DE2436479A1 - METHOD OF CLEANING UP STEROID HETEROSIDES - Google Patents

Description

PATENT LAWYERS

Reg. No. 124 419 Dr-Ing. Wolff H Bartels Dipl.-Chem. Dr. Brandes Dr.-Ing. Hero Dipl.-Phys. Wolff

8 Munich 22, Thierschstrasse

Tel. (089) 293297 Telex 0523325 (patwod) Telegram address: wolffpatent, munich postal check account Stuttgart 7211 (BLZ 60010070) Deutsche'Bank AG, 14/28630 (BLZ 60070070) Office hours: 8 a.m. to 12 p.m., 1 p.m. to 4.30 p.m. except Saturdays

July 25, 1974

Pierre Fabre S ₉ A ₉ , 75016 Paris / France Process for the purification of steroid heterosides

Method for purifying steroid heterosides

The invention relates to a method for purifying steroid heterosides, in particular of steroid heterosides, which are obtained from root stocks of Rubcus aculeatus L. (also called "petit houx") are extracted to obtain extracts of Ruscus aculeatus L. with a defined content of steroid heterosides.

The rhizomes of Ruscus aculeatus L contain various active substances, the most important of which are steroid heterosides with venotonic action, which through hydrolysis lead to the prosapogenins and the genins.

The pharmacopoeias do not state any active substance extracts, and so there are considerable fluctuations in the content of steroid heterosides of liquid extracts of the Ruscus codex type (Pharmacopoeia). Depending on the origin of the plant and harvest time, for example, the deviations can vary from 0.45 to 1.20 g of steroid heterosides per 100 g of codex type extracts, resulting in corresponding fluctuations in the therapeutic activities of these extracts.

To remedy these disadvantages, it seems essential to use liquid extracts, Adjust moist extracts and dry extracts from Ruscus to a content of active substances, their effects and Pharmacological properties are well defined and constant.

Such adjusted extracts can be easily achieved with the help of the purification process according to the invention, which leads to steroid heterosides in powder form that are practically 100 % pure, as well as to solutions or intermediate products of a precisely defined composition, in particular to extracts with a content of 20, 50 or 80 % steroid heterosides.

Numerous processes are already known which make it possible to use the steroid heterosides of Ruscus aculeatus L. in more or to obtain less pure form, in particular precipitation processes by changing the solvent, processes for absorption on substances such as B. silicon dioxide, aluminum oxide, magnesium oxide, kieselguhr or animal charcoal, method for solid-plush extraction using less polar solvents, such as' z. B. benzene, petroleum ether or chloroform, and finally, processes for liquid-liquid extraction.

None of the known methods has so far made it possible to achieve this of very pure steroid heterosides in good yield and under conditions that are easy to implement on an industrial scale. This is particularly due to the fact that the steroid heterosides because of their chemical structure are endowed with not insignificant surface-active properties, which is very easy to enter of compounds with a wide variety of substances, and especially in the liquid-liquid type extractions the presence of a surfactant can delicately interfere with the extraction process, e.g. B. · by modification the interface properties.

The present invention relates to a purification process in which a liquid-liquid extraction is carried out under such conditions that a very large yield of steroid heterosides, the purity of which is of the order of 100 io, is obtained.

The present invention therefore relates to a method for cleaning of steroid heterosides, in particular of steroid heterosides from Ruscus aculeatus L., by liquid-liquid extraction, which is characterized in that an organic phase containing at least one solvent for steroid heterosides (butanol) contains, extracted with an aqueous phase, the pH of which is adjusted to 13 or above with the aid of a base,

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wherein the steroid heterosides are in solution prior to extraction in one of the two phases, and that, after decanting, the steroid heterosides obtained are separated off from the organic phase.

The present invention is based on the finding that, in comparison to methods in which the steroid heterosides containing aqueous phase is extracted, and whose tdH value is usually close to the neutral point, the invention Use of an aqueous phase with a pH of over 13 has an excellent separation coefficient on steroid heterosides allows to preserve while at the same time considerably increasing the solubility of the steroid hetero3ids usually associated impurities in the aqueous phase.

With the foregoing in mind, it is easy to see that the present invention is capable of both includes those methods according to which the steroid heterosides are in aqueous solution and through the organic Phase are extracted while the impurities are retained in the aqueous solution, as well as those processes according to which the steroid heterosides in the organisdhen Solvents are in solution and the impurities are extracted by the aqueous solution, the steroid heterosides be held back in the organisdsn phase.

According to a preferred embodiment of the method of the invention, the aqueous phase which contains the steroid heterosides used at the time of the Plüssig-Plüssig extraction is that solution which is obtained by treating the raw or preferably finely chopped roots of Ruscus aculeatus L. with water under pressure and with heating after separation of the spent grain and any concentration of the supernatant solution to a level of 50 i »dry substance, wherein the pH value of the solution thus obtained at 13

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or set a value above this with the aid of a base will.

According to a further preferred embodiment of the invention, the organic phase, which is that of the liquid-liquid extraction Contains steroid heterosides to be subjected to, obtained by extraction of the raw roots of Ruscus aculeatus L. with water under pressure and with heating, as indicated above, decanting of the supernatant phase, possible concentration and subsequent extraction of this phase or the concentrate one or more times with a solvent for steroid heterosides, without the pH of the aqueous solution being adjusted to be basic. The organic phase that the purification through Liquid-Plüssig extraction is subjected to is obtained by Combination of the organic phases from the previous extraction »

According to other advantageous embodiments of the present invention are made 20 or 80 powder i "steroid heterosides containing and then optionally dissolved in water or a solvent for the steroid heterosides to form in this way the aqueous or organic phase, which then for cleaning the Liquid-liquid extraction is subjected after adjusting the pH to 13 or above, if it is an aqueous phase. According to one of these embodiments, a 20 % steroid heteroside-containing powder is prepared from a concentrated ₉ containing about 50 % dry matter ^ solution obtained as indicated above by atomization of the same. According to another embodiment, a powder containing 80 $ steroid heterosides is produced by extraction of Ruscus aculeatus L. roots with water under pressure and with heating, separation of the spent grains, possible concentration ₉ single or multiple extraction of the supernatant solution with a solvent for Steroid-Heterosid ^ Concentration of the combined organic extracts by evaporation

iind atomizing the concentrated solution to achieve the Powder containing $ 80 steroid heterosides.

It also proves advantageous to concentrate the organic phases obtained and treat them with water, to one of organic solvents, e.g. B. butanol, free aqueous phase is obtained, and by atomization of this aqueous phase a powder with a content of 50 ^ steroid heterosides to manufacture.

The various solutions obtained in this way or Powders representing intermediate products are also essential to the invention.

The bases used to adjust the pH of the aqueous solution to 13 or above are preferred alkali metal or alkaline earth metal hydroxides, especially sodium or potassium hydroxide, but other bases are also possible can be used, provided that they are able to lead to a pH value in the order of magnitude of 13 in aqueous solution.

The solvent for steroid heterosides is preferably aliphatic alcohols which are immiscible or only slightly miscible with water (or an aqueous solution with a pH of more than 13), in particular n-butanol, but all the others are also miscible known solvents for steroid heterosides can be used for carrying out the method of the invention.

According to the invention, the solvent for steroid heterosides is preferably used in the form of the pure solvent, but it can also be used in a mixture with an inert or weakly polar solvent, or a mixture of solvents for steroid heterosides can also be used.

603830/0822

Of course, the liquid-liquid extraction according to of the invention can also be repeated, i.e. H. it is possible to use one aqueous phase containing the steroid heterosides Male to extract with an organic solvent, and it is also possible to use one containing the steroid heterosides To extract aqueous phase and extract the organic phase obtained in this way again with a basic solution of pH 13 »to complete the cleaning, and all combinations are possible and are within the scope of the present invention.

After decanting, the purified steroid heterosides are made out. the organic phase separated according to customary known Methods. In all cases, however, it proves to be expedient to provide a water wash for the organic phase to separate off the base which was inevitably introduced during the extraction, which in certain cases is necessary can prove to separate the existing traces of base with the help of an ion exchange column, for example an acidic resin of the type "Amberlite IRC 50" contains.

As soon as the organic phase has been freed from the base carried along, it is preferably concentrated until a precipitate begins to form, whereupon the concentrate is left to stand at low temperature for several hours and then filtered, washed and dried, preferably by heating under vacuum; if the solvent used is n-butanol, for example a dry ^{temperature on the order of 100 °} C. at 15 mm Hg can be used. The powder obtained under these conditions shows a steroid heteroside content close to $ 100. The overall yield of the process is on the order of $ 50 to $ 80

According to a variant of the concentration process step can increase the concentration of the organic phase with the water

- St ~

acts and the aqueous solution of steroid heterosides obtained in this way can then be atomized in such a way that a powder is obtained with a yield and an active substance content comparable to those described above is.

For therapeutic purposes, it is particularly advantageous to prepare an ethanolic solution with a content of 2 % steroid heterosides. Such a solution can be easily obtained from purified powders containing 100 i <> steroid heterosides by simple dilution, but it is also possible to obtain such a solution from the intermediates forming powders.

It is also possible to prepare an ethanolic solution with a content of about 2 fo steroid heterosides directly by receiving the 50 i <> dry substance containing concentrate in ethanol at 96 °, followed by decantation.

In general, it is advantageous to titrate the therapeutic solutions prepared, even if they were obtained by dilution of a powder containing typically 100 i <> steroid heterosides.

The application of the following experimental measures has therefore proven to be particularly advantageous.

dosage

A vial calibrated to the nearest 100 cm got an accuracy of 0.020 g weighed sample of steroid heterosides was introduced and this sample was dissolved in concentrated sulfuric acid, whereupon up was filled up to the calibration mark. The resulting solution was left to stand for at least 1 hour (hereinafter referred to as this solution is labeled I).

509830/0022

2436A79

In another vial, calibrated at 100 cm, were approximately 0.050 g precisely weighed sample of Ruscus extract (the obtained by drying an aqueous or organic solution). This sample was concentrated Sulfuric acid was added and the mixture was stirred until it dissolved, whereupon the volume was made up to 100 cm. The received The solution was left to stand for at least 1 hour (hereinafter this solution is referred to as II).

The following dilution series was prepared with the aid of solution I: i

1 2

Solution I 0 5 ml 12.5 ml 20 ml 25 ml

with conc. H SO.

made up to ¹⁰ ° ^ml

mg steroid heteroside 01?
fo ml sulfuric acid- ⁹

solution

The optical density of each Staadard solution was compared to a The blank sample was measured and the optical density values found were plotted graphically as a puncture of the concentration « The concentration of steroid heterosides was determined from this by measuring the optical density of solution II Conversion determines that of the extract.

If the concentration of the extract was known, it was then easy to adjust its steroid heteroside content to, for example, 2 fo.

The following examples are intended to explain the invention and the advantages that can be achieved in more detail, but not to constitute a restriction represent.

Example 1 ; Production of a concentrate with a content of 50 fo dry extract

a) Extraction:

In a stainless steel reactor, the extraction of 150 kg finely ground (particle size 1 mm) Ruscus roots nit was / ater performed demineralized 2000 1 V at 110 ⁰ C. The pressure was 0.5 bar; the stirring speed was 50 UoLI; the extraction time was 1 hour.

After cooling, a decanter from Tyn "Westfalia SDA 2 30" with a speed of 5500 rpm Decanted for 45 to 50 minutes.

The broth was then clarified in a "SAMR 3036" clarifier until a clear solution was obtained.

b) Concentrationϊ

It was then concentrated under vacuum in a double effect apparatus carried out until a concentrate with a content of $ 50 dry extract (concentrate A) is obtained.

Example 2: Preparation of a purified steroid-heteroside powder

The pH of a concentrate obtained according to Example 1 was adjusted to 13 with the aid of an N sodium hydroxide solution.

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The basic solution obtained in this way was extracted by volume or volume 3 times in succession with n-butanol. The strongly colored impurities appeared after decanting in the aqueous phase, while the steroid heterosides remained in the organic phase. The combined butanol phases were washed with water and then with animal charcoal treated to remove the last colored impurities.

The organic layer obtained was concentrated until the start of precipitation, and the concentrate was stirred at ⁰ +5 C for 4 hours left standing, and then filtered, washed, and 100 mm in ⁰ O under a vacuum of 15 Hg was dried.

The analyzes carried out led to the following results:

Content of the steroid heteroside powder: 98 to 100? S, Process yield: $ 50 to $ 70

Example 3 ? Manufacture of a powder containing about $ 20 steroid heterosides

A concentrated extract obtained according to Example 1 was brought to dryness by atomization while observing the following Conditions:

Inlet temperature of the air: 200 ^{0 C,}
Outlet temperature of the air: 95 ⁰ C,
Turbine speed: 16,000 rpm

In this way, an atomized product containing about $ 20 steroid heterosides was obtained with a yield of about $ 18 to $ 20 based on the weight of the Ruscus roots.

Example 4: Production of a powder containing
about $ 80 steroid heterosides

A concentrate obtained according to Example 1 was subjected to three successive butanol extractions at room temperature.

The butanol extract obtained was then concentrated in a plate evaporator before being subjected to atomization
Brought to dryness compliance with the following conditions
became:

Inlet temperature of the air: 200 ^{0 C,}
Outlet temperature of the air: 100 ⁰ C,
Turbine speed: 22,000 rpm

Example 5: Production of a powder containing
about 50 # steroid heterosides

A concentrate obtained according to Example 1 was subjected to four successive extractions with butanol. The butanol phases obtained were combined and concentrated in water until an aqueous solution free of butanol was obtained
Phase. By atomizing this aqueous phase, an extract containing 50% steroid heterosides was obtained.

Example 6: Preparation of a purified steroid-heteroside powder

The butanol extract obtained in Example 4 became a
Plüssig-Fltiaeig is subjected to extraction with a sodium hydroxide solution of pH 13, which dissolved the impurities present in the steroid heterosides. The organic phase thus obtained was washed with water to remove it

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To remove dissolved sodium hydroxide solution, whereupon the organic solution is passed through a column with ion exchange resin of the type "Amberlite IRC 50 "was sent. The eluate obtained was then to concentrated at the beginning of a precipitate and then filtered, washed and, as described in Example 2, dried.

The white powder obtained contained di ^ steroid heterosides in pure form. The process yield was on the order of $ 55 to $ 75.

Example 7

The procedure described in Example 2 was repeated with the exception that instead of that obtained in Example 2, the procedure was repeated Concentrate last organic phase, a concentration in water has been effected; the aqueous obtained in this way Solution was then atomized.

The steroid heterosides in the nebulized product were obtained in pure form ($ 98 to $ 100).

It thus follows that the various process stages of the process according to the invention under industrial conditions are easy to carry out and that in particular the liquid-liquid extractions with the help of one of the numerous known, apparatus commonly used industrially for this purpose are feasible, in particular z. B. with extractors of the mixer-decanter type or with countercurrent extractors.

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The continuous implementation of these operations does not pose any problems whatsoever, which is the case with the method according to the invention makes it even more interesting from an industrial point of view.

The steroid heterosides obtained by the process of the invention are useful as medicaments, especially as venotonica.

Pharmacological and clinical trials

The liquid extracts used for these experiments had a content of 2 % steroid heterosides.

The acute toxicity of extracts containing 2% steroid heterosides is very low and practically equal to that of a 50% alcoholic solution.

After administration orally over a period of 1 month (subchronic toxicity) could by autopsy (Wistar rats) macroscopically and histologically no abnormalities whatsoever were found in the viscera. The mean of the Weight curves of treated animals were almost identical to those of untreated control animals.

Ant i inf laminate or i see effect

In the rat, Garraghenin produced edema on the paw; in the animals treated with 0.5 to 1 ml / kg extract from Ruscus aculeatus L. (administration per os) animals edema resolved 2 to 3 hours after administration; the percentage reduction was found to be proportional the administered doses and in all cases statistically verifiable was.

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lie in the clinic in the form of oral and rectal preparations The extracts tested gave very beneficial results in the treatment of varicose and ovarian syndromes and the one under the French name "jambes lourdes" known syndrome. The mean posology was 1.5 ml of extract per day. In acute hemorrhoidal crisis, excellent Results obtained with an offensive posology of 1.5 to 2 ml times a day.

Very good results have also been obtained with compound formulations in which the extract from Ruscus aculeatus is used L. at the same time the vitamin pair P and C and a salidiuretic were administered.

The various products used never led to incompatibilities and always gave constant therapeutic results.

$ 09030/082 ^

Claims (15)

Claims Process for the purification of steroid heterosides, in particular those from Ruscus aculeatus L ,, by liquid-liquid extraction, characterized in that an organic phase containing at least one solvent for steroid heterosides contains, with an aqueous phase, the pH of which is adjusted to 13 or above with the aid of a base is extracted that the steroid heterosides before extraction in one of the two phases in solution are, and that after decanting the steroid heterosides are separated from the organic phase. 2. The method according to claim 1, characterized in that the steroid heterosides before the extraction in the aqueous phase are in solution. 3 »Method according to claim 1, characterized in that the Steroid heterosides are in solution in the organic phase prior to extraction. 4. The method according to claim 2, characterized in that a solution is used as the aqueous phase, which is obtained by extraction of the rhizomes of Ruscus aculeatus L. with water under pressure and with heating, then Separation of the spent grains and subsequent adjustment of the pH of the resulting solution to 13 or above with Help from a base. 5. The method according to claims 1 and 2, characterized in that that an aqueous concentrate with a content of about 50 $ dry matter is prepared by extraction of the rhizomes of Ruscus aculeatus L. with water under pressure and with heating, separation of the spent grain and concentration 509830 / Ü822 tion of the solution up to a content of 50 ^a / o dry matter, and that the concentrate obtained is used as an aqueous phase for the extraction. 6. The method according to claim 1, characterized in that a powder with a content of $ 20 steroid heterosides is prepared is made by extracting the rhizomes of Ruscus aculeatus L. with water under pressure and with heating, separation of spent grain, concentration of the supernatant to a dry matter content of $ 50 and subsequent atomization of the concentrate to obtain a powder with a steroid heteroside content of $ 20, and that the obtained Powder then dissolved in water under a solvent for steroid heterosides and used as a phase to be extracted is used. 7. The method according to claim 3, characterized in that an organic phase is used which is obtained by extraction of raw rhizomes of Ruscus aculeatus L. with water under pressure and with heating, and by subsequent Extraction of the resulting aqueous solution with a solvent for steroid heterosides, and that the obtained Solvent phase is used as the organic phase for the extraction. 8 »Method according to claim 1, characterized in that the rhizomes of Ruscus aculeatus L _{8 are} extracted with water under pressure and with heating and the supernatant is concentrated so that the concentrate obtained is subjected to a liquid-liquid extraction with a solvent for steroid heterosides that the combined organic phases are atomized to form a powder with a content of steroid heterosides of 80 fo _t and that the powder obtained is dissolved in water or a solvent for steroid heterosides and used as the phase to be extracted. 9830/0822 JO- 2436A79 Process according to claim 1, characterized in that the rhizomes of Ruscus aculeatus L. are extracted with water under pressure and with heating, that the supernatant is concentrated, that the concentrate obtained is a liquid-liquid extraction with a solvent for steroid Heterosides is subjected that the solvent phase obtained is concentrated in water to obtain a solvent-free aqueous phase, that the aqueous phase obtained is atomized to give a powder with a steroid heteroside content of 50 "/ <>, and that the obtained Powder dissolved in water or a solvent for steroid heterosides and used as an au extracting phase. 10. The method according to Ansnruch 1, characterized in that din rhizomes of Ruscus aculeatus L. are extracted with water under pressure and with heating, that the supernatant is concentrated, and that the concentrate obtained is extracted with ethyl alcohol at 96 ° to obtain a alcoholic solution with a content of steroid heterosides of 2 1o. 11. The method according to one of the claims 1 to 10, characterized in that that an alkali metal or alkaline earth metal hydroxide is used as the base. 12. The method according to claim 11, characterized in that sodium hydroxide solution or potassium hydroxide solution is used as the base. 13. The method according to any one of claims 1 to 12, characterized in that that n-butanol is used as a solvent for steroid heterosides. 14. The method according to any one of claims 1 to 13, characterized in that that the steroid heterosides from the organic S09830 / 0822 Phase can be separated by concentrating the organic phase until the steroid heterosides precipitate. 15. Therapeutic agent with a content of steroid heterosides purified according to claims 1 to 14 as the active component. 509830/0822 ORIGINAL INSPECTED