KR930003073B1 - Process for preparation of sylibine without silibinin - Google Patents

Abstract

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Description

Method for preparing silybinin free of isosilbin

The present invention relates to a method for producing isolicillin-free silybinin and a pharmaceutical composition containing the same.

Lady Thistle (Silybum marianum (L.) Gaireton = Silybum marianum (L.) Gaertn. (Carduus marianus L.) has long been known as a medicinal plant. R.Munster has isolated a component called silybin (see f. Dissertation R. Munster, Munchen, 1966.) The chemical structure of this compound was elucidated by A. Pelter and Al. Hansel (Tetrahedron Letters, London 25, 2911-2916 / 1968).

It has also been found that Silibin, formerly named Silymarin, is effective as an intervening material (see German Federal Patent Application No. 1767666). A process for the preparation of silybin (Silymarin I) has been presented, for example, in patent application 1923082 of the Federal Republic of Germany.

Around 1974, H. Wagner, P. Diesel, and M. seitz (Arzneimittelforschung, 24 (4), 466-471) deduced the existence of two positional isomers for silybin, namely, silybin and isosilbin. have. This reasoning was experimentally confirmed by A. Arnone, L. Merlini and A. Zanarotti (J. Chem. Soc. Chem. Comm., 16, 696-697 / 1979). Accordingly, the known silicides consist of two different compounds, ie compounds represented by the following structural formulas (A) and (B).

From these structural formulas it can be seen that these compounds are positional isomers. The compound of formula (A) has recently been named silbinin in the INN nomenclature. In this specification, the name by this nomenclature is used for compound (A).

The two compounds, (A) and (B), have so far been separated and prepared only in analytical quantities, and nothing is known about the pharmacological action of each of the isomers.

It is an object of the present invention to provide a method for preparing siliviinin free of isosilbin.

According to the present invention, there is provided a method for preparing the silybinin of the above formula (A) free of isosilinine, which is caused by bursting the dried fruit of Ladybug, Silybum marianum L. Geartn, with a high mechanical pressure. To remove a large amount of fatty oil, the remaining 5 to 10% of the remaining oil content at the pressure is completely extracted with ethyl acetate, and the dried residue obtained by evaporating the ethyl acetate, 95 parts by weight of methanol, 5 parts by weight After dissolving to 2% by weight in a lower layer of methanol and water in a third solvent mixture of 3 parts water and 100 parts by weight of petroleum ether (bp 40-60 ° C.), centrifugation until the aggregated solid particles are removed and clear. And uniformly distribute the dry residue thereof several times in the solvent system, maintaining the total flow volume ratio of the upper layer / lower layer to 1: 1, and then let the lower layer flow out under vacuum Crude evaporation separates 70 to 80% of a mixture of polyhydroxy phenyl chromanone (Silymarin I-IV = Silymarin I-IV group) into this brown powder, which is three to five times the weight. The suspension obtained by suspending in methanol is heated with boiling until stirring, thereafter, one third to two thirds of methanol is removed under reduced pressure, and the concentrate is left at ambient temperature, and then the obtained precipitated product is subjected to 1 3 to 3 small portions were washed with cold methanol, dried and dissolved in 40 to 60 times the weight of ethyl acetate by heating, then treated with activated charcoal under reflux, and the filtrate was filtered off and the filtrate was dried under reduced pressure. Evaporate to about one-tenth of the original volume and mix the resulting concentrate with methanol, about one tenth the volume of the concentrate volume, under vigorous stirring, and then at ambient temperature. While a method of stirring by a powerful mixer, after allowed to stand, and filtered off by suction and, after washing the suction and filtration of the product with 1 to 3 times, a small amount of ethyl acetate, dried in vacuo the product was precipitated.

Silybinin free of isosilbin is also a) suspended in 1 part by weight of the brown powder in ethyl acetate saturated with 0.7 to 1.5 parts by weight of water, and the precipitate obtained by standing the suspension for 1 to 2 days by suction filtration and b) The precipitate is washed with cold ethyl acetate saturated with 0.07 to 0.15 parts by weight of water and then dried under vacuum at 30 to 50 ° C. c) the product is dissolved in 30 to 50 parts by weight of anhydrous ethyl acetate at its boiling temperature Treated with 0.2-0.4 parts by weight of active charcoal under reflux for 2 hours, filtered and the filtrate was concentrated to 1/12 volume of the solvent added to dissolve it under vacuum at 30-50 ° C., d) Ethyl acetate saturated with 0.5 to 0.8 parts by weight of water was added to the concentrate, and after 5 to 10 hours, the precipitated silybinin was filtered out. It may also be prepared by suspending 0.9 to 1.8 parts of acetate, predrying in vacuo at 30 to 50 ° C, then grinding the predried product and again vacuum drying at 30 to 50 ° C.

The brown powder used as starting material in step a) is crude silymarin, which is a mixture consisting of silymarin (I)-(IV). Treatment of the crude silymarin with water-saturated ethyl acetate results in substantial amounts of silymarin (II) to (IV) (silibinin is silymarin (I)), and ancillary substances present in the crude silymarin by about 20 to 30%. Some isosilbin is isolated. In step (b) of this method, depending on the quality of the crude silymarin used, a crude silybinin is obtained in a yield of 80 to 85% and an 80 to 84% content with respect to the content of the silyvanin in the crude silymarin. As mentioned above, the coarse silbinin is a mixture of isosilbin and silivinin at a ratio of about 1: 4.

In steps c) to e), the residues of the other components described above are separated, and also the silybinin and the large amount of isosilbin are separated from each other.

A particular advantage of the separation process of the invention is the use of only one solvent, ie ethyl acetate, although the water content is different. It is therefore important to use anhydrous ethyl acetate in step c) and ethyl acetate saturated with water in step d). By the process according to the invention, the silivinin is obtained in a yield of 79 to 85% and in a 96 to 98% silibinine content with respect to the siliviin content in the coarse silbinin.

According to a preferred embodiment of the process according to the invention, a) 1 part by weight of the brown powder is suspended in 0.9 parts by weight of ethyl acetate immersed in water, and allowed to stand at ambient temperature for 48 hours, after which the precipitate obtained is suction filtered and b) this precipitate was washed with 0.09 parts by weight of cold ethyl acetate saturated with water and dried in vacuo at 40 ° C. for 48 hours, and c) the resulting product was dissolved in 36 parts by weight of anhydrous ethyl acetate at its boiling temperature and refluxed for 2 hours. After heating with 0.36 parts by weight of active charcoal, it is filtered and concentrated to 1/12 volume of solvent added to dissolve it at 30 to 50 ° C. under vacuum, d) 0.6% of ethyl acetate saturated with water in this concentrate. Part was added at ambient temperature and left to stand for 12 hours to filter the precipitate product obtained, e) the product twice, respectively Was suspended in 0.9 to 1.8 parts by weight of ethyl acetate, then filtered and dried in vacuo then pre-dried under vacuum at 40 ℃ for 24 hours, triturated again in 40 ℃ for 48 hours.

The inventors have found that silybinin free of isosilbin is very suitable for pharmaceutical purposes. Surprisingly, this has been found to be quite beneficial compared to any other known ingredients of Silybum marianm extract. It is particularly suitable for the treatment of cirrhosis of the liver and toxic metabolic liver damage. It can also be used prophylactically so that the above damage does not occur.

As a result, the present invention also provides a pharmaceutical composition containing silybinin in a mixture with a solid or liquid pharmaceutical diluent or carrier. These compositions are generally used systemically, for example in the form of pills, capsules and solvents together with conventional carriers.

Adult daily doses are generally about 50 to 500 mg, depending on the patient's condition and the severity of the disease symptoms.

The following examples are presented for the purpose of illustrating a method for preparing isolicillin free of isosilbinin by the present invention.

Example 1

500 g of a polyhydroxy-phenyl clomanone mixture obtained from patent application 1923082 of the Federal Republic of Germany (see lines 8, lines 14-19) (silimarin I-IV = silymarin I-IV group; content about 70%) Is suspended in 2 kg (about 2.53 liters) of methanol and heated until boiling for 15 minutes with stirring. Thereafter, some silybinin precipitates from this solution. Subsequently, 0.75 to 1.25 kg (about 0.96 to 1.58 liters) of methanol are removed under vacuum and the resulting residue is left at ambient temperature for 10 to 28 days. The precipitated silybinin is filtered off and washed twice with 50 ml of cold methanol. After drying under vacuum at 40 ° C., the crude crude silybinin was purified again as follows.

60 g of crude silybinin is dissolved in 3 liters of industrial quality ethyl acetate while heating, then mixed with 20 g of active charcoal and stirred under reflux for 2 hours. Thereafter, the solution is filtered and clarified, and the solution is evaporated to 50 ° C. under reduced pressure to about 250 ml. This concentrate is mixed with 25 ml of methanol with stirring on an Ultra-Turrax apparatus for 15 minutes. The mixture is then allowed to stand overnight at ambient temperature. The solution is stirred again in an Ultra-Turrax apparatus for 5 minutes prior to suction filtration of the precipitated silybinin. The suction filtered precipitate is washed twice with 50 ml of ethyl acetate and dried overnight in a vacuum drying cabinet at 40 ° C. The product is triturated and dried again under the same conditions for 48 hours.

Example 2

The 1 kg polyhydroxy phenyl clomanone mixture used as starting material in Example 1 is suspended in 1 liter of ethyl acetate saturated with a Turrax stirrer. After standing at ambient temperature for 48 hours, the precipitate obtained is suction filtered, washed with 100 ml of cold ethyl acetate saturated with water and vacuum dried at 40 ° C. for 48 hours. The yield of this intermediate product, ie, coarse silybinin, is 80 to 85% and 80 to 84% of the content of the coarse silymarin, depending on the quality of the coarse silymarin used.

The resulting crude silybinin was dissolved in 40 liters of anhydrous ethyl acetate at its boiling temperature, boiled with 360 g of active charcoal under reflux for 2 hours, filtered and 1/12 of the solvent added to dissolve it in a vacuum of 30 to 50 ° C. Concentrate to volume. To this solution, 667 ml of ethyl acetate saturated with water are added with vigorous stirring at ambient temperature. After 1 to 3 hours, crystallization of silybinin begins. After standing overnight, the precipitated silybinin is filtered off, suspended twice in 5-10 minutes each in 1200 ml of industrial purity ethyl acetate, filtered again and pre-dried for 24 hours at 40 ° C. vacuum. After grinding, it is again vacuum dried at 40 ° C. for 48 hours. The yield of the silinbinin relative to the content of the coarse silinin was about 79 to 85% depending on the quality of the coarse silinin, and the content of the silininin was 96 to 98.5%.

[Clinical Experiment with Silybinin]

In the last decade, addictive liver damage has increased dramatically. As before, the most common cause of liver damage is alcohol.

Controlled studies have demonstrated the superiority of silybinin by comparing it with placebo or other comparative therapeutic agents. In a double blind study, 66 patients with alcohol-addicted liver injury were randomized using placebo (n = 35) and silybinin (n = 31), respectively.

Statistically evaluated results show that it is remarkable that it is much better than the placebo of silybinin and generally has a shorter treatment period than that placebo.

In the second blind study, 39 of 76 patients received silybinin and 37 received control treatments, with the difference being much more pronounced on the silybinin side.

In the case of anesthesia associated with surgery of the abdominal organs, it has been demonstrated that in the case of liver-addictive conditions, administration of silibinin prior to surgery significantly reduces the increase in blood enzyme levels after surgery. It is frequently caused by certain medicines and is effectively prevented with liver damage and silybinin. This proved, for example, that in the case of hepatosis caused by phenylhydantoin, despite the additional regimen of idiopathic spasms-preventive medicine, all experimental values returned to normal in a very short period of time by simultaneous administration of silybinin. .

In patients with severe schizophrenia and severe liver damage from chloropromazine, further studies have demonstrated the positive effect of silybinin. Other experiments have been carried out, for example, on the prevention of liver damage of chloroquinine and asparaginase. In all cases of liver damage due to occupational factors, the experimental studies that were initially pathologically increased in all treated patients, regardless of the specific cause of the disease, were greatly improved.

It has also been fully demonstrated that silybinin can provide significant improvement in the case of chronic inflammatory liver disease. For example, patients with liver cirrhosis were studied and long-term trials of silibinin were administered to placebo in a double randomized blind test. The criterion for the success of the treatment was especially survival. This test demonstrated that silibinin was clearly superior compared to placebo treatment.

A comparative study of the action activity between silybinin, isosilinine and silybin (silbinin / isosilinine mixture) was administered intravenously, using a model of phalloidin and proseodymium in rats. It carried out by doing.

Silybinin, Isosilin and Silybin were tested for anti-hepatotoxic activity in the form of N-methylglucamine salts. The experiment was conducted by intravenous administration of a dose of mg / kg ⁻¹ . The test substances were administered 1 hour prior to paloidine and 1 hour before and 6, 24 and 48 hours after praseodymium. In the case of paloidine poisoning, this survival rate was measured, and in the case of praseodymium poisoning, various serum and liver parameters were measured after 72 hours of poisoning.

In the case of paloidine poisoning, the survival rate after silybinin administration was 100% in both doses, whereas the survival rate was not more than 40% for the untreated, impaired control with other test substances.

Since isocillin was found to be incompatible with praseodymium poisoning, the dose of 100 mg / kg ⁻¹ had to be divided into two divided doses.

Claims (3)

The dry fruit of Silybum marianum (L.) Gaertn. Was popped with high mechanical pressure to remove the large amount of fatty oils present in it, The remaining 5-10% residual oil is extracted completely with ethyl acetate and the dry residue obtained by evaporating the ethyl acetate is extracted with 95 parts by weight of methanol, 5 parts by weight of water and 100 parts by weight of petroleum ether (bp 40- 60%) in a lower layer of methanol and water in a ternary solvent mixture in an amount of 2% by weight, centrifuged until the aggregated solid particles are removed and cleared, and the dried residue is washed several times in the solvent system. Distribute, but evenly distribute so that the total flow volume ratio of the upper and lower layers is 1: 1, then drain off the lower layer and evaporate to dryness in vacuo to give 70 to 80% of the polyhydroxyphenylclomanone horn. Water (Silymarin I-IV = Silymarin I-IV Group) is separated into a brown powder, suspended in three to five times its methanol, and the suspension is heated until boiling with stirring, stirring, and under reduced pressure. One-third to two-thirds of methanol is removed and the concentrate is left at ambient temperature, then the precipitate is washed with 1 to 3 small portions of cold methanol, dried and 40 to 60 times the weight of ethyl. Dissolve with heating in acetate, treat with active charcoal under reflux, filter this charcoal, then evaporate the filtrate under reduced pressure to about 1/10 of its initial volume, and concentrate this concentrate about 1/10 of that. After mixing with methanol corresponding to the volume and allowing to stand for several hours at ambient temperature, the mixture was stirred with a strong mixer, the precipitate was filtered off with suction, and the suction-filtered product was subjected to 1 to 3 small amounts of ethyl acetate. And washing with a trope, followed by drying in vacuo.

The method of claim 1, wherein the dried fruit of Silybum marianum (L.) Gaertn. Is popped with high mechanical pressure to remove a large amount of fatty oil present therein. The remaining 5-10% oil still under pressure was extracted completely with ethyl acetate and the dry residue obtained by evaporating the ethyl acetate was extracted with 95 parts by weight of methanol, 5 parts by weight of water and 100 parts by weight of petroleum ether (bp 40- 60 ° C.), dissolved in a lower layer of methanol and water in a mixture of ternary solvents in an amount of 2% by weight, followed by centrifugation until the aggregated solid particles are removed and cleared. Several times so that the total flow volume of the lower layer is evenly distributed in a 1: 1 ratio, and then the lower layer is run off and dried and evaporated to dryness in vacuo to 70 to 80% of the polyhydroxyphenylclomanone mixture (silimarin Group I-IV = silymarin group I-IV) is separated into a brown powder, a) 1 part by weight of this brown powder is suspended in ethyl acetate saturated with 0.7 to 1.5 parts by weight of water, and the suspension is allowed to stand for 1 to 2 days. The precipitate was suction filtered and b) the precipitate obtained was washed with cold ethyl acetate saturated with 0.07 to 0.15 parts by weight of water and then dried under vacuum at 30 to 50 ° C., and c) the product was dried in 30 to 50 parts by weight of anhydrous ethyl acetate. After dissolving at its boiling temperature, the mixture was treated with 0.2 to 0.4 parts by weight of active charcoal under reflux for 2 hours, and then the filtrate obtained by filtering the charcoal was dissolved in 1/12 parts of the solvent added to dissolve it in a vacuum at 30 to 50 ° C. D) to the concentrate was added ethyl acetate saturated with 0.5 to 0.8 parts by weight of water, and after 5 to 10 hours, the precipitated silybinin was filtered off, e) Suspended in 0.9 to 1.8 parts by weight of ethyl acetate in a commercial product, pre-dried under vacuum at 30 to 50 ° C., and then crushed the pre-dried product, followed by drying again in a vacuum at 30 to 50 ° C. Method for preparing silybinin free of isosilbin. A precipitate according to claim 2, wherein a) 1 part by weight of brown powder is suspended in 0.9 parts by weight of ethyl acetate saturated with water and allowed to stand at ambient temperature for 48 hours, then the precipitate obtained is suction filtered and b) the precipitate is cold saturated with water. 0.09 parts by weight of ethyl acetate was washed and dried in vacuo at 40 ° C. for 48 hours, c) the resulting product was dissolved in 36 parts by weight of anhydrous ethyl acetate at its boiling temperature and heated with 0.36 parts by weight of activated charcoal under reflux for 2 hours. The charcoal was then filtered and concentrated in vacuo to 1/12 volume of the solvent added to dissolve it at 50 ° C., d) To this concentrate, 0.6 parts by weight of ethyl acetate saturated with water were added at ambient temperature for 12 hours. The precipitated product obtained by standing at this temperature for a period of time was filtered off, and e) the product was subjected to twice, each from 0.9 to 1. ethyl acetate of industrial quality. 8 parts by weight of suspension, followed by filtration, predrying for 24 hours at 40 ° C. vacuum, followed by grinding and further drying for 48 hours at 40 ° C. vacuum.