CS223986B2 - Method of gaining the silymarine - Google Patents

Description

The invention relates to a process for obtaining pure silymarin by extracting the fruit of the milk thistle (Silybum marianum Gaertn.).

The term silymarin means according to the state of the art a group of silymarins, i.e. polyhydroxyphenylchromanones (silymarin I-IV), which have already been isolated from the milk thistle fruit in 70 to 80% purity, depending on the drug (German Offenlegungsschrift 1 923 082). .

Silymarin X to silymarin III are isomers of the summary formula ^ 25 ^ 22 ^ 10 ' ^{in the} literature have been characterized structurally chemically as silybin, silydianin and silichristin (German Offenlegungsschrift 1 923 082; A. Pelter, R. Hansel: Tetrahedron Letters, London 25. 2 911 (1968), H. Wagner, L. Horhammer, 0. Seligmann and NR Farnsworth: Tetrahedron Letters 31.

675-2678 (1970); H. Wagner, O. Seligmann, L. Horhammer, and M. Seitz: Tetrahedron Letters 22, 1895-1889 (1971). Silymarin IV is taxifolin (German Offenlegungsschrift No. 1 923 082).

Silymarin is a valuable liver therapeutic and the silymarin mixture is more effective than the sum of the effects of the individual isomers due to synergism (German Laid-Open Publication No. 1,923,082).

German Offenlegungsschrift No. 1 923 082 discloses a process for obtaining silymarin (silymarin I-IV), wherein the dried fruit of a milk thistle Gaertn. mechanically pressed to get rid of the main amount of fatty oil. Exhaustive extraction of the residue after pressing with ethyl acetate by evaporation of the ethyl acetate solution, taking the dry residue into aqueous methanol, defatting with a uniform multiple countercurrent separation and evaporating the fat-free methanolic solution under vacuum to dryness yields 70-80% polyhydroxyphenylchromanone-IV (silymarinin). The ratio of silymarin I-III is 3: 1: 1; the content of silymarin IV, taxifoline is about 2 to 3% of the total silymarin content.

A further state of the art is German Patent Publication No. 2,020,407. The process of this publication is concerned with the isolation of silymarin without prior defatting by total extraction of solvent mixtures of alcohol-water-ester of lower fatty acid or hydrocarbon and separation of fat fractions by subsequent distillation. up to 40% of the volume of the obtained extraction solution, followed by weaning and removal of the fat deposited above. According to the application, this process leads to only 40% silymarin extract; the patent application further recommends recrystallization from methanol, but without further details of the process. In an attempt to reproduce the process, the process was found to be unrepeatable and about 13% of the original fat remained in the final product.

Another method is known from German Patent Publication No. 2,017,789. The end products of this process should contain 40 to 70% silymarin and the yield of the final product is 0.8 to a maximum of 2.4% (at 40% silymarin). Its reproduction has shown that this method is also unrepeatable.

French patent No. 7 214 042, which relates to the production of milk thistle extract without defatting, has long been disclosed in the prior art. The final product is said to contain about 82% polyhydroxyphenylohromones; silymarins should be 50% of the reported 82%. It should be noted that silymarin is assigned to another group of compounds, namely polyhydroxyphenylchromanones, but not polyhydroxyphenylchromones. Also, this method is not reproducible.

The method of German Unexamined Patent Publication No. 1 923 082 has not yet been substantially improved. The difficulty lies in the separation of the active ingredients from the drug while preserving the existing silymarin mixture and in its separation from about 30% of the fatty oils and the difficult to remove accompanying substances. These difficulties were only partially overcome by German Unloading Document No 1 923 082.

Since silymarin is a valuable liver therapeutic, there is a need for highly concentrated silymarin, but which is above the state of the art.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a novel process for obtaining pure silymarin (silymarin I-IV) which is largely free of accompanying substances and contains all the natural isomers. This new process should simply lead to a very pure product and higher yields.

Surprisingly, a process has been found which overcomes the disadvantages of the known processes and results in economically pure silymarin (silymarin I-IV) (purity 90-96%).

Process for obtaining silymarin (silymarin I-IV), wherein the milk thistle is deprived of most of the fatty oil by cold pressing, the compressed meat is crumbled, the pressing residue is extracted with ethyl acetate and processed, characterized in that:

a) extraction of the residue after pressing is carried out two to four times, preferably three times, in each case for 0.5 to 1.5 times, preferably for 1 hour, with anhydrous ethyl acetate at a temperature of 65 to 76 ° C, preferably at a temperature of 75 ° C, in a ratio of 1: 8 to 1:12, preferably 1:10 with vigorous stirring, and the extracted drug is separated.

b) either the combined ethyl acetate solutions are evaporated at a temperature of 40 to 55 ° C, preferably at 50 ° C under a vacuum of 1.5 kPa, the residue after evaporation a) is defatted by a three-minute to twenty-minute, preferably fifteen-minute solid-liquid extraction using petroleum ether having a boiling range of 60 to 70 ° C in a ratio of 1:15 to 1:25, preferably 1:20, or bb) evaporation residue a) is dissolved in a 6 to 10 times, preferably 8 times the amount of methanol undissolved the remaining fractions are separated, defatted with a triple ten to twenty minute, preferably fifth minute solid-liquid extraction with light petroleum (boiling range 60/70 ° C), in a ratio of 1: 1.5 to 1: 2.5, preferably 1 : 2,

c) dissolving the defatted residue b) in a 6 to 10-fold, preferably 8-fold amount of methanol and separating the remaining insoluble fractions, or cc) the defatted methanolic solution bb) is separated from the petroleum ether,

d) chloroform and water are added in equal volumes in methanol solution c) or cc) and the mixture is vigorously stirred for about 15 minutes and decanted,

(e) the chloroform phase is mixed five times with methanol and water in a 1: 1: 1 ratio by volume, for about 5 minutes with vigorous stirring, and then the chloroform phase is separated,

f) the combined methanolic phases are evaporated at a temperature of 40 to 50 ° C, preferably at 40 ° C under a vacuum of 1.5 kPa,

g) the evaporation residue is suspended in methanol in a ratio of 1: 4 to 1: 6, preferably 1: 5, and the suspension is stirred in an eight to twelve, preferably tenfold volume of water, after centrifugation the precipitate is washed several times with water,

h) drying the precipitate for 20 to 30, preferably for 24 hours at 30 to 50 ° C, preferably at 40 ° C under a vacuum of 1.0 kPa, then grinding and the milled product optionally dried at a temperature of 30 to 50 ° C, preferably at 40 ° C under a vacuum of 1.0 kPa.

The products isolated in this way have a silymarin (silymarin I-IV) content of 90 to 96% based on the anhydrous substance.

From the technical point of view of carrying out the process according to the invention, the individual steps and specific measures are chosen in such a way that a perfectly active drug mixture is practically isolated. If these embodiments are deviated, one or the other compound would be lost in whole or in part. This difficulty increases with the degree of enrichment of the active compound mixture, especially in the advanced stages of the process, since the desired active compounds may have been partially recovered as individual compounds or as a total mixture with the separate accompanying substances.

The process of the invention takes this into account, so that it results in the pure product in optimal yield while maintaining the isomer ratio that is in the drug.

Surprisingly, only a combination of the measures of the process according to the invention, certain solvents or solvent systems, and adherence to physical or chemical conditions, is achieved by a suitable product.

Cold pressing of the milk thistle fruit prevents chemical degradation or polymerization of silymarins and prevents the formation of an inert emulsion of silymarin and the concomitant drugs in the fatty oil, which could be disadvantageous for subsequent subdivision. By pressing, the drug is partially defatted. About 25 to 30% of the oil is pressed.

The provision of fine grinding allows rapid subsequent extraction, as the extraction agent can penetrate more readily into the drug particles.

In this process, it is critical that the ethyl acetate extraction is carried out with anhydrous ethyl acetate. The water content may not exceed 0.5%. If the water content exceeds 0.5%, it is no longer possible to separate more accompanying substances in the following stages. This surprising fact, in combination with other measures of the process according to the invention, is decisive for the success of the process, namely obtaining pure silymarin.

The drug / solvent ratio in the ethyl acetate extraction of 1: 8 to 1:12, preferably 1:10, is selected such that it provides a specific dissolution of the silymarin without unnecessary excess. Two to four times, preferably three times, extraction allows perfect recovery of silymarin from the drug. Temperatures in the range of 60 to 80 ° C, preferably 75 ° C, are favorable for the good dissolution of the active ingredients, while not leading to the decomposition of the chemical compounds present therein.

Mixing with an efficient stirrer brings about mechanical non-fermentation of the drug, which allows extraction. The chosen short time for each of the multiple extractions from 0.5 to 1.5, preferably 1 hour, however, allows exhaustive extraction.

The combined ethyl acetate extracts were evaporated gently at low temperature in vacuo.

The subsequent multiple solid-liquid defatting with petroleum ether at a relatively high ratio of residue to petroleum ether, for example 1:20, has the advantage of separating the fatty oil in an exhaustive manner in a short time and without much expense. Optionally, the evaporation residue can be dissolved in about eight times by weight, the undissolved fractions can be separated, and then the solid-liquid extract is extracted several times with petroleum ether in a ratio of about 1: 2. The results are practically the same.

Methanol as a relatively inexpensive solvent is suitable for dissolving the defatted residue and is particularly suitable for its great solvating ability. Silymarin (silymarin I-IV) dissolves relatively well in it. A high residue / solvent ratio is preferred for silymarin (silymarin I-IV) to be completely removed. No silymarin remains in the remainder.

New and particularly intensive is the use of chlorinated, organic solvents, in particular chloro-substituted lower alkanes, such as chloroform, methylene chloride, 1,2-dichloroethane, preferably chloroform, to separate the remaining silymarin accompanying substances (silymarin I-IV) without silymarin suffering altogether and without loss of isomers. This separates other lipophilic accompanying substances which are not eliminated in the defatting stage together with the oil and petroleum ether, as well as other less polar accompanying substances which, although initially dissolved in methanol, behave appropriately in the phase separation system. The solubility equilibrium - the state diagram of the selected solvent system - both in kind and in quantity, in this case meta-, nol-water-chloroform 1: 1: 1 is particularly suitable in the presence of silymarin (silymarin I-IV) and the accompanying substances present, since this solvent system achieves a good separation into two phases. The metering area of the state diagram shows the specified solvent ratios.

The partition coefficient of silymarin (silymarin I-IV) between the two phases is very high. After six shots were run in methanol / chloroform / water 1/1/1 (v / v / v), the ratio of silymarin in the combined aqueous methanolic phases to the silymarin remaining in the chloroform phase was 13: 1.

In this way, virtually all silymarin (silymarin I-IV) can be recovered from the methanolic phase.

Surprisingly, it has been achieved by the invention that silymarin I-IV remain fractionated both in the aqueous methanolic phase and as individual compounds, so that the isomer ratio is maintained.

The aqueous methanolic phases are evaporated gently at low temperature and under vacuum to dryness and the residue is suspended several times in water for washing. This washing procedure has proven to be suitable for removing the occlusion caused by product impurities occurring in conventional filtration.

Compared with the prior art, the process according to the invention has the advantage that it results in a simple way to 90 to 96% pure silymarin (silymarin I-IV) in a yield of 1.7%. Surprisingly, only a combination of the selected measures of the invention using anhydrous ethyl acetate and a chlorine-substituted alkane such as chloroform leads to the recovery of very pure silymarin from the milk thistle fruit.

The effect of anhydrous ethyl acetate extraction of the drug should be highlighted as particularly inventive. The hydrophilic substances thus remain in the remainder. In the subsequent stages of the process as separation in solvents, these hydrophilic accompanying substances can no longer be separated from the silymarin.

Further, the separating effect of solvents of the chlorinated lower alkane type, in particular chloroform in the second stage of the process, is particularly surprising due to the specific selectivity, virtually eliminating all of the accompanying substances present at this stage, so that silymarin remains very pure in the methanolic solution.

He did

To 300 kg of finely ground and cold pressed fruit of the cilantro, add 3 000 liters of anhydrous ethyl acetate and heat to 75 ° C. At this temperature, the mixture was stirred vigorously for 1 hour with a powerful stirrer. It is then filtered off and the remainder of the drug is extracted twice more with ethyl acetate as described.

The combined, clarified ethyl acetate solutions are evaporated to dryness at 50 ° C under vacuum at 1.5 kPa. For defatting, the residue is stirred at room temperature three times with 300 liters of petroleum ether (b.p. 60 DEG-70 DEG C.) for 15 minutes and then freed of solvent under vacuum. The crude product thus obtained is taken up to four times the amount of methanol, the non-solution solid being centrifuged, washed twice with half of said amount of methanol and finally, since virtually no silymarin is present, it is separated.

To the combined methanolic solutions was first added chloroform, then water in a 1: 1: 1 by volume ratio and transferred to a decanter tank.

After 15 minutes of vigorous stirring and phase separation, the lower phase (chloroform phase) was separated and extracted five more times in a 1: 1: 1 by volume methanol-water mixture. The chloroform phase is then separated.

The combined methanolic phases are evaporated at 50 ° C under a vacuum of 1.5 kPa and the residue is suspended in a 5-fold amount of methanol. The suspension is added by a thin stream to a ten-fold volume of water with stirring and then stirred vigorously for 15 minutes. It is then centrifuged and the residue in the centrifuge is washed repeatedly with water to form a suspension. The product was dried in an oven for 24 hours at 40 ° C, grinded and dried at 40 ° C for a further 48 hours.

A light colored powder is obtained which contains 90 to 96% silymarin (determined by the method using 2,4-dinitrophenylhydrazine [DNF-method] analogously to that of silymarin in the milk thistle fruit DAB VII, edition 1968, appendix 2, 1975, p. 198, as well as a quantitative evaluation of the draw curves). Demonstration of identity was performed by thin layer chromatography. The ratio of the individual isomers was determined by quantitative evaluation of the draw curves (see Fig.). The yield is 4.9 kg ~ 1.7% based on dry drug.

Example 2

To 300 kg of finely ground cold-pressed milk thistle fruit was added 3000 liters of anhydrous ethyl acetate and heated to 75 ° C. At this temperature, the mixture was vigorously stirred for 1 hour using a high-performance stirrer. It is then filtered and the remainder of the drug is extracted twice as described.

223986 6

The combined, clarified ethyl acetate solutions are evaporated to dryness at 50 ° C under 1.5 mm Hg.

The evaporation residue is dissolved in 120 liters of methanol. The methanolic solution was subjected to solid-liquid extraction (135 liters) of petroleum ether three times over 15 minutes.

To the methanolic solution was first added chloroform, then water in a 1: 1: 1 by volume ratio, and the solution was transferred to a decanter tank.

When the mixture was vigorously stirred for 15 minutes and phase separation was achieved, the bottom (chloroform phase) was separated and extracted five times in a 1: 1: 1 by volume methanol-water mixture for 15 minutes. The chloroform phase is then separated.

The combined aqueous methanolic phases are evaporated at 50 ° C under a vacuum of 1.5 kPa and the residue is suspended in a 5-fold amount of methanol. The suspension is added by a thin stream to a ten-fold volume of water with stirring and then stirred for 15 minutes. It is then centrifuged and the centrifuge residue is washed several times with water to form a suspension. The product is dried for 24 hours at 40 ° C in an oven and dried for a further 48 hours at 40 ° C.

The process results in 90 to 96% silymarin of the same properties as in Example 1.

Example 3

The same embodiment of 94% silymarin. The yield is

Example 4

The same embodiment of 93% silymarin. Yielding using methylene chloride instead of chloroform yields 90-1.7%.

using 1,2-dichloroethane instead of chloroform gave 90-1.8%.

Valid for all examples:

The percentages of purity and small variations in yield depend on the drug.

Claims (1)

A process for obtaining silymarin, i.e. silymarin I-IV, wherein the milk thistle is deprived of most of the fatty oil by cold pressing, the compressed meat is crushed, the rest of the pressing is extracted with ethyl acetate, the ethyl acetate extract is evaporated and processed, that a) extraction of the residue after pressing is carried out two to four times, preferably three times, in each case for 0.5 to 1.5 times, preferably for 1 hour, with anhydrous ethyl acetate at a temperature of 65 to 76 ° C, preferably at a temperature of 75 ° C, in a ratio of 1: 8 to 1:12, preferably 1:10 with vigorous stirring, and the extracted drug is separated, b) either the combined ethyl acetate solutions are evaporated at a temperature of 40 to 55 ° C, preferably at 50 ° C under a vacuum of 1.5 kPa, the residue after evaporation a) is defatted three-tenths of a tenth to twenty minutes, 5 preferably by fifteen-minute extraction with solids; a liquid with petroleum ether having a boiling range of 60 to 70 ° C in a ratio of 1:15 to 1:25, preferably 1:20, or bb) evaporation residue a) is dissolved in a 6- to 10-fold, preferably 8-fold amount of methanol, the undissolved residuals are separated, defatted with a triple ten to twenty minute, preferably fifteen minute solid-liquid extraction with light petroleum, boiling range 60 to 70 ° C, in a ratio of 1: 1.5 to 1: 2.5, preferably 1: 2. (c) either the defatted residue (b) is dissolved in a 6 to 10-fold, preferably eight-fold, amount of methanol and the remaining insoluble fractions are ‘separated, or cc) the defatted methanolic solution bb) is separated from petroleum ether, d) chloroform and water are added in equal volumes to methanol solution c) or cc) and the mixture is vigorously stirred for 15 minutes and decanted, (e) the chloroform phase is mixed five times with methanol / water (1: 1: 1 by volume) for 15 minutes with vigorous stirring, and then the chloroform phase is separated; f) evaporating the combined methanolic phases at a temperature of 40 to 50 ° C, preferably at 40 to 50 ° C. a temperature of 40 ° C under a vacuum of 1.5 kPa, g) the evaporated residue is suspended in methanol in a ratio of 1: 4 to 1: 6, preferably 1: 5, and the suspension is admixed 8 to 12 times, preferably 10 times by volume of water, after centrifugation the precipitate is washed several times with water, h) drying the precipitate for 20 to 30, preferably for 24 hours at 30 to 50 ° C, preferably at 40 ° C under a vacuum of 1.0 kPa, then grinding and the milled product optionally dried at a temperature of 30 to 50 ° C, preferably at 40 ° C under a vacuum of 1.0 kPa. 1 drawing