WO2017194629A1 - Process for extraction of antioxidants from plant material - Google Patents

Abstract

A process for isolation of at least one antioxidant agent from a plant substrate is disclosed which comprises a) contacting a plant substrate with an aqueous soap extraction solution comprising an aqueous medium and at least one soap compound R-C(=O)O-M⁺or⁺M^-O(O=)C-R-C(=O)O^-M⁺, wherein R is saturated or unsaturated linear, branched or cyclic C₃to C₁₇alkyl or C₆to C₁₈aryl, wherein the alkyl or aryl group can be substituted with up to 5 functional groups, and wherein M⁺is a monovalent cation, to obtain a soap extract and b) isolating an antioxidant mixture comprising at least one antioxidant agent from the soap extract obtained in step a).

Description

Process for Extraction of Antioxidants from Plant Material

The present invention is concerned with a process for extraction of plant material.

The demand for natural antioxidants will become more important in the future, as some synthetic antioxidants like butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have found to have cancerogenic potential, and therefore, might be banned.

Many plants contain antioxidants which are highly desirable as food additives, additives in animal feedstuff and for cosmetic preparations. The antioxidants are mainly present in leaves, blossoms, bark, seeds, fruits, and other tissues of a plant. They are valuable com- pounds that are stable at high and low temperature, but can be destroyed soon in alkaline medium or in contact with compounds having a redox potential.

For example, the plants of the Labiatae family are rich in valuable antioxidant compounds like carnosic acid and derivatives thereof like carnosol, in particular Rosmarinus officinalis L. and Salvia officinalis L. are rich in antioxidants. Although carnosol is a derivative of carnosic acid which is built by oxidation, it still has activity as antioxidant. Rosemary comprises hydrophobic antioxidants, like carnosic acid, as well as hydrophilic antioxidants, like rosmarinic acid. These antioxidants are present in rosemary in an amount of about 4 to 30 mg/g plant of carnosic acid (see B below) and derivatives thereof, and in an amount of about 2 to 25 mg/g plant of rosmarinic acid see A below).

Rosemary extracts are used commercially as flavours, antioxidants and oil-soluble preservatives for lipid containing food like meat, poultry, fish, oil, fat, sauce, and gravy. Methods for isolating antioxidants from plants are known, usually antioxidants are obtained either by steam distillation or by solvent extraction. Steam distillation can be used for extracting antioxidants from plants. However, the problem when using steam distillation for extracting antioxidants from plants is the long duration of pre-soaking and distillation, low yield and high cost. Steam distillation requires the use of higher temperature, which requires more energy than methods carried out at room temperature or slightly above. Moreover, no specificity or selective extraction is possible and hydro- philic substances cannot be extracted.

It is well-known to use organic solvents like acetone, ethanol, or hexane, for extraction. However, as the antioxidants shall be used as food or feed additive or in cosmetic preparation, it is necessary to remove any solvent from the extract as most solvents are undesired or even harmful. Moreover, solvent extraction is laborious. Furthermore, nowadays there is a high demand for "green technology", i.e. technology that uses as few resources as possible, where possible endogenous material, most preferable only renewable resources, and does not waste energy. Obtaining an extract by use of organic solvents can also preclude the use of this extract in products that are in contact with human skin, such as in cosmetic products or in food. Moreover, extraction over long time periods can result in most of the extracted product being deteriorated. Therefore, it is desirable to provide a method for obtaining antioxidants from plants with no or hardly any„foreign" or undesirable ingredient, a method that allows extraction of the antioxidants under mild conditions, in short time and with reasonable yield and at the same time results in a product having high quality in and reasonable quantity without wasting any resources.

A number of plants of the family Labiatae are known to contain antioxidants that have very favourable properties. A major percentage thereof is found in the leaves, but also in buds, branches, seeds, fruits, blossoms and other plant tissue, although in a lower amount. The predominant compound is carnosic acid and the derivatives thereof, in particular carnosol. Both are hydrophobic compounds and are found in solvent extracts. Further compounds having antioxidant activity are contained in minor amounts. Rosemary comprises additionally a hydrophilic antioxidant - rosmarinic acid. In US-A 5,859,293 a method for isolating carnosic acid is disclosed, wherein plant parts are extracted with a solvent for up to 36 hours, a base is added to the extract to precipitate un- desired compounds, the supernatant comprising carnosic acid is then subjected to distilla- tion, to remove essential oils, and eventually the solution is acidified to cause carnosic acid to precipitate. A crystallization step can follow for further purification.

Furthermore, Herrero, M., Plaza, M., Cifuentes, A., Ibafiez, E. in Journal of Chromatography A 1217 (16), pp. 2512-2520 http://dx.doi.Org/10.1016/j.chroma.2009.1 1 .032 describe three different processes to obtain antioxidants from plants. Their goal was to improve methods using solvent extraction or extraction using supercritical carbon dioxide and to provide a "green" process. They tested the performance of three different extraction procedures for extracting antioxidants from rosemary (Rosmarinus officinalis L), i.e. pressurized liquid extraction (PLE), using water and ethanol as solvents, supercritical fluid extraction (SFE), using pure supercritical C0₂ and supercritical C0₂ modified with ethanol, as well as a novel extraction process called WEPO (Water Extraction and Precipitation On-line). Extraction time for all procedures was 5 hours, high pressure (between 400 and 100 bar) was used and a temperature of 100 to 200°C. Although the newly proposed method allowed to use water as sole ex- tractant, high pressure, high temperature and long extraction times are necessary with this method.

All these known processes use solvents, need long time periods for extraction and/or are time consuming and laborious. As many other plants, the plants of the family Labiatae comprise fatty acids and their esters, for example myristic acid or myristic acid ester, and only about 0.33 to about 5% of the valuable antioxidants.

It was the object of the present invention to provide a process for obtaining antioxidants from plant substrates, such as rosemary or sage leaves, in an efficient, environmentally friendly, process using mild conditions and to obtain valuable products in a high quantity and quality without using detrimental harmful conditions, and/or undesirable, flammable or combustible solvents. In particular, it was the object of the present invention to provide a method for extraction of natural antioxidants from plant material in short time, using room temperature or slightly increased temperature, ambient pressure and/or endogenous extraction agents. This object is achieved by using a process for isolation of antioxidants as defined in the claims. It has been found that by using soap components, as defined in the claims and below, for extraction, antioxidants can be isolated in short time, in high yield and in excellent quality. Surprisingly it is possible to extract hydrophobic as well as hydrophilic antioxidants from a plant substrate in shorter time and at lower temperature than with the methods known from the prior art, for example at room temperature or slightly increased temperature and within very short time, such as about 1 to about 60 minutes. One explanation for this could be that by using the soap extracting solution of the present invention the cells in the plant parts are broken up; allow easy access for the soap extracting solution, and result in fast release of the desirable compounds. Definitions

In this description the following definitions are used.

"Plant" or "plant parts" refers to any type of plant that comprises compounds active as antiox- idant.

The term "plant substrate" comprises any part of a plant that contains antioxidants such as leaves, buds, needles, blossoms, seeds, fruits, and other plant tissues. The plants or plant parts are harvested as it is known to the skilled person. Plant substrate shall refer to any plant or plant part that has at least some antioxidant content and includes plant powder or plant granules. This term also comprises a plant substrate that has been pretreated, for example by steam distillation to remove at least part of odoriferous agents, but still comprises a major part of antioxidants. "Plant powder" refers to plants or plant parts that have been crushed or comminuted to a size that is useful for extraction. Methods for crushing plant parts are known in the art, cryogenic milling being one example. The method has to be such that the valuable compounds are not destroyed or impaired. Usually the plants or plant parts are prepared before extraction for example by drying or storing under controlled conditions, or are pretreated to remove undesired parts. The plants are comminuted to a size small enough to be extracted and usually plant powders or plant granules are used.

"Antioxidant" refers to any material that hinders, at least partially, or delays oxidation of other compounds, for example natural compounds, such as food, feed, or cosmetic ingredients, like any type of lipids, such as oils, fats, essential oils etc. This term shall comprise any compound found in plants that has an activity as antioxidant.

"Aqueous soap extracting solution" (also referred to as extracting medium) refers to a composition for extracting antioxidants that comprises an aqueous solvent or solvent mixture and dissolved therein at least one soap compound as defined below. The soap extracting solution is a thermodynamically stable solution of one or more soap compounds and optionally one or more hydrotropic additives and/or buffer agents, wherein soap and, if used, any additive molecules are dispersed homogenously and wherein soap molecules form micelles or micelle-like structures, which can also be like liquid crystalline phases and/or wherein soap and additive molecules together act as surfactants. An aqueous soap extracting solution as used for the present invention shall have an alkaline pH value, preferably a slightly alkaline pH value, such as up to about 13, or about 10 or lower. If necessary a buffer can be added to adjust the pH value, for example to not higher than 13 or not higher than 10.

"Aqueous solvent" refers to water or a mixture of water with a minor amount of a water misci- ble or hydrophilic solvent, in particular pure water. A water miscible solvent can be added, but only in an amount that does not interfere with the isolation of the antioxidant.

A "soap compound" is a salt of a fatty acid and a monovalent cation. A soap compound is in particular a salt of a fatty acid and a monovalent cation that in an aqueous medium forms micelle-like structures.

The term "fatty acid" as used in this description refers to an alkyl or aryl carboxylic acid having a linear, branched, or cyclic chain of 4 to 18 carbon atoms, which can carry functional groups, for example up to five functional groups, like hydroxy groups, and can be saturated or mono- or poly-unsatu rated. Examples are linear or branched carboxylic acids or dicarbox- ylic acids having a chain of 4 to 18 carbon atoms, which can be substituted with functional groups like hydroxy or alkyl or aryl groups. The term fatty acid shall also comprise fruit acids like hydroxy carboxylic acids or dicarboxylic acids.

A "monovalent cation" can be any positively charged metal or group, in particular any mono- valent metal or group that is naturally present in plants. Examples are positively charged alkali metal ions, like potassium and sodium, or nitrogen containing charged units like ammonium or cholinium.

"Krafft temperature" (also known as Krafft point, or critical micelle temperature) is the mini- mum temperature at which surfactants form micelles. Below the Krafft temperature micelles cannot form.

"Critical micelle concentration" (CMC) is the concentration of surfactants above which micelles form and all additional surfactants added are integrated to the micelle structure.

The term "endogenous" when used in this application refers to compounds that are found in those plants that are extracted, such as myristic acid, but is not used to imply that a compound referred to as endogenous has to be obtained from this plant. In other words, a compound such as myristic acid is endogenous to plants, but for the method of the present inven- tion any type of myristic acid can be used, whether produced and obtained from a plant or from another origin or synthetically.

It has been found that natural antioxidants can be obtained from plants or plant parts of the Labiatae family by using the following two steps:

a) contacting a plant substrate with an aqueous soap extraction solution and

b) precipitating fatty acid and antioxidant.

Thus, a process for extracting at least one antioxidant from a plant substrate is provided which comprises the steps:

a) contacting a plant substrate with an aqueous soap extraction solution comprising an aqueous medium and at least one soap compound R-C(=0)0-M+ or +M-0(0=)C-R-C(=0)0- M+, wherein R is saturated or unsaturated linear, branched or cyclic C3 to C17 alkyl or C6 to C18 aryl, wherein R can be substituted with alkyl, aryl, at least one functional group such as up to 5 hydroxy groups, and wherein M+ is a monovalent cation, for 1 to 60 minutes to obtain a soap extract and b) adding an organic or anorganic acid to lower the pH value to obtain a precipitate and a supernatant, wherein precipitate and/or supernatant comprise at least one antioxidant from the soap extract obtained in step a). It has been found that hydrophilic antioxidants like rosmarinic acid are nearly completely extracted by the soap extracting medium of the present invention and after lowering the pH value such as by addition of an acid are found in the aqueous supernatant, and that hydrophobic antioxidants like carnosic acid and carnosol are extracted by the soap extracting medium of the present invention in a high amount and after lowering the pH value are found in the precipitate. Both, the hydrophilic antioxidants comprising supernatant and the hydrophobic antioxidant comprising precipitate can be directly used as antioxidant in food, feed and/or cosmetic preparations or can be isolated from or enriched in the precipitate or supernatant, respectively. By using the method of the present invention a very valuable high quality plant extract can be obtained, which comprises natural antioxidant compounds of a plant, in very short time and using "green" substances. Moreover, it has been found that using the method of the present invention can provide hydrophobic as well as hydrophilic antioxidants, that are not available by prior art methods like steam distillation or solvent extraction. Thus, antioxidants from plants can be provided that are useful for aqueous based as well as fat based products.

Parameters that are important for the present method and enable the isolation of antioxidants are the number and type of fatty acids soaps which determine Krafft temperature and CMC, amount of soap, pH value and time period during which the antioxidants are subjected to alkaline conditions, temperature, among others. The skilled person can find the best combination of these parameters with the information provided in the description.

It has been found that by using an aqueous soap extracting solution according to the present invention short extraction times, high yield and an extract of high quality can be achieved because of the mild extraction conditions and in addition specificity can be obtained when necessary. Furthermore, the soap compounds used according to the present inventions are compounds of fatty acids that are naturally in the plant. Therefore, with the method of the present invention it is avoided to include an undesired amount of foreign substances or toxi- cally or environmentally detrimental substances. Mostly or even only naturally occurring compounds are part of the antioxidant composition, which contributes to the high quality of the product.

In one embodiment the soap compounds used are compounds that are based mainly or only on fatty acids and cations that are naturally occurring in plants, or within the plant that is extracted. For example when extracting rosemary or sage leaves, an extraction medium is used which comprises at least a soap of myristic acid, i.e. a fatty acid that is present in these plants and has favourable properties. The method of the present invention comprises two steps which both are important for obtaining the valuable antioxidant extract. The first step is an extraction step wherein antioxidants are extracted from the plant substrate, i.e. plants or parts thereof, by an aqueous soap extracting solution which comprises at least one soap compound dissolved or dispersed in an aqueous medium, such as water. In a second step the extracted antioxidants are recov- ered by acidification.

The starting material for step a) can be any plant material obtained from plants of the family Labiatae. It is preferred to use a plant substrate in particulate form, such as a powder, or granules etc.. The plant material to be extracted can be freshly obtained material or pretreat- ed material. It can for example be a plant material that has been subjected to a pretreatment for removal of odoriferous and other undesired compounds, for example to a steam distillation. In such a pre-step compounds like essential oils are removed that are sometimes undesired when the extracted substance or the extract per are for use as antioxidant. When a pre- step is used, for example a steam-distillation, this step is carried out for a time period to allow to remove a majority of odoriferous and/or other undesired substances but not so long that antioxidants are deteriorated. The best conditions and time period can be found by routine experimentation. The plant substrate can also be from plants that have been dried, frozen, and/or stored before extraction. The extraction step is carried out just by contacting the plant substrate with an aqueous soap extracting solution of the present invention. The duration of the extracting step depends on the plant substrate used, the size of the substrate, the concentration of the soap and the pH value. Good results are obtained when extraction is carried out for about 1 to about 60 min, preferably about 3 to about 30 min. It has been found, that extracting longer than 30 min does not provide more of the desirable compounds, but longer extraction can result in oxidation of the antioxidant. Therefore, contacting times of 3 to 15 min are most preferable.

Surprisingly, the extraction medium of the present invention allows very short extraction cy- cles. It has been found that an extraction period of 30 minutes or less allows to extract all valuable compounds from plant material. For example, if powdered rosemary or sage leaves are used as plant substrate, 3 to 15 minutes of contact with the soap composition is enough to more or less fully extract all valuable components. A very rich antioxidant mixture is obtained if potassium myristate is used. The short extract time and subsequent acidification avoids oxidation or deterioration of the antioxidants.

The size of the plant particles has an influence on the duration and yield of the extraction. A particle size in the range of about 0.1 to about 10 mm, such as about 0.5 to about 5 mm, or 0.8 to 3 mm, has been found useful for efficient extraction. In one embodiment the plant par- tides are in the form of a powder or of granules, which allow highly efficient extraction within contact times of only a few minutes, such as 2 to 1 0 min or even 3 to 7 min are sufficient to extract the valuable compounds.

Depending on the type of soap component the extraction can be carried out at room temper- ature or at slightly increased temperatures. A useful temperature range is 15 to 45°C, preferably 20 to 30°C. The temperature for extraction depends on the Krafft temperature of the soap used for extraction, i.e. on the solubility of the soap compound in the extracting medium. Sometimes a mixture of two or more different soap compounds is useful. Moreover, when the solubility of a fatty acid soap in the aqueous medium is not sufficient, a hydrotropic additive can be added to the solution as described below.

Soap compounds that are useful for the extraction according to the present invention are soaps of fatty acids as defined above, i.e. generally fatty acids, such as saturated or unsaturated linear, branched or cyclic carboxylic acids or dicarboxylic acids wherein the carboxylic acid can be an alkyl or aryl carboxylic acid. The fatty acid can also carry functional groups, in particular hydroxy groups. Fatty acids having one hydroxy group are preferred, up to five hydroxy groups can be present. Furthermore, the chain of the carboxylic can be substituted with alkyl and aryl groups. The counter ion is a monovalent cation, preferably a cation , which is naturally present in the plant to be extracted. Examples for useful cations are sodium, po- tassium, ammonium and cholinium. Any carboxylic acid salt as defined above can be used as soap compound of the present invention as long as it has soap properties, i.e. is dissolved in an aqueous medium and forms micelles or micelle-like structures in an aqueous medium.

The concentration of soap in the extracting solution according to the present invention is at least the CMC, but is preferably higher. A suitable extraction solution comprises at least one soap in an amount of about 2 to about 6 % by weight. It has been found that although for example in the case of sodium myristate the CMC is much lower, i.e. about 0.2 %, an amount of 2 to 6 % by weight results in an increased yield of carnosic acid. Without being bound by theory it is assumed that both the soap and the antioxidant cooperate in forming a micellar structure.

It is one object of the present invention to use endogenous substances as far as possible. Therefore, at least one soap used for extraction preferably comprises one or more fatty acids that are endogenous for the plant to be extracted. Myristic acid is such a fatty acid and, therefore, is a preferred part of the soap. Furthermore, the Krafft temperature of the soap determines the temperature of the extraction. Thus, by choosing a soap having a lower Krafft temperature, the need for using higher temperatures can be avoided. Another option is to use a combination of two or more fatty acids to achieve a convenient temperature range for extraction. Sodium myristate having a Krafft temperature of 45°C requires to apply this tem- perature for extraction and also for this reason is very useful. It was found that there can be a problem if a long-chain soap, for example a Cie-soap, is used as this can result in a hardly compatible mixture that requires higher temperatures to get dissolved. Surprisingly, it was found that this problem can be overcome by adding another soap having a medium chain length which provides for a better solubility of both in water.

It has been surprisingly found that a combination of soaps of differing length can provide an extracting soap solution that can be used at room temperature or slightly higher temperatures, such as below 40°C. The reason is that a combination of soaps of different lengths can have a solubilising effect if they are carefully combined. A ternary composition of short-chain, medium-chain and long-chain fatty acid soaps has been found to lower the dissolution temperature and, therefore, can be considered if a low temperature for extraction is desired.

Another approach to overcome the incompatibility is to use an extracting medium of the present invention that also contains one or more hydrotropic additives to improve the solubility of soap components, to improve extracting properties of the solution or to tailor the extracting properties. In particular, when a long chain fatty acid soap is used as soap component and the solubility is not sufficient at room temperature, the addition of an additive is favorable. The type of hydrotropic additive depends on the soap compound used in the extracting medium and the temperature desired for extraction. If for example the main soap component is a long-chain fatty acid which can be dissolved in the extraction medium only at a higher tem- perature, the additive can be used to lower the dissolution temperature. Hydrotropic additives that are useful in the method of the present invention are in particular compounds that are based on fatty acids or fatty alcohols, preferably fatty acids or alcohols that are naturally occurring in the plant to be extracted. Examples for additives are carboxylic acids, salts and esters thereof for example mono- and dicarboxylic acids having alkyl and/or aryl groups. Useful are for example compounds selected from fatty alcohols, C₆-Ci₂ dicarboxylic acids or salts or esters thereof, C₆-Ci₂ hydroxycarboxylic acids or salts or esters thereof, branched or cyclic C₆-Ci₈ fatty acids or salts or esters thereof or mixtures of any of the mentioned compounds. The counter ion also has an influence on the solubility of the soap component in the extraction system. It has been found that cholinium soap components are particularly useful because cholinium as counter ion increases the solubility of the soap component in the extraction medium. Other counter ions which have a lower basicity are also contemplated. Suitable the soap components that are used in the present extraction medium are sodium or potassi- urn salts of fatty acids.

To accelerate the extraction of plant material and in particular to enhance the extraction efficiency, the contacting step can be assisted by ultrasound. As can be seen in the examples, the use of ultrasound during the contacting step a) can increase the yield of antioxidant.

Another important parameter is the time period of the extraction step and the pH value of the extracting solution. The soap solution used has an alkaline pH-value and carnosic acid is a pH sensitive compound, i.e. it is destroyed significantly at a pH-value above 10 and by contact with basic agents for a longer time. By extracting only for a short time, oxidation and thus loss of yield can be avoided. Therefore, it is preferable to use a soap extracting solution having a pH value below 13, or even below 10 and to carry out the extraction step only for a few minutes. Is has surprisingly been found that carnosic acid is extracted in a high amount within a time period of 1 to 30 minutes when using the extracting solution of the present invention, whereas with the extracting agents of the prior art long time periods were necessary. Moreover, to protect the antioxidants during extraction, the use of buffer agents to lower the pH value can be useful. Another measure, which can additionally or alternatively be used to protect the antioxidants during the extraction is the use of a protective gas, such as nitrogen, to avoid oxidation and, thus, loss for example of carnosic acid. When using myristate as soap in an amount of about 2 to about 6 % for an extraction for 3 to 15 minutes up to 75 % by weight of carnosic acid from plant substrate can be obtained. Furthermore, it has been found that potassium myristate is particularly useful as extractant as an increased yield of carnosic acid can be obtained and the extraction can be carried out at room temperature, i.e. at about 20 to 25°C because of the lower Krafft temperature of potas- sium myristate compared to sodium myristate.

The extraction step provides a solution or dispersion of the extraction medium with the plant material. Before step b) is carried out, optionally the extract solution/dispersion is subjected to a filtration or separation step to remove any solid plant material from the solu- tion/dispersion. Any method for separation can be used such as filtration, centrifugation etc.

The second step of the method of the present invention, i.e. step b) provides for the recovery of antioxidants from the extract obtained in step a). By lowering the pH value of the extraction solution by adding an acid, a precipitate and an aqueous supernatant are obtained, which both can comprise valuable antioxidants.

To recover or isolate desirable compounds from the extract obtained in step a), an acid is added to the solution to precipitate hydrophobic antioxidants, fatty acids are also precipitated. Any acid can be used, such as inorganic and organic acids, but for using green technolo- gy it is preferred to use an acid that is present in organisms, such as formic acid, acetic acid , citric acid, phosphoric acid, or hydrochloric acid. By lowering the pH value of the extract solution, i.e. by acidifying the extract solution, both fatty acid and hydrophobic antioxidants, such as carnosic acid and carnosol, precipitate. Thus, a hydrophobic antioxidant can be obtained in form of a precipitated composition that comprises also a major amount of fatty acid, such as myristic acid. This composition can directly be used as food, feed or cosmetic additive, in particular when it shall be used for lipid containing products.

In other words the at least one hydrophobic antioxidant is isolated by lowering the pH of the extract, thereby breaking the micelles, so that the hydrophobic compounds can migrate to the fatty phase or oily phase whereas watersoluble compounds, such as hydrophilic antioxidants, remain in the aqueous phase.

Without being bound by a theory it is assumed that the soap compounds used according to the present invention wet or coat the antioxidant in the plant substrate and enclose it within micelles. The hydrophobic antioxidants are attracted by the hydrophobic part of the soap, i.e. the fatty acid part or alkyl chain part, and, therefore, end up in the center of the micelle. The lipophobic part of the soap, i.e. the acid part or carboxylic group is disposed such that it forms an outer layer which is in contact with the aqueous solution that is part of the extraction medium.

As soon as the pH value of this medium is changed, i.e. lowered, protons migrate to the carboxylic group of the fatty acid and protonate it. The neutralized fatty acid becomes water insoluble and migrates into the oily phase. Thus, the hydrophobic antioxidant together with the protonated fatty acids separates as oily precipitated phase.

By changing the pH value the micelles break up and phase separation occurs such that the extract medium separates in an aqueous phase and an oily phase. All hydrophobic components migrate to the oily phase, whereas watersoluble parts and hydrophilic compounds mi- grate to the aqueous phase. The most valuable components of the extract, i.e. the antioxidants separate, the hydrophobic ones migrate together with the fatty acids into the oily phase and can be separated together with the oily phase. Thereby, an antioxidant composition can be isolated wherein the antioxidants are embedded in an oily phase which is formed primarily by the fatty acids which have been added as soap components and fatty acids which are native part of the plant substrate.

When using rosemary as plant material to be extracted, the supernatant obtained in step a) comprises hydrophilic antioxidant, in particular rosmarinic acid. Rosmarinic acid is extracted by the soap extraction medium of the present invention in a high amount up to nearly quanti- tatively and will remain in solution when the extract is acidified. Thus, hydrophilic antioxidant can be isolated directly from the supernatant. Alternatively, the supernatant can be directly used as additive for food, feedstuff or cosmetic preparations. For example, supernatant having antioxidant activity can be used as an alternative to conventional antioxidants, like ascorbic acid, in beverages. By using those soap compounds that are derived from fatty acids that are naturally in the plant or plant substrate to be extracted, the obtained mixture will be comprised only of those compounds that are naturally part of the antioxidant containing plant. A further option to isolate the antioxidant from the soap extract is the use of an organic solvent or an organic solvent mixture to enrich desirable compounds from the extract. Organic solvents that are useful for this approach are known and can be those solvents that are used for extraction methods known in the prior art. A solvent extract obtained by extracting the soap extract of the present invention differs from a solvent extract, obtained by a method known in the prior art, because in the soap extract the valuable antioxidants have been enriched and undesirable components are not or hardly present in the soap extract and, thus, cannot be extracted by the solvent.

The method of the present invention is very valuable because it is possible to tailor the ex- tract obtained from the plant substrate. By varying the soap components used for extraction the temperature and pH value can be controlled.

Optionally, in a further step carnosic acid can be enriched from the mixture with fatty acid as obtained after step b). In this embodiment the precipitated composition comprising the fatty acid and carnosic acid and carnosol is dissolved in a solvent, preferably an alcohol such as ethanol, if necessary with slight warming, until fatty acids as well as antioxidants are dissolved. The solution is then cooled down to the critical temperature of the fatty acid, such that the fatty acid is precipitated and can be removed, whereas the antioxidant is still in dissolved form. For myristic acid cooling to -20°C or less is a preferable step. Thereby the anti- oxidant can be enriched. After precipitation of the fatty acid the antioxidant can be isolated by removing the solvent, for example by rotating or distilling. This additional step also removes remaining flavourant that is undesired.

Any precipitated composition that has been obtained as described above, can be used either directly or after enrichment of the antioxidant by one or more solvent/cooling steps, as antioxidant preparation. These preparations are particularly useful as food, feed, and cosmetic additive.

Thus, a further aspect of the present invention is an antioxidant composition comrpising at least one fatty acid, such as myristic acid, and at least one antioxidant, such as carnosic acid and/or carnosol. Such a composition can be obtained as described above. Thus, the antioxidant composition can be a composition obtainable with the method of the present invention. The antioxidant composition can be a composition comprising compounds, in particular carnosic acid and/or carnosol, isolated from Labiatae plant substrates, in combination with myr- sitic acid. For example, an antioxidant composition of the present invention can be the product obtained by extracting a plant substrate with at least one soap compound to obtain a soap extract and by adding an acid to the soap extract to obtain a precipitate and a supernatant, as described above, or it can be the precipitate or the supernatant. Thus, the antioxidant composition can be the soap extract after precipitation, the precipitate, the supernatant or a mixture thereof.

For example a hydrophobic antioxidant can be obtained in form of a precipitated composition that comprises also a major amount of fatty acid, such as myristic acid. A hydrophilic antioxidant an be obtained in the supernatant of the precipitating solution. Thus, a composition comprising the precipitate and optionally the supernatant can directly be used as food, feed or cosmetic additive, in particular when it shall be used for lipid containing products.

The inventors found that it is possible to extract water-soluble and water-insoluble antioxidants from plant material, in particular from rosemary leaves, by using an aqueous soap so- lution. A simultaneous extraction of the watersoluble antioxidant rosmarinic acid and of the water-insoluble carnosic acid is possible with a soap solution. Furthermore, it was found that degradation of the compounds with time due to an alkaline pH value can be avoided with the method of the present invention, as it is possible to decrease the extraction time to about 1 to 10 minutes. This is a great improvement compared to common extraction methods. As can be seen in the experiments that are described in the following examples, the method of the present invention allows to extract rosmarinic acid exhaustively from rosemary leaves, and to recover a high amount of the total carnosic acid content. By adjusting extraction period and by optionally using ultrasound assisted extraction and/or an adjusted particle size of the rosemary leaves the extraction yield of carnosic acid can be further increased. In summary, a method is provided to obtain plant antioxidants in short time, under mild conditions, using natural components and, thus, in an environmentally friendly manner.

Some embodiments of the present invention are further explained in the following examples and in the Figures, wherein Figure 1 shows HPLC chromatograms of a sodium myristate and acetone extract. Peak identification: (1 ) rosmarinic acid; (2) carnosol; (3) gemfibrozil (IS); (4) carnosic acid;

Figure 2 shows mass concentration of rosmarinic acid and carnosic acid yields obtained by Soxhlet extraction lasting 4 h with three different solvents: water, methanol and acetone. Concentrations are given in mg of antioxidant per 1 g of rosemary leaves

Figure 3 shows the mass concentration of rosmarinic acid and carnosic acid yield obtained by micellar extraction for different durations. A 3 wt% solution of sodium myristate was used for every extraction.

Figure 4 shows the mass concentration of rosmarinic acid and carnosic acid yields obtained by micellar extraction with varying concentrations of sodium myristate. Extraction time was always 5 min at 45 °C stirred in a water bath.

Figure 5 shows the influence of pH value on the extraction yield of the antioxidants rosmarinic acid and carnosic acid. Extractions were carried out within 5 min at 45 °C stirred in a water bath. Figure 6 shows an overview of different investigated surfactants and the corresponding extraction yield of rosmarinic acid and carnosic acid. For the experiments a 4 wt% solution of sodium myristate (NaC14), a technical sodium myristate solution (NaC14 (techn.)), potassium myristate (KC14), and potassium stearate (KC18) was used. All extractions were carried out for 5 min and 45 °C in an ultrasonic bath.

Figure 7 shows the influence of the rosemary leaves particle size and ultrasound-assisted (US) extraction on the extraction yield of antioxidants. All extractions were carried with sodium myristate solutions (3 wt%) for 5 min at 45 °C. Figure 8 shows the influence of pH value on the extraction yield of the antioxidants rosmarinic acid and carnosic acid. Extractions were carried out within 5 min at 45 °C stirred in a water bath. Example 1

It was tested if carnosic acid could be recovered selectively from rosemary leaves by an extraction with aqueous sodium myristate solutions. For this reason, extractions of normal rosemary leaves were realized with a 4 wt% sodium myristate solution in an ultrasonic bath for 5 min at 45 °C. The suspensions were subsequently centrifuged for 10 min. A defined volume of the supernatant was acidified with formic acid. The low pH value of the aqueous solution results in the precipitation of a white solid. The suspension was filtrated, obtaining a brown solution and a white solid. The precipitate was dried for 1 h in the compartment drier at 40 °C. Also, extractions with acetone were carried out to compare the selectivity of both solvents. To this purpose, normal rosemary leaves were extracted with acetone in an ultrasonic bath for 5 min at 45 °C. The solvent was evaporated under a nitrogen stream after the filtration of the suspension, obtaining a green solid. The white solid and brown solutions of the micellar extraction, as well as the green solid obtained by acetone extraction were ana- lyzed by HPLC/UV.The results are shown in Figure 1 .

Example 2

In preparation for experiments the total content of rosmarinic acid and carnosic acid in leaves of rosemary was determined. Dried rosemary (Rosmarinus officinalis L.) leaves were obtained from the company Phytotagante/France. The plants were cultivated in Morocco and dried there in the sun after harvest.

To determine the total content of rosmarinic acid and carnosic acid in the leaves, various Soxhiet extractions were carried out. About 6 g of ground rosemary leaves were extracted for

4 h with approximately 50 mL of solvent. Three different solvents were tested: water (milli- pore), methanol (HPLC-grade, Merck) and acetone (p. a., Merck). After extraction, the volume of the extract was readjusted at room temperature to 50 mL with the corresponding solvent.

Then 0.5 mL of the extract solution was mixed with 0.5 mL of methanol (90%) and afterwards 1 mL of the internal standard solution (see below) was added. The solution was filtrated through a 0.2 μηη PTFE-syringe filter and then analyzed by HPLC/UV. All extractions were carried out in triplicate.

Different concentrations (1 , 2, 3, 4, 5 wt%) of aqueous sodium myristate solutions were pre- pared. For this purpose, sodium myristate (99%, Sigma-Aldrich) was weighed in a lockable glass envelope and the appropriate amount of water was added to obtain a total weight of 5 g. The mixture was stirred at 45 °C in an oil bath to dissolve sodium myristate. After a clear solution was obtained, about 0.25 g of ground rosemary leaves were added and the mixture was put in the oil bath at 45 °C again. A control sample was prepared in the same way, but with a 0.1 N NaOH-solution (Merck) instead of sodium myristate as aqueous extraction solvent. At 5, 30, and 60 minutes samples were taken, each sample, in the form of a suspension, was centrifuged at 4000 rpm for 10 min. Then 500 μΙ_ of the supernatant were taken, acidified with 100 μΙ_ formic acid, and 500 μΙ_ of the internal standard solution (see below) was added. The solution was filtrated through a 0.2 μηη PTFE-syringe filter and then ana- lyzed by HPLC/UV. All extractions were carried out three times.

HPLC/UV analysis

The contents of rosmarinic acid (RAc) and carnosic acid (CAc) in the extracts were determined by HPLC/UV. The analyses were performed on a "Waters HPLC System" with two Waters 515 HPLC Pumps, Waters 717plus Autosampler and Waters 2487 UV/VIS-Detector. Separation was achieved on Knauer Eurosphere C18-column (100 A, 250 x 4.6 mm). The injection volume was each 10 μί. The compounds were eluted at a flow rate of 1 .0 mL/min and a temperature of 30 °C. The solvents for gradient HPLC consisted of 0.1 % formic acid (A) and acetonitrile (B) (Merck, HPLC-grade). The composition of the mobile phase started at 10% B, it was increased to 40% B 4 within 40 min, further increased to 100% B within 20 min and hold then for 20 min. The detection wavelength was 204 nm. Analysis of each sample was carried out three times.

The content of the antioxidants (AO) was determined quantitatively by internal standard (IS) calibration. To this purpose, stock solutions (1 mg/mL) of rosmarinic acid (99%, Sigma- Aldrich) and carnosic acid (99%, Phytolab) in methanol (90%) were prepared. These primary stock solutions were diluted to concentrations of 1 .0, 0.8, 0.6, 0.4, 0.2 mg/mL. To 1 mL of each sample, 1 mL of a 1 mg/mL solution of the internal standard gemfibrozil (98%, Cayman) was added. The solutions were filtrated through 0.2 μηη PTFE-syringe filters and then meas- ured by HPLC/UV. The detection wavelength in further measurements was set at 204 nm. The concentration of gemfibrozil in all samples was kept constant at 0.5 mg/mL. The response factor of gemfibrozil and both antioxidants is determined by plotting the ratio of the antioxidant peak area and gemfibrozil area against the ratio of antioxidant concentration and gemfibrozil concentration and a linear trend was observed. All samples were analyzed three times. Afterwards, the response factor K was calculated, which is K(CAc) = 1 .36 for carnosic acid and K(RAc) = 0.84 for rosmarinic acid. For the analysis of the extracts, a gemfibrozil solution (1 mg/mL) was added to every sample and the concentration of the antioxidants was estimated with the response factors.

Soxhlet extraction is an exhaustive method to determine the total amount of antioxidants. Rosmarinic acid is soluble in water, whereas carnosic acid is not. Figure 2 shows the three different investigated solvents for Soxhlet extractions. As expected, water, a polar protic solvent, can extract the highest amount of rosmarinic acid with 8.90 mg per 1 g rosemary, but only a negligible amount of carnosic acid. The extraction behaviour of the aprotic polar solvent acetone is vice versa and the highest amount of carnosic acid with 23.62 mg/g can be extracted. Methanol is also a protic solvent, but less polar than water. It combines the extraction efficiency of both, water and acetone. Therefore, methanol is a suitable solvent for Soxhlet extraction to determine the total amount of both antioxidants, rosmarinic acid and carnosic acid, from the rosemary leaves.

It was found that the extraction conditions, in particular pH value and procedure have a negative influence on the desired antioxidants. According to that, the extraction method had to be adjusted. After extraction and centrifugation, 500 μΙ_ of the supernatant were taken and im- mediately acidified with 100 μΙ_ formic acid to decrease the pH value. This results in the precipitation of myristic acid and carnosic acid, which are both insoluble in water at room temperature. Therefore, 500 μΙ_ of the internal standard in methanol (90%) was added to ensure the complete solubilization of the antioxidants. This adapted procedure was used for all further experiments.

Example 3

The influence of extraction time was evaluated. The extraction time has a strong influence on the antioxidant yield. For all experiments, 3 wt% solutions of sodium myristate were used. It was observed that the colorless solution immediately turned to green after the addition of rosemary. After a few minutes, the mixtures became brown and with increasing extraction time steadily darker. The extraction time refers to the maceration time at 45 °C in the oil bath. It has to be mentioned that extraction is not promptly stopped because of the subsequent centrifugation which lasts additionally 10 min. Figure 3 shows the influence of time on the extraction yield of the antioxidants. The recovery of rosmarinic acid is almost complete after 5 min of extraction. It only slightly increases within 30 min, and an amount of 10.33 mg rosmarinic acid per 1 g of rosemary can be determined. A longer extraction time does not influence the yield significantly. By contrast, carnosic acid shows a different behaviour. The highest amount of 9.94 mg/g can be extracted after 5 min, which is 42% of the total content in the leaves. With longer extraction times, the yield of carnosic acid decreases significantly to 4.67 mg/g after 60 min, which is due to the decomposition of the antioxidant generated by the alkaline pH value. After 4 h of extraction carnosic acid is no longer detectable. In summary, in this test an extraction time of 5 min was found very useful. Figure 3 shows mass concentration of rosmarinic acid and carnosic acid yield obtained by micellar extraction for different durations. A 3 wt% solution of sodium myristate was used for every extraction.

Example 4

The influence of concentration of soap in the extraction medium was evaluated. Besides the extraction time, also the concentration of sodium myristate solution influences the yield of antioxidants significantly. The extraction time for these experiments was always 5 min. Also a control sample was prepared with a NaOH solution and a pH value of 10.

Figure 4 shows the influence of the sodium myristate concentration on the extraction yield of rosmarinic acid and carnosic acid. It can be seen that the yield of rosmarinic acid rises with increasing concentration of sodium myristate. A nearly exhaustive recovery of 8.97 mg rosmarinic acid per 1 g of rosemary leaves can be achieved with a 4 wt% aqueous solution of sodium myristate within 5 min. Carnosic acid shows quite the same behaviour. The yield of carnosic acid increases with rising soap concentration. The maximum amount which can be extracted with a 4 wt% sodium myristate solution is 12.57 mg carnosic acid per 1 g rosemary. This is about 53% of the total carnosic acid amount in the leaves. The extraction of both antioxidants, the water-soluble rosmarinic acid and the water insoluble carnosic acid, might be related to the formation of structures, such as formation of mixed micelles (soap plus antioxidants) in the soap solution. It is assumed, that this is the reason why a concentration higher than the critical micellar concentration (cmc) of sodium myristate, which is about 0.1 wt%, is more useful. The control sample with sodium hydroxide as extraction solvent showed an unexpected result. Here, about 4.04 mg/g of the commonly non water-soluble carnosic acid can be extracted. This is 20 times the amount, which can be extracted with pure water. On the one hand the high pH value enhances the extraction efficiency, but on the other hand the yield of the antioxidants decreases over time.

Example 5

The influence of pH value and type of base was evaluated.

The previous results showed that the pH value of the sodium myristate influences the stability of the antioxidants. For this reason it has been investigated if the high pH value (9.7) of the micellar soap solution has a significant influence on the extraction yield of the antioxidants, especially carnosic acid. To this purpose, extractions of grinded rosemary leaves were carried out with sodium hydroxide solutions with pH values of 9, 1 1 , 13, and 14. The experi- ments were also carried out for 5 min at 45 °C to make them comparable to the micellar extraction. Right after the addition of rosemary leaves to the sodium hydroxide solution, the first difference can be observed. The small rosemary particles swim initially at the water surface and the color of the solutions turns from yellow to brown. The color of the sodium hydroxide extract solution is also getting darker with rising pH. This is in contrast to the sodium myristate solutions, where the rosemary leaves are immediately in solution and the color becomes green. Furthermore, sodium hydroxide solutions form stable foams after 5 min of extraction. Also, a solid precipitates after the neutralization of the extract solution with formic acid. The extracts were also analyzed by HPLC/UV to determine the exact extraction yield of the antioxidants. The influence of the pH value on the extracted concentration of rosmarinic acid and carnosic acid is plotted in Figure 5. The diagram shows that the extraction yield of rosmarinic acid increases with rising pH until a value of 13 is reached. In contrast at higher pH values the yield decreases slightly. In turn, the extraction yield of carnosic acid shows an unexpected trend. The extracted concentration increases significantly at pH values higher than 1 1 . For instance, at a pH value of 13, about 4.04 mg/g of the commonly non-water- soluble carnosic acid can be extracted. This is 20 times the amount, which can be extracted with pure water. The extraction yield can be increased up to 7.68 mg/g at a pH value of 14. Figures 5 and 8 show the influence of pH value on the extraction yield of the antioxidants rosmarinic acid and carnosic acid. Extractions were carried out within 5 min at 45 °C stirred in a water bath. The macroscopic observations and the HPLC results indicate again that the alkaline pH value of the extraction solution results in a certain transformation of the com- pound. A possible explanation can be the formation of carnosic acid to carnosate at high pH. This molecule has a strong similarity to the cholic acid respectively the salt molecule cholate. Sodium cholate, a bile salt, is soluble in water. Above the cmc of 15 mM, cholate can form micelles in solution. Thus, it seems that carnosate is soluble in water and can form micelles. These micelles are able to extract and solubilize other compounds. This behavior is also described for bile acids and their salts. The solubility of a bile acid can be enhanced by the addition of a bile salt above the cmc. After the neutralization with an acid, carnosic acid is formed. The compound is not anymore soluble in the aqueous solution and finally precipitates as a solid. In addition, the formation of choline salts of phenolic acids was reported in literature. The solubility of these choline-based salts in water is three orders of magnitude higher than the one of the respective acidic precursors]. For this reason, extractions with choline hydroxide were carried out for 5 min at 45 °C. It is assumed that the extraction with choline hydroxide leads to the formation of choline carnosate and hence increases the yield of carnosic acid. The behavior of the extraction solution after the addition of rosemary leaves is quite similar to the sodium hydroxide samples. Only the color of the solution becomes darker brown and it seems to be more viscous. The extract also shows foam formation. The extraction yield of both antioxidants is higher with choline hydroxide than sodium hydroxide at a pH value of 14. In detail, 4.68 mg/g of rosmarinic acid and 10.31 mg/g of carnosic acid can be extracted with choline hydroxide. These higher values can indicate the formation of choline carnosate. But the yields with bases as extraction solvent are still below the values of micellar extraction with sodium myristate. On the one hand, the high pH value enhances the extraction efficiency, but on the other hand, the yield of the antioxidants decreases over time. In summary, the extraction process is somewhat dependent on pH, but the extraction yield is significantly influenced by the sodium myristate concentration and the duration.

Example 6

Due to the fact that especially carnosic acid cannot be extracted exhaustively with aqueous sodium myristate solutions, alternative surfactants respectively soaps were investigated. Ex- periments were carried out with potassium myristate (KC14), potassium stearate (KC18), and a self-prepared sodium myristate solution (NaC14 (techn.)). Sodium myristate can be easily produced by adding an appropriate amount of sodium hydroxide to an aqueous myristic acid solution at 60 °C. The reaction is completed at the point when a homogenous clear solution is obtained. The pH value of a 4 wt% solution was measured to be 9.5. Potassium stearate was obtained from the company Stearinerie Dubois. The purity of the fine white powder is 97.5%. An advantage of this compound is the lower prize compared to sodium myristate. The potassium stearate as used in this example is a technical mixture of potassium salts of saturated fatty acids myristic acid (C14), palmitic acid (C16), and stearinic acid (C18). The white powder contains 60% potassium stearate and 30% salts of the other fatty acids. The trade name of this product is Ligastar KA M from the company Peter Greven. In addition, further extractions were carried out with sodium stearate, but the soap is not soluble in water at 45 °C. A clear homogenous solution can be obtained only at more than 65 °C. This high temperature can accelerate the decomposition of the antioxidants. For this reason, stearate is not preferred for the extraction of rosemary leaves. The results are shown in Figure 6.

Example 7

The influence of particle size and ultrasound-assisted extraction was evaluated. In order to increase the extraction yield of the antioxidants the influence of the particle size was tested. Therefore, a fine and homogenous powder of rosemary leaves was prepared by grinding the leaves in liquid nitrogen. Additionally, the influence of ultrasound-assisted extraction was investigated. It is expected that ultrasound enhances the extraction efficiency by accelerating the extraction equilibration. Therefore, the samples were put in the ultrasonic bath for 5 min at 45 °C. Sodium myristate solutions with a concentration of 3 wt% were used for all these experiments.

Figure 7 shows the influence of the particle size of the rosemary leaves and the influence of ultrasound-assisted extraction on the extraction yield of rosmarinic acid and carnosic acid. It is illustrated that the extraction efficiency of rosmarinic acid is more or less neither influenced by the particle size nor by ultrasound-assisted extraction. The yield of carnosic acid is more influenced by these factors. If normal grinded rosemary leaves are used, ultrasound can increase the extraction yield for carnosic acid from 9.55 mg/g up to 13.44 mg/g. A smaller particle size of the rosemary leaves actually decreases the extracted amount of carnosic acid. Ultrasound-assisted extraction of the fine powder does not significantly change the extraction yield. This behavior can possibly explained by the fact that carnosic acid is more adsorbed on the rosemary leaves surface due to the smaller particle size. For this reason, the mass transfer kinetics of the antioxidant to the solvent is slowed down. In summary, normally grinded leaves can be used to extract rosemary with aqueous sodium myristate solutions. Ultrasound-assisted extraction can be used to improve extraction.

Claims

Claims

Process for extraction of at least one antioxidant from a plant substrate comprising

a) contacting a plant substrate with an aqueous soap extraction solution comprising an aqueous medium and at least one soap compound R-C(=0)0^"M⁺ or ⁺MO(0=)C-R-C(=0)0^"M⁺, wherein R is saturated or unsaturated linear, branched or cyclic C₃ to Ci₇ alkyl or C₆ to Ci₈ aryl, wherein the alkyl or aryl group can be substituted with up to 5 functional groups, and wherein M⁺ is a monovalent cation, for 1 to 60 minutes to obtain a soap extract, and

b) adding an organic or inorganic acid to lower the pH value to obtain a precipitate and a supernatant, wherein precipitate and/or supernatant comprise at least one antioxidant from the soap extract obtained in step a).

Process according to claim 1 , wherein the precipitate comprises at least one hydrophobic antioxidant and/or the supernatant comprises at least one hydrophilic antioxidant.

Process according to claim 1 or 2, wherein the plant substrate comprises leaves, buds, needles, blossoms, seeds, fruits, or other plant tissue, or a mixture thereof, from plants of the Labiatae family.

Process according to one of the preceding claims, wherein the plant substrate comprises leaves of rosemary, sage, thyme, oregano, and/or mixtures thereof; and/or wherein the substrate is in particulate, crushed or powdered form.

Process according to one of the preceding claims, wherein the antioxidant comprises carnosic acid, carnosol or any mixture thereof.

Process according to one of the preceding claims, wherein M⁺ is sodium, potassium, ammonium or cholinium.

Process according to one of the preceding claims, wherein the aqueous soap dispersion comprises at least one soap compound R-C(=0)0^"M⁺, wherein R is CH₃(CH₂)n, wherein n is 2 to 16 and wherein M⁺ is a monovalent cation.

8. Process according to one of the preceding claims, wherein step a) is carried out at Krafft temperature of the soap or above.

9. Process according to one of the preceding claims, wherein the aqueous soap extracting solution of step a) comprises at least water, sodium myristate and/or potassium myristate, wherein optionally the concentration of sodium myristate and/or potassium myristate is in a range between 2 and 6 % by weight.

10. Process according to one of the preceding claims, wherein the extraction step is carried out for 1 to 30 minutes.

1 1 . Process according to one of the preceding claims, wherein in step b) an acid for lowering the pH value is selected from formic acid, acetic acid, citric acid, phosphoric acid, or hydrochloric acid.

12. Process according to one of the preceding claims, comprising an additional step c) for enriching antioxidant by

c) dissolving the precipitate obtained in step b) in a solvent selected from an alcohol, ketone or alkane, and cooling the solution to a critical temperature of the soap to precipitate at least part of the fatty acid and thereby enriching the antioxidant.

13. Process according to one of the preceding claims, wherein an aqueous soap extracting solution comprising soaps of those fatty acids that are present in in the plant substrate is used.

14. Solid antioxidant composition comprising myristic acid and carnosic acid and/or carnosol.

15. Use of a composition comprising myristic acid and carnosic acid and/or carnosol as main ingredients, as obtained in a process according to one of claims 1 to 13, as antioxidant in food, feedstuff, or a cosmetic preparation.