CH666906A5 - PROCESS FOR THE PRODUCTION OF VITIS VINIFERA L. GRAPES ANTHOCYANOSIDE POLYMERS - Google Patents

Description

DESCRIPTION

The invention relates to the pharmaceutical field. More specifically, it relates to a process for obtaining polymers of grape anthocyanosides (Vitis vinifera L.) which can be used for various therapeutic purposes. Said polymers are obtained by fermentation in an aqueous acidic medium of anthocyanosides of grape monomers, free of aglycones, fermentation carried out by means of a strain acclimated to Aspergillus niger. It is defined in the claims.

Certain mixtures of products containing anthocyanoside polymers have been proposed as medicinal substances, in particular for ophthalmic use. To date, such mixtures are obtained from plant extracts subjected to relatively complex treatment and purification processes. Isolated with a low yield, they are therefore relatively expensive.

The process of the present invention has the advantage of making it possible to obtain grape anthocyanoside polymers with yields greater than those known hitherto and with a degree of purity sufficient for the intended therapeutic use. Said method also has the advantage of using as starting material the anthocyanosides of grape monomers, present on the market in abundant quantities and easily accessible: these anthocyanosides are in fact used as food colors.

According to the invention, the anthocyanosides of grape monomers are used in the form of an aqueous solution, free of aglycones. Such a solution is obtained by dissolving said monomers in an acid medium (pH preferably between 3.3 and 3.5), followed by elimination of the undissolved solid phase, for example by filtration or centrifugation.

The actual fermentation is carried out using microorganisms from a strain acclimatized to Aspergillus niger. The acclimatization of said strain is carried out according to the usual techniques, after selection on agar medium, subsequent culture in an appropriate medium, called Karow, and finally culture in the presence of a portion of the solution of monomeric anthocyanosides. In practice, the strain used is cultivated for approximately 8 days in Karow medium, then from 4 to 7 days in the presence of the monomer solution, until the number of spores per volumetric unit has reached the desired level.

Once the preculture stage of the strain is completed, the microorganisms are introduced into a fermentation tank, the latter being supplemented with the acid solution of grape anthocyanoside monomers. During fermentation, good agitation of the mixture is maintained and sterile air is introduced into the medium at regular intervals. During the reaction, the temperature of the reaction medium is maintained at a value below approximately 35 ° C. using external cooling, preferably at a value between 27 and 32 ° C.

The pH of the reaction medium is constantly monitored and adjusted, so as not to exceed approximately 3.7.

The pH can be adjusted by adding mineral or organic acid compatible with the fermentation medium, such as HCl, H2SO4 or acetic acid for example.

Once fermentation is complete, the resulting mixture can be filtered or centrifuged; the clear solution obtained is then concentrated and finally evaporated to dryness, most generally at a temperature close to or less than about 50 ° C. The dry product thus obtained io consists essentially of polymers of desired anthocyanosides; it is obtained with a sufficient degree of purity for its therapeutic application.

The polymers obtained by means of the process according to the present invention can then be incorporated into the appropriate solvents or excipients, thus serving as a basis for the preparation of various medicaments.

The invention will be illustrated using the example below, without being limited (temperatures in degrees centigrade).

Example:

The anthocyanosides of raw grapes (Vitis vinifera L.) used as starting materials are commercial products (source: Société cooperative agricole de distillation du Languedoc-Roussil-lon, avenue Pierre-de-Coubertin, Béziers, France). A) Preparation of the solution of monomeric anthocyanosides

11.25 kg of crude monomeric anthocyanosides were introduced into 901 of water and the pH of the mixture was brought from 3.3 to 3.5 by addition of hydrochloric acid. After stirring and centrifuging the acidified mixture, 3.15 kg of insoluble products (aglyco-30 born, etc.: approx. 28%) were eliminated. The resulting clear solution was used as it was for fermentation.

b) Acclimatization of the strain

An Aspergillus niger strain was first selected on agar medium, then cultivated for 8 days in 21 approx. of a Karow medium having the following composition:

- Sucrose 150 g

- Urea 1 g

- MgSO * 0.5 g

- KH2P04 0.08 g 40 - KCl 0.15 g

- MnS04 0.02 g

- ZnS04 0.01 g Water q.s.p. 1 litre

4s At the end of the above-mentioned period, a 10% aqueous solution of monomeric anthocyanosides (letter a) was added to the culture medium, at a rate of! / Io of the total volume of said culture. The development of the strain was continued at a temperature of the order of 27 to 32 ° C., until 3-109 to 3-1010 spores were obtained within the medium. This phase lasted almost 5 days. The culture mentioned above was carried out in a previously sterilized medium.

c) Fermentation itself

The culture resulting from the above step was ground with a ho-homogenizer (ULTRA-TURAX type) and introduced into the fermentation tank 55 previously cleaned. After all of the monomeric anthocyanoside solution has been introduced (step a), the mixture is kept stirred by means of a turbine, at approx. 150-300 rpm, during the whole fermentation period: the volume introduced represents 80-90% of the capacity of the tank.

60 After 24, respectively 48 h of fermentation, a stream of sterile air was passed through the reaction medium, at a rate of 1 m 3 / h, for 5 to 10 min each time. During the fermentation, the temperature of the reaction medium was maintained between 27 and 32 ° using an external cooling system and the pH stabilized at 65 a value less than or equal to 3.7 by repeated additions of HCl . Fermentation was considered complete after approximately 72 h.

The resulting mixture was then drawn off from the tank, filtered through a filter press and the clear solution obtained concentrated under pressure.

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reduced, to approx. 50 ° C. The syrupy mass thus obtained was finally dried in a vacuum oven, at a temperature at most equal to 50 °, to give the desired anthocyanoside polymers. It was observed that the purity of the product thus obtained was acceptable as regards the therapeutic uses envisaged. 5

The purity of the fermentation product was confirmed by means of thin layer chromatography analysis, as shown below.

A 2.5% solution of the finished product is prepared as follows:

Dissolve the product in the minimum amount of water at pH 3.3 to 3.7 and add methanol and water so as to have a methanol-water solution at 50% (v / v).

Apply 40 to 50 µl of the following solution in a 20 mm line centered at an angle 3 cm from the edge. 15

Thin film plates of 20 × 20 cm cellulose, Macherey & Nagel MN 300, thickness 0.75 mm, are used.

Allow to migrate for about 2 hours (3 to 4 cm from the upper edge of the plate) using as migration solvent: amyl alcohol-formic acid-HCl N / 10 (8-4-1.5). 20

Dry with a stream of air at room temperature for about 1 hour.

Carry out the second migration using a mixture: glacial acetic acid-water (50/50) (v / v). Before carrying out the 2nd migration, the cellulose layer is humidified by placing capsules filled with water at about 80 ° at the bottom of the tank so as to moisten the cellulose.

The development is stopped at 2 to 3 cm from the upper edge of the plate, which requires approximately 1 hour 30 min. It is dried by a stream of air at room temperature. 30

There is an irregular main spot of very high Rf, towards the front of the solvent, and 2 or 3 small spots of lower Rf.

The main stain corresponds to the anthocyanoside polymers (red), the small stains to the remaining monomers. At the front of the solvent, there is the presence of a practically zero spot of Rf corresponding to a few more condensed polymers.

d) Analysis by pressure liquid chromatography (HPLC) A sample of the product resulting from the fermentation described above was subjected to analysis, under the following conditions:

- sample in 1% solution in methanol

- LICHROSORB RP 18 column

- eluents: A) 5% formic acid in H20

B) methyl alcohol

- flow rate: 2.5 ml / min

During the first 8 minutes of elution, a mixture of eluents A / B 50:50 (volume) was used; beyond 8 min of elution, an A / B 20:80 (volume) mixture was used.

The resulting chromatogram indicates the presence of 3 peaks (polymers) defined as follows:

Retention time% of total

1) 0.95 min |

approx. 99% approx. 1%

2) 1.38-1.42 min

3) 5.60 min e) Partial chemical analysis

A sample of the product resulting from fermentation, subjected to acid hydrolysis under the usual conditions, indicates that the dominant genine is malvidine.

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Claims (5)

666,906 1. Process for obtaining polymers of anthocyanosides from grapes Vitis vinifera L., characterized in that anthocya-nosides of grape monomers, free of aglycones, are subjected to fermentation in an acidic aqueous medium carried out with using microorganisms from an acclimatized strain $ Aspergillus niger. 2. Method according to claim 1, characterized in that the fermentation is carried out at a pH less than or equal to 3.7. 2 o 3. Method according to one of claims 1 or 2, characterized in that the fermentation is carried out at a temperature less than or equal to about 35 ° C, preferably between 27 and 32 ° C. 4. Method according to one of claims 1 to 3, characterized in that the fermentation is carried out in an aerobic medium. 5. A drug containing, as a pharmacologically active ingredient, polymers of anthocyanoside from grapes Vitis vinifera L. obtained by means of the process according to one of claims 1 to 4.