AU662343B2 - Method of extracting sennosides A, B and A1 - Google Patents

Abstract

The invention concerns a method of extracting sennosides A, B and A1. The method comprises the following steps: a sennoside mixture is reduced to the corresponding anthrone-8-glucosides, the aloe-emodin components are removed by liquid-liquid separation of the anthrone-8-glucosides, and the rheine-9-anthrone-8-glucoside obtained after the separation step is oxidized again to sennosides A, B and A1. The invention also concerns sennosides A, B and A1 obtained by this method and pharmaceutical agents containing them.

Description

OPI DATE 25/01/93 AOJP DATE.25/03/93 APPLN. ID 21654/92 jfj"''sI111Illllllli PCT NUMBER PCT/EP92/01428 111111 111111111111111111111111111 AU9221654 (51 InerntioalePatntkassfiktio 5(11) Intemnationale Veroffentlichungsnummer: WO 93/00350 C07H 15/244, A61K 35/78 A (43) Internationales, Veroffentlichungsdatum: 7. Januar 1993 (07.01.93) (21) Intenationales Aktenzeichen: PCT/EP92/0 1428 (74) Anwalt: CARCASONA, Alfons; Patentabteilung, Madaus AG, Ostmerheimer Str. 198, D-5000 K65In 91 (DE).

(22) Intemnationales; Anmeldedatum: 24. Juni 1992 (24.06.92) (81) Bestimmungsstaaten: AU, CA, CS, Fl, HU, JP, PL, RU, Priorititsdaten: US, europiiisches Patent (AT, BE, CH, DE, DK, ES, FR, P 4120 991.5 25. Juni 1991 (25.06.9 1) DE GB, GR, IT, LU, MC, NL, SE).

(71) Anmelder (ffir alle Bestimmungsstaaten ausser US): MA- Veroffentlicht DAUS AG [DE/DE]; Ostmerheimer Stralle 198, D-5000 Mit internationalem Recherchenbericht.

91 Vor Ablauf der fir .4nderungen der Anspriiche zugelassenen Frist. Verdffentlichung wird wiederholt falls Anderun- (72) Erfinder; und gen eintreffen.

Erfinder/Anmelder (nur fur US) C kRCASONA, Alfons [ES/DE]; Frankfurter Strage 192, D-5000 Ko1n 80 (DE).

GRIMMINGER, Wolf [DE/DE]; Friedrich-Rosengarth-Stralle 31, D-5060 Bergisch-Gladbach I 624 3 HIETALA, Pentti [Fl/Fl]; Alkutie 5, SF-00660 Helsinki 66 (Fl1). ZAESKE, Helga [DE/DE]; Platanenweg 20, D- 5063 Overath 3 WITTHOHN, Klaus [DE/DE]; H4$henstrage 26, D-5063 Overath 3 (DE).

(54) Title: METHOD OF EXTRACTING SENNOSIDES A, B and Al (54) Bezeichnung: VERFAHREN ZUR GEWINNUNG DER SENNOSIDE A, B UND Al (57) Abstract The invention concerns a method of extracting sennosides A, B and Al. The method comprises the following steps: a sennoside mixture is reduced to the corresponding anthrone-8-glucosides, the aloe-emodin components are removed by liquid-liquid separation of the anthrone-8-glucosides, and the rheine-9-anthrone-8-glucoside obtained after the separation step is oxidized again to sennosides A, B and Al. The invention also concerns sennosides A, B and Al obtained by this method and pharmaceutical agents containing them.

(57) Zusainmenfassung Die Erfindung betrifft emn Verfahren zur Gewinnung der Sennoside A, B und Al. Das Verfahren besteht darin, dag emn Sennosid-Gemisch einer Reduktion zu den entsprechenden Anthron-8-glucosiden unterworfen wird, eine Fliissig-Fliissig-Vertei-' lung der Anthron-8-glucoside zur Entfemnung der Aloeemodin-Komponenten erfolgt und das nach der Verteilung erhaltene Rhein-9-anthron-8-glucosid wieder zu den Sennosiden A, B und AlI oxidiert wird. Die Erf indung betrifft auch die nach diesemn Verfahren erhifitlichen Sennoside A, B und Al und pharmazeutische Mittel, das diese enthlt.

I-

I

obtaining of sennosides A, B and Al which are substantially free from the sennosides C, D and D1 and from aloe-emodin components, as well as the sennosides obtainable according to this process and pharmaceutical agents containing these sennosides.

The sennosides are laxative-acting substances which occur in dried drugs of the genus Cassia and Rheum. The senna drugs consists of the dried leaves and pods of the senna plant, for example of Indian senna (Cassia acutifolia).

The laxative-active sennosides are dianthrone glucosides derived from rhein and aloe-emodin. The most important ones are the sennosides A, B, Al, C, D and Dl. They correspond to the formula:- %H210o o Oil

COOH

R

1H 115 0

OH

In the case of the sennosides AT B and Al, R stands for COOH and in the case of the sennosides C, D and DI, R stands for CH OH. Sennosides A, B i; -3and Al and D and Dl,, respectively, are stereoisomers and differ from one another by the configuration on the C-atoms 10 and Besides the sennosides, the crude drug also contains aglycones (sennidines), semi-glycosilated senLiidines, polymers,, decomposition products of the sennosides, aloe-emodin and derivatives thereof etc.

which can bring about undesired side effects, such as ill-feeling, vomiting,, flatulence and colid.

Processes for the obtaining of the sennosides from the senna drug are described., for example in DE-B-16 17 667, FR-M. 6611, GB-A-852017i and DE-A- 200 131., Depending upon the drug,. the sennosides, obtained according to these known processes contain .a sennoside mixture with 1.5 5% of the sennosides C, D and DI. As already shown above,, these contain in their molecule a part derived from alie-emodin of the formula:- HO OIL ca oH 0. 2 It would be desirable if one could obtain sennosides which are substantially free from sennosides C, D and Dl.

A. substantially complete separation of the sennosides C, D and Dl. from a sennoside mixture is

ALL

not known from the present state of the art.

Therefore, the task forming the basis of the invention is to make available a process for obtaining sennosides A, B and Al which are substantially free from undesired accompanying materials, especially from sennosides C, D and Dl.

This task is solved by the process according to the invention which is characterised in that A) a sennoside mixture is subjected to a reduction to rhein-9-anthrone-8-glucoside, B) a liquid-liquid partitioning is carried out of the compounds obtained between a polar organic solvent only partly miscible with water and an aqueous phase and C) the rhein-9-anthrone-8-glucoside contained after the partitioning in the aqueous phase is oxidized to the sennosides A, B and Al which are then recovered.

Step A As starting material for the process according to the invention, there are, in general, used sennoside mixtures 20 such as are obtained in the case of the extraction of the senna drug according to the above-mentioned processes. For example, as starting material, a sennoside mixture is useable which one obtains according to the process described in DE-A- 32 00 131. Thereafter, one first extracts the senna drug 25 with aqueous methanol. The concentrate remaining after complete removal of the methanol 04,p\op* .t.r\dab16r4-92.185,4 Shnni III. a.

y\ C 95O74,p\opr«db,265a92.85, contains ithe sennosides in the form of the potassium salts.. This concentrate can be used as starting material for the process' according to the invention.

One can also purify the concentrate by liquidliquid extraction with alcohols or ketones partly soluble in water butanol-2,. 2-butanone, acetone) (raffinate). The raffinate is acidified to a pH of about 1..5 to 2..0 and the sennosides are brought to crystallisation by seeding. The crude sennoside mixture obtained is also useable as starting product for the process according to the invention. If desired, one can also recrystallise the crude sennoside mixture, Alternatively, as starting material one can also i 15 use the concentrate mixed with an alcohol or ketone partly soluble in water, especially butanol-2, v In the case of the extraction of the senna drug, the ratio of drug to extraction solvent preferably jj amounts to l: to especially l:4 to 1:.10, The! extraction is preferably carried out in the presnce of,a buffer, for example trisodium citrate, glycine,: sodium bicarbonate or saccharose.

According to the ,nvention, these starting material are subjected to a complete reduction to the corresponding rhein-9--anthrone-8-glucoside (R COOH) and to the corresponding aloe-emodin- S anthrone-8-glucoside i =CI O 2 H) of the formula:,- L i- c 0 O -6-

R

Reducing agents with a suitable reduction potential are tin-II chloride, sulphur dioxide, alkali metal boron hydrides and preferably alkali metal dithionites, especially sodium dithionite.

For the carrying out of the reduction, one can take the starting material in aqueous solution oa suspension and add the reducing agent thereto in solid form or dissolved in water. Especially in the case of the use of senna fruit primary extract, according to DE-A-52 00 131 (aqueous concentrate), one can also work in a 2-phase mixture in that one: adds a polar organic solvent partly miscible with water,, especially 2-butanol or acetone.

One can reduce at ambient temperature or higher temperature. The reduction is expediently carried out at 40 to 60 0 C, especially at 50 to 5500. One works at weakly acidic to weakly alkaline pH value of the starting sennoside solution or suspension, preferably at pH 5 10.5. If desired,, one can carry out the reduction several times, especially two to ten times.

One precipitates out the 9-anthrone-8-glucoside formed by addition of an acid, for example sulphuric acid, up to a pH of about 2 to 4.5. The temperi -7ature should thereby expediently amount to not more.

than 400., In the case of the precipitating out of the anthrone glucosides and in the case of their isolation (for example by filtration), one expediently works under nitrogen in order to prevent an uncontrolled oxidation of these compounds.

It is important that the reduction proceeds to completion.. Therefore, one expediently uses the reducing agent in large excess. In gener&l, one uses dithionites, especially sodium dithionite, in the 1 to 4 fold wt.. amount, referred to the content of sennosides in the starting material., Furthermore, one, allows the reducing agent to act- for at least 2 h, preferably at least 3 h. In general, the reduction takes place for not longer than 10 ho One preferably carries out a post-reduction under said conditions.

Before its use in step B, the product obtained is preferably reprecipitated in that one brings it into aqueous solution by addition of a base (NaOH,, KOH) up to about pH 6 7, extracts the aqueous solution with 2-butanol, 2-butanone or acetone and again precipitates out the product by addition of an acid to about pH 2 4.

Step B 25 In this step, the aloe-emodin components, especially aloe-emodin-- anthrone-8-glucoside, are removed. For this purpose, a liquid-liquid partitioning of the product obtained is carried out in a polar *4 1 0^ y 1I i 1 Li 1 'f -8organic solvent only partly miscible with water and an aqueous phase. Suitable polar organic solvents are C4-CQ 5 -alkanols and di-C--C 3 -alkyl ketones, such as 1-butanol, 2-butanol, 2-butanone and acetone. One preferably uses 2-butanol or acetone, To the aqueous phase one preferably adds a reducing agent in order to impart to the aqueous phase during the whole of the liquid-liquid partitioning a redox potential of -210 mV or more negative. Expediently, one uses the same reducing agent as in step In the case of using an alkali metal dithioniLte as reducing agent, in general a, 2 to 4 wt.% solution at a pH value of 7 to 10.5 is sufficient in order to maintain the mentioned potential conditions. The pH value is expedien;ly maintained in this range by addition of a buffer..

The volume ratio of a.queous phase (heavy phase) to organic phase (light phase) generally lies in the range of from 1:,5 to The liquid-liquid extraction preferably takes place in countercurrent.. The mixture of the anthrone compounds is thereby introduced in the form of the solution obtained after the reduction or,, when the anthrone compounds have been isolated, in the form of a 3 to 15 wt., solution..

After the partitioning, the desired rhein-9anthrone-8-glucoside is present in the aqueous phase.

It is precipitated by addition of an acid up to a i -9pH value of about 2 to 4 and reovered in the usual way.

Step C In this step, the rhein-9-anthrone-8-glucoside is again oxidised to the corresponding sennoside compounds. Suitable oxidation agents herefor are hydrogen peroxide, manganese dioxide,. permangante, manganese-III acetonylacetonate.

However, one preferably carries out the oxidation with oxygen. As oxygen source, there can serve, for example, air.

Since rhein-9-anthrone-8-glucoside is insoluble in water, for the oxidation it is converted into a soluble form.. This takes place, for example, in that, by addition of a suitable base up to a pH value of about 7, it is converted into an alkali metal salt or the calcium salt. If desired, a small amount (up to about 30 volume of a solvent only partly miscible with water,, especially 2-butanol, can be added to the solution.

The oxidaition is carried, out in a solution which is as concentrated as possible becQuse, in this way,, the formation of the desired sennosides is favoured..

One expediently carries out the oxidation with a solution which contains about 250 to 300 g of rhein- 9-anthrone-8-glucoside per litre of solvent. In the case of the use of oxygen as oxidation agent, one.

expediently passes this through the solution.

t i One can facilitate the oxidation with oxygen by use of a catalyst.. Suitable catalysts are eg., palladium black or iron-III salts, especially iron- III chloride. In general, the amount of catalyst lies in the range of 0.2 to 2 referred to the amount of rhein-9-anthrone-8-glucoside, and especially in the range of 0.5 to 1 Alternatively, one can carry out the oxidation with iron-III salt, Fe.2 (S)3 or FeC;13, at a pH value of 8 8.5. One thereby preferably works at 30 50QC and in the presence of trisodium citrate, The oxidation is carried out until no more rhein- 9-anthrone-8-glucoside is detectable (absence of the UV fluonescence of the anthrone compounds).

The sennosides are obtained in the usual way by acdification of the solution obtained. Before addition of an acid, one expediently dilutes the solution to 2 to 5 times the volume present with the solvent used water/2-butanol).. In this way, it is achieved that the rhein-8-glucoside formed as by-product remains substantially in solution in the case of the -precipitating out of the sennosides, The separation of the rhein-8-glucoside can also take place via the calcium salt because the calcium salt of rhein-8-glucoside is insoluble and precipitates- out, whereas: the calcium salt of the sennosides remains in solution, 0 I I question substantially of the sennosides A, B and Al.

They are substantially free from sennosides C, D and Dl and other aloe-emodin contaminations. The content

H

of sennosides C: and D1 in the product obtainoale according to the invention amounts to less than 100 ppm (determined according to the analysis method givene in the Examples), The invention also concerns the mixture of sennosides A, B and Al obtainable according to the .ivention, as well as a pharmaceutical agent which contains the mentioned mixture.

The fiid. of use, the dosage to be administered and the suitable forms of dosage are known from the initially mentioned publications.

The following Examples explain the invention..

Example I.

Obtaining the sennoside mixture used as starting f material..

In each case, one places40 kg of senna drug into two percolators, connected in series, with a volume of 250 1 and covered with a perforated steel plate, As solvent for the extraction,; one uses 70% methanol which is passed to the drug in the first percolator.

One passes the solution formed in the first percolator S-12to the drug which is present in the second percolator.

One thereby allows the solvent to flow freely through the first percolator.

For the extraction of 40 kg of senna drug, one uses, in all, 160 1 of solvent., After one has passed this volume of 70% methanol through the two percolators and has collected the corresponding amount of percolate, one couples the emptying pipe of the percolator with a post-percolate container and then additionally passes 60 1 of 70% methanol though the percolators. Thereafter, one passes the remaining free solvent from the first percolator into the upper part of the second percolator and collects the postpercolate until it makes up a total of 120 1. One then empties the first percolator, again fills it with of senna drug and pumps,t.e post-percolate on to the drug, whereby 120 1 of post-percolate suffice in order to cover the drug in the percolator.

Subsequently, one brings the temperature of the solution to +30C,. Thereafter, one leaves to stand overnight, One connects this percolator with the one previously extracted and carries out the extraction as described above..

In each case,; for 40 kg of drug one collects 160 1 of percolate from which one removes the methanol in a vacuum rotary evaporator which is equipped with a packed column. One obtains about UA ic r r, rfir -13- 1 of bottom product.

This concentrate is extracted with the equal volume of 2-butanol which is saturated with water.

The phases are separated and the aqueous phase is further worked up.

Step A Reduction of the sennosides to rhein-9-anthrone-8glucosides 1 of the extracted concentrate are brought to pH 7.5 with 48% caustic sod solution. One heats to 60 0 C and, while stirring, qdds 90 g of sodium dithionite in solid form to the solution over the course of half an hour. After completed addition, one stirs for a further hour. Subsequently, while stirring, one adds concentrated sulphuric acid thereto up to a pH of 2. In the course of two hours, one cools to ambient temperature, filters off the separated crystalline precipitate and washes it with sulphur dioxide-containing water.

If desired, crude rhein-9-anthrone-8-glucoside is reprecipitated. The still moist filter cake is so dissolved in a mixture of 15 vol. parts of 2butanol and 85 vol. parts of:water which contains O .5 wt.% of sodium pyrosulphite that, by addition of a 48% aqueous solution of sodium hydroxide to a pH of 7, one obtains a 10% solution The solution is acidified with concentrated hydrochloric acid to pH 2.8 or below and left to stand

I

rl

L-

i I- -14.for 2 h. The precipitate obtained is filtered off and washed with sulphur dioxide- or sodium pyrosulphite-containing water and dried.

Yield One carries out a renewed reduction (postreduction) with the product obtained in this manner as follows: One dissolves 3.0 g of the crude, dried rheinanthrone-8-glucoside or the corresponding amount of the moist product in 15 ml of water, together with 1.4 g of sodium dithionite and 2.3 ml 5N NaOH.

Subsequently, one makes up with water to 24 ml and heats the solution for 20 minutes at 55 0 C. Thereafter, one adds a further 1.5 g of sodium dithionite to the solution and heats to 55 0 C for 20 minutes. Subsequently, one adds thereto 0.9 ml. of 5N NeOH and g of sodium dithionite. After heating for minutes at 5500, one again adds 0.9 ml of 5N NaOHi.

The solution obtained is then introduced directly into the following ligqid-liquid extraction, Step

B:

Separatof te an TheSeparating off of the aloe-emodin components T he separating off of the aloe-emodin components takes place by liquid-liquid partitioning of the anthrone-8-glucosides in countercurrent with the ,i help of an apparatus of 60 mixer-separator units S (mixer-settler apparatus)., As' aqueous, heavier S phase, one uses a solution of 310 g sodium dithionite, Ah yi 1 I- M. ST 1 5- i in 5.5 ml 5N NaOH and. 96 ml of water.. As organic, lighter phase, one uses (water-saturated) 2-butanol or acetone, The two phases are so supplied to the apparatus that the volume ratio of the heavier phase to the lighter phase amounts to The mixture to be sepataed is supplied to the apparatus in the form of the freshly reduced solution or in the form of a solution of appropniate pH value and of appropriate concentration which contains the anthrone-8-glucosides obtained from step A, namely, in such a manner that 30 vol.. parts of the organic phase are use per part by volume of the mixture to be separated.

The pH. of the solution containig the mixture is maintained at 9 9..5 With the help af a glycine, buffer. The buffer of 3 vol. parts of a 7.5% glycine; solution and 1 vol. part of 1N NaOH is introduced in an amaunt pf 240 ml of buffer solution per 150 g of crude rhein-9-anthrone-8-glucoside. The undesired aloe-emodin compounds enrich in the organic phase, whereas the rhein-9-anthrone-8-glucoside remains in the aqueous phase., The aqueous phase is aidiified with sulphuric acid to pH 2.8, the precipitate formed is filtered off and washed with water and.

acetone and dried in the air at ambient temperature.

In this way,, one obtains rhein-9-anthrone-8-glucoside with a content of aloe-emodin components of 49 ppm (determined as aloe-emodin)..

-16- Yield: referred to rhein-9-anthrorie-8-glucoside.

Step C: Oxidation of the rhein-9-anthrone-8-glucosides One dissolves 18.8 g of the rhein-9-anthrone-8glucoside obtained in 56 ml of water and 11 ml of 2-butanol, whereby one adds thereto 17N NaOH up to a pH of 6.5. While stirring, air is blown through this solution for 5 hours in a cylindricallyshaped vessel with the help of a glass frit. The rate of flow of the air amouits to 40 ml/min. The; course of the oxidation is monitored by means of

HPLC.

When no noe rhein-9-anthrone-8-glucoside is detectable, the solutio o is diluted to about 200 ml with water/butanol 56/11. One adds thereto concentrated hydrochloric acid up to pH 1.5 to 2, stirs for 2 hours at ambient temperature, filters off the precipitated crystals and washes: them with water and acetone and dries. One obtains 14.4 g of pure sennoside mixture with a content of 41 ppm aloeemodin components (determined as aloe-emodin) according to the analysis procedure given in step C, Example 2.

Example 2 One repeats the process described in Example 1, whereby, however, one carries out the oxidation step C as follows:: iA" r h F-l 150 g of the pure rhein-9-anthrone-8-glucosides and 75 g iron-III chloride hexahydrate are dissolved in 480 ml of water and 120 ml of 2-butanol. A 48% sodium hydoxide solution is added thereto until pH 6.5 is reached and the rhein--anthrone-8-glucoside has dissolved.. The solution is introduced into a vessel with a sinter bottom plate. Subsequently, a vigorous air current is passed through the solution.

The oxidation is finished after about 30 minutes.

Subsequently, one dilutes the solution with a mixture of 120 ml 2-butanol and 480 ml of water, adds thereto g sodium dithionite and adjusts the pH of the solution to 2.,0 by addition of concentrated hydrochloric acid.The solution is stirred for 18 hours., Subsequently, the separated precipitate is filtered off, washed with 600 ml of water and 800 ml of acetone and dried. The content of anthranoid compounds in the product lies between 94 and The product is taken up in 200 ml of 2-butanol and precipitated with 800 ml of water with the addition of 5.5 g sodium pyrosulphite. After filtering off and drying of the precipitate obtained, one obtains 95.4 g of a product of anthranoid compounds of the following composition (according to HPLC, analysis of a typical experiment); SI 950704,p:\opc\dab,2.1654-92.1 8 5 ,4 eI -18rhein-8-glucoside sennoside B 49.7% sennoside Al 13.3% sennoside A 33.6% sennidine monoglucosides 1.1% rhein 0.02% 99.22% Sennosides C and D and aloe-emodin glucoside could not be detected by HPLC. The total content of aloe-emodin and of its derivatives was determined as being 30 ppm according to the following method Sennosides C and D and aloe-emodin-8-glucoside can no longer be dependa:bly determined as sennosides in the ppm range by means of HPLC chromatography.

Therefore, it is necessary to convert the substance to be investigated by oxidation with iron-III chloride with simultaneous hydrolysis with hydrochloric acid in a 2-phase mixture of aqueous solution/ carbon tetrachloride into rhein or aloe-emodin. The rhein is then converted into a salt so that it can be extracted into the aqueous phase and the aloeemodin in the organic phase can be determined by means of HPLC.. In this way, there can be given the total content of sennosides C, D, aloe-emodin-8-glucosides and other aloe-emodin components, expressed as aloeemodin..

44 Example. 3 One repeats the extraction of the senna drug and the reduction of the sennosides described in Example 1.

One then carries out the post-reduction as follows: One dissolves 14.0 g of saccharose, 4.5 g of sodium dithionite and 13.3 g of potassium acebate in 133 ml of water and adds thereto 1.3 ml of 48% sodium hydroxide solution and 17.3 g of potassium carbonate. Subsequently, one mixes with 293 ml of 1D acetone and 50 ml of water. One shakes the mixture in a separating funnel and separates the phases, whereby one obtains 375 ml of upper phase (acetone phase) and 130 ml of lower phase.

One dissolves 1.4 ml of 48% sodium hydroxide solution and 10 g of crude rhein-9-anthrone-8glucoside in 98 ml of the lower phase. One warms to to 50

Q

C and maintains at this temperature for to 50 minutes. Subsequently, one adds thereto 1.0 ml of 48% sodium hydroxide solution and 5.4 g sodium dithionite and heats for a further 20 to 30 minutes' at 45 to 50°C. Subsequently, one again adds 1.0 ml of 48% sodium hydroxide solution and 3.4 g sodium 'i 1 dithionite thereto and heats for 20 to 30 minutes' to 45 to The separation of the aloe-emodin components takes place by liquid-liquid partitioning of the reduced solution in countercurrent against the abovei!,mentioned upper phase (acetone phase)., The raffinate phase flowing off containing r-hein-9-anthrone-8glucoside is concentrated to 400 ml and mixed with ml butanol-2 or acetone. One adds thereto hydrochloric acid or sulphuric acid up to pH 4.0 to The precipitate formed is filtered off, washed with 40 ml of water and 30 ml of acetone and subsequently dried. The subsequent oxidation takes place as described in Example 2., Example 4 The concentrate obtained after the extraction of the senna drug is mixed with about 2 1 of butanol-2.

The reduction of the senna fruit concentrate/butanol- 2 mixture is then carried out in 7 steps under nitrogen as protective gas. After the reduction step I, there follows a precipitation of the crude rhein- 9-anthrone-8-glucoside.

Reduction step I 100 1 of senna fruit concentrate/butanol-2 mixture containing about 4 kg of sennosides, are placed in a stirrer container and covered with nitrogen. While stirring,, 6 1 of 20% caustic soda solution, thereafter 350 1. of water-saturated butanol-2 from step II), are added successively thereto and stirred for 15 min.. The batch is heated to 42. to 50 C and mixed with 7 kg sodium dithionite. It is further stirred for 45 min. The pH vabue is maintained at 7.5 8 With 20% (wt.) caustic soda solution. The reduction potential t w^ .il r -21- (against Ag/AgC. electrode) if necessary,: maintained bel8w -630 mV by sodium dithionite addition. After cooling to 30 350C, it is precipitated within 1.5 hrs. with 10% sulphuric acid to pH 4, The resultant suspension is stirred, for about 10 hrs. at (25 0 C with slow stirring speed.

The resultant precipitate is filtered off. The precipitate is suspended in 60 1 of 15% (wt.).butanol-2, stirred for 30 min. at 50 to 60 0 C and subsequently filtered.. The residue is washed with 100 1 of demineralised water.. The crude yield of rhein-9anthrone-8-glucoside lies above 82%, referred to sennosides used.

Reduction step II 3.3 kg crude rhein-9-anthrone-8-glucoside from step I are suspended in a mixture of ~2 1 of demineralised water and 7.4 1 butanol-2.. The suspension is brought into solution with 2 1 of 20% caustic soda solution and 9.,9 kg trisodium citrate and thereafter mixed with 3..3 kg sodium dithionite and 350 I of water-saturated butanol-2 from step III).

The batch is heated to 42 4500. The pH value is maintained at 8.5 to 9 with 20% caustic soda solution. The reduction potential (against an Ag/AgGl.electrode) is, if necessary, maintained below.

750 mV by sodium dithionite addition.

After a standing time of 50 min, the upper phase is removed and the lower phase further worked up Sin step IITI a i 1_ ii :i if i; i J I- .i i Reduction step III The reduction/extraction process described in step II is repeated with the lower phase from step II, with the addition of the following chemicalsz 1,65 kg sodium dithionite 0..8 1 of 20% caustic soda solution 350 1 of water-saturated butanol-2 from step IV).

Reduction steps IV VII The reduction/extraction process described in step II is repeated with, in each case, the lower phase from the preceding step with the addition of the following chemicals: 0.825 kg sodium dithionite 0.4 1 of 20% caustic soda solution 350 1 of water-saturated butanol-2 in each case from the following steps countercurrent principle).

The lower phase separated off in step VII is cooled to 50 35C and the rhein-9-anthrone-8glucoside precipitated out as described in step I.

The resultant precipitate is filtered off and washed Swith 100 1 of demineralised water.. Subsequently, it is covered with 10 1 of iron-III sulphate solution (preparation see step B, Example 1).

4 1

T,'

1 i -s i i -23-

I

i

I

i iii: The rhein-9-anthrone-8-glucoside is then converted into the sennosides as described in Example 1 or 2, Example The oxidation of the rhein-9-anthrone-8-glucoside can also take place according to the following process:.

kg of filter-moist rhein-9-anthrone-8glucoside are mixed with 12..6 kg trisodium citrate.

This mixture is dissolved in 7.0 1 of 1 NaOH with vigorous stirring and mixed with 0.7 1 of butanol-2.

Subsequently, it is mixed with 8.8 1 of iron-III sulphate solution (28 kg Fe 2

(SO

4 1 3 in 100 1 of demineralised H20) and so much 20% NaOH added that the pH value amounts to about 8.3. One leaves to react for 3 4 hours at about 40°C, then acidifies with 52% H 2

SO

4 to pH 1.8 2.0 and works up as described in Example 1.

Example 6 Alternatively, one brings rhein-9-anthrone-8glucoside into solution in 50 ml of water by addition 20 of a calcium hydroxide-saccharose solution (prepared by suspending of 7.0 g calcium hydroxide in a solution of 30.0 g saccharose in 100 ml of H20 and removal of the undissolved calcium hydroxide). One adds 20 ml butanol-2 thereto and passes a vigorous ccrrent of air through the solution over the course of 90 minutes.

One adds 5.,0 g COCI,.2HWO thereto and adjuststhe pH to 8.5 with the calcium hydroxide-saccharose solution.. The precipitate formed is filtered off and Pi 1 sides remains in solution.

L-i i C I -r L

I

t ;r r -24-, the filtrate is diluted with H20 to 340 ml,, mixed with 60 ml butanol-2 and adjusted to pH 2.0 with concentrated hydrochloric acid., The further working up takes place as described in Example 1, Pharmacological investigations Laxative action The laxative effect of the sennoside mixture according to the anvention was determined on mice.

Ma-le NMRI i-mice were used which were kept during the experiment in Plexiglas cages and received standard feed mix with tap water of mushy consistency. A separate supply of drinking water didnot take place during the experiment.

The animals received 100, 200 and 400: mg/kg of the sennoside mixture in 10 ml of 0.5% NaHCO3/kg by stomach probe. After administration of the compounds to be tested, faeces and urine of the animals were collected for 24 hours and then determined. The results obtained, referred to kg of body weight, are summarised in the following Table..

It can be seen that the sennosides display a good laxative action which commences relatively quickly.

The time up to the appearance of the first soft faeces (2 hours.) is, however, also to be combined.

with a previous transit to the large intestine and a *breakdown of the sennosides by the flora of the large intestine. A dosage-action relationship is present..

r I I One passes the solution formed in the first percolator 4 P":Za; Acute toxicity In each case, 10 male and female Wistar rats were given sennosides once in dosages of 200 to 25,000 mg/ kg with a stomach probe.

Macroscopic organ damage caused by the administered substances could not be observed. The determined

LD

50 values ares male rats: 5200 8 mg/kg 720 female rats:. 3530 380mg/kg 340 mg/kg In the case of male and female mice (n 8, strain NMRI), the maximum administerable dose of 5000 mg/kg did not result in any deaths. In all mice,, diarrhoea occurred,, although to a lesser extent than in the case of rats. For both sexes, the LD 50 values amounted to 5000 mg/kg.

T.a b 1. e Laxative effect of the sennoside mixture according to invention on mice dosage number number of number of soft faeces (mg/kg) of normal faecal. soft as of the animals pellets faecal, total faecal pellets excretion 0 50 1265 0 0 100 40 587 144 28,0 200 3,0 223 239 56..0 400 30 236 282 60,0 j -li; i-l- ;r-l -I I

Claims (15)

1. A process for obtaining sennosides A, B and Al of the formula: CeH 1 0 5-O 0 OH COOH COOH C, HiOs-- O O OH which are substantially free from sennosides C, D and D1 and aloe-emodin components, characterised in that A) a sennoside mixture is subjected to a reduction to rhein-9-anthrone-8-glucoside and aloe-emodin-9-anthrone-8- glucoside, B) a liquid-liquid partitioning is carried out of the compounds obtained between a polar organic solvent only partly miscible with water and an aqueous phase and 25 C) the rhein-9-anthrone-8-glucoside contained after i !I r partitioning in the aqueous phase is oxidized to the corresponding sennosides which are then recovered.

2. A process according to claim 1, characterised in that the sennoside mixture is obtainable by extraction of the senna drug with aqueous methanol.

3. A process according to claim 2, characterised in that the senna drug is extracted with aqueous methanol in the presence of a buffer. 950704,p:\opcr\dab,2654-92.185,26 L i I i' chloric acid to pH 2.8 or below and left to stand iir;; f: -27-

4. A process according to any one of the preceding characterised in that an alkali metal dithionite is reducing agent in step A. claims, used as

5. A process according to claim 4, characterised in that process is carried out at a pH value of 5 to 10.5.

6. A process according to any one of the preceding characterised in that 2-butanol is used as a polar solvent in step B. claims, organic 4t 4 *6t r:I 4 C C r

7. A process according to any one of claims 1 to characterised in that acetone is used as a polar organic solvent in step B.

8. A process according to any one of the preceding claims, characterised in that, in step B, an aqueous phase the redox potential of which is -210 mV or more negative is used. 20 9. A process according to any one of the preceding claims, characterised in that the liquid-liquid partitioning is carried out in step B in countercurrent. A process according to any one of the preceding claims, 25 characterised in that the oxidation is carried out in step C with oxygen or an iron-(III) salt.

11. A process according to claim 10, characterised in that the oxidation is carried out with oxygen at weakly acidic pH value. tr C t C C C t V C C C C C .4

12. A process according to claim 10 or 11, characterised in that the oxidation is carried out with oxygen in the presence of a catalyst.

13. A process according to claim 12, characterised in that the catalyst is an iron-(III) salt. 950704,p:\opcr\dab,21654-92.185,27 I' 1 .A:"0 1 :d I 1 i-i I::r N V 11 l m s m i A ii it-- 28

14. Sennosides A, B and Al as defined in claim 1 which are substantially free from sennosides C, D and D1 and aloe- emodin components.

15. Pharmaceutical agent containing the sennosides according to claim 14, together with usual pharmaceutical carriers and adjuvant materials.

16. Sennosides A, B and Al which are substantially free from sennosides C, D and Dl and aloe-emodin components, processes for obtaining them or pharmaceutical agents containing them, substantially as hereinbefore described with reference to the Examples. 4 t; I Dated this 4th day of July, 1995 MADAUS AG By Its Patent Attorneys S DAVIES COLLISON CAVE r 4-4 t 4 I4l IL J 1 950704,p:\op\dab,21654-92.185,28 L -iii.-l. -29- Summary, The invention concerns a process for the obtaining of the sennosides A, B and Al, The process consists in that a sennoside mixture is subjected to a reduction to the corresponding anthrone-8-glucosides, a liquid-liquid partioning of the anthrone-8-glucosides takes place for the removal of the aloe-emodin components and the rhein-9-anthrone-8-glucoside obtained after the partitioning is again oxidised to the sennosides A, B and Al. The invention also concerns the sennosides A, B and Al obtainable according to this process and pharmaceutical agent which contains these. NNmyi Process for the obtaining of sennosides A, B and Al The invention concerns a process for the' obtaining of sennosides A, B and Al which are substantially free from the sennosides C, D and D1 and from aloe-emodin components, as well as the sennosides obtainable according to this process and pharmaceutical agents containing these sennosides. The sennosides are laxative-acting substances which occur in dried drugs of the genus Cassia and Rheum. The senna drugs consists of the dried leaves and pods of the senna plant, for example of Indian senna (Cassia acutifolia). The laxative-active sennosides are dianthrone glucosides derived from rhein and aloe-emodin. The most important ones are the sennosides A, B, Al, C, D and D1. They correspond to the formula:- C OL OR C 6 11 0 5 O O 0 OH In the case of the sennosides A, B and Al, R stands for COOH and in the case of the sennosides 20 C, D and Dl, R stands for CH OH. Sennosides A, B /-^aAOH.N lensie A, 2B 1 E ii :r :-ir; I IB and Al. and D and D1, respectively, are stereo- isomers and differ from one another by the config- uration on the C-atoms 10 and Besides the sennosides, the crude drug also contains aglycones (sennidines), semi-glycosid.ated sennidines, polymers, decomposition products of the sennosides, aloe-emodin and derivatives thereof etc. which can bring about undesired side effects, such as ill-feeling, vomiting, flatulence and-colid. Processes for the obtaining of the sennosides from the senna drug are described, for example in DE-B-16 17 667, FR-M 6611, GB-A-832017 and DE-A- 3 200 131.. Depending upon the drug, the sennosides obtained according to these known processes contain a sennoside mixture with 1.5 55 of the sennosides' C, D. and Dl. As already shown above, these contain in their molecule a part derived from alle-emodin of the formula:- HO OH 0 It would be desirable if one could obtain sennosides which are substantially free from sennosides 0, D and Dl, A substantially complete separation of the sennbsides C, D and Dl from a sennoside mixture is L i-i I I i:.L r not known from the present state of the art. Therefore, the task forming the basis of the invention is to make available a process for the obtaining of sennosides A, B and Al which are substantially free from undesired accompanying materials, especially from sennosides C, D and Dl. This task is solved by the process according to the invention which is characterised in that one A) subjects a sennoside mixture to a reduction to rhein-9-anthrone-8-glucoside, B) carries out a liquid-liquid partitioning of the compounds obtained between a polar organic solvent only partly miscible with water and an aqueous phase and C) oxidises the rhein-9-anthrone-8-glucoside contained after the partitioning in the aqueous phase to the sennosides A, B and Al and recovers these, Step A As starting material for the process according to the invention, there are, in general, used sennoside mixtures such as are obtained in the case of the extraction of the senna drug according to the above-mentioned processes, Eor example, as starting material, a sennoside mixture is usable which one obtains according to the process described in DE-A-52 00 131. Thereafter, one first extracts the senna drug with aqueous methanol.. The concentrate r. emaining after complete removal of the methanoL h.. -z 3 t -i contains -the sennosides in the form of the potassium salts.. This concentrate can be used as starting material for the process according to the invention. One can also purify the concentrate by liquid- liquid extraction with alcohols or ketones partly soluble in water butanol-2,. 2-butanone, acetone) (raffinate). The raffinate is acidified to a pH of about 1.5 to 2.0 and the sennosides are brought to crystallisation by seeding. The crude sennoside mixture obtained is also useable as starting product for the process according to the invention. If desired, one can also recrystallise the crude sennoside mixture., Alternatively, as starting material one can also use the concentrate mixed with an alcohol or ketone partly soluble in water, especially butanol-2. In the case of the extraction of the senna .drug, the ratio of drug to extraction solvent preferably amounts to 1:4 to 1:15, especially 1:4 to 1:-10. The extraction is preferably carried out in the presence of ,a buffer, for example trisodium citrate, glycine, sodium bicarbonate or saccharose. According to the invention, these starting materias are subjected to a complete reduction to the corresponding rhein-9--anthrone-8-glucoside (R COOH) and to the corresponding aloe-emodin- 9 anthrone-8-glucoside (R CH2OH) of the formula:- /2 i i 1 i caustic soda- solution., The reduction potential r'' rilHIIIIBHH-----;----- M-.ili"-IYMtf iIII_« C 6 I 1 0 5 0- O1. Reducing agents with a suitable reduction potential are tin-II chloride, sulphur dioxide, alkali metal boron hydrides and preferably alkali metal dithionites, especially sodium dithionite. For the carrying out of the reduction, one can take the starting material in aqueous solution or suspension and add the reducing agent thereto in solid form or dissolved in water. Especially in the case of the use of senna fruit primary extract, according to DE-A-32 00 131 (aqueous concentrate), one can also work in a 2-phase mixture in that one adds a polar organic solvent partly miscible with water,. especially 2-butanol or acetone. One can reduce at ambient temperature or higher i temperature. The reduction is expediently carried out at 40 to 60°C, especially at 50 to 550C. One works at weakly acidic to weakly alkaline pH value of the starting sennoside solution or suspension, preferably at pH value5 10.5. If desired, one can carry out the reduction several times, especially two to ten times. One precipitates out the 9-anthrone-8-glucoside formed by addition of an acid, for example sulphuric acid, up to a pH vaueof about 2 to 4.5. The temper- a i 1 ft is removed and the lower phase further worked up i I SA'" in step III., 7 7 -I ature should thereby expediently amount to not more than 40 C. In the case of the precipitating out of the anthrone glucosides and in the case of their isolation (for example by filtration), one.exped- iently works under nitrogen in order to prevent an uncontrolled oxidation of these compounds. It is important that the reduction proceeds to completion. Therefore, one expediently uses the reducing agent in large excess.. In general, one uses dithionites, especially sodium dithionite, in the 1 to 4 fold wt. amount, referred to the content of sennosides in the starting material. Furthermore, one allows the reducing agent to act for at least 2 h, preferably at least 5 h. In general, the reduction takes place for not longer than 10 h. One preferably carries out a post-reduction under said conditions. Before its use in step B, the product obtained is preferably reprecipitated in that one brings it into aqueous solution by addition of a base (NaOH, KOH) up to about pH value 6 7, extracts the aqueous i solution with 2-butanol, 2-butanone or acetone and again precipitates out the product by addition of an acid to about pH value2 4. Step B In this step, the aloe-emodin components, especially aloe-emodin-9-anthrone-8-glucoside, are removed. For. this purpose, a liquid-liquid partition- I i, \ing of the product obtained is carried out in a polar 1 i. ;i -i~ilC_-r iX organic solvent only partly miscible with water and an aqueous phase. Suitable polar organic solvents are C-C0 5 -alkanols and di-C -C -alkyl ketones, such as 1-butanol, 2-butanol, 2-butanone and acetone. One rJeferably uses 2-butanol or acetone. To the aqueous phase one preferably adds a reducing agent in order to impart to the aqueous phase during the whole of the liquid-liquid part- itioning a redox potential of -210 mV or more negative. Expediently, one uses the same reducing agent as in step A. In the case of using an alkali metal dithionite as reducing agent, in general a 2 to 4 wt.% solution at a pH value of 7 to 10.5 is sufficient in order to maintain the mentioned potential conditions. The pH value is expediently maintained in this range by addition of a buffer. TMe volume ratio of aqueous phase (heavy phase) to organic phase (light phase) generally lies in i the range of from 1:5 to 1:40. The liquid-liquid extraction preferably takes place in countercurrent, The mixture of the anthrone compoundis thereby introduced in the form of the solution obtained after the reduction or, when the of a 3 to 15 wt.% solution.. After the partitioning, the desired rhein-9- anthrone-8-glucoside is present in the aqueous phase. It is precipitated by addition of an acid up to a L I I?- i: i -i pH value of about 2 to 4 and reovered in the usual way. Step C In this step, the rhein-9-anthrone-8-glucoside is again oxidised to the corresponding sennoside compounds. Suitable oxidation agents herefor are hydrogen peroxide, manganese dioxide, permangante, manganese-III acetonylacetonate. However, one preferably carries out the oxidation with oxygen. As oxygen source, there can serve, for example, air. Since rhein-9-anthrone-8-glucoside is insoluble in water, for the oxidation it is converted into a soluble form.. This takes place, for example, in that, by addition of a suitable base up to a pH value of about 6 7, it is converted into an alkali metal salt or the calcium salt. If desired, a small amount (up to about 30 volume of a solvent only partly miscible with water, especially 2-butanol, can be added to the solution. The oxida.tion is carried, out in a solution which is as concentrated as possible becquse, in this way, the formation of the desired sennosides is favoured.. One expediently carries out the oxidation with a solution which contains about 250 to 300 g of rhein- 9-anthrone-8-glucoside per litre of solvent.. In the case of the use of oxygen as oxidation agent, one expediently passes this through the solution. (ij r B One can facilitate the oxidation with oxygen by use of a catalyst.. Suitab? 0 catalysts are e.g., palladium black or iron-IiI salts, especially iron- III chloride, In general, the amount of catalyst lies in the range of 0.2 to 2 referred to the amount of rhein-9-anthrone-8-glucoside, and espec- ially in the range of 0.5 to 1 wt.%. Alternatively, one can carry out the oxidation with iron-III salt, e.g. Fe2(SO) or FeCl1, -t a pH value of 8 One thereby preferably works at 30 500 and in the presence of trisodium citrate. The oxidation is carried out uintil no more rhein- 9-anthrone-8-glucoside is detectable (absence of the UV fluonescence of the anthrone compounds). The sennosides are obtained in the usual way by acidification of the solution obtained. Before addition of an acid, one expediently dilutes the solution to 2 to 3 times the volume present with the solvent used water/2-butanol). In this way, it is achieved that the rhein-8-glucoside formed as by-product remains substantially in s lution in the case of the precipitating out of the sennosides.' The separation of the rhein-8-glucoside can also take place via the calcium salt because the calcium salt of rhein-8-glucoside is insoluble and precip- itates out, whereas the calcium salt of the senno- sides remains in soLution. Ii l ,39: The sennosides are precipitated out by addition of an acid to a pH value of about 2 to 4 and then recovered in the usual way. SI the case of the sennosides obtained, it is a question substantially of the sennosides A, B and Al. They are substantially free from sennosides C, D and Dl and other aloe-emodin contaminations. The content of sennosides C, D. and Dl in the product obtainable according to the invention amounts to less than 100 ppm (determined according to the analysis method given in the Examples). The invention also concerns the mixture of senno- sides A, B and Al obtainable according to the invention, as well as a pharmaceutical agent which contains the mentioned mixture. The fie]d of use, the dosage to be administered and the suitable forms of dosage are known from the initially mentioned publications. The following Examples explain the invention. Example 1 Obtaining the sennoside mixture used as starting material. In each case, one places 40 kg of senna drug into two percolttors, connected in series, with a volume of 250 1 and covered with a perforated steel plate. As solvent for the extraction, one uses 70% methanol which is passed to the drug in the first percolator. One passes the solution formed in the first percolator 'N 7 950704,p:\oper\dab,21654-92. 185,26 to the drug which is present in the second percolator. One thereby allows 'the solvent to flow freely through the first percolator. For the extraction of 40 kg of senna drug, one uses, in all, 160 1 of solvent, After one has passed this volume of 70% methanol through the two percol- ators and has collected the corresponding amount of percolate, one couples the emptying pipe of the percolator with a post-percolate container and then additionally passes 60 1 of 70% methanol though the percolators. Thereafter, one passes the remaining free solvent from the first percolator into the upper part of the second percolator and collects the post- percolate until it makes up a total of 120 1. One then empties the first percolator, again fills it with of senna drug and pumps the post-percolate on to the drug, whereby 120 1 of post-percolate suffice in order to cover the drug in the percolator, Subsequently, one brings the temperature of the solution to +300C. Thereafter, one leaves to stand overnight,. One connects this percolator with the one previously extracted and carries out the extraction as described above. In each case, for 40 kg of drug one collects 160 1 of percolate from which one removes the methanol in a vacuum rotary evaporator which is equipped with a packed column. One -obtains about v^ r m i, j- i I the catalyst is an iron-(III) salt. 950704,p:\opcr\dab,21654-92.185,27 K-- 1 of bottom product. This concentrate is extracted with tho equal volume of 2-butanol which is saturated with water. The phases are separated and the aqueous phase is further worked up.. Step A Reduction of the sennosides to rhein-9-anthrone-8- glucosides 1 off the extracted concentrate are brought to pH value7.5 with 48% caustic soda solution. One heats to 600C and, while stirring, adds 90 g of sodium dithionite in solid form to the solution over the course of half an hour. After completed addition, one stirs for a further hour. Subsequently, while stirring, one adds concentrated sulphuric acid thereto up to a pH value of 2. In the course of two hours, one cools to ambient temperature, filters off the separated crystalline precipitate and washes it with sulphur dioxide-containing water. If desired, crude rhein-9-anthrone-8-glucoside is reprecipitated. The still moist filter cake is so dissolved in a mixture of 15 vol. parts of 2- butanol and 85 vol. parts of water which contains 0 .5 wt.b of sodium pyrosulphite that, by addition of a 48t aqueous solution of sodium hydroxide to a pH value of 7, one obtains a 10% solution The solution is acidified with concentrated hydro- I A -7 chloric acid to pH 2.8 or below and left to stand L c- I i: for 2 h. The precipitate obtained is filtered off and washed with sulphur dioxide- or sodium pyro- sulphite-containing water and dried. Yield One carries out a renewed reduction (post- reduction) with the product obtained in this manner as follows: One dissolves 3.0 g of the crude, dried rhein-9- anthrone-8-glucoside or the corresponding amount of the moist product in 15 ml of water, together with 1.4 g of sodium dithionite and 2.3 ml 5N NaOH. Subsequently, one makes up with water to 24 ml and heats the solution for 20 minutes at 550C. Thereafter, one adds a further 1.5 g of sodium dithionite to the. solution and heats to 5500 for 20 minutes. Sub- sequently, one adds thereto 0.9 ml of 5N NaOH and g of sodium dithionite. After heating for minutes at 550C, one again adds 0.9 ml. of 5N NaOH. The solution obtained is then introduced directly into the following liquid-liquid extraction. Step B: Separating of of fthe aloe-emodin components The separating off of the aloe-emodin components takes place by liquid-liquid partitioning of the 9_ anthrone-8-glucosides in countercurrent with the help of an apparatus of 60 mixer-separator units A (mixer-settler apparatus). As aqueous, heavier /1 phase, one uses a solution of 3.0 g sodium dithionite I- y to the lighter phaseamounts to 1:10 The mixture to be sepa ated is supplied to the apparatus in the form of the freshly reduced solution or in the form of a solution of approprniate. pH value and of appropriate concentration which contains the 9- i anthrone-8-glucosides obtained from step A, namely, in such a manner that 50 vol. parts of the organic phase are use per part by volume of the mixture to be separated. The pH.value of the solution containing the mixture is maintained at 9 9.5 With the help af a glycine buffer. The buffer of 5 vol. parts of a 7.5% glycine solution and 1 vol.. part of IN NaOH is introduced in an amcaunt of 240 ml of buffer solution per 150 g of crude rhein-9-anthrone-8-glucoside. The undesired aloe-emodin compounds enrich in the organic phase, whereas the. rhein-9-anthrone-8-glutoside remains in the aqueous phase. The aqueous phase is SCidified with sulphuric acid to pH value 2 the precipitate formed is filtered off and washed with water and acetone and dried in the air at ambient temperature. In this way, one obtains rhein-9-anthrone-8-glucoside with a content of aloe-emodin components of 49 ppm f (determined as aloe-emodin). Yield: referred to rhein-9-anthrone-8-glucoside. Step C: Oxidation of the rhein-9-anthrone-8-glucosides One dissolves 18.8 g of the rhein-9-anthrone-8- glucoside obtained in 56 ml of water and 11 ml of 2-butanol, whereby one adds thereto 17N NaOH up to a pH value of 6.5. W hile stirring, air is blown through this solution for 5 hours in a cylindrically- shaped vessel with the help of a glass frit. The rate of flow of the air amouhts to 40 ml/min. The course of the oxidation is monitored by means of HPLC. When no: noe rhein-9-anthrone-8-glucoside is detectable, the solutio# is diluted to about 200 ml with water/butanol 56/11. One adds thereto concent- rated hydrochloric acid up to pH value 1.5 to 2, stirs for 2 hours at ambient temperature, filters off the precipitated crystals and washes them with water and acetone and dries. One obtains 14.4 g of pure sennoside mixture with a content of 41 ppm aloe- 'i emodin components (determined as aloe-emodin) according to the analysis procedure given in step C, Example 2. Exampl.e 2 i 25 One repeats the process described in Example 1, whereby, however, one carries out the oxidation step 0 as follows: i. "4 la, C 1 .JI (SL.V1- LJ 4.tJ1 C V Wl 150 g of the pure rhein-9-anthrone-8-glucosides and 75 g iron-III chloride hexahydrate are dissolved in 480 ml of water and 120 ml of 2-butanol. A 48% sodium hydfoxide solution is aded thereto until pH 6.5 is reached and the rhein-9-anthrone-8-glucoside has dissolved. The solution is introduced into a vessel with a sinter bottom plate. Subsequently, a vigorous air current is passed through the solution. The oxidation is finished after about 30 minutes. I0 Subsequently, one dilutes the solution with a mixture of 120 ml 2-butanol and 480 ml of water, adds thereto g sodium dithionite and adjusts the pH value of the solution to 2.0 by addition of concentrated i hydrochloric acid.The solution is stirred for 18 hours. Subsequently, the separated precipitate is filtered off, washed with 600 ml of water and 800 ml of acetone and dried. The content of anthranoid compounds in the product lies between 94 and The product is taken up in 200 ml of 2-butanol and precipitated with 800 ml of water with the addition of 5.5 g sodium pyrosulphite. After filtering off and drying of the precipitate obtained, I one obtains 95.4 g of a product of anthranoid compounds of the following composition (according to HPLC, analysis of a typical experiment); i l 'I 1 *I 1 nemaining after complete removal of the methanoL rhein-8-glucoside sennoside B 49.7% sennoside Al 13. sennoside A 33.6% sennidine monoglucosides 1.1% rhein 0.02%

99.22% Sennosides C and D and aloe-emodin glucoside could not be detected by HPLC. The total content of aloe-emodin and of its derivatives was determined as being 30 ppm according to the following method: Sennosides C and D and aloe-emodin-8-glucoside Ican no longer be dependably determined as sennosides in the ppm range by means of HPLC chromatography. Therefore, it is necessary to convert the substance to be investigated by oxidation with iron-III chloride with simultaneous hydrolysis with hydro- chloric acid in a 2-phase mixture of aqueous solution/ carbon tetrachloride into rhein or aloe-emodin. The rhein is then converted into a salt so that it can be extracted into the aqueous phase and the aloe- emodin in the organic phase can be determined by means of HPLC. In this way,, there can be given the total content of sennosides C, D, aloe-emodin-8-glucosides and other aloe-emodin components, expressed as aloe- emodin. 1 Example 3 One repeats the extraction of the senna drug and the reduction of the sennosides described in Example 1. One then carries out the post-reduction as follows:. One dissolves 14.0 g of saccharose, 4.5 g of sodium dithionite and 13.3 g of potassium acetate in 133 ml of water and adds thereto 1.5 ml of 48% sodium hydroxide solution and 17.3 g of potassium carbonate. Subsequently, one mixes with 293 ml of 1O acetone- and 50 ml of water. One shakes the mixture in a separating funnel and separates the phases, whereby one obtains 375 ml of upper phase (acetone phase) and 130 ml of lower phase. One dissolves 1.4 ml of 48b sodium hydroxide solution and 10 g of crude rhein-9-anthrone-8- glucoside in 98 ml of the lower phase.-One warms to to 50°C and maintains at this temperature for to 30 minutes. Subsequently, one adds thereto 1.0 ml of 48% sodium hydroxide solution and 3.4 g sodium dithionite and heats for a further 20 to 30 minutes- at 45 to 50°C. Subsequently, one again adds 1.0 ml of 48%. sodium hydroxide solution and 3.4 g sodium dithionite thereto and heats for 20 to 30 minutes to 45 to 500C. The separation of the aloe-emodin components takes place by liquid-liquid partitioning of the reduced solution in countercurrent against the above- /J mentioned upper phase (acetone phase). The raffinate phase flowing off containing rhein-9-anthrone-8- glucoside is concentrated to 400 ml and mixed with ml butanol-2 or acetone. One adds thereto hydro- chloric acid or sulphuric acid up to pH value 4.0 to 4.2. The precipitate formed is filtered off, washed with 40 ml of water and 30 ml. of acetone and sub- sequently dried. The subsequent oxidation takes place as described in Example 2. Example 4 The concentrate obtained after the extraction of the senna drug is mixed with about 2 1 of butanol-2, The reduction of the senna fruit concentrate/butanol- 2 mixture is then carried out in 7 steps under nitrogen as protective gas. After the reduction step I, there follows a precipitation of the crude rhein- 9-anthrone-8-glucoside. Reduction step I 100 1 of senna fruit concentrate/butanol-2 mixture containing about 4 kg of sennosides, are i 20 placed in a stirrer container and covered with nitrogen. While stirring, 6 1 of 20% caustic soda solution, thereafter 350 1 of water-saturated butanol-2 from step are added success- ively thereto and stirred for 15 min. The batch is heated to 42 to 50°C and mixed with 7 kg sodium dithionite. It is further stirred for 45 min. The pH value is maintained at 7.5 8 With 20% (wt.) S caustic soda solution. The reduction potential A /61/0i I 4 1 (against Ag/AgCl electrode) is, if necessary, maintained blo6w -630 mV by sodium dithionite addition. After cooling to 30 35 C, it is pre- cipitated within 1.5 hrs, with 10% sulphuric acid to pH The resultant suspension is stirred for about 10 hrs. at <25 0 C with slow stirring speed. The resultant precipitate is filtered off. The precip- itate is suspended in 60 1 c.f 15% butanol-2, stirred for 30 min. at 50 to 60 0 C and subsequently filtered. The residue is washed with 100 1 of demineralised water. The crude yield of rhein-9- anthrone-8-glucoside lies above 82%, referred to sennosides used. Reduction step II 3.3 kg crude rhein-9-anthrone-8-glucoside from step I are suspended in a mixture of 42 1 of demin- eralised water and 7.4 1 butanol-2. The suspension is brought into solution with 2 1 of 20% caustic soda solution and 9..9 kg trisodium citrate and there- after mixed with 3.3 kg sodium dithionite and 350 1 of water-saturated butanol-2 from step III). The batch is heated to 42 45 0 C. The pH value is maintained at 8.5 to 9 with 20% caustic soda solution. The reduction potential (against an Ag/AgC1 electiode) is, if necessary, maintained below 750 mV by sodium dithionite addition. After a standing time of 30 min, the upper phase S is removed and the lower phase further worked up Sin step III. c I. i- It is precipitated by addition of an acid up to a 1 Reduction step III The reduction/extraction process described in step II is repeated with the lower phase from step II, with the addition of the following chemicals: 1.65 kg sodium dithionite 0.18 1 of 20% caustic soda solution 350 1 of water-saturated butanol-2 from step IV). Reduction steps IV VII The reduction/extraction process described in step II is repeated with, in each case, the lower phase from the pneceding step with the addition of the following chemicals: 0..825 kg sodium dithionite 0.4 1 of 20% cadstic soda solution 350 1 of water-saturated butanol-2 in each case from the following steps counter- current principle). The lower phase separated off in step VII is cooled to 30 3500 and the rhein-9-anthrone-8- glucoside precipitated out as described in step I The resultant precipitate is filtered off and washed with 100 1. of demineralised water. Subsequently,, it is covered with 10 1 of iron-III sulphate solution (28 kg Fe 2 (S0 4 3 in 100 1. of demineralised H 2 0). 4" i. F 1~ r _4111' The rhein-9-anthrone-8-glucoside is then converted into the sennosides as described in Example 1 or 2. Example The oxidation of the rhein-9-anthrone-8-glucoside can also take place according to the following process: kg of filter-moist rhein-9-anthrone-8- glucoside are mixed with 12.6 kg trisodium citrate, This mixture is dissolved in 7.0 1 of 1N NaOH with vigorous stirring and mixed with 0.7 1 of butanol-2, Subsequently, it is mixed with 8.8 1 of iron-III sulphate solution (28 kg Fe 2 (SO 4 3 in 100 1 of demineralised H20) and so much 20% NaOH added that the pB value amounts to about 8.35 One leaves to react for 3 4 hours at about 40 C, then acidifies with 52% H2SO4 to pH valuel.8 2.0 and works up as described in Example 1. Example 6 Alternatively, one brings rhein-9-anthrone-8- glucoside into solution in 50 ml of water by addition of a calcium hydroxide-saccharose solution (prepared by suspending of 7.0 g calcium hydroxide in a solution of 50.0 g saccharose in 100 ml of H20 and removal of the undissolved calcium hydroxide). One adds 20 ml butanol-2 thereto and passes a vigorous carrent of air through the solution over the course of 90 minutes. One adds 5.0 g CaC1 2 .2H 2 0 thereto and adjuststhe pH value to 8.5 with the calcium hydroxide-saccharose solution. The precipitate formed is filtered off and Ci y[- -l -i I i S-52- the filtrate is diluted with H20 to 340 ml,, mixed with 60 ml butanol-2 and adjusted to pH value 2.0 with concentrated hydrochloric acid. The further working up takes place as described in Example 1. Pharmacological investigations Laxative action The laxative effect of the sennoside mixture according to the invention was determined on mice. Male NMRI i-mice were used which were kept during the experiment in Plexiglas cages and received stand- ard feed mix with tap water of mushy consist- ency. A separate supply of drinking water didnot take place during the experiment. The animals received 100, 200 and 400 mg/kg of the sennoside mixture in 10 ml of 0.5% NaHCO 3 /kg 3 by stomach probe. After administration of the compounds to be tested, faeces and urine of the animals were collected for 24 hours and then determined. The I results obtained, referred to kg of body weight,, are summarised in the following Table, It can be seen that the sennosides display a good 4 laxative action which commences relatively quickly, The time up to the appeaance of the t first soft faeces (2 hours) is, however, also to be combined with a previous transit to the large intestine and a breakdown of the sennosides by the flora of the large intestine. A dosage-action relationship is present.. h .r i i i i ~I Acute toxicity In each case, 10 male and female Wistar rats were given sennosides once in dosages of 200 to 25,000 mg/ kg with a stomach probe. Macroscopic organ damage caused by the administered substances could not be observed. The determined LD 50 values are: male rats: female rats:. 5200 840 5200 20 mg/kg 530 3 0 mg/kg O0 In the case of male and female mice (n 8, strain NMRI), the maximum administerable dose of 5000 mg/kg did not result in any deaths. In all mice, diarrhoea occurred,. although to a lesser extent than in the case of rats. For both sexes, the LD 50 values amounted to 5000 mg/kg. Table effect of the sennoside mixture accordirn to Laxative I 4" invention on mice dosage number number of number of soft faeces (mg/kg) of normal faecal soft as of the animals pellets faecal total faecal pellets excretion 0 30 1265 0 0 100 40 587 14L 28,0 200 30 223 239 56.0 400 30 256 282 60 .0 I I INTERNATIONAL SEARCH REPORT International application No. PCT/EP 92/01428 A. CLASSIFICATION OF SUBJECT MATTER Int.C1. 5 C 07 H 15/244, A 61 K 35/78 According to International Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) Int.Cl. 5 C 07 H; A 61 K Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched Electronic data base consulted during the international search (name of data base and, where prac cable, search terms used) C. DOCUMENTS CONSIDERED TO BE RELEVANT Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. X Fortschritte der Chemie organischer Naturstoffe, 1-13 Vol. 599, 1950 A. Stoll et al.: "Sennosides A and B, the Active Principles of Senna", see page 248 page 269 A FR, Al, 2594337 (OPPAG 21 August 1987, 1-13 see the whole document A DE, Al, 3200131 (DR. MADAUS CO.) 1-13 14 July 1983, see the whole document A Chemical Abstracts, Vol. 108, No. 14, 1-12 4 April 1988, (Columbus, Ohio, US) Iwata Masana et al: "Purification of sennosides as laxatives", abstract 118961r, Jpn. Kokai Tokkyo Koho JP 62,178,598, August 1987 \Ci S Further documents are listed in the continuation of Box C. O See patent family annex. S Special categories of cited documents: laterdocumentpublishedaftertheinternational filingdateorpriority document defning the general state of the art which is not considered ate and oin co t it appiio ut cit to understand to he of particular relevance the principle or theory underlying the invention earlier document but published on or after the international filing date document of particular relevance; the claimed invention cannot be document which ay throw doubts on priority claim(s) or which considered novel or cannot be considered to involve an inventive document which may throw doubts on priority claim(s) or which is the document is taken alone cited to establish the publication date of another citation or other twen t cument i ta special reason (as specified) documentof particular relevance; the claimed invention cannot be document referring to an oral disclosure, use, exhibition or other considered to involve an inventive step when the document is means combined with one or more othersuch documents,such combination document published prior to the international filing date but later than being obvious to a person skilled in art the priority date claimed document member of the came patent family Date of the actual completion of the international search Date of mailing of the international search report 9 November 1992 (09.11.92) 26 November 1992 (26.11.92) Name and mailing address of the ISA/ Authorized officer EUROPEAN PATENT OFFICE Facsimile No. Telephone No. Form PCT/ISA/210 (second sheet) (July 1992) ,i INTERNATIONAL SEARCH REPORT International application No. PCT/EP 92/01428 C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. A GB, A, 2018588 (SYNTHELABO) 24 October 1979, 1-12 see the whole document A GB, A, 1135528 NATTERMANN CIE, GMBH) 1-12 4 December 1968 see the whole document A GB, A, 555450 (SANDOZ LIMITED) 24 August 1943, 1-12 see the whole document Form PCT/ISA/210 (continuation of second sheet) (July 1992)