FR2691974A1 - Sepg. non-saponifiable(s) from plant oils - by enzymatic hydrolysis of tri:glyceride(s) solvent extn. and anion exchange treatment to recover prod. in natural, esterified form, useful in cosmetics and pharmaceuticals - Google Patents

Abstract

Sepn. of a plant-based oil, fatty material or lipid extract, consisting mainly of triglycerides (TG) and a small amt. of non-saponifiables (A) comprises (1) subjecting the material to enzymatic hydrolysis by adding it to an aq. suspension of TG-specific lipase, and stirring the mixt. for as long as required to give the desired yield, (2) adding organic solvent, (3) sepg. the aq. phase (enzymes and glycerol) from the organic phase (fatty acids from TG and (A)), (4) extracting fatty acids by retention on an ion-exchange resin and (5) recovering (A) from the eluate. Also new is (A) prepd. by this process. USE/ADVANTAGE - (A), consisting of sterols, terpene alcohols, tocopherols and aliphatic alcohols, is useful in topical cosmetics and human or veterinary pharmacy, generally incorporated at 0.1-5 wt.%. This method recovers components of (A) in their natural, i.e. esterified forms.

Description

FRACTION SEPARATION PROCESS
UNSAPONIFIABLE FREE OR ESTERIFIED OF AN OIL, A
FATTY BODY OR AN ORIGINAL LIPID EXTRACT
PLANT, INSULATED PRODUCT THUS OBTAINED AND
COMPOSITIONS CONTAINING IT.

 The present invention relates to a separation process without saponification of the non-glyceridic minor constituents which do not react with a base to give a soap and remain soluble in solvents, called "unsaponifiable fraction", contained in an oil, a fatty substance or an extract. lipid of vegetable origin, in particular a vegetable oil; it also relates to the isolated product thus obtained and the compositions containing said product.

 It is known that oils and natural fatty substances are mainly constituted by tri-esters of fatty acids and glycerol but also contain, in a smaller amount, constituents such as hydrocarbons and hydroxylated compounds other than glycerol, these compounds being partly in esterified form with fatty acids and partly in free form. All of the compounds other than the simple, partial or complex triglycerides contained in the oils or fatty substances are generally designated by the name of "unsaponifiable" or of "unsaponifiable fraction". This unsaponifiable material generally represents up to 1.5% by weight of the natural oil or fatty substance, but may reach higher contents, for example 7%, in certain oils or fatty substances such as avocado oils, butter. shea and egg oils.

 The hydroxylated compounds contained in the unsaponifiable material are sterols, terpene alcohols, tocopherols and alphatic alcohols.

 The composition of the unsaponifiable material is characteristic of a specific oil or fatty substance. This composition is characteristic as a whole, that is to say that the nature, the number and the relative proportions of the constituents present in each of the categories of constituents (hydrocarbons, sterols, terpene alcohols, tocopherols and alphatic alcohols) are characteristic.

For the same category of hydroxylated compounds, for example sterols, the number and nature of the compounds present as well as their relative proportions are also characteristic of each oil or fatty substance.

 Oils and fatty substances are very widely used in cosmetics and in human or veterinary pharmacy, and it has often been hypothesized that their beneficial action was, in large part, due to the presence of hydroxylated compounds other than glycerol, in the form esterified and / or in free form, and that the particularly favorable action of certain oils or fatty substances was due to the particular composition of their unsaponifiable matter. We have therefore sought to isolate the unsaponifiable in order to benefit as much as possible from its favorable effects.

 Unfortunately, the known processes for separating unsaponifiable matter do not make it possible to collect the corresponding hydroxylated compounds in their esterified form. According to a first method, a saponification is carried out by heating the oils or fatty substances with a base, such as potassium hydroxide, and by extracting on the one hand, using an alcoholic solution, the glycerol and the saponified fatty acids. obtained, and, on the other hand, unsaponifiable using a solvent, such as hexane or ether. The unsaponifiable material thus obtained consists of hydroxylated compounds in free form, that is to say that the OH functional group (s) of these constituents is (are) not esterified (s) since by the action of the base being saponified, the esters of the hydroxylated compounds have been split to give rise to the hydroxylated compounds themselves. According to another process for extracting the unsaponifiable matter, the oil or fatty substance is treated with steam entrainment and the entrained compounds are separated from the unsaponifiable matter by decantation. This process is conventionally implemented during refining and more especially during the deodorization of oils or fatty substances; but this steam entrainment only collects the hydroxylated compounds present in free form and not those present in the form of fatty acid esters. Until recent times, unsaponifiable fractions have been used in cosmetic or dermatological compositions in which the hydroxylated compounds were in free form. These compounds have a beneficial action but which generally does not have the nature and amplitude that one could hope in view of the action of natural oils and fatty substances from which this unsaponifiable matter is extracted.

 In French patent application 89-07437, it has been proposed to re-esterify the hydroxylated compounds of the unsaponifiable material extracted in free form and it has been found that this re-esterification leads to an improvement in efficiency compared to the use of the compounds hydroxylated in free form of the unsaponifiable fraction.

These re-esterified hydroxylated compounds, however, do not have exactly the same composition as in the natural unsaponifiable material since the esterification is not carried out with the fatty acids used in the exact proportion of their presence in the oil or fatty substance. natural.

In Japanese patents 55-139119 and 52-041215, compositions have also been described containing the re-esterification product of an unsaponifiable fraction extracted from an oil; in the first of these documents, the re-esterification of a soy oil deodorization distillate is proposed and the esterified fraction contains 40 to 50% of plant sterols, 18 to 22% of tocopherol and approximately 30% of a fatty acid unsaturated; in the second of these documents, a composition is proposed containing a higher fatty acid ester such as palmitic acid, and a sterol of animal or vegetable origin.

 However, in the state of the art, the re-esterification products of the non-breathable fractions extracted are always chemically different from the natural unsaponifiable matter, both by the association of fatty acids and hydroxylated compounds and by the weight distribution of the various esters in the reesterification product.

 The object of the present invention is to describe a process which makes it possible to isolate the unsaponifiable fraction of a natural oil or fatty substance while preserving it in its natural form both in the nature of the esters which it contains and in their weight proportions between them and vis-à-vis the hydroxylated compounds in the free state of the unsaponifiable fraction. The separation of such a product allows, when it is used in cosmetic or pharmaceutical compositions intended for humans or animals, to benefit from all of the favorable effects of these unsaponifiable fractions without any distortion due to modifications subsequent to the extraction process.

 The present invention therefore relates to a process for the separation of the non-glyceridic minor constituents making up the unsaponifiable matter of an oil, of a fatty substance, or of a vegetable lipid extract consisting, for the most part, of triglycerides and , for a small part, of said unsaponifiable, characterized in that the oil, the fatty substance or the lipid extract is subjected to an enzymatic hydrolysis by means of at least one lipase specific for triglycerides, said hydrolysis being carried out by adding an aqueous suspension of enzyme (s) to the oil, the fatty substance or the lipid extract, the mixture obtained being kept under stirring for a time sufficient to obtain the desired yield; that an organic solvent is then added; that one separates, on the one hand, an aqueous phase containing the enzyme (s) and glycerol and, on the other hand, an organic solvent phase containing a hydrolyzed mixture consisting of fatty acids from triglycerides and unprocessed unsaponifiable fraction; and that the fatty acids are extracted from the said hydrolyzed mixture by passage over an ion exchange resin which retains them, the natural unsaponifiable fraction being found in the eluted organic phase.

It is preferred to use, as an enzyme, lipase L-1734 type
VII of Candida cylindracea; the enzymatic hydrolysis is advantageously carried out away from oxygen, since it has been found that the yield of the hydrolysis was affected when the oil or the fatty substance contains peroxides or self-oxidation products: proposes therefore to carry out the enzymatic hydrolysis under a neutral gas sweep, for example under an argon sweep. It is advisable that the hydrolysis be as far as possible so that the unsaponifiable fraction extracted contains the least possible residual triglycerides; to do this, it is advantageous to use a volume ratio (aqueous suspension / oil) of between 1 and 1.5; it is also possible to provide that the weight ratio (enzyme (s) / oil) is between 0.02 and 0.1; in this way, the hydrolysis can be carried out up to a mole yield of at least 85%, and preferably at least 95% with a hydrolysis time of between 2 and 4 days, at a temperature of between 20 and 300 C. In the reports given above the term "oil" was used alone to facilitate the description, but it should be understood that the term "oil" also covers fatty substances and lipid extracts of vegetable origin.

 After the hydrolysis, at least one organic solvent is added to the hydrolysis mixture in a volume ratio (solvent (s) / hydrolysis mixture) of between 2.5 and 4; the organic solvent can advantageously be chosen from the group formed by ethyl oxide and hexane.

 After addition of an organic solvent, the enzyme (s) and the glycerol resulting from the hydrolysis pass into the aqueous phase whereas the fatty acids resulting from the hydrolysis and the unsaponifiable fraction pass into the solvent phase; the two phases are preferably separated by decantation (s) with possible washing (s) with water and with solvent (s) so that the entire unsaponifiable fraction is found in the solvent phase; the solvent is then removed, for example by evaporation under reduced pressure, to obtain the hydrolyzed mixture.

 The fatty acids are separated from the hydrolyzed mixture obtained by passing over an anionic resin, in particular that known under the trade name "DOWEX 1 x 8 (FLUKA)"; the passage over this ion exchange resin is carried out in a column with a solution solvent such as methyl-t. butyl ether.

 The natural unsaponifiable fraction is then separated from the elution solvent by evaporation of the solvent under reduced pressure.

 The separation process according to the invention gives particularly advantageous results when it is applied to oils of vegetable origin such as corn oil, sunflower oil, rapeseed oil, avocado oil, hazelnut oil or the like.

 The subject of the invention is also the natural unsaponifiable fraction of an oil, a fatty substance or a lipid extract of vegetable origin isolated by the process as defined above.

 The invention finally relates to a composition for human or veterinary use, characterized in that it contains, in an appropriate carrier, in an effective amount, a natural unsaponifiable fraction isolated according to the process as defined above. In such a composition, the effective amount of unsaponifiable fraction is generally between 0.1 and 5 5G by weight. Such a composition can advantageously be used, in cosmetics or in human or veterinary pharmacy, by topical application.

 To better understand the invention, we will now describe, by way of example, purely illustrative and not limiting, an embodiment.

EXAMPLE
3.5 g of corn oil, 3.5 g of sunflower oil or 3.5 g of rapeseed oil are successively placed inside a 50 ml two-neck flask and To this is added a homogeneous suspension of 80.3 mg of enzyme and 4.8 ml of water. The enzyme is Candida cylindracea lipase L-1734 type VII (SIGMA). The reagents are mixed gently by magnetic stirring, the flask being connected to a stream of argon. The temperature is maintained at 250 C.

The hydrolysis reaction is stopped after 72 hours by adding 30 ml of ethyl oxide, stirring being maintained for a few minutes. The whole is poured into a 100 ml separating funnel. After decantation, the aqueous phase is removed, which contains the enzyme and glycerol. Two extractions of this aqueous phase are repeated using each time 20 ml of ethyl oxide so as to extract practically all of the hydrolysed mixture consisting of fatty acids and the unsaponifiable fraction. The ethereal phases are combined and washed twice with 5 ml of water. We dry on
Na2SO4. The organic phase is filtered on filter paper and then subjected to evaporation under reduced pressure. The hydrolyzed mixture is thus obtained.

 The hydrolysis rate was measured by measuring the fatty acids by a titrimetric method. To do this, 150 mg of hydrolyzed mixture dissolved in 10 ml of ethanol are introduced into a 100 ml Erlenmeyer flask. A few drops of phenolphthalein are added, then a 0.1N potassium hydroxide solution is added dropwise until the color changes. When the hydrolysis is carried out as indicated above, it can be seen that, for corn oil, the hydrolysis rate is 96.4%, for sunflower oil 97.5% and for 98.2% rapeseed.

 In a subsequent step, the fatty acids are separated from the hydrolyzed mixture. To do this, a column 25 mm in diameter and 50 cm high is used which is filled with 100 g of an anionic resin sold under the trade name "DOWEX I x 8 (FLUKA)". The resin particles have a particle size corresponding to the retention between two sieves having respectively 20 and 50 meshes for 2.54 cm.

This resin has, per gram of dry resin, 3.3 milliequivalents (groups - (R) 3N + Cl-). Before being introduced into the column, the 100 g of resin are placed in a 2 liter erlenmeyer flask to which 750 ml of 2N aqueous potassium solution are added. The whole is stirred magnetically for 15 hours then is poured into the column.

The column is then washed neutrally with water; then the water is displaced by washing with methyl-t.butyl-ether (MTBE).

 1.5 g of hydrolyzed mixture are weighed and diluted in 3 ml of MTBE. The solution is poured into the top of the column and eluted with 300 ml of MTBE. The recovered solution is washed with sodium hydrogen carbonate to remove all traces of acid and then with water until neutral. After drying over Na2SO4, the eluent is filtered on filter paper and the MTBE is evaporated under reduced pressure. The natural unsaponifiable fraction is thus obtained.

To extract, if desired, the fatty acids retained on the resin, poured into the column head as eluent 300 ml of hydrochloric methanol; the flow is very slow to allow a good action of the eluent. The alcoholic phases recovered are brought to the boil for 30 minutes. After dilution with a solution of
Saturated NaCl (2 volumes for 1 volume of alcohol), the aqueous phase is extracted 3 times with 200 ml of hexane. The organic phases are combined, washed with water and then with neutral sodium carbonate. There is no emulsion, the fatty acids being all methylated. After washing with neutrality, the hexane phase is dried over neutral sodium sulfate, filtered through filter paper and evaporated under reduced pressure.

 The resin is regenerated by eluting with water to remove any trace of acid. After which it is eluted with 300 ml of 2N potassium hydroxide and then washed with water until neutral and the column is again ready for use as previously indicated.

Claims (19)

 1 - Process for the separation of an oil, a fatty substance or a vegetable lipid extract consisting, for the most part, of triglycerides and, for a small part, of said unsaponifiable matter, characterized in that one subjects the oil, fatty substance or lipid extract for enzymatic hydrolysis by means of at least one lipase specific for triglycerides, said hydrolysis being carried out by adding to the oil, the fatty substance or the lipid extract of a suspension aqueous enzyme (s), the mixture obtained being maintained, with stirring, for a time sufficient to obtain the desired yield; that an organic solvent is then added; that one separates, on the one hand, an aqueous phase containing the enzyme (s) and glycerol and, on the other hand, an organic solvent phase containing a hydrolyzed mixture consisting of fatty acids derived from triglycerides and the unprocessed unsaponifiable fraction; and that the fatty acids are extracted from the said hydrolyzed mixture by passage over an ion exchange resin which retains them, the natural unsaponifiable fraction being found in the eluted organic phase.  2 - Method according to claim 1, characterized in that one uses, as an enzyme, lipase L-1734 type VII of Candida cylindraceae.  3 - Method according to one of claims 1 or 2, characterized in that the enzymatic hydrolysis is carried out away from oxygen.  4 - Process according to claim 3, characterized in that the enzymatic hydrolysis is carried out under a neutral gas sweep.  5 - Method according to one of claims 1 to 4, characterized in that one chooses a volume ratio (aqueous suspension / oil) during the enzymatic hydrolysis between 1 and 1.5.  6 - Method according to one of claims 1 to 5, characterized in that one chooses a weight ratio (enzyme (s) / oil) between 0.02 and 0.1.  7 - Method according to one of claims 1 to 6, characterized in that the hydrolysis is carried out to a yield in moles of at least 85% and, preferably, at least 95%.  8 - Method according to one of claims 1 to 7, characterized in that the hydrolysis is maintained for a time between 2 and 4 days, at a temperature between 20 and 30 "C.  9 - Method according to one of claims 1 to 8, characterized in that after hydrolysis, at least one organic solvent is added in a volume ratio (solvent (s) / hydrolysis mixture) between 2, 5 and 4.  10 - Process according to claim 9, characterized in that the organic solvent is chosen from the group formed by ethyl oxide, hexane, and chloroform.  11 - Method according to one of claims 1 to 10, characterized in that the separation is carried out (aqueous phase / organic solvent phase) by decantation (s) with possible (s) washing (s) with water and solvent (s), the solvent then being removed to obtain the hydrolyzed mixture.  12 - Method according to one of claims 1 to 11, characterized in that the fatty acids are extracted during the hydrolysed mixture by passage over an anionic resin.  13 - Method according to one of claims 1 to 12, characterized in that one carries out the passage over an ion exchange resin in a column with an elution solvent.  14 - Method according to one of claims 1 to 13, characterized in that the natural unsaponifiable fraction is separated from the elution solvent by evaporation under reduced pressure.  15 - Method according to one of claims 1 to 14, characterized in that the oil is an oil of vegetable origin.  16- Natural unsaponifiable fraction of an oil, a fatty substance or a lipid extract of vegetable origin isolated by the process according to one of claims 1 to 15.  17- Composition for human or veterinary use, characterized in that it contains, in an appropriate carrier, in an effective amount, at least a fraction according to claim 16.  18 - Composition according to claim 17, characterized in that the effective amount is between 0.1 and 5% by weight.  19 - Composition according to one of claims 17 or 18, for use in cosmetics or in human or veterinary pharmacy.