WO2001003713A1 - METHOD FOR EXTRACTING RESVERATROL AND/OR ε-VINIFERIN - Google Patents

Abstract

The invention concerns a method for extracting resveratrol and/or epsilon -viniferin from vine stems wherein the vine stems used are dry vine stems. Said method comprises an extraction step, a primary purification step preferably followed by a secondary purification step. The resulting purified extract is characterised by its particularly high resveratrol and/or epsilon -viniferin content.

Description

 Method of extracting resveratrol and / or ε-viπiferine

The present invention relates to a process for the preparation of an extract of the vine concentrated in resveratrol and / or in ε-viniféπne as well as an extract obtained by this process

Resveratrol, or 3, 5, 4'-tπhydroxystιlbène, is found in very low contents, of the order of a few mg / l, in red wines The resveratrol present in the skin and in the seeds of grapes is extracted during vinification and is found in the final product, the wine, especially the red wine for which the skin of the grapes macerates in the must of the wine

Resveratrol exists in the cis and trans form II is generally present in the monomer form but also exists as an oligomer comprising up to four monomers The term "resveratrol" below relates to the cis or trans forms of 3, 5, 4'- trιhydroxystιlbène Resveratrol is often associated with ε-vιnιféπne which is the dimer of resveratrol This product is therefore generally extracted at the same time as resveratrol

Resveratrol is particularly interesting because it has many properties with therapeutic applications. It is in particular to this molecule that we attribute the effects of the “French Paradox”. First studies have indeed shown its effects in reducing of the risk of cardiovascular diseases Resveratrol improves the fluidity of the blood, and therefore the circulation of blood, and reduces the risks of blockage of the vessels by clots II also decreases the level of lipids and cholesterol in the blood

The properties and benefits of resveratrol go beyond, since first studies have shown effects in the prevention of cancer and certain neurological diseases, as well as its antioxidant activity

Finally, revesratrol which is produced by the vine in response to bacterial attacks by the bacterium Botrytis cmerea, responsible for gray rot disease, has fungicidal and bactericidal properties

Resveratrol is also found in other plant sources such as peanuts, blackberries, and also a plant used by Chinese medicine, Polygonum Cuspitadum

Extracts from the vine from the skin and grape seeds are not very concentrated in resveratrol The maximum concentration of around

500ppm is not compatible with certain industrial applications The concentration of resveratrol in most other plants, does not allow the production of more concentrated extracts

Only extracts from Polygonum Cuspitadum roots can have resveratrol contents greater than 20% However, these extracts have the drawback of also containing emodin The presence of this active principle does not allow the use of these extracts in some industrial applications

The publication following the conference from 15 to 18 July 1996 at

Bordeaux concerning polyphenols (pages 39-40 "isolation and characterization of trans-resveratrol and ε-vιnιférιne from the vine shoot" by Boukharta, Girardin and Metche) indicates that it is possible to extract trans-resveratrol and ε-viniféπne of lignified vine shoots

Document WO-99/03816 reveals a method for obtaining monomeric and / or oligomeric resveratrol fractions from grape vines

The extracts obtained by the processes described in these documents do not contain emodin, or other annoying active ingredients, but their yield of resveratrol and ε-viniferin is low.

The subject of the present invention is a process for the manufacture of resveratrol and / or ε-vinifenne, which does not contain an active principle such as emodin and has a good extraction yield. Preferably, this process will be easy to implement and will not require the use of advanced purification techniques

To this end, the process it offers is a process for extracting resveratrol and / or ε-vinifenne from vine shoots

According to the invention, the vine shoots used to implement this process are dry shoots

Surprisingly, it has been noticed that the use of dry shoots allows a much higher yield of resveratrol and ε-viniferine. to that obtained with “fresh” branches For the moment, no explanation, even a posteriori, can explain this result

Preferably, the branches used to extract resveratrol and ε-vιnιféπne have been dried in the open air for at least two months. The branches dried in the open air for three months give very good results.

The branches used advantageously have a humidity level of less than 20%, preferably less than 5%

The invention also proposes to extract resveratrol and / or ε-vιnιférιne using a process in which, after the stage of drying the branches, an extraction stage using a solvent chosen from the group comprising the products having a carbonyl radical, such as for example esters and ketones, and alcohols is provided In this process, the extraction is advantageously carried out in the absence of water This is contrary to all known processes of the prior art where the solvent used to extract resveratrol is always an aqueous solvent. The solvent used to carry out this extraction can be, for example, ethanol or else acetone, which both give very good results, acetone being however the preferred solvent.

To improve the extraction yields, several successive contacts can be made. The extraction can also be done continuously with a counter-current circulation of the ground branches and the extraction solvent.

Preferably, the method according to the invention comprises, after an extraction step, a primary purification step in which a hydroalcoholic mixture is used as the primary purification solvent. The solvent used is for example a mixture of water and d ethanol

To obtain an even more concentrated product, the process can include an additional secondary purification step in which a hydroalcoholic mixture having a lower alcohol content than that of the primary purification solvent is used as the secondary purification solvent. The purification solvent secondary used can be simply water

After the extraction step and / or after the primary purification step, the solvent used in this step is for example evaporated. realization provides that this solvent is distilled in the presence of water so as to precipitate the solid

A preferred extraction process from branches dried three months in the open air is described below with an indication of some numerical values obtained

To carry out an extraction of resveratrol and / or ε-viniféπne, the mass solvent rate, which must be sufficient to impregnate the whole of the solid and allow the mixture to be stirred, is generally between 5 and 15 The contact is made at a temperature between room temperature and the boiling temperature of the solvent The contact time between the plant material from the vine and the extraction solvent is between a few hours and 24 hours The contact time can be reduced up to 'at% of hour

The solvent and the plant material from the vine are then separated by filtration and the solvent is eliminated by evaporation. The crude extract obtained has a resveratrol content of between 1 and 5% and has a greenish gum texture.

The crude extract is then purified to obtain a less colored product in the form of a powder. The primary purification is carried out by dissolution in a solvent which is a hydro-alcoholic mixture. The alcohol is an alcohol miscible with water. It is for example ethanol The alcohol content is between 10 and 80% The dissolution is carried out at a temperature between 20 and 40 ° C. The impurities insoluble in this solvent are separated from the supernatant by centπfugation Filtration could be performed HERE instead of, or in addition to, centrifugation

The solid obtained after removal of the solvent from the supernatant by evaporation is a light brown powder, having a resveratrol content of between 7 and 20% The ratio between the mass content of resveratrol and the mass content of ε-viniféπne in the extract purified is around 1

This extract is then purified a second time by dissolving certain impurities in a secondary purification solvent. This solvent is water or a hydro-alcoholic solvent having a lower alcohol content than primary purification solvent Dissolution is carried out at a temperature between 20 and 40 ° C. The supernatant is separated from the insoluble part by centrifugation Here too, filtration can be envisaged The solid obtained after removal of the solvent from the supernatant by evaporation is a powder light brown in color, having a resveratrol content of between 1 and 3% The ratio of the resveratrol content to the content of ε-vinifenne is between 0.25 and 1 The purified extract obtained after drying of the insoluble part has a light brown color and is in the form of a powder Its resveratrol content is between 10 and 50%, and the ratio between the resveratrol content and the ε-vinifenne content is between 0.25 and 4

The present invention also relates to a natural extract of the vine obtained by a process as described above. This natural extract of the vine has for example a content of resveratrol greater than 1%, preferably greater than 10% Likewise, the invention relates more particularly to a natural extract of the vine whose ε-viniferin content is greater than 1%, preferably greater than 10%

In a natural extract of the vine according to the invention, the mass content of ε-viniféπne is for example between 0.25 and 4 times its mass content of resveratrol

The term vine represents in this document in particular the vine families Vitis Viniféra and Vitis Rupestris Without this list being exhaustive, we can cite HERE as examples the varietals Merlot, Sauvignon, Chardonnay, Cabernet, Syrah, Tanat, Gamay, etc. The invention allows advantages compared to the prior art, in that it allows the obtaining of vine extracts from 20 to 1000 times more concentrated in resveratrol than the extracts currently marketed and obtained from the vine This invention allows the preparation of liquid, powder or tablet formulations having high contents of resveratrol extracted from the vine, compatible with the usual industrial applications for these products. It applies in particular to uses in the pharmaceutical, dietetic fields. and cosmetics Various particular embodiments of the invention are described in more detail below. This description below is in no way limiting.

The method according to the invention can be implemented as follows.

Example 1:

One kilogram of vine shoots, coming from a Cabernet grape variety, dried in the open air for four months and having a humidity rate of 4% is ground to a particle size of less than 4 mm. The obtained crushed solid is mixed with 7 liters of ethanol. The extraction is carried out for 20 hours at a temperature of 30 ° C, with moderate stirring.

The solid and the supernatant are separated by filtration. After evaporation of the ethanol, 28 grams of crude extract are obtained, having a greenish color, a gummy appearance, and a resveratrol content of 3%.

The crude extract is taken up with 225 milliliters of a water / ethanol mixture at 50% by volume. The mixture is maintained at 25 ° C., with stirring for one hour, then the undissolved solid and the supernatant are separated by centrifugation.

After removing the solvent from the supernatant by evaporation, 8 grams of a purified extract are obtained having a resveratrol content of 10% and an ε-viniferin content of 7%. The purified extract has a light brown color and is in the form of a powder.

The purified extract is taken up in 225 milliliters of water. The mixture is kept at a temperature of 25 ° C with stirring for one hour. The undissolved solid and the supernatant are separated by centrifugation. After drying the undissolved solid, 2.5 grams of an extract are obtained having a resveratrol content of 28% and an ε-viniferin content of 12%. The purified extract has a light brown color and is in the form of a powder. After removing the water from the supernatant, 5.5 grams of an extract having a resveratrol content of 2% and an ε-viniferin content of 5% are obtained. The purified extract has a light brown color and is in the form of a powder. Example 2

One kilogram of vine shoots, coming from a Cabemet grape variety, dried in the open air for three months and having a humidity rate of 12% is ground to a particle size of less than 4 mm The ground solid obtained is mixed with 7 liters of acetone

The extraction is carried out for 20 hours at a temperature of 25 ° C, with moderate agitation

The solid and the supernatant are separated by filtration. After evaporation of the acetone, 25 grams of crude extract are obtained, having a greenish color, a gummy appearance, and a resveratrol content of 3%.

The crude extract is taken up with 250 milliliters of a water / ethanol mixture at 50% by volume. The mixture is maintained at 25 ° C., with stirring for one hour, then the undissolved solid and the supernatant are separated by centrifugation.

After removal of the solvent from the supernatant by evaporation, 7 grams of a purified extract are obtained having a resveratrol content of 11% and an ε-viniféπne content of 10%. The purified extract has a light brown color and is present in the powder form

The purified extract is taken up in 250 milliliters of water The mixture is kept at a temperature of 25 ° C. with stirring for one hour The undissolved solid and the supernatant are separated by centrifugation After drying the undissolved solid, 2 is obtained, 5 grams of an extract having a resveratrol content of 25% and an ε-vinifenne content of 15% The purified extract has a light brown color and is in the form of a powder After removal of the water from the supernatant, 6.5 grams of an extract having a resveratrol content of 2% and an ε-viniferin content of 4% are obtained. The purified extract has a light brown color and is in the form of a powder.

Example 3

Tests were carried out on the following four grape varieties Syrah, Cabemet Franc, Merlot and Gamay For each of these grape varieties, an extraction of resveratrol and ε-viniféπne was carried out according to the same operating protocol Tests were carried out on A) fresh branches pruned for less than a fortnight and having a humidity level of around 50%,

B) fresh branches pruned for less than two weeks, ground and dried in an oven at 40 ° C to a humidity level of less than 3%, and C) branches dried naturally in the open air for three months and with a humidity level between 3 and 12%.

A total of twelve separate extractions were performed. The extraction process is described below for 500 g of branches.

The branches are first crushed to a particle size of less than 4mm. Of course, the ground branches and dried in the oven were not ground a second time. The grinding of fresh branches which have been parboiled is the same as that of fresh branches and dried branches.

The ground branches are then macerated in 3 liters of acetone at 30 ° C for 12 hours. The agitation during this maceration is moderate. The mixture thus obtained is then filtered and the solid material obtained undergoes dry evaporation of the acetone.

The crude extract then obtained is taken up in 150 ml of a mixture of water and ethanol at 50% by volume of ethanol. This mixture is kept at 25 ° C for one hour and with stirring. The solid then not dissolved and the supernatant are then separated by centrifugation.

By evaporation of the solvent, a solid is then obtained containing resveratrol and ε-viniferine.

This solid obtained was then ground to carry out its analysis. The results of this analysis are summarized in the table below. It is clear from the table below that the highest yields are obtained by the processes using dry shoots. They are significantly higher when the branches have been dried in the open air for three months. The drying in an oven allows to have an interesting concentration of ε-viniferine. These results are surprising and are only empirical.

Example 4

This variant consists in not evaporating to dryness after the extraction phase and the first purification, but in distilling the solvent in the presence of water so as to precipitate the solid.

After preconcentration of the extraction solvent, water is added to the mixture, and the removal of the solvent is continued by distillation so that at the end of the operation, the solvent content in the mixture is less than 5 % and the volume of the mixture is between 25 and 500 milliliters per kilogram (ml / kg) of vine shoot An equivalent volume of purification solvent is then added to the mixture The mixture is stirred for one hour, then the undissolved solid is separated from the mixture by filtration or centrifugation

The purification solvent is then removed from the supernatant by distillation, water is added to the mixture during the distillation, so that at the end of the operation, the content of solvent in the mixture is less than 5%, and the volume between 25 and 500 milliliters per kilogram of shoot The precipitated solid is separated from the mixture by filtration An aqueous phase and a wet solid are obtained After drying and grinding of the solid, a brown powder is recovered, having a resveratrol content between 15 and 50%, with a ratio between the content of resveratrol and ε-viniféπne between 0.25 and 1 After evaporation of the water from the liquid mixture, a powder of brown in color, having a resveratrol content of between 1 and 3% with a ratio between the content of revesratrol and ε-viniferin between 0.25 and 1

Many variations can be considered depending on the circumstances of use, with regard to the operating parameters of the different stages of the process.

The present invention is not limited to the details of the above embodiments considered by way of example, but on the contrary extends to modifications within the scope of the skilled person within the scope of the claims below

Claims

1. Process for extracting resveratrol and / or ε-viniferine from vine shoots, characterized in that the vine shoots used are dry shoots. 2. Method according to claim 1, characterized in that the moisture content of the branches used is less than 20%, preferably less than 5%. 3. Method according to one of claims 1 or 2, characterized in that the branches have been dried in the open air for at least two months. 4. Method according to one of claims 1 to 3, characterized in that after the stage of drying the branches, it comprises an extraction stage using a solvent chosen from the group comprising the products having a carbonyl radical, such as for example esters and ketones, and alcohols. 5. Method according to claim 4, characterized in that the extraction is carried out in the absence of water. 6. Method according to one of claims 4 or 5, characterized in that the solvent used is ethanol. 7. Method according to one of claims 4 or 5, characterized in that the solvent used is acetone. 8. Method according to one of claims 4 to 7, characterized in that after the extraction step, a primary purification step in which a hydro-alcoholic mixture is used as the primary purification solvent. 9. Method according to claim 8, characterized in that the solvent used is a mixture of water and ethanol. 10. Method according to one of claims 8 or 9, characterized in that it comprises a secondary purification step in which a hydroalcoholic mixture having an alcohol content lower than that of the primary purification solvent is used as solvent secondary purification. 11. Method according to claim 10, characterized in that the secondary purification solvent used is water. 12. Method according to one of claims 1 to 11, characterized in that the solvent used in the primary extraction and / or purification step is evaporated. 13. Method according to one of claims 1 to 9, characterized in that the extract obtained after the extraction step and / or the primary purification step is distilled in the presence of water so as to precipitate the solid. 14. Natural extract of the vine, characterized in that it is obtained by a process according to one of claims 1 to 13. 15. Natural extract of the vine according to claim 14, characterized in that its resveratrol content is greater than 1%, preferably greater than 10%. 16. Natural extract of the vine according to one of claims 14 or 15, characterized in that its ε-viniferin content is greater than 1%, preferably greater than 10%. 17. Natural extract of the vine according to one of claims 14 to 16, characterized in that its mass content of ε-viniferin is between 0.25 and 4 times its mass content of resveratrol. 18. Application of natural extracts according to one of claims 14 to 17 in the therapeutic, cosmetic and dietetic fields.