FR2775686A1 - Production of neutral lipids useful in cosmetic, dermatological or pharmaceutical compositions - Google Patents

Abstract

New method (I) for the production of neutral lipids from grape (Vitis vinifera) skins. (I) comprises extracting the lipids with organic solvents at 0-90 deg C for between 1 minute and 24 hours. An Independent claim is also included for new neutral lipids extracted from grape skins by the above process.

Description

DESCRIPTION
The present invention relates to new neutral lipids extracted from the cuticular membrane of the Vitis vinifera L berry. These lipids can be used in a cosmetic, dermatological or pharmaceutical composition. The neutral lipids, extracted from whole berries and / or from the grape skins recovered after pressing the berries, consist of the compounds present in epicuticular waxes, intracuticular waxes and cutin.

 It is recalled that the cuticular membrane constitutes the most superficial structure of the Vitîs vinifera L bay. It forms a protective film on the external walls of the epidermal cells of the berries, limiting water losses and controlling gas exchanges.

The broad cuticle or cuticular membrane of the Vitis vinifera L. berry is made up of different areas, respectively
- epicuticular waxes consisting essentially of free and esterified fatty acids (cerides), terpenoic acids, mainly ursolic and oleariolic acid and glycosidic derivatives, terpenols and their esters, fatty alcohols, aldehydes, and d 'hydrocarbons (paraffins, olefins).

 - of the cuticle sensu stricto formed by the cutin, an insoluble polymer of esters of hydroxylated and / or epoxylated fatty acids (substituted aliphatic fatty acids) essentially comprising 16 or 18 carbon atoms.

 - the cuticular layer formed by cutin and intracuticular waxes (essentially free and esterified fatty acids, fatty alcohols, aldehydes, hydrocarbons).

 Cerides are high molecular weight esters (C36 to C72), resulting from the combination of a fatty acid and a monoalcohol. The free fatty acids can be normal, hydroxylated and / or ethylenic, short series (C 12 to C 18) or long series (C20 to C32). Aldehydes and primary alcohols contain compounds of 22 to 32 carbon atoms, hydrocarbons contain compounds of 17 to 35 carbon atoms, secondary alcohols contain compounds of 21 to 33 carbon atoms and ketones contain compounds of 23 to 33 carbon atoms.

 The composition of the waxes varies according to the varietals of Viiis vinifera and the stage of maturation of the grape berry. In the late maturation stage (vintage), epicuticular waxes generally contain 20 to 80% of terpenoic acids (mainly oleanolic and / or ursolic acid and glycosidic derivatives), 2 to 30% of alcohols, 2 to 30% cerides, 2 to 30% free fatty acids, 2 to 20% aldehydes, 2 to 20% hydrocarbons. Intracuticular waxes contain from 2 to 40% of alcohols, from 2 to 40% of cerides, from 2 to 80% of free fatty acids, from 2 to 40% of aldehydes, from 2 to 40% of hydrocarbons.

 The main fatty acids constituting the grape berry cutin are 16-hydroxyhexadecanoic acid, (9, 16) -dihydroxyhexadecanoic acid, (10, 16) -dihydroxyhexadecanoic acid, 18-Hydroxyoctadecanoic acid, L '18-hydroxyoctadécè-9-oïque acid, cis 9, 10 -époxy- 1 8-hydroxyoctadécanoïque acid and (9, 10, 18) -trihydroxyoctadécanoïque acid.

 Cuticular waxes (epicuticular and intracuticular waxes) are extracted in organic solvents or solvent systems (chloroform, dichloromethane, ethyl ether, petroleum ether, ethyl acetate, methanol, non-exhaustive list). As an example, epicuticular waxes can be partially extracted in a chloroform bath for 5 minutes, most of the intracuticular waxes can be extracted at reflux in chloroform for 24 hours. Mixed extractions (epi and intracuticular waxes) can be obtained in different organic solvents or solvent systems for an extraction time greater than or equal to 30 minutes (example, 1 hour in a dichloromethane-methanol mixture 2: 1, v / v).

 The cutin is recovered in a pure form after extraction of the epi- and intracuticular waxes and after digestion of the underlying cellular foundations (epidermal cells) in maceration pads. The fatty acids or fatty acid salts of the cutin can be obtained by chemical hydrolysis of the cutin.

 The grape berries (Vitis vinifiera L.) can constitute a material of choice for the extraction of compounds of interest for the cosmetic, dermatological and pharmaceutical industries. Thus, the grape skins can be recovered in large quantities after pressing the berries. Currently, grape skins are mainly used for animal feed. Regarding the use of whole berries, depending on the wine region, harvests exceeding the authorized quotas (yield by weight or volume by area) are either bought at a ridiculously low price, or simply left on the ground. Grape berries or grape skins can therefore prove to be inexpensive equipment.

The extraction of neutral lipids from the cuticular membrane presents a double interest; quantitative (volumes that can be treated are significant), and qualitative (properties of extractable lipid compounds).

 The applications of neutral lipids extracted from epicuticular waxes, intracuticular waxes and fatty acids resulting from the hydrolysis of grape berry cutin are numerous with regard to their physico-chemical properties and / or their biological activities.

Thus, and by way of nonlimiting examples, most cosmetic and dermatological compositions, and certain pharmaceutical compositions, contain, at a more or less high concentration, a fatty phase which can be constituted by a vegetable wax. Cerides (fatty acid esters), free fatty acids and fatty alcohols can be used as basic products (excipient) and as emulsifying agents, for example for the formulation of creams, ointments, soaps and lip stick. Hydrocarbons can be exploited for the extraction of perfumes. Ursolic and oleanolic acids and their glycosidic derivatives in particular have many properties of pharmacological interest.

Different publications report their hepatoprotective, anti-inflammatory, anti-radical, anti-tumor, anti-hyperlipidemic, anti-ulcer, anti-microbial, hypoglycemic or anti-cariogenic effects. These terpenoic acids are generally extracted from medicinal plants. None of these documents suggests or describes the use of terpenoic acids and their derivatives extracted from the cuticular membrane of Vitis vinifera L.

 Also, the invention relates to any cosmetic, dermatological and pharmaceutical preparation containing the lipid compounds (epicuticular waxes, intracuticular waxes, cutin) extracted from the cuticular membrane of grape berries (whole berries or grape skins), and used either in mixing either after purification.

 The invention also relates to the processes for obtaining the lipids extracted in organic solvents such as chloroform, ethanol, ethyl ether, petroleum ether, ethyl acetate, methanol, dichloromethane or hexane, for extraction times between 1 minute and 24 hours, at temperatures between OOC and 900C.

 The subject of the invention is also a process for the separation of terpenoic acids and their glycosidic derivatives from a crude lipid mixture by phase partition according to the description developed in Example 5.

Examples
In the following paragraph, we will describe examples dealing with the extraction of epicuticular waxes, endocuticular waxes and cutin, from whole grape berries or from the grape skins recovered after pressing the berries, then techniques for separating lipid compounds. Many variations (nature of the solvents used, extraction time, fractionation methods, etc.) can be carried out, these examples are therefore not limiting.

Example 1
Extraction of epicuticular and intracuticular waxes from whole grape berries. The berries are dipped successively:
- in a petroleum ether bath for 3 minutes (extraction mainly of the terpenoic acid fraction),
- in a chloroform bath for 5 minutes (extraction of most of the other constituents of epicuticular waxes),
- in a chloroform-methanol mixture (2: 1, v / v) for 1 hour (extraction of residual epicuticular waxes and endocuticular waxes).

Example2
Extraction of epicuticular waxes, endocuticular waxes and cutin from grape skins recovered after pressing the berries.

 - The grape skins are brought to the boil in an oxalic acid buffer (0.4%, w / v) ammonium oxalate (1.6%, w / v) to separate the raw cuticles from the underlying tissues.

 - Maceration of the raw cuticles for 18 hours in a sodium acetate buffer (100 mM, pH 4.5) containing 0.5% (w / v) of cellulases and 0.1% (w / v) of pectinases.

 - The cuticles are collected and rinsed with water.

 - Epicuticular waxes are extracted with dichloromethane for 10 minutes.

 - The cuticles are immersed in a methanol bath for 30 minutes then in a dichloromethane-methanol mixture (2/1, v / v) for 30 minutes (extraction of residual epicuticular waxes and part of the intracuticular waxes).

 - The cuticles are extracted at reflux in dichloromethane for 24 hours (extraction of intracuticular waxes). Insoluble residues constitute the pure cutin.

 - The cutin is depolymerized by chemical hydrolysis. The cutin reduced to powder is subjected to boiling under reflux in 300 ml of a 3% methanolic potassium solution (w / v).

After 24 hours, the methanolic filtrate is dried. The dry residue, fatty acid salts from saponification, is taken up in water. Fatty acids can be recovered by acidifying the solution with hydrochloric acid. The fatty acids released are extracted with ethyl ether.

Example3
The grape skins are treated directly after dehydration. The epi and endocuticular waxes are extracted as a mixture in a solvent system (dichloromethane-methanol) for 1 hour at 4 "C.

Example4
The recovered extracts can be used as a mixture (crude extract) or separate
The different lipid categories are fractionated on silica gel, by selective crystallization or by phase partition.

 The lipid compounds are dissolved in hexane and the extract is chromatographed on a silica column. The different elution solvents are: hexane, hexane-ethyl ether 99: 1 (v / v), hexane-ethyl ether 95: 5 (v / v), hexane-ethyl ether 95: 5 (v / v), hexane - ethyl ether 50:50 (v / v), ethyl ether, chloroform, methanol. The hydrocarbons are found in the fraction eluted with the hexane-ethyl ether mixture 99: 1 (v / v), the cerides in the hexane-ethyl ether fraction 95: 5 (v / v), the free fatty acids in the chloroform fraction. .

Example 5
The purification of terpenoic acids can be carried out by phase partition. The partition is based on the differences in affinity of the compounds for more or less polar immiscible solvents (hexane and methanol).

 In a separating funnel, add 1 volume of methanol to 2 volumes of hexane containing epicuticular waxes. After gentle stirring, the extract is decanted until the formation of two phases. The upper phase (hexane phase) is recovered and washed several times with methanol. The lower phase (methanolic phase) is washed several times with hexane. The phases containing either methanol or hexane are combined.

 The methanolic phase consists of the most polar compounds of epicuticular waxes, in this case terpenoic acids. By this technique, terpenoic acids are recovered in more than 90% pure form. The hexane phase consists of the other wax compounds, and in particular by fatty alcohols, fatty acids and fatty acid esters.

Claims (7)

Vites vinifera L. characterized in that the lipids are extracted with organic solvents, for an extraction time between 1 minute and 24 hours, for a temperature between OOC and 900C.  CLAIMS 1) Process for the preparation of neutral lipids extracted from the cuticular membrane of the bay 2) Preparation process according to claim 1 characterized in that the organic solvents used consist of chloroform, dichloromethane, ethanol, ethyl ether, petroleum ether, ethyl acetate, hexane, methanol. 3) Process for the treatment of neutral lipids obtained by the process according to either of Claims 1 or 2, characterized in that the terpenoic acids (oleanolic acid, ursolic acid and glycosidic derivatives) which are in the crude mixture are separated neutral lipids extracted, by methanol / hexane phase partition, the terpenoic acids being found in the methanol phase. 4) New neutral lipids extracted from the cuticular membrane of Vitis vinifera L., obtained according to the method of one of claims 1 to 3.  5) Use of the neutral lipids obtained by the process according to one of claims 1 to 3 for the manufacture of a cosmetic, dermatological or pharmaceutical composition in combination with a cosmetically, dermatologically or pharmaceutically acceptable excipient. 6) Use of the acids obtained in the methanolic phase (oleanolic acid, ursolic acid and glycosidic derivatives) by the method according to claim 3, for their hepatoprotective, anti-inflammatory, anti-radical, anti-tumor, anti-hyperlipidemic, anti -ulcerous, anti-microbial, hypoglycemic, anti-cariogenic. 7) Use of fatty alcohols, fatty acids and fatty acid esters obtained in the hexane phase by the process according to claim 3, as the fatty phase in a cosmetic, dermatological or pharmaceutical composition.