of free suberin acids .

3. Suberin acids isolation by solvent fractionation

Starting from the extracts recovered from the depolymerisation reactions, fractions enriched in the targeted suberin acids are obtained by a two-step solvent fractionation process. In the first step, a non-polar solvent is used, and two fractions are separated by differential solubility in the cold or room temperature solvent; in the second step, a polar solvent is used, and each of the former first-step fractions gives rise to two new fractions. This two-step solvent-fractionation process is applied to the suberin acid mixtures both in the form of methyl esters (from the methanolysis depolymerisation reaction, Figure 1) , as well as in the form of free suberin acids (from the alkaline hydrolysis reaction, Figure 2) .

3.1. Isolation of suberin acids methyl esters by solvent fractionation

The following fractions showed in the flowchart in Figure 1 below were obtained by the different steps of the process of the present invention:

3.1.1 First isolation step: solvent f actionation in a non-polar solvent

Suberin acids methyl esters are recovered from the methanolysis suberin depolymerisation products by extraction with a hot low polarity solvent, typically a short-chain n-alkane or iso- alkane. The semi-solid extract from the methanolysis reaction is mixed with the solvent in a temperature controlled reaction vessel, and heated to a temperature close to the solvent boiling point, with stirring. The extraction can be made either in reflux conditions, or carried out at a temperature a few degrees below the solvent boiling point. The ensuing solution carrying the extracted suberin acids methyl esters is either decanted or filtered to separate the non-solubilised matter. The decanted/filtrated solution is cooled as described below to isolate soluble and insoluble fractions in the cold solvent.

Isolation of the S60_Me fraction

The suberin acids methyl esters solution is cooled to 0°C, by means of a surrounding ice bath or chilling fluid, and a precipitate is formed and separated by filtration. The white- yellowish powder thus recovered is called "Fraction S60_ e" (suberin acid methyl esters soluble at ca. 60°C in the non-polar solvent, but insoluble at 0°C) . This fraction is enriched in n- alkanols, ω-hydroxyalkanoic acids higher than C20 and Cis 9,10- diol acids. This fraction is the starting point for the isolation of 22-hydroxydocosanoic acid methyl ester (Hid22_Me) , 9, 10, 18-trihydroxyoctadecanoic acid methyl ester (Hidl8diol_Me) and 9, 10-dihydroxyoctadecane-l, 18-dioic acid dimethyl ester (Dil8diol_Me) .

Isolation of the S0_Me fraction

After removal of the products that precipitate at 0°C above described, the remaining solution is evaporated by means of a rotary evaporator. A whitish viscous solid is recovered, called "Fraction S0_ e" (suberin acid methyl esters soluble at ca . 0°C in the non-polar solvent) , which is enriched in the methyl esters of alkanoic acids, Ci 6 ω-hydroxyalkanoic and α,ω- alkanedioic acids, Ci 8: i ω-hydroxyalkenoic and a, ω-alkenedioic acids, and C22 a, ω-alkanedioic acid. This fraction is the starting point for the isolation of the 18-hydroxyoctadec-9- enoic acid methyl ester (Hidl8:l_Me) and octadec-9-enedioic acid dimethyl ester (Dil8:l_Me).

3.1.2 Second isolation step: fractionation by solubility in a polar solvent

Fractions S60_Me and S0_Me recovered as described above are further extracted with a hot polar solvent, typically a short- chain alcohol like methanol, or other solvent of equivalent polarity at a temperature close to the solvent boiling point. After cooling, a precipitate is formed, affording cold solvent- soluble and insoluble fractions.

Isolation of S60/lM0_Me and S60/SM0_Me fractions

Fraction S60_Me is mixed with the polar solvent, heated to a temperature close to its boiling point, with stirring. The extraction can be made either in reflux conditions, or carried out at a temperature a few degrees below solvent evaporation. The reaction mixture is cooled to approximately 0°C, by means of a surrounding ice bath or chilling fluid; a precipitate is formed and separated by filtration in a glass G3-porosity filter. The recovered insoluble fraction is called "S60/IM0_Me" and is enriched in Hid22_Me (80-85%) . The solvent of the remaining filtrated solution is removed by evaporation, giving rise to the "S60/SM0_Me" fraction. This fraction is enriched in Hidl8diol_Me and Dil8diol_Me, and is to be used as the starting point for their purification.

Isolation of S0/lM0_Me and S0/SM0_Me fractions

The same polar solvent extraction procedure is applied to the S0_Me fraction, giving rise to the "S0/IM0_Me" and "S0/SM0_Me" fractions. The S0/IM0_Me is the fraction insoluble at low temperature in the polar solvent, recovered as the precipitate. This fraction is enriched in C 2 z ω-alkanoic and a, ω-alkanedioic acids (docosanoic acid methyl ester, 26%, docosanedioic acid dimethyl ester, 19%), and can be used as a starting point for their further purification. The S0/SM0_Me is the soluble fraction at low temperature, which is recovered after solvent evaporation. This fraction is enriched in 18-hydroxyoctadec-9- enoic acid methyl ester (Hidl8:l_Me, 61%) and octadec-9-enedioic acid dimethyl ester (Dil8:l_Me, 14%), and is the starting point for the purification of these acids.

3.2 Isolation of free suberin acids by solvent fractionation

A similar process to the one described above for the suberin acid methyl esters is applied for the isolation of suberin acids in the form of free carboxylic acids (FA, for "free acids") . An overview flowchart is presented in Figure 2. The free suberin acids are extracted from the mixture recovered after the alkaline hydrolysis reaction followed by acidification and solvent removal (Detailed description, 2.2). The recovered hydrolysate is extracted with a low polarity organic solvent, as described in 3.1.1. The solubilised material is decanted or filtered, and allowed to cool to room temperature (20 °C) . A precipitate is formed, and two fractions are separated by filtration: the precipitate material gives rise to the N S60_FA" fraction and the soluble material, after solvent removal by evaporation, gives rise to the "S20_FA" fraction. The S60_FA fraction is enriched in Hid22_FA (in a purity of ca. 65%) and Hid 24_FA (ca. 9%) and is used as the starting point for the isolation of Hid22_FA; This fraction can be used as such in the reversed-phase chromatography purification step, or the concentration of Hid22_FA firstly raised up to 80% by re- crystallization with the non-polar solvent used for its initial isolation. The S20_FA fraction is enriched in Hidl8:l_FA (40%) and Dil8:l_FA (20%) and is used to isolate these two suberin acids. This fraction is applied as such in the first purification step by normal-phase chromatography.

4. Suberin acids purification by dual chromatography

4.1 First purification step: medium pressure normal-phase

chromatography

The suberin acids obtained as enriched fractions by fractional solubility, are first purified by liquid medium pressure column chromatography in normal-phase (NP-MPLC) .

Each of the fractions described above, which include the target suberin acids methyl esters, namely S0/SM0_Me, S60/SM0_Me and S60/IM0_Me, and free suberin acids, namely S20_FA, are dissolved in a mixture of non-polar/polar organic solvents, in varying relative volume proportions depending in the fraction compounds polarity. The solubilised suberin acid fractions are applied to a silica-gel column, and eluted isocratically under pressure with solvent mixtures. Typically the fractions made up of suberin acids methyl esters are eluted in n-hexane/ethyl acetate mixtures, and the free suberin acid fractions are eluted in n- hexane/2-propanol mixtures, the later slightly acidified with a weak acid. Small volume fractions are collected at the outlet of the column. Composition and compound purity of the collected fractions are controlled by gas-chromatography-mass spectrometry, after appropriated solvent drying and derivatization of sampled aliquots. Collected fractions with the compounds of interest in purities above the targeted level are combined and the solvent removed by evaporation.

In this manner purified "isolates" of the suberin acids are obtained, which are afterwards subjected to the reversed-phase chromatography step for final purification: from the S0/SM0_Me fraction a Dil8:l_Me isolate" (compound purity ≥ 75%) and a "Hidl8 : l_Me isolate" {≥ 85%) are obtained [Example 7]; from the S60/IM0_Me fraction a "Hid 22_Me isolate" (>92%) is obtained [Example 8]; from the S20_FA fraction a "Dil8:l_FA isolate" (>70%) and a "Hidl8:l__FA isolate" (60-70%) are obtained. In this first purification step Dil8diol_Me and Hidl8diol_Me are obtained from the S60/SM0_Me fraction both in a purity ≥ 97%, and are no further purified.

4.2 Second purification step: high-performance reversed-phase chromatography

Suberin acid isolates obtained in the first purification step as described above in 4.1 are further purified by preparative high- performance liquid chromatography (prep-HPLC) in reversed-phase (RP) mode. The suberin acid isolates are applied to non-polar stationary phase columns, typically Cis or C 8 bonded silica, and eluted in gradient mode with mixtures of strong/weak solvents, typically acetonitrile or methanol/water mixtures. The gradient solvent mixtures start with weak solvent volume proportion of 10-30% and change gradually to the pure strong solvent. Compound elution is monitored splitting the outflow between the collector (for instance 9999/10000 parts) and a . Light Scattering Detector (1/10000 parts) . The time window in the detector signal, where the compound of interest has purity above the targeted level is defined, and the corresponding eluted material collected separately. Most of the solvent mixture is evaporated in a rotary evaporator, and residual solvent is removed by gentle heating (< 40°C) under nitrogen flow, and further in a vacuum oven. The purity and proof of structure of the final purified compounds are checked by GC-MS, FTIR, and ID (¾, 13 C) and 2D (COSY, HSQC, HMBC) NMR.

Final purities achieved for the targeted suberin acids are: Hidl8:l_Me, >99.9%; Dil8:l_Me, >99.9%; Hid22_Me, >99.8%; Hidl8:l_FA, >99.5%; Dil8:l_FA, >95.0%; Hid22_FA, >97.0%.

EXAMPLES

Materials and reagents

All materials and reagents were commercially acquired. Solvents used in solvent fractionation isolation steps, and normal-phase and reversed-phase chromatography purification steps were of HPLC-grade quality.

Example 1 :

This example illustrates how the removal of non-polymeric extractives from cork powder may be performed in a Soxhlet apparatus . Cork powder with particles of size < 0.25 mm is obtained by grinding raw or boiled cork, by means of a mill, followed by sieving, or as a by-product of the cork industry. Then the powder is placed in a Soxhlet apparatus and extracted sequentially with the following solvents and extraction times: Dichloromethane is used for extraction during 6 hours and the yield is 4.5-6.5% (w/w) ; Ethanol is used for extraction during 12 hours and the yield is 3.5-4.3% (w/w); Water is used for extraction during 18 hours and the yield is 3.0-8.7%.

Example 2 :

This example illustrates the cork suberin depolymerisation by methanolysis .

A sodium methoxide solution is prepared by dissolving the corresponding molar proportions of either metallic sodium or sodium hydroxide in methanol. 500 g of air-dried extractive-free cork powder is reacted under reflux, at a temperature of approximately 65°C, in 11 litres of a 0.25 M sodium methoxide/methanol solution, for 3 hours.

The methanolysis mixture is then filtered in a glass filter ' of Gl porosity, and washed with methanol. About 12 litres of filtrate are recovered and acidified to pH 6 with approximately 0.5 litres of a 3M H 2 SO 4 in methanol solution. The soluble matter in the acidified solution is taken to dryness by solvent evaporation under reduced pressure in a rotary evaporator, giving a viscous solid mixture of suberin acid methyl esters.

Example 3 :

This example illustrates the cork suberin depolymerisation by alkaline hydrolysis. In a 5 litre round flask with a reflux condenser, 2 litres of a 0.5 M NaOH solution of ethanol/water 9:1 are added to 150 g of extractive-free cork powder, and the mixture refluxed for 2 hours with stirring. After the alkaline hydrolysis reaction, the hydrolysate solution is separated from the solid residue by filtration in a glass Gl-porosity filter, and acidified to pH 6 with 3 M H 2 S0 4 in water. The acidified hydrolysate solution is evaporated close to dryness in a rotary evaporator, giving 45-55 g of a semi-solid mass composed of suberin acids in free acid form.

Example 4 :

This example illustrates the first isolation step of suberin acid methyl esters with a low polarity solvent, starting from the solid methanolysis depolymerisation extract, giving rise to the S60_Me and S0_Me fractions.

4 litres of n-hexane are added to ca. 430 g of the recovered solid matter after methanolysis depolymerisation (Example 2) and the mixture is heated in an oil bath to a temperature of 63°C, with stirring, for 2 hours. The mixture is then cooled close to 0°C, by immersing the reaction vessel in an ice/water bath. The precipitated matter is recovered by filtration in a G3 filter, giving ca. 42 g of fraction S60_Me. The n-hexane is evaporated from the remaining filtered solution affording ca. 48 g of fraction S0_Me .

Example 5 :

This example illustrates the second isolation step of suberin acid methyl esters with a polar solvent, starting from the S60_Me and S0_Me fractions of Example 4.

Fraction S60_Me (42 g) is mixed with 1 litre of methanol, heated to 62 °C and stirred for two hours. The reaction mixture is then cooled to 0°C in an ice/water bath. The precipitated matter is filtered in a G3 glass filter, with a yield of 24 g (S60/IM0_Me) ; methanol is evaporated from the filtrate solution in a rotary evaporator, with a yield of 17 g (S60/SM0_Me) . The same procedure is applied to the S0_Me (48 g) fraction with the following yields: precipitated matter, 9.3 g (S0/IM0_Me) ; soluble matter, 38.1 g (S0/SM0_Me) .

Example 6 :

This example illustrates the isolation step of free suberin acids with a low polarity solvent, starting from the alkaline hydrolysis depolymerisation products.

N-hexane (500 ml) is mixed with ca. 50 g of the recovered material after alkaline hydrolyis (Example 3), in a flask with a reflux condenser, and the mixture heated to the boiling point of the solvent for 2 hours. The soluble material, a mixture of free suberin acids, ca. 20-25% of the initial mass, is separated from the non-soluble material by decantation. The decanted n-hexane solution is cooled to room temperature, and the precipitate formed removed by filtration in a G3 filter, giving a white- yellowish powder, the S60_FA fraction, which accounts for 35% of all recovered suberin acids; the soluble material at room- temperature is recovered as a whitish solid paste after the evaporation of the n-hexane, the S20_FA fraction.

Example 7 :

This example illustrates the purification of Hidl8:l_Me and Dil8:l_Me by normal-phase chromatography (NP-MPLC) starting from the S0/SM0_Me fraction.

Ca. 38 g of S0/SM0_Me (Example 5) are dissolved in 50 ml of n- hexane/ethyl acetate 7:3, applied to two coupled silica-gel (45- 75 ]im particle size) VersaPak™ (80x75 mm plus 80x300 mm) columns, in a VersaFlash™ apparatus. The S0/SM0_Me fraction is eluted with n-hexane/ethyl acetate 7:3, at a flow rate of about 80 ml/min, for 1 hour. Successive fractions of 100 ml are collected and analysed in their composition by GC- S. A "Dil8:l_Me isolate" (6.3 g) is recovered in a purity ≥ 75% in the fractions eluted at the volumes interval [400-900 ml] ; A "Hidl8:l_Me isolate" (11.7 g) is recovered in a purity ≥ 85% in fractions eluted in volumes [1100-2900 ml] .

Example 8 :

This example illustrates the purification of Hid22_Me by normal- phase chromatography (NP-MPLC) starting from the S60/IM0_Me fraction .

The same procedure and chromatographic system described in Example 7 are applied, with the following differences: injected solution: 24 g of S60/IM0_Me (Example 5) dissolved in 40 ml of n-hexane/ethyl acetate 7:3; successive fractions of 80 ml are collected; a "Hid 22_Me isolate" (10.0 g) is recovered in a purity ≥ 92% in fractions eluted at volumes [3360-3920 ml] .

Example 9:

This example illustrates the purification of Dil8diol_Me by normal-phase chromatography (NP-MPLC) starting from the S60/SM0_Me fraction.

The same procedure and chromatographic system described in Example 7 are applied, with the following differences: ca. 17.5 g of S60/SM0__Me (Example 5) are dissolved in 50 ml of dichloromethane, applied to the silica-gel columns and eluted with n-hexane/ethyl acetate 1:1, at a flow rate of 75 ml/min. Successive fractions of 75 ml are collected, and a "Dil8diol_Me isolate" (1.1 g) is recovered in a purity ≥ 97% in- the fractions eluted in volumes [2250-2775 ml] . Example 10 :

This example illustrates the purification of Hidl8diol_Me by normal-phase chromatography ( NP-MPLC). , starting from the S60/SM0_Me fraction.

After recovery of the Dil8diol isolate (Example 9) the chromatographic columns are washed with 1 litre of dichloromethane/2-propanol 2:8, yielding 2.4 g with 17% of Hidl8diol_Me . 1.1 g of this mixture including Hidl8diol_Me is dissolved in 8 ml of dichloromethane/2-propanol 6:4 and applied to a silica-gel (45-75 μπι particle size) VersaPak™ (80x75 mm) column, in a VersaFlash™ apparatus and eluted with n~ hexane/ethyl acetate 1:1, at a flow rate of 20 ml/min. Successive fractions of 30 ml are collected, and Hidl8diol_Me (114 mg) is recovered in a purity ≥ 97% in the fractions eluted in volumes [1050-1950 ml] .

Example 11:

This example illustrates the purification of Hidl8 : 1_FA and Dil8:l_FA by normal-phase chromatography (NP-MPLC) starting from the S20__FA fraction.

The same procedure and chromatographic system described in Example 7 are applied, with the following differences: ca.

30 g of S20_FA fraction (Example 6) are dissolved in 50 ml of n- hexane/2-propanol/formic acid 85:15:1 and eluted isocratically with the same solvent at a flow-rate of 75 ml/min. A fraction is collected between 1500-2000 ml of eluted material, with 35% Dil8:l_FA; and a fraction with 60-70% of Hidl8:l_FA, the "Hidl8:l_FA isolate", is collected between volumes 2000-2500 ml. The former fraction enriched in Dil8:l_FA (35%) is again applied to the same chromatographic system, and eluted with n-hexane/2- propanol 95:5; a fraction with 70% Dil8:l_FA is collected between 3.5 and 4.2 litres, and named the "Dil8:l_FA isolate".

Example 12 :

This example illustrates the purification of Hidl8:l_Me by reversed-phase chromatography (RP-HPLC) , starting f om the Hidl8:l_Me isolate.

Ca. 250 mg of Hidl8:l_Me isolate (Example 7), dissolved in 2 mL of acetonitrile, are injected in the following prep-HPLC system: XTerra® C8 column, 50xl50mm, 10 μπι of particle size, equipped with a Waters 2525 binary gradient pump. The sample is eluted at a flow rate of 150 ml/min with a gradient mixture of acetonitrile/water, starting with 70% acetonitrile and reaching 100% acetonitrile in 10 minutes. In the time window of 6.10-6.70 min ca. 155 mg of Hidl8:l_Me are recovered with purity in excess of 99.9%.

Example 13:

This example illustrates the purification of Dil8:l_Me by reversed-phase chromatography (RP-HPLC) , starting from the Dil8:l_Me isolate.

Ca. 250 mg of Dil8:l_Me isolate (Example 7), dissolved in 2 mL of methanol, are injected in the prep-HPLC system described in Example 12 and eluted at 150 ml/min with a gradient mixture of methanol/water, starting with 70% methanol and reaching 100% methanol in 10 minutes. In the time window of 10.75-11.20 min ca. 60 mg of Dil8:l__Me are recovered with purity above 99.9%. Example 14 :

This example illustrates the purification of Hid22_Me by reversed-phase chromatography (RP-HPLC) , starting from the Hid22_Me isolate.

Ca. 200 mg of Hid22_Me isolate (Example 8), dissolved in 2 mL of methanol/dichloromethane 1:1, are injected in the prep-HPLC system described in Example 12 and eluted at 150 ml/min with a gradient mixture of acetonitrile/water, starting with 80% acetonitrile and reaching 100% acetonitrile in 10 minutes. In the time window of 8.50-9.00 rain ca. 107 mg of Hid22_Me are recovered with purity above 99.8%.

Example 15:

This example illustrates the purification of Hidl8:l_FA by reversed-phase chromatography (RP-HPLC) , starting from the Hidl8:l_FA isolate.

Ca. 172 mg of Hidl8:l_FA isolate (Example 11), dissolved in 2 mL of methanol/dichloromethane 3:1, are injected in the prep-HPLC system described in Example 12, and eluted at a flow rate of 120 ml/min with a gradient mixture of methanol/water, starting with 85% methanol and reaching 100% methanol in 10 minutes. In the time window of 2.50-2.83 min ca. 62 mg of Hidl8:l_FA are recovered with purity above 99.5%.

Example 16:

This example illustrates the purification of Dil8:l_FA by reversed-phase chromatography (RP-HPLC) , starting from the Dil8:l FA isolate. Ca. 250 mg of Dil8 : 1_FA isolate (Example 11), dissolved in 2 mL of methanol/dichloromethane 3:1, are injected in the prep-HPLC system described in Example 12, and eluted at 120 ml/min with a gradient mixture of methanol/water, starting with 90% methanol and reaching 100% methanol in 20 minutes. In the time window of 3.50-4.30 min ca . 120 mg of Dil8:l_FA is recovered with purity above 95.0%.

Example 17 :

This example illustrates the purification of Hid22_FA by reversed-phase chromatography (RP-HPLC) , starting from the S60_FA fraction.

Ca. 200 mg of S60_FA fraction (Example 6), dissolved in 2 mL of methanol/dichloromethane 3:1, are injected in the prep-HPLC system described in Example 12 and eluted at 120 ml/min with a gradient mixture of methanol/water, starting with 90% methanol and reaching 100% methanol in 12 minutes. In the time window of 5.50-6.00 min ca. 109 mg of Hid22__FA is recovered with purity above 97.0%.

Lisbon, December 12, 2012.

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