FR2652743A1 - Composition based on proanthocyanidols. Their pharmacological application - Google Patents

Abstract

The invention relates to a composition based on proanthocyanidols, in particular of vegetable origin, characterised by a weight proportion of at least 50% of molecules having apparent molecular weights above 2000, this proportion consisting of at least 40% of proanthocyanidols, this percentage being expressed in terms of tanning function, the aforementioned composition being essentially free from: - aromatic oils, and in particular essential oils, resins and other lipophilic substances, - polymerised sugars such as starch, gums, cellulose derivatives, - oligomeric sugars such as sucrose, glucose, levulose. It also relates to the application of this composition in pharmaceutical compositions.

Description

 The invention relates to proanthocyanidol compositions and their application in pharmaceutical compositions.

 Proanthocyanidols belong to the family of polyphenols known for their ability to attach, at very high levels, to proteins.

 Polyphenols are generally involved in the formation of tannins present in particular in certain plants.

 The invention relates to compositions containing specific classes of tannins, called catechin tannins (synonymous with proanthocyanidins).

It relates more specifically to polymers of catechin tannin.

 Generally speaking, the ability of certain crude vegetable extracts comprising various tannins to have antiseptic and sometimes even anti-viral activity has been known to date.

 Some tannins present as oligomers were used for their veinotonic properties.

Until now, catechin tannins or other pharmaceutically usable tannins were generally constituted by oligomers (of molecular weight generally less than 1000). High tannins of high molecular weight (MW), especially polymers, were systematically discarded. For example, it is recalled that for gallic tannins (another family of tannins present for example in plants) cytotoxicity increases with molecular weight, which does not promote their use.

 The inventors have now found that catechin tannins of high molecular weight, in the form of polymers, may be of interest for their pharmacological properties.

 The invention relates in this respect to a composition based on proanthocyanidols, in particular of plant origin, characterized by a weight proportion of at least 50% of molecules having apparent molecular weights greater than 2000, this proportion being composed of at least 40% of proanthocyanidols, this percentage being expressed as a tanning function, and in that said composition is essentially free of: - aromatic essences and in particular essential oils, resins and other lipophilic substances, - polymerized sugars such as starch, gums, cellulosic derivatives, oligomeric sugars such as sucrose, glucose, levulose.

 Particularly suitable methods for the selection of compositions comprising proanthocyanidols and as defined above are for example the following: a / To characterize the level of the tanning function of the proanthocyanidols of the above compositions, it is possible to use the following dosage which comprises on the one hand an assay of the polyphenolic function, on the other hand, a dosage of the tanning function.

10 / -Dosage of polyphenolic function:
In this assay the property of polyphenols is used to reduce phosphotungstic acid. The assay is performed according to the standards of the French Pharmacopoeia 90 edition.

Phosphotungstic reagent (A):
10 g of sodium tungstate are refluxed with 8 ml of orthophosphoric acid and 75 ml of deionized water for 3 hours. Let cool and complete to 100ml with deionized water.

15% sodium carbonate solution in deionized water: reagent B.

 50 mg of the sample containing the proanthocyanidol to be assayed are weighed out and dissolved in 10 ml of exactly measured water.

 2 ml of this solution is taken and diluted to 25 ml in a volumetric flask (solution C).

In a volumetric flask of 50ml, place:
Solution C: 5 ml
Reagent A: îml
Reagent B: qs50ml.

 The optical density (OD) is read at 715 nm, 2 minutes exactly cleared of the mixture of the last reagent. The same procedure is followed with a range of increasing amounts of pyrogallol (0 to 50 mg) as references and the optical density obtained with each of them is measured. The proanthocyanidol content of the sample studied is expressed by reference to the amount of pyrogallol (measured quantity) which would lead, under the same analysis conditions, to the same OD measurement. The determination of this amount of pyrogallol is carried out by reference to the results of the measurements made under the conditions indicated above with the increasing quantities of pyrogallol used as references.

20 / -Dosing of the tanning function:
An aqueous solution of proanthocyanidol prepared as solution C above. The determination of the polyphenolic function is then carried out as indicated above. 5 ml of the solution are then stirred for one hour at room temperature with 1 g of pre-chromated skin powder (Merck No. 4332); then the solution is filtered on paper.

 An assay of the total polyphenolic function on the filtrate is then carried out as above.

The tanning function represents the difference between the value found for the total polyphenols and the value found on the filtrate, separated by the skin powder.

b / To determine the fraction of proanthocyanidols whose apparent molecular weight is greater than 2000, the following molecular weight determination method may be used:
Firstly, a high performance exclusion chromatography on pBondagel 125 gel (Waters) in an aprotic solvent against polystyrene controls (Aldrich) is carried out.
Flow: 0.5 ml / minute isocratic.

Solvent: Chloroform-dimethylsulfoxide 50-50 (v / v)
Detection: DO at 283nm
Concentration of the controls: lmg / ml in the same solvent
Concentration of proanthocyanidol: 0.05 mg / ml in the same solvent
Molecular weights of controls: 184,200, 50,000, 24,150, 7,025, 4,136 and 2,000.

 The 184.200 molecular weight control is used as a total exclusion control on the gel. Indeed, this type of gel does not retain molecular weights greater than 50,000. The relative retention time of each sample is then calculated in relation to the totally excluded control.

 The correspondence curve between the decimal logarithms of the molecular weights of the controls and the corresponding relative retention times is plotted. The molecular weight of the proanthocyanidols present is appreciated by comparing the retention time measured for them and the retention times of the controls. These methods are given only as an example.

 Other methods for determining the proportion of proanthocyanidols in a given composition and also leading to the weight distribution of the proanthocyanidols present, are naturally adapted to the embodiment of the invention.

 The invention also relates to a composition based on proanthocyanidols as defined above, characterized in that it is furthermore essentially free of: - vegetable proteins, - gallic acid and gallic tannins, - flavonoids not belonging to to the family of flavanediols, oligomeric flavan diols, and phenolic acids.

 According to a first advantageous embodiment of the invention, the compositions of the invention are characterized in that they consist of at least 95% by dry weight of proanthocyanidols.

 According to another advantageous aspect of the invention, these compositions based on proanthocyanidols are characterized in that the proanthocyanidols have a molecular weight of between about 2000 +500 and about 55000 + 5000.

 A particularly useful composition preferably comprises proanthocyanodols of molecular weight of about 45,000 ± 5,000.

 Another particularly useful composition comprises proanthocyanidols having a molecular weight of about 5000 + 500.

 Determination of the purity of the polymeric proanthocyanidol, in a range of selected molecular weight, can be carried out using gel chromatography, using an organic membrane having a determined cutoff threshold, for example a polyethersulfone membrane or of polyvinylidine difluoride. Details concerning the protocol used are given in the examples.

dans laquelle n est un entier supérieur ou égal à 3 et de préférence compris entre 3 et 350,
R1, R2, R3, R4 et Ra sont ensemble ou séparément, l'hydrogène, un groupement hydroxyle ou un groupement méthoxy, et de préférence en ce qu'il s'agit de polymères de procyanidols dans lequel Ri, Rz, R4 et Ra sont des groupements hydroxyle et R3 est de l'hydrogène, ou de polymères de prodelphinidols dans lequel R1, R2, R3, R4 et Ra sont des groupes hydroxyle ou en ce qu'il s'agit de polymères de promalvidols dans lequel Ri et R3 sont des groupes méthoxy et R2 , R4 et Ra sont des groupements hydroxyle.The compositions adapted to the embodiment of the invention are according to another definition, characterized in that they comprise proanthocyanidols in the form of polymers, corresponding to the following formula:

in which n is an integer greater than or equal to 3 and preferably between 3 and 350,
R1, R2, R3, R4 and Ra are, together or separately, hydrogen, a hydroxyl group or a methoxy group, and preferably in that they are procyanidol polymers in which R1, Rz, R4 and Ra are hydroxyl groups and R3 is hydrogen, or prodelphinidol polymers in which R1, R2, R3, R4 and Ra are hydroxyl groups or in that they are promalvidol polymers in which R1 and R3 are are methoxy groups and R2, R4 and Ra are hydroxyl groups.

 The proanthocyanidol polymers of the invention can be either in linear form or organized in networks. The compositions of the invention may be formed from one or more types of proanthocyanidols, especially when these compositions are extracts obtained from a plant material, several types of polymers may be present in the composition and their distribution is not not necessarily constant depending on the initial plant material used.

 In a particularly interesting way, the compositions of the invention comprise proanthocyanidols having a certain solubility in water, a function of the molecular weight and the nature of the molecule.

 When these compositions are plant extracts, they can be obtained from any plant material plant rich in proanthocyanidol molecular weight greater than 2000 and likely, after the implementation of the methods of assaying polyphenolic and tanning functions, to to give compositions having the characteristics defined above.

Thus, by way of example of plant species that can be used as a source of proanthocyanidols, mention is made of conifers such as Cupressus sempervirens, Pinus maritima, and zingiberaceae such as Alpinia.
Galanga, plants with procyanidols, with prodelphinidols, such as Vitis vinifera, Vaccinum myrtillus, Fragaria vesca and plants with rarer proanthocyanidols such as plants with profisetinidols of the genus Schinopsis and with proguibourtinidols extracted from various Acacia.

 There may also be mentioned plants of the family Mimosaceae.

 The invention naturally aims, in a preferred embodiment, compositions comprising several. types of plant extracts that meet the definitions given.

 The invention also relates to a process for the preparation of a proanthocyanidol composition, from a plant part containing proanthocyanidols with a molecular weight greater than 2000, this process being characterized in that it comprises the following steps: decoction or maceration in an alcohol, in particular methanol or ethanol, to eliminate polysaccharides, in particular starch, - recovery of hydroalcoholic extracts and evaporation to dryness, under reduced pressure, optionally followed by washing and recovery of the dry residue, - recovery dry residue and filtration to remove a portion of the aromatic fraction of the extract, - countercurrent extraction, using successively at least 3 solvents of increasing polarity, including ethyl ether, sodium acetate, , butanol or methanol, in particular to eliminate the remaining aromatic species, as much as possible of the oligomers of 2000 to 2000 and proanthocyanidol monomers and to recover in the alcoholic phase a plant extract containing the proanthocyanidols, molecular weight greater than 2000, - if necessary, the purification of the extract obtained to remove the remaining solvents.

 The purification step may consist of dyafiltration with deionized water to remove most of the solvent. This operation is for example carried out by cooling the liquid continuously at low temperature, in particular at 40C.

 For the preservation of the product, once the solvent has been removed, it is possible to concentrate as much as possible the solution-aqueous suspension of polymeric proanthocyanidols and then lyophilize it. The preservation can also be carried out as a crystallized product from the aqueous suspension solution.

 The extraction is preferably carried out so as to avoid the degradation of proanthocyanidols. To do this, it is preferable to avoid too high temperatures and to reduce the amount of oxygen in contact with the proanthocyanidolic compounds.

Thus, the extraction and the subsequent steps can be carried out under an inert atmosphere, for example under nitrogen.

 In order to eliminate proanthocyanidols with a molecular weight of less than 2000, the following method may be used: the fraction extracted using the various solvents used is ultrafiltered on an organic membrane and in particular of the polyether sulfone membrane type, or a difluoride membrane of polyvinylidene ~~~~ whose cutoff threshold is chosen according to the fraction of proanthocyanidol that it is desired to eliminate. At the end of the operation, a concentrated solution of polymers retained on the membrane is obtained, and a dilute solution of products of molecular weight lower than that of the cutoff threshold.

 These steps may optionally be followed by a washing step until a content of less than 10%, advantageously less than 5%, and preferably 1% of light products, relative to the final substance obtained is obtained.

 Another particularly advantageous process for the preparation of proanthocyanidol compositions, from a plant part containing proanthocyanidols of molecular weight greater than 2000, comprises the following steps: decoction or maceration in an alcohol, in particular methanol or ethanol, for eliminating polysaccharides, in particular starch; ultrafiltration of hydroalcoholic solutions on an organic membrane of a porosity such that its cut-off threshold would be 20,000 Daltons for a globular protein ultrafiltered in an aqueous solution.

- Recovery of hydroalcoholic extracts and evaporation to dryness, under reduced pressure, optionally followed by washing and recovery of the dry residue.

 According to an advantageous embodiment of the invention, the process described above can be carried out starting from an initial plant material constituted by cones of Cupressus sempervirens, or from fresh rhizomes of Alpinia galanga.

 The compositions of the invention are particularly useful in that they lack cytotoxicity and hence can be used in pharmaceutical compositions.

In addition, the inventors have found the ability of proanthocyanidols of these compositions to bind to the cellular receptors of viruses. In this regard, the invention relates to pharmaceutical compositions comprising as active ingredients, a proanthocyanidol composition as defined above, in association with an acceptable pharmaceutical vehicle.

 Such compositions may be used according to different modes of administration.

 By way of example, suitable routes of administration include the topical route (dermal or ocular), the rectal route and the oral route.

 Particularly advantageously, they will be applied in topical form, in this case they include excipients or adjuvants usually used for this mode of administration.

 These pharmaceutical compositions advantageously comprise between about 0.1% and about 5% of active ingredients.

The use of the proanthocyanidols of the invention in pharmaceutical compositions is of interest for the antiviral properties of these molecules. Such compositions are particularly suitable for combating viruses such as that of herpes type I, herpes type II, varicella zoster, influenza A, the virus and the like.
Echo 25 and the HIV virus, rotavirus and cytomegalovirus.

 An advantage of the compositions of the invention lies in the fact that they cause few adverse side effects, and are of very low toxicity.

 A dosage suitable for oral administration is to administer 0.01 / kg / 24 hours of composition at 0.2 / kg / 24 hours of composition and preferably about 0.1 g / kg / 24 hours.

 The invention also relates to pharmaceutical compositions comprising as active principle, proanthocyanidols in combination with one or more substance (s) chosen from antibiotics, antiseptics, anti-inflammatory and antipyretic, since these substances are chemically compatible with proanthocyanidols.

 The invention is further directed to the use of the above proanthocyanidol compositions for the manufacture of a medicament with antiviral activity.

Other features and advantages of the invention will appear in the examples given without limitation in the following
Example 1: Hydromethanic extract of cones
Cupressus sempervirens
200g of crushed dry cones of Cupressus sempervirens are decocted three times in succession for half an hour per 2000ml of 80% methanol. The hydromethanic extracts are combined and evaporated to dryness under reduced pressure. This residue is taken up in 200 ml of distilled water at about 200 ° C. We filter. The insoluble material is taken up in 200 ml of distilled water at approximately 200 ° C. It is filtered. The filtrates are combined. The residue of this solution is approximately 33 g, ie a yield of 16.5%.

1.2 - Procyanidolic Fraction
The aqueous solution obtained above is adjusted to 400 ml with distilled water and extracted countercurrently with 5 times 100 ml of ethyl ether. The ether phase is removed, the ethyl ether is removed from the aqueous phase by evaporation under reduced pressure. The volume of the aqueous solution is made up to 400 ml with distilled water, and the mixture is then extracted with 5 times 100 ml of ethyl acetate.

The organic phase is eliminated. Ethyl acetate is removed from the aqueous solution by evaporation under reduced pressure. The volume of the solution is made up to 400 ml with distilled water and is then extracted countercurrently with 5 times 100 ml of n-butanol. The butanol phase is washed twice with 100 ml of distilled water and then evaporated to dryness under reduced pressure. This residue is dissolved in 20 ml of methanol and the methanolic solution is filtered and then evaporated to dryness. The residue is 780 mg, a yield of 0.39%. The product thus obtained, chromatographed on a silica gel plate, reveals the absence of substances capable of migrating into the solvent ethyl acetate, formic acid, water (8-1-1
V / V), indicating the absence of low molecular weight substances (<103). This technique makes it possible to monitor the purification of the product. On the other hand, this residue dissolved in an alcohol and treated with hot with hydrochloric acid gives rise to the formation of a red color. Plaque chromatography against one control revealed the formation of cyanidine, excluding other anthocyanins. Determination of the molecular weight of this gel permeation residue in a nonaqueous medium reveals a molecular weight distribution of about 1000 to 5000, with the minority presence of high molecular weight polymers (> 5.103).

EXAMPLE 2 Hydroethanolic Extract of Rhizomes of Alpinia Galanga 2.1 - Soluble Raw Extract
1kg of fresh rhizomes of Alpinia galanga containing 88% of water are crushed and then subjected to a first decoction of half an hour in 9.2 liters of ethanol at 65.8% in order to actually make a decoction of l equivalent of 220 g of dry rhizomes per 10 liters of 60% ethanol. Then the rhizomes are decocted twice in succession by 10 liters of 60% ethanol for half an hour. The extracts are combined and evaporated to dryness under reduced pressure. This residue is then taken up in 220 ml of distilled water at approximately 200 ° C. and filtered. Insoluble is taken up in 220ml of distilled water at 200C. The filtrates are combined. The residue of this aqueous solution is 38.4 g is a yield of 3.84% compared to fresh rhizomes and 17.45% compared to dry rhizomes.

2.2 - Proevanidolia fraction
The preceding aqueous solution is treated in the same manner as in the case of Example 1.2 until the evaporation of the butanol phase. The residue is then washed at about 200 ° C. with twice 20 ml of distilled water. The final residue is 104 mg, a yield of 0.01% compared to fresh rhizomes and 0.4% compared to dry rhizomes. This fraction has the same qualitative characteristics as those of Cupressus sempervirens cones. The measurement of the molecular weight reveals an average weight of 4000.

Example 3 Insoluble Procvanidolia Fraction of
Cupressus Sempervirens
200g of crushed dry cones of Cupressus sempervirens are decocted three times in succession for half an hour per 2000ml of 80% methanol. The hydromethanic extracts are combined and evaporated to dryness under reduced pressure. This residue is washed twice with 200 ml of distilled water at approximately 200 ° C. Wash water is removed. The residue is dried under vacuum in the desiccator and then washed at reflux with twice 200 ml of ethyl ether. The ethereal phasing is eliminated. The residue is then washed twice with 200 ml of ethyl acetate. The soluble phase in ethyl acetate is removed. The residue is then taken up in 20 ml of methanol at approximately 200 ° C. The methanolic solution is filtered. The residue of this methanolic solution is 1.74 g, ie a yield of 0.87%. This water-insoluble fraction has the same qualitative characteristics as the soluble fraction obtained in Example 1. The determination of the molecular weights indicates the very prominent presence of molecules of high molecular weight (5.104).

 An additional step may be added after the final dyafiltration with 80% methanol: another dyafiltration is carried out with deionized water in order to eliminate almost all the methanol. This operation is done by refrigerating the liquid continuously at + 40C. Once the methanol is removed, the polymeric procyanidol aqueous suspension solution is concentrated as much as possible and lyophilized.

 Crystallization can also be carried out at this stage from the aqueous suspension solution. Polymeric procyanidol crystallizes into dark mahogany colored needles.

EXAMPLE 4 Assay Methods and Determination of Purity
The dosage of procyanidols obtained from
Cupressus sempervirens can use their polyphenolic structure and therefore their reducing property phosphotungstic acid (not very specific) or their nature of proanthocyanidol that is to say their property of giving an anthocyanin by heat treatment by the strong acids.

 In both cases we obtain a value relative to a control more or less distant from the molecule itself.

 It is also possible to use the tanning property of procyanidols in aqueous solution: it is in this case to determine the fixation on pre-chromed skin powder.

10 / -Dosage of polyphenolic function:
10 g of sodium tungstate are refluxed with 8 ml of orthophosphoric acid and 75 ml of deionized water for 3 hours. Let cool and complete to 100ml with deionized water.

15% sodium carbonate solution in deionized water: reagent B.

 50 mg of procyanidol are weighed and dissolved in 10 ml of exactly measured water.

 2 ml of this solution is taken and diluted to 25 ml in a volumetric flask (solution C).

In a volumetric flask of 50ml, place:
Solution C: 5 ml
Reagent A: lml
Reagent B: qs50ml
The OD is read at 715 nm, exactly 2 minutes after mixing the last reagent. The same is true with a range of pyrogallol (0 to 50 mg) as a reference.

 The result for the polymeric procyanidol is 43.5% of total polyphenols expressed as pyrogallol.

20 / -Dosing of the tanning function:
A solution of procyanidol prepared as solution C above is stirred for one hour at room temperature with 1 g of pre-chromed skin powder (Merck P / N 4332).

 An assay of the total polyphenolic function is then carried out as above. The tanning function represents the difference between the value found for the total polyphenols and the value found after treatment with the skin powder.

 The result for the polymeric procyanidol is 80% expressed as a tanning function.

30 / -Dosage of the procyanidolic function proper:
10 mg of polymer procyanidol measured approximately exactly are dissolved in 20 ml of 75% ethanol.

 A test tube is prepared with 1 ml of this solution, 5 ml of 75% ethanol and 1 ml of concentrated hydrochloric acid. Place in a boiling water bath for 30 minutes.

 The control tube is made by mixing the same constituents in the same proportions but at room temperature and just before reading on the spectrophotometer.

 The OD of the test solution is read at 540 nm against the control solution.

 The result for the polymeric procyanidol is 0.60.

40 / -Determination of the molecular weight of polymeric procyanidol:
WuBondagel 125 (Waters) gel exclusion high performance chromatography was performed in an aprotic solvent against polystyrene controls (Aldrich)
Flow: 0.5 ml / minute isocratic.

Solvent: Chloroform-dimethylsulfoxide 50-50 (v / v)
Detection: DO at 283nm
Concentration of the controls: lmg / ml in the same solvent
Concentration of proanthocyanidol: 0.05 mg / ml in the same solvent
Molecular weights of controls: 184,200, 50,000, 24,150, 7,025, 4,136 and 2,000.

 The 184.200 molecular weight control is used as a total exclusion control on the gel. Indeed, this type of gel does not retain molecular weights greater than 50,000. The relative retention time of each sample is then calculated in relation to the totally excluded control.

 The correspondence curve between the decimal logarithm of the molecular weight and the relative retention time is plotted.

 The result for the polymeric procyanidol obtained by applying the technique described above is 45,000.

50 / -Determination of purity of polymeric procyanidol by gel chromatography:
The chromatography technique above provides a plot that includes two peaks. One corresponds to impurities with a molecular weight of less than 5000. Indeed, by this chromatography technique it is found that the molecular weights of less than 5000 are collected in a single peak at the end of the chromatogram.

 The method of preparation, which uses a final ultrafiltration on a membrane with a cut-off point of approximately 20,000, makes it possible to obtain at the end of the operation a concentrated solution of polymer retained on the membrane and a dilute solution of products of lower molecular weight. to 20,000 and which does not contain procyanidol (verified by the method of 30).

 The total solids concentration of the two solutions is then determined. Then their absorption in U.V. is determined at 283 nm. It is thus possible to calculate the E% 1cm: Absorption capacity of the light of the product in solution at 1% on 1cm of optical path of each product.

 The ratio of the areas of the two peaks is then calculated on the chromatogram and the purity of the polymeric procyanidol is concluded.

 The result obtained by this method is 98%.

Example 5: In Vitro Antiviral Trials
The antiviral trials were performed on the type I herpes simplex virus implanted on human embryonic lung fibroblasts.

 The antiviral efficacy of the different proanthocyanidolic products obtained, that is to say the amount of product per night, was determined on the same batch of viral dilution corresponding to an infectious dose of 50 (ID 50) 10 5 virus per night. that is 100u1 medium which causes a 50% decrease in the ID 50.

By way of comparison, the oligomers (PM 103) of Pinus maritima obtained from dry Pinus maritima bark were also tested, as in the case of Example 1. These oligomers are found in solution in sodium acetate. which is normally used for washing the higher molecular weight residue.

<tb><SEP> EXTRACT <SEP> QUANTITY <SEP> PROVOQUING
<tb><SEP> THE <SEP> DECREASE <SEP> OF
<tb><SEP> HALF <SEP> FROM <SEP> THE <SEP> DI <SEP> 50
<tb> Procyanidols <SEP> soluble <SEP> of
<tb> Cupressus <SEP> sempervirens <SEP> 4 <SEP> 9/100 <SEP> pl
<tb> Insoluble <SEP> Procyanidols <SEP> of
<tb> Cupressus <SEP> sempervirens <SEP> 7.2 <SEP> erg / 100 <SEP> pl
<tb> Soluble Procyanidols <SEP>
<tb> of Alpinia <SEP> galangal <SEP> 1 <SEP> ut / 100 <SEP> ul <SEP>
<tb> Procyanidolic Oligomers <SEP>
<tb> of <SEP> Pinus <SEP> maritima <SEP> 15.6 <SE> pg / 100p1
<Tb>

Example 6: Animal Toxicity Tests of the Proanthocyanidols Obtained in the Previous Examples
The oral toxicity of all proanthocyanidols obtained, sought in the mouse could not be evaluated, the doses to be ingest exceeding the possibilities of the animals and being greater than 2g per kg of body weight.

 The primary dermal irritation tests of the same products as well as the eye irritation tests carried out in the rabbit in 5% aqueous solution for the soluble fractions made it possible to define the products as non-irritating. Primary cutaneous irritation tests of 5% solution insoluble protanthocyanidols in a 50% monopropylene glycol / water mixture defined these products as non-irritating compared to a pure mixture of monopropylene glycol and 50% water. .

Example 7: Clinical Trials on Procyanidols of Alpinia qalanqa
The clinical trials were carried out on the proanthocyanidols of Alpinia galanga obtained in Example 2.

The study included 140 patients with herpetic symptoms beginning less than 24 hours, divided as follows:
.24 women between 17 and 47 years of age with recurrent herpes related to the menstrual cycle.

Among them, there were 18 cases of herpes labialis and 6 cases of herpes affecting various areas of the body non-mucous (wings of the nose, buttocks, cheeks).

 116 men between the ages of 19 and 54, all with cold sores.

The women were divided into four groups:
a control group of recurrent herpes labialis,
a test group of the same condition is each time 9 people,
a control group of various herpes,
a trial group of various herpes, in each case 3 people.

 The men were divided into two equal groups of 58 people, one trial group and one control group as homogeneous as possible.

The witnesses are treated until the disappearance of the lesions at the rate of one application at 7h, 9h, 11h, 13h, 15h, 17h and 19h during three days then of an application morning, noon and evening until the end, d a placebo gel of the following composition:
Crosslinked sodium polyacrylate: 0.75%
Caramel: qs for coloring
Preservatives: propyl and methyl paraoxybenzoate: 0.35%.

 Distilled water: qs 100%.

The test groups are treated under the same conditions as the controls with a gel of the following composition:
Crosslinked sodium polyacrylate: 0.75%
Proanthocyanidol fraction: 2%
Preservatives: propyl and methyl paraoxybenzoate: 0.35%.

 Distilled water: qs 100%.

Results
We appreciate the time that the lesion disappears completely, leaving only a cicatricial trace completely closed and dry.

Men's control group: 5th day: 1
6th day: 4 and 1 abandon
7th day: 4
8th day: 16 and 2 dropouts
9th day: 11
10th day: 14
11th day: 2
12th day: 3
Men's test group: 1st day: 1
2nd day: 5
3rd day: 37
4th day: 12
5th day: 1
6th day: 0
7th day: 1 abandon
8th day: 1
First control group of women:
5th day: 1
6th day: 1
7th day: 4
8th day: 0
9th day: 2 abandons
10th to 15th day: 0
16th day: 1
First group test of women:
1st day: 4
2nd day: 2
3rd day: 2
4th day: 1
Second control group of women:
13th day: 2
17th day: 1
Second group test of women:
4th day: 3
In addition to a high efficacy of the product, we note in all treated subjects excellent tolerance to treatment, no side effects have been reported, discontinuations during the trial are not related to intolerance to the product.

Example 8
Trial of procyanidols of Cupressus sempervirens soluble in water on the virus acquired human immunodeficiency syndrome (HIV-1).

a / Antiviral effect
The assay is performed on H9 cells infected with successive decimal dilutions of HIV-1 for 90 minutes.

 After infection of the cells, they are treated with decreasing concentrations of procyanidols at doses lower than the cytotoxicity observed on this cell line (150 μg / ml).

 After 10 days of culture, the cells are centrifuged and the viruses are searched by radioimmunoassay (search for the p24 protein) on the supernatant.

Results:
Soluble procyanidols of cypress obtained according to Example 1 provide an inhibition of viral expression of 97% at a concentration of 50 μg / ml.

b / Virucidal effect
The viral suspension (HIV1) is treated for 30 minutes at 370C with decreasing concentrations of soluble procyanidols cypress. Then the cells
H9 are infected by successive decimal dilutions of the previous suspension.

Results:
Soluble procyanidols in cypress provide a reduction in viral titer of 1.5 log (initial titer: 3.104) at a concentration of 50 μg / ml, and a reduction of 2.5 log at a concentration of 150 μg / ml.

Example 9
Trial of soluble procvanidols in water of
Cupressus sempervirens on the herpes virus
Simplex type II.

 Antiviral trials were performed on herpes simplex virus type II implanted on human embryonic lung fibroblasts.

 A batch of virus suspension corresponding to an infectious dose (ID 50) of 2.5 × 10 4 viruses per well was determined, the antiviral efficacy of the soluble procyanidols of Cupressus sempervirens, that is to say the amount of product per well. (ie in 100u1 of medium) which causes a halving of the DI50.

 The value found is 3.3pg / 100p1.

Example 10
Trial of soluble procvanidols in water of
Cupressus sempervirens on the Varicella-Zona virus
The antiviral trials were performed on the varicella virus and shingles implanted on human embryonic lung fibroblasts.

 A batch of virus suspension corresponding to an infectious dose 50 (ID50) 5 × 10 3 viruses per well, the anti-viral efficacy of soluble procyanidols of Cupressus sempervirens, i.e. the amount of product per well (c that is, 1001 of medium) which causes a 50% decrease in the ID50.

The value found is 6ug / 100u1
Example 11
Trial of procyanidols soluble in water of
Cupressus sempervirens on Influenza A virus
Antiviral tests were performed on Influenza A virus implanted on human colon adenocarinoma cells.

 A batch of virus suspension corresponding to an infectious dose 50 (ID50) of 7.103 viruses per well was determined, the antiviral efficacy of the soluble procyanidols of Cupressus sempervirens, that is to say the amount of product per well (c '). that is to say in 100 μl of medium) which causes a halving of the DISO.

 The value found is 12pg / 100> 1.

Example 12
Trial of procyanidols soluble in water of
Cupressus sempervirens on the ECHO 25 virus
Antiviral assays were performed on the EC11O virus implanted on human embryonic lung fibroblasts.

 A batch of virus suspension corresponding to an infectious dose (ID50) of 104 viruses was determined per well, the antiviral efficacy of the soluble procyanidols of Cupressus sempervirens, i.e., the amount of product per well (e.g. that is to say in 100 μl of medium) which causes a halving of the DISO.

 The value found is 31pg / 100p1.

Example 13
Preparation of a fraction of nolecular weight 45,000 daltons of procyanidols cones of Cupressus sempervirens
20kg of Cupressus sempervirens cones are macerated 3 days three times in a row in 300 liters of 80% methanol (20% water by volume). The extractive solutions are combined to form about 850 liters.

 The solution is ultrafiltered on polyethersulfone tubular membranes having a cut-off threshold of 50000 daltons, to remove very highly polymerized polymers greater than about 5.104.

 The filtrate is then ultrafiltered on membranes of the same type but having a cutoff threshold of 20,000 daltons. The product retained on these membranes is washed by diafiltration until the low molecular weights only represent 1% of the total weight of dry substance.

Example 14
Activity of the procyanidolic fraction of molecular weight 45000 on Rotavirus
Antiviral trials were performed on a wild rotavirus strain implanted on MA104 rhesus monkey embryonic kidney cells.

 On a lot of virus suspension corresponding to an infectious dose 50 (ID50) 2 × 10 3 virus per well, the antiviral efficacy of this procyanidolic fraction, that is to say the quantity of the product per well (ie ie in 100u1 of medium) which causes a halving of the DISO. The value found is 8pg / 100p1.

Claims (14)

1 / Composition based on proanthocyanidols, in particular of plant origin, characterized by a weight proportion of at least 50% of molecules having apparent molecular weights greater than 2000, this proportion consisting of at least 40% of proanthocyanidols, a percentage being expressed as a tanning function, and in that said composition is essentially free of: - aromatic essences and in particular essential oils, resins and other lipophilic substances - polymerized sugars such as starch, gums, derivatives cellulosic, oligomeric sugars such as sucrose, glucose, levulose. 2 / composition based on proanthocyanidols according to claim 1, characterized in that it is essentially free of: - plant proteins, - gallic acid and gallic tannins, - flavonoids not belonging to the family of flavanes- diols, oligomeric flavan diols, phenolic acids. 3 / A composition according to claim 1 or claim 2, characterized in that it consists of at least 95% by dry weight of proanthocyanidols. 4 / Composition according to any one of claims 1 to 3, characterized in that the proanthocyanidols have a molecular weight of between about 2000 + 500 and about 55000 + 5000. 5 / A composition according to any one of claims 1 to 4, characterized in that it comprises proanthocyanidols of molecular weight about 45000 t 5000. 6 / A composition according to any one of claims 1 to 4 characterized in that it comprises proanthocyanidols of molecular weight about 5000 + 500. 7 / Composition according to any one of claims 1 to 6, characterized in that they comprise proanthocyanidols in the form of polymers corresponding to the following formula:

 in which n is an integer greater than or equal to 3 and preferably between 3 and 350. R1, R2, R3, R4 and Ra are together or separately, H, OH or OCH3, and preferably in that it is a procyanidol polymer in which R1, R2, R4 and Ra are hydroxyl groups and Ra is hydrogen, or in that it is is preferably a prodelphinidol polymer in which R 1, R 2, R 3, R 4 and R 6 are hydroxyl groups or in that it is a promalvidol polymer in which R 1 and R 3 are methoxy groups and R 2,, R4 and Ra are hydroxyl groups. 8 / plant extract according to any one of claims 1 to 7, as obtained from a plant selected from conifers such as Cupressus sempervirens, Pinus maritima, acacias, mirnosacees. 9 / Process for the preparation of a composition based on proanthocyanidols from a plant part containing proanthocyanidols of PM greater than 2000 according to any one of claims 1 to 8, characterized in that it comprises the steps following: - decoction or maceration in an alcohol, especially methanol or ethanol, to eliminate polysaccharides including starch. - Recovery of hydroalcoholic extracts and evaporation to dryness, under reduced pressure, optionally followed by washing and recovery of the dry residue, - recovery of the dry residue and filtration to remove a portion of the aromatic fraction of the extract, - extraction against the current using, successively, at least 3 solvents of increasing polarity, in particular ethyl ether, ethyl acetate, butanol or methanol, in particular to eliminate the remaining aromatic species, as much as possible of the oligomers of MW less than 2000 and proanthocyanidol monomers and to recover in the alcoholic phase a plant extract containing the proanthocyanidols of molecular weight greater than 2000, - if necessary, the purification of the extract obtained to remove the remaining solvents. 10 / Process for the preparation of a proanthocyanidol-based composition from a plant part containing proanthocyanidols of PM greater than 2000 according to any one of Claims 1 to 8, characterized in that it comprises the steps following: - decoction or maceration in an alcohol, in particular methanol or ethanol, to eliminate polysaccharides including starch, - ultrafiltration of hydroalcoholic solutions on an organic membrane of a porosity such that its cut-off threshold would be 20000 Daltons for a globular protein ultrafiltered in an aqueous solution, - recovery of hydroalcoholic extracts and evaporation to dryness, under reduced pressure, optionally followed by washing and recovery of the dry residue, 11 / Process according to claim 9 or 10, characterized in that the Recovered plant extract is concentrated and then lyophilized or crystallized. 12 / A method according to claim 9 or claim 10, characterized in that the initial plant material is constituted by cones of Cupressus sempervirens. 13 / Composition according to any one of claims 1 to 8, characterized in that it is non-cytotoxic. 14 / Pharmaceutical composition, characterized in that it comprises as active ingredient, a proanthocyanidol composition according to any one of claims 1 to 8, in association with an acceptable pharmaceutical vehicle. 15 / Use of a proanthocyanidol composition according to any one of claims 1 to 8, for the manufacture of a medicament with antiviral activity.