Process for the purification of a red fruit extract...

Details Process for the purification of a red fruit extract... Process for the purification of a red fruit extract...

2001-08-14

2002-10-08

Lilling, Herbert J. (Department: 1651)

Drug, bio-affecting and body treating compositions

Plant material or plant extract of undetermined constitution...

Containing or obtained from a fruit , including berry

Reexamination Certificate

active

06461648

ABSTRACT:

The invention relates to a process for purifying a red fruit extract containing anthocyanosides. It also relates to the extract obtained by said process and its use.
In the remainder of the description and in the claims, “red fruit” denotes in particular elderberry (
Sambucus nigra
), blackcurrant (
Ribes nigrum
), redcurrant (
Ribes rubrum
), mulberry (
Morus nigra
), grape (
Vitis vinifera
), and more particularly bilberry (
Vaccinium myrtillus
and other species of the genus Vaccinium), this being without limitation.
In general, the term “anthocyanosides” denotes a heterosaccharide resulting from the combination of an anthocyanic genin with one or more glycosidic groups. These anthocyanosides exist in the form of monomers, but also of dimers, oligomers and polymers according to an increasing degree of polymerization. The anthocyanoside-anthocyanoside bond occurs between the C4 of the C nucleus and the C8 of the A nucleus.
In the present case, anthocyanosides denote the structures in the form of monomers whose molecular weight varies from 400 to 600 Daltons. Certain forms of dimers may also be present whose molecular weight may be up to about 1000 Daltons.
Red fruits are rich in pigments called anthocyanosides which have been demonstrated to have an effect on blood microcirculation by acting in particular as vitamin P factor, as antioxidant, as platelet aggregation inhibitor and as anti-free radical agent. These properties have been recognized for the treatment of disorders involving retinal circulation, in the treatment of functional disorders of hair brittleness and visual disorders of vascular origin. These properties have also been demonstrated for the treatment of hesperanopia and myopia, in the proprietary medicinal products marketed under the trade marks DIFRAREL® 100 and DIFRAREL® E by LEURQUIN MEDIOLANUM laboratories.
Several processes for extracting anthocyanosides from plants or from plant portions have been proposed.
Document FR-A-2 299 385 thus describes a process for extracting anthocyanins from grape marc comprising an extraction step proper, followed by a step for concentrating the extract obtained.
According to this process, the extraction step consists in treating the marcs with an acidic aqueous extraction solution (pH=2) supplemented with SO
2
in the hot state (between 40 and 55° C.). The clear solution obtained, containing the anthocyanosides, but also the acids, salts, polyphenols and proteins, is then concentrated. To do this, it is loaded onto a resin. The resin is then eluted with an eluting solution containing either a ketone, an amide or an aqueous solution of an alkali or alkaline-earth metal hydroxide. The anthocyanins are finally separated from the eluate obtained.
It is evident from the examples described, in particular example 1, that starting with an extract containing 120 mg of anthocyanins per liter, an extract is obtained at the outlet of the resin which comprises 800 mg per liter of anthocyanins, that is to say an extract concentrated 6.6 fold.
However, even if the process used makes it possible to markedly increase the concentration of anthocyanosides in the extract, this concentration is not at all indicative of a purification of the anthocyanosides.
Furthermore and in particular, the initial extraction of the anthocyanosides with a solvent supplemented with SO
2
leads to the attachment of the anthocyanosides to the resin in a modified form, which is therefore capable of disrupting the physicochemical characteristics of the anthocyanoside and therefore its activity.
More recently, a process for extracting and then purifying anthocyanosides from bilberries was proposed in the document EP-A-412 300.
The extraction step proper consists in placing frozen fruits in contact with an aqueous solution of methanol, each extraction extending over a period of four hours.
The extract obtained is then purified. To do this, it is first concentrated under vacuum, the resulting concentrate then being supplemented with sodium bisulfite. A bond is then formed between the anthocyanosides and the bisulfite ions. After stirring for three hours and neutralization by adding a sodium hydroxide solution, the extract obtained is loaded onto a column of a nonionogenic polymer resin and then the column is eluted with purified water. The eluate is then acidified to pH=1 with concentrated hydrochloric acid (HCl). To remove the SO
2
, nitrogen is then bubbled through the solution obtained so as to dissociate the anthocyanosides-bisulfite complex. This dissociation leads to the release of sulfur dioxide. The aqueous solution is then extracted with butanol. The butanolic solution is supplemented with 14 volumes of ethyl acetate. After allowing to stand overnight, the precipitate is dried at 40° C.
As above, the process used still requires the use of bisulfite ions leading to the formation of an anthocyanoside-bisulfite complex capable of altering the physicochemical properties of the desired anthocyanosides.
Furthermore, the use of nitrogen for regenerating the anthocyanosides results in emissions of sulfur dioxide which can create environmental problems and problems of cumbersome treatments of the discharges.
It is finally also important to emphasize the multitude of solvents used (methanol, butanol and ethyl acetate) and the cumbersome chemical treatments which this extraction process involves: use of SO
2
, NaOH, acidification to pH=1 with concentrated HCl and use of nitrogen.
A process for extracting coloring substances and more particularly a process for extracting anthocyanosides from the berries of fruits, bilberries, blackcurrants or cranberries requiring in particular the use of a weakly polar supercritical solvent which is subsequently distilled off is known from patent FR 2641983. The extraction residue is purified by the customary physical means, in particular by chromatography on a polyamide column and then elution with hydrochloric methanol.
The anthocyanosides thus obtained and in particular those of bilberry are active ingredients of medicaments.
From this prior state of the art, the invention relates to a process for purifying an extract of red fruits containing anthocyanosides capable of solving the following problems:
dispensing with the use of any SO
2
-type additive which is likely to modify the structure and the physicochemical characteristics of the anthocyanosides extracted;
avoiding any emission of harmful substances and in particular of sulfur dioxide;
increasing the concentration of anthocyanosides in the purified extract obtained;
carrying out a purification without structural modification of the anthocyanosides;
reducing as much as possible the quantity of residual solvents contained in the purified extract;
obtaining an extract in which the residual solvents are clearly identified, and the low quantity of which complies with the ICH (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use) guidelines.


REFERENCES:
patent: 5200186 (1993-04-01), Gabetta et al.
patent: 0 412 300 (1991-02-01), None
patent: 2 299 385 (1976-08-01), None
patent: 2 456 747 (1980-12-01), None
patent: 2 641 283 (1990-07-01), None
patent: 1 235 379 (1971-06-01), None

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