IL34381A - Process for obtaining polyhydroxy-phenylchromones which stabilise the cell structure and the cytometabolism - Google Patents

Description

Process for obtaining polyhydroxyphenyl- chromones which stabilise the cell structure and the cyt©metabolism DR. MADAtfS & CO Ct 32651 Process for obtaining polyhydroxyphenyl-chromones which stabilise the cell structure and the cytometabolism.

The present invention is concerned with a process for obtaining polyhydroxyphenyl-chromones which stabilise the cell structure and the cytometabolism. The present invention is also concerned with pharmaceutical compositions containing these polyhydroxyphenyl-chromones.

We have' found that certain polyhydroxyphenyl-chromones, for example silymarin, exercise a protective and stabilising-effect on the intracellular biomembranes, which are sites of cytometabolism, and, because of this novel action, are valuable pharmaceuticals.

The previous suggestions for the isolation of silymarin on a laboratory scale are not suitable for use on a large scale · because the starting material, the fruit of Silybum marianum Gaertn., contains up to 30 of fatty oils and accompanying materials which are difficult to separate and only greasy concentrates are obtained, which give rise to exceptional difficulties in the further working up thereof. Furthermore, working according to these previous proposals on a technical scale would be difficult because of the extremely high expenditu of time and because of the many and various solvents which are used individually and successively one after the other for the removal of the ineffective accompanying materials and of the fatty oils.

Thus, in the case of production according to the usual methods, several days are required for defatting by extraction and, in most cases, even this is not sufficient.

If, on the other hand, as is provided by the process of the present invention, a complete defatting is omitted and the process is altered in such a manner that a starting material containing 5 - 10 fat can be used, then the defatting can be carried out under high pressure, preferably with the use of screw presses, such as are used for oil seeds, whereby 20 - 25$ of the fatty oil can be removed, without loss of active material By means of this defatting method, the otherwise necessary grinding step can also be omitted because the intercellular bonds are substantially broken down by the high pressure and by the attrition in the screw press to such an extent that the active materials can then be quickly and completely removed by an extraction agent.

Because of the residual content of fatty oil, there must first be found an extraction agent in which not only the fatty oils but also the active materials are particularly readily soluble. In a series of investigations, ethyl acetate has been found to be optimally suitable for this purpose. It enables an > exhaustive extraction to be achieved in onljr 24 hours, whereas in the case of a laboratory scale extraction with acetone, an extraction time of 14 days has been reported to be necessary.

Although, after evaporation of the ethyl acetate, an oily- greasy, partially lumpy product is obtained v/ith an active material content of about 20 - 30$, it was necessary to find, in order to avoid repeated treatments with various solvents, a method for the separation of the active materials from the accompanying materials and fatty oils with the help of a · partition process "between two liquid phases of limited miscibility.

The finding of a solvent system suitable for this purpose was unsuccessful for a long time because, in the case of all phase pairs investigated, the partition factor of the mixture of the accompanying materials and of the fatty oils and the partition factor G,y of the active materials gave such a low separation factor β that the number of partition steps necessary would have required an inadmissably high expenditure on apparatus.

In the case of the finally investigated phase pair methanol/petroleum ether (b.p. 40 - 60°C . ) » there was calculated from the partition factors = 0. 25 and = 0.038 given for a substance concentration of 2 , the separation factor β of 6 .5 , which theoretically would still have required, for a 70 - 80$ enrichment, about 20 partition steps. However, when carrying out the partition, we observed a very strong phase displacement within the system. Experiments to reduce it by the addition of water were successful.

The then further investigated ternary system methanol/water petroleum ether proved to be useful. As optimum, we finally foun a methanol-water-petroleum ether mixture consisting of methanol/ water ( 95 s 5) as the lower phase in equilibeium with pure petrole um ether (b.p. 0 - 60°C . ) as upper phase. We have also found that good results are obtained when the ratio by volume of the upper phase to the lower phase is about 1 : 1. Furthermore, the system in a state of equilibrium showed the advantage of an unexpected displacement of the partition factors in favour of the desired purification process for the active materials.

In the case of the practical utilisation of this ternary system to he used according to the present invention, we found, surprisingly, that as few as six individual partitions sufficed in order to oh ain a 70 - QCfi enrichment of materials.

In the case of a multiplicative partition, which can he carried out intermittently or uniformly and is preferahly carried out with the two phases in countercurrent , the active materials "become enriched in the lower phase and can he removed at the exit of the apparatus used with the lower phase.

As far as apparatus is concerned, for a uniform multiplicative partition, there is preferably used a hattery comprising six individual steps, each with, for example, a centrifugal separator and an emulsifier connected one behind the other in such a manner that the purified active material runs off continuously with the lower phase, whereas the upper phase, passed in countercurrent against the lov/er phase, runs off at the opposite end of the battery after pass^age through the six emulsifying and separation steps.

A comparable degree of effectiveness is achieved with a Ziehl's column apparatus having a theoretical plate number of 10 - 12 (calculated from HTU = height for one transfer unit).

In the case of both methods of carrying out the process, there has proved to be useful not only the introduction of the substance at the beginning of a Jantzen battery but also in the middle of a van Dyk battery. The supply of substance preferably takes place in the form of an approximately 2$ solution in the lower phase.

After the partition, one can obtain from the loweii phase , evaporation thereof, as a beige-colored dry product the purifie polyhydroxyphenyl-chromones which show a positive reaction wit 2,4-dinitrophenylhydrazineo This dry product contains 70-80$ silymarin and can be used directly in this form or also after further purification or separation.

The upper phase contains also 2,4-DNPH-positive zones which are not used.

By thin layer chromatographic separation of the obtained silymarin on silica gel plates with the elution agent carbon tetrachloride : acetonitrile 1:1 (v : v) , with a io solution of 2,4-dinitrophenylhydrazine there can be detected, seven substances named silymarin I, II, Ilia, Illb, IVa, IVb, IVc which are clearly separated from one another.

The substance having the greatest value is Silymarin I. The proposals concerning its structure and conformation are not yet sufficently secured. It has the empirical formula ^25¾2^10 and ^s ' a ^-substituted dihydroxyphenyl-chromone .

The melting point is 167° C.

The other substances II, Ilia, Illb, IVa, IVb and IVc can be separated by column chromatography and by cristallisatio They are likewise polyhydroxyphenyl-chromones. In the same way as silymarin I, they all exhibit the same effects ift-eif-e-c^-in pharmacological tests.

Consequently, because of the great pharmacological similarity between these seven compounds,, it is justifiable to retain for the product consisting of a mixture of substances I, II, Ilia, Illb, IVa, IVb, IVc, the term silymarin and to call the individual components silymarin I, silymarin II, silymarin Ilia and b, and silymarin IV a, b and c. (also Silymarin I can be separated" directly from the lower phase running off from the partition apparatus "by crystallising out. Tor this purpose, the lower phase, after treatment with activated charcoal preferably in an amount of about 0.5$» is evaporated to such an extent in a vacuum of about 0.5 mm.Hg. that, after removal of the active charcoal, upon further concentration the silymarin I separates out in the form of crystals.

We have found that good results are thereby obtained when the lower phase is. mixed with 0. $ activated charcoal, evaporated at about 20 mm.Hg. to about one third of the initial volume, clarified in a filter press while still hot, and then further evaporated to form one sixth to one tenth of the initial volume, silymarin I crystallising out in the form of white to ochre-coloured crystals.

Thus, the process for obtaining polyhydroxyphenyl-chromones especially of silymarin or of its components, comprises, according to the present invention, the following steps: 1. Pressing off of the main amount of the fatty oils under high pressure, preferably in a screw press, 2. Exhaustive extraction of the press residues, which still contain oil, with ethyl acetate, followed by evaporation of the solvent to give an oily-greasy primary extract, 3· Uniform multiplicative partition of the primary extract in the solvent system according to the present invention, followed by evaporation of the lower phase thereof to give a dry product with a silymarin content of 70 - 80$, 4. Optionally a previous separation of silymarin I by crystallising out the lower phase, the other polyhydroxyphenyl-chromones being obtained from the mother liquor.

The following Example is given for the purpose of illustrating the present invention: -Example. 1. 100 kg. dried fruit from Silybum marianum are freed from the main amount of the fatty oils in a screw press of the type used for oil seeds, operating under a high pressure.

There is obtained about 75 - 80 kg. of press residue with an oil content of 5 - 10 . 2. 80 kg. of the press residue are exhaustively extracted with ethyl acetate. After evaporation of the ethyl acetate, there are obtained about 5 - 6 kg. of an oily-greas ,· :. partially lumpy dry residue with an active material content of 20 - 30$. 3· The dry residue is dissolved in the form of a 2$ solution in the lower phase of the system methanol/water ( 95 : )/petroleum ether (p.p.. 0 - 60°C.) which had been brought into equilibrium and then centrifuged from flocculent solids to give a clear solution. The final volume of the lower phase is about 300 litres.

Six individual centrifugal separators for liquid/liquid separation are, with the intercalation of an emulsifying step between each one, connected in series in such a manner that the lower phase flows through the upper one in countercurrent , whereby, in each emulsifying stage, both phases are emulsified with one another for the purpose of effecting a material exchan In the following separator step, the emulsion is again separate into a heavy and a light phase. The flowings are conducted in such a manner that the separated upper phase passes in counter-current into the lower phase of the preceding emulsifying step.

The upper and lower phases are first passed continuousely in countercurrent , ' without the addition of substance, through the partition batter in order to obtain phase equilibrium.

Into the flowing lower phase, there is then continuously passed in a 2$ solution of the substance. When feeding in at the inflow of the lower phase, care must be taken that the total flow volume ratio upper phase: lower phase of 1 : 1 does i change .

The flow velocity of the two phases depends substantially upon the efficiency of the emulsifiers and of the separators. The optimum adjustment can be determined gravimetrically by quantitative determinations of the material transfer factor.

The lower phase (about 300 litres) flowing off from the partition battery is evaporated to dryness in a vacuum at mm.Hg. to give a beige-coloured to brownish powder in a total yield. of 3 - 1 kg* The active material content (silymarin) is between 70 and 80 , i.e. the yield of active material is 2. 2 , calculated on the amount of starting material used. 4. Modification for obtaining silymarin I directly from the lower phase: The lower phase (about 300 litres) flowing off from the partition battery, is collected, mixed with 1. 5 kg activated charcoal and concentrated in a vacuum at 20 mm.Hg. to 100 litr The still hot solution is clarified in a filter press and subsequently further concentrated. In the region of a residual volume of about 50 ~ 30 litres, an increasing amount of crystallisation commences. Depending upon the degree of concentration, there is obtained a white to pchre-coloured crystallisate .

The crystallisate has a melting point of 167 - 169°C. It consists of silymarin I and has a degree of purity of 95 - 100 .

The mother liquor remaining after separation of the crystallisate, which contains silymarins II, III a and b and IV a - c, is evaporated to dryness by further concentrati in a vacuum at 20 mm.Hg.

The polyhydroxyphenyl-chromones otained "by the process according to the present invention showed, even in the case of the first animal experimental investigations, an outstandi protective action against influences which are dangerous to the liver.

Electron-microscopical investigations have evidenced that the massive damages of the- ultra structure of the liver parenchym cell caused "by liver poisons (for instance carbon tetrachloride) can be prevented by silymarin.

Even the considerably damaged structure of the mitochondria and of the endoplasmic reticulum which occured after the administration of cx-amanitine was completely overcome by silymarin. The restoration of the destroyed integrity means that silymarin exercised not only protection but also a stabilising action upon the structural elements of the liver cells and therewith also upon the cytometabolism.

Since the same electron-optical findings resulted when bioptic material from human livers was investigated,- there presents itself now a completely new therapeutic principle, namely the direct action upon the structure elements of the liver cells.

Especially impressive are the clinical results on toxic metabolic liver damages, chronic persistent hepatitis and acute hepatitis with cholestatic trend.

Incompatibilities and side effects v/ere not observed, e- not in cases of high dosage and of prolonged therapy.

Claims (8)

What we claim is :- ^

1. Process for obtaining polyhydroxyphenyl-chromones '. which stabilise the cell structure and the cytometabolism, wherein the dried fruit of Silybum marianum Gaertn. is subjected to high mechanical pressure in order to. free it from the main amount of the fatty oil present therein, the press residue, which contains still 5 - 10 residual oil, is exhaustively extracted with ethyl acetate, the ethyl acetate extract is evaporated, the oily-greasy, partially lumpy dried residue obtained, which contains about 20 - 30 of active material, is dissolved, to give an approximately 2 o by weight solution, in the lower phase of a ternary solvent system for. the separation of undesired accompanying materials, said ternary system consisting of water, methanol and petroleu ether, centrifuged until clear for the removal of flocculent solid material particles, subjected in this solvent system to a multiplicative partition in countercurrent and the lower phase separated off and. evaporated in a vacuum to give a brownish powder containing 70 - 80 polyhydroxyphenyl- chromones.

2. Process according to claim 1, wherein the dried fruit is subjected to high mechanical pressure in a screw press.

3. Process according to claim 1 or 2, wherein the ternary system used for carrying out the partition consists of methanol ( 5 ) , water (5) and petroleum ether (b.p. 40 - 60°C.) (100).

4. Process according to any of the preceding claims, wherein the partition is carried out in a battery ..consisting of a plurality of pairs of emulsifiers and separators, the flow through said batter being such that the upper phase separated in one separator passes in oountercurrent to the flow of the lower phase in the preceding emulsifier and into the flowing lov/er phase there is continuously fed in the approximately 2 solution of material containing 20 - 30 of active substance, the ratio of total flowing volume of upper phase/lower phase being kept at 1 : 1.

5. Process according to any of the preceding claims, wherein the lov/er phase flowing off from the partition stage mixed with about 0.5$ activated charcoal, first evaporated in a vacuum at about 20 mm.Hg, to about one third of the initial volume, the still hot solution clarified through a filter press and further evaporated to form one sixth to one tenth of the initial volume, in which concentration range a white to ochre-coloured crystallisate , silymarin I, separates out and is separated off.

6. Process according to any of the preceding claims for obtaining polyhydroxyphenyl-chromones , substantially as hereinbefore described and exemplified. '

7. Polyhydroxyphenyl-chromones, whenever obtained by the process according to any of claims 1 - 6.

8. Pharmaceutical compositions containing at least one polyhydroxyphenyl-chromone obtainable by the process according to any of claims 1 - 6, in admixture with a solid or liquid pharmaceutical carrier or diluent.