Method of preparing diacetyl rhein

Diacetylrhein which is substantially free of aloe-emodin components, is prepared by a process wherein PA0 a) rhein-9-anthrone-8-glusoside containing aloe-emodin components is subjected to a liquid-liquid partitioning of the compounds obtained is carried out between a polar organic solvent which is only partly miscible with water and an aqueous phase, PA0 b) the rhein-9-anthrone-8-glucoside contained after the partitioning in the aqueous phase is oxidized to rhein-8-glucoside, PA0 c) the glucose residue in the 8-position of the rhein-8-glucoside is split off in an acidic medium and, PA0 d) the rhein obtained is acetylated and the diacetylrhein recovered.

The present invention is concerned with a process for the preparation of 
diacetylrhein of pharmaceutically usable purity with a residual content of 
undesired aloe-emodin derivatives of, in all, less than 20 ppm, the 
diacetylrhein obtainable according to this process and a pharmaceutical 
composition which contains this compound. 
Diacetylrhein of the formula: 
##STR1## 
is a medicinally-active compound which possesses anti-arthritic, 
anti-inflammatory, antipyretic and analgesic activity. Therefore, 
diacetylrhein is used for the treatment of arthritic diseases (cf., for 
example DE-A-27 11 493 and U.S. Pat. No. 4,244,968). 
Diacetylrhein can be prepared, for example, by the acetylation of barbaloin 
and oxidation of the peracetylated barbaloin obtained with chromium 
trioxide. Furthermore, diacetylrhein can be prepared by the acetylation of 
rhein which can be obtained, for example, from senna drug. 
Diacetylrhein obtained according to these processes contains undesired 
accompanying aloe-emodin derivatives which result from an incomplete 
oxidation with chromium trioxide or are co-extracted in the case of the 
extraction of senna drug. These accompanying materials are present in 
relatively small amounts and can, therefore, only be separated with great 
difficulty by means of well-known purification procedures. Furthermore, in 
the case of the first of the above-mentioned processes, chromium residues 
are present which have to be removed in appropriate manner. 
Therefore, it is an object of the present invention to provide a process 
for the preparation of diacetylrhein which is simple to carry out and 
gives high yields and in which diacetylrhein is obtained of 
pharmaceutically usable purity with a residual content of undesired 
aloe-emodin derivatives of, in all, less than 20 ppm. 
Thus, according to the present invention, there is provided a process for 
the preparation of diacetylrhein, wherein 
a) rhein-9-anthrone-8-glucoside containing aloe-emodin components (i. e. 
aloe-emodin and/or derivatives thereof) is subjected to a liquid-liquid 
partitioning between a polar organic solvent which is only partly miscible 
with water and an aqueous phase, 
b) the rhein-9-anthrone-8-glucoside contained after the partitioning in the 
aqueous phase is oxidised to rhein-8-glucoside, 
c) the glucose residue in the 8-position of the rhein-8-glucoside is split 
off in an acidic medium and 
d) the rhein obtained is acetylated and diacetylrhein recovered. 
Important sources of rhein-9-anthrone-8-glucoside are the sennosides 
contained in senna drug. A preferred embodiment of the present invention 
is, therefore, a process for the preparation of diacetylrhein which is 
substantially free of aloe-emodin components wherein 
a) a sennoside mixture is subjected to a reduction to the corresponding 
rhein-9-anthrone-8-glucoside and aloe-emodin-9-anthrone-8-glucoside 
compounds, 
b) a liquid-liquid partitioning of compounds obtained is carried out 
between a polar organic solvent which is only partly miscible with water 
and an aqueous phase, 
c) the rhein-9-anthrone-8-glucoside compounds contained in the aqueous 
phase after partitioning are oxidised to the corresponding anthraquinone 
compound, 
d) the glucose residue in the 8-position of the anthraquinone compound is 
split off in an acid medium and 
e) the 1,8-dihydroxyanthraquinone compound obtained is acetylated and the 
diacetylrhein recovered. 
Reduction of the Sennosides 
The sennoside mixture used as starting material can be obtained, for 
example, from senna drug. The senna drug consists of the dried leaves and 
fruits of the senna plant, for example of the Indian senna (Cassia 
angustifolia) and the of Egyptian senna (Cassia acutifolia). The senna 
drug contains dianthrone glucosides of rhein and aloe-emodin. The most 
important ones are sennosides A, B, Al, C, D and Dl. The sennosides 
correspond to the general formula: 
##STR2## 
In the case of sennosides A, B and Al, R stands for COOH and in the case 
of sennosides C, D and Dl, R stands for CH.sub.2 OH. The sennosides A, B 
and Al and the sennosides C, D and Dl are stereoisomers and differ from 
one another by the configuration on carbon atoms 10 and 10'. 
The obtaining of sennosides from senna drug is described, for example, in 
DE-A-32 00 131, reference to which is here made to the complete 
specification. According to this, the senna drug is first extracted with 
aqueous methanol. The concentrate remaining after complete removal of the 
methanol contains the sennosides in the form of the potassium salts. This 
concentrate can be used as starting material for the process according to 
the present invention. 
The concentrate can also be purified by liquid extraction with alcohols or 
ketones which are partly soluble in water, for example butan-2-ol or 
butan-2-one (raffinate). The raffinate is acidified to a pH of about 1.5 
to 2.0 and the sennosides are brought to to crystallisation by seeding. 
The crude sennoside mixture obtained can also be used as starting material 
for the process according to the present invention. If desired, the crude 
sennoside mixture can also be recrystallised. 
Alternatively, the concentrated mixed with an alcohol or ketone which is 
partly soluble in water, especially butan-2-ol, can be used as starting 
material. 
In the case of the extraction of the senna drug, the ratio of drug to 
extraction solvent is preferably 1:4 to 1:15 and 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 process of the present invention, these starting materials 
are subjected to a complete reduction to give the corresponding 
rhein-9-anthrone-8-glucoside (R=COOH) and the corresponding 
aloe-emodin-9-anthrone-8-glucoside (R=CH.sub.2 OH) of the general formula: 
##STR3## 
wherein R is COOH or CH.sub.2 OH. 
Reducing agents with an appropriate reducing potential include, for 
example, stannous chloride, sulphur dioxide, alkali metal borohydrides and 
preferably alkali metal dithionites, especially sodium dithionite. 
For carrying out the reduction, the starting material can be present in 
aqueous solution or suspension and the reducing agent is added thereto in 
solid form or dissolved in water. It is also possible to work is a 
two-phase mixture by adding thereto a polar organic solvent which is 
partly miscible with water, especially butan-2-ol. 
The reduction can be carried out at ambient or higher temperature. The 
reduction is preferably carried out at 40.degree. to 60.degree. C. and 
especially at 50.degree. to 55.degree. C. Working is carried out at a 
weakly acidic to weakly alkaline pH value of the starting sennoside 
solution or suspension, preferably at pH 7 to 9. If desired, the reduction 
can be carried out several times and especially 2 to 10 times. 
The 9-anthrone-8-glucosides formed are precipitated out by the addition of 
an acid, for example of sulphuric acid, to a pH value of 4 to 4.5. The 
temperature should thereby preferably be not more than 40.degree. C. In 
the case of the precipitating out of the 9-anthrone-8-glucosides and in 
the case of the isolation thereof, for example by filtration, it is 
preferable to work under an atmosphere of nitrogen in order to avoid an 
uncontrolled oxidation of these compounds. 
It is important that the reduction proceeds to completion. Therefore, it is 
preferable to use the reducing agent in large excess. In the case of using 
sodium dithionite, in general there is employed a 1 to 4 fold amount by 
weight of sodium dithionite, referred to the content of sennosides in the 
starting material. Furthermore, the reducing agent is allowed to act for 
at least 2 hours and preferably for at least 3 hours. In general, the 
reduction takes place for not more than 10 hours. A post-reduction is 
preferably carried out under the said conditions. 
Before use in the next step, the product obtained is preferably 
reprecipitated by bringing it into solution by the addition of a base, for 
example sodium hydroxide or potassium hydroxide, up to a pH of about 6 to 
7, the aqueous solution is extracted with butan-2-ol, acetone or 
butan-2-one and the product is again precipitated out by the addition of 
an acid to a pH of about 2 to 4. 
Liquid-Liquid Partitioning 
In this step, the aloe-emodin components and especially 
aloe-emodin-9-anthrone-8-glucoside are removed. For this purpose, there is 
carried out a liquid-liquid partitioning of the product obtained between a 
polar organic solvent which is only partly miscible with water and an 
aqueous phase. Appropriate polar organic solvents include C.sub.4 -C.sub.5 
-alkanols and di-C.sub.1 -C.sub.3 -alkyl ketones, for example acetone, 
butan-1-ol, butan-2-ol and butan-2-one, the use of butan-2-ol or acetone 
being preferred. 
To the aqueous phase is preferably added a reducing agent in order to 
impart a redox potential of -210 mV or more negative to the aqueous phase 
during the whole of the liquid-liquid partitioning. It is preferable to 
use 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% by weight 
solution at a pH value of from 7 to 11 is sufficient in order to maintain 
the mentioned potential conditions. 
The volume ratio of aqueous phase (heavy phase) to organic phase (light 
phase) is generally in the range of from 1:5 to 1:40. 
The liquid-liquid extraction preferably takes place in countercurrent. The 
mixture of anthrone compound is thereby isolated 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% by weight solution. 
After the partitioning, the desired rhein-9-anthrone-8-glucoside is present 
in the aqueous phase. It is precipitated out by the addition of an acid to 
give a pH value of about 2 to 4 and recovered in the usual manner. 
Oxidation of the Rhein-9-Anthrone-Glucoside 
The rhein-9-anthrone-8-glucoside is now oxidised to rhein-8-glucoside of 
the general formula: 
##STR4## 
Oxidation agents which can be used for this purpose include, for example, 
oxygen, peroxide compounds, such as hydrogen peroxide, and manganese, 
chromium and iron compounds in high oxidation states. It is preferred to 
use a ferric salt and especially ferric sulphate. It is preferable to work 
at an elevated temperature but at one below 60.degree. C. In this way, the 
formation of undesired and undefined oxidation products is avoided. When 
the oxidation is complete, the rhein-8-glucoside formed is isolated in the 
usual manner. 
Splitting off of the Glucose Residue 
The glucose residue in the 8-position is split off in acidic solution. It 
is preferable to work at a temperature of about 85.degree. to 95.degree. 
C. The product obtained is isolated in the usual manner. 
It is known to convert sennosides, after acidic hydrolysis, by reaction 
with ferric chloride directly into rhein (see, for example DE-A-27 11 
493). However, the yield is thereby only about 10% and, in addition, the 
rhein formed is difficult to separate. 
In the case of the process according to the present invention, the 
reductive cleavage of the sennosides, the oxidation of the anthrone 
compounds formed to the corresponding anthraquinone compounds and the 
splitting off of the glucose residue in the 8-position of the 
anthraquinone compounds are, in each case, carried out in separate steps. 
Subsequent to the reductive cleavage, all compounds which, in the further 
course of the process, could lead to the formation of aloe-emodin or 
derivatives thereof are quantitatively removed by liquid-liquid 
partitioning. Furthermore, it is possible to carry out the oxidation at 
modest temperatures so that the formation of undesired and undefinable 
oxidation products is avoided. Furthermore, when carrying out the 
reaction, the iron salt used can be recovered almost quantitatively and, 
after reoxidising, can be used again. The separation of oxidation step and 
hydrolysis step permits, on the basis of the greater water solubility of 
the anthrone glucosides in comparison with the aglycones in question, the 
gentle carrying out of the oxidation at ambient temperature or below 
60.degree. C., the otherwise unavoidable formation of undefined 
by-products thereby being avoided. 
Acetylation of the 1,8-Dihydroxyanthraquinone Compound 
The acetylation of the 1,8-dihydroxyanthraquinone compounds obtained takes 
place in the usual manner. For example, acetylation can be carried out 
with acetic anhydride in the presence of sodium acetate in the manner 
described in Arch. Pharm., 241, 607/1903. However, the acetylation can 
also take place by means of other methods known to the expert, for example 
by reaction with acetyl chloride or the like. 
The diacetylrhein obtained in this manner is substantially free from 
aloe-emodin and derivatives thereof. The content of these impurities 
thereby still amounts to about 50 ppm (determined by the analysis process 
described in the following Examples). The content of these impurities can 
be further reduced when the diacetylrhein obtained is recrystallised in 
the following manner: The diacetylrhein is converted into an alkali metal 
salt by treatment with an appropriate base, an appropriate base being, for 
example, an alkali metal acetate and preferably potassium acetate. It is 
preferable to use equimolar amounts of base and an aqueous C.sub.1 
-C.sub.3 -alcohol, for example 80 to 90% ethanol, as reaction medium. The 
alkali metal salt of diacetylrhein is allowed to crystallise out in the 
cold, taken up in an aqueous C.sub.1 -C.sub.3 -alcohol and precipitated 
out by the addition of an acid to a pH value of about 3. The diacetylrhein 
precipitated out is then isolated in the usual manner and worked up. 
The product thus obtained contains less than 20 ppm of the above-mentioned 
impurities. Furthermore, the product is present in the form of 
needle-shaped crystals which are especially appropriate for galenical 
formulation. 
The product can be dried in the usual manner. It is preferable first to 
carry out the drying in a vacuum at a relatively low temperature, for 
example of not more than 40.degree. C., until the water content of the 
product has decreased to about 3% or less. Subsequently, the temperature 
can be increased to 70.degree. to 110.degree. C. 
The present invention is also concerned with the substantially pure 
diacetylrhein obtainable according to the present invention, as well as 
with pharmaceutical compositions which contain this compound. The fields 
of use, the dosage to be administered and appropriate forms of dosaging 
are known from and described, for example, in U.S. Pat. Nos. 4,244,968, 
4,346,103, and 4,950,687 and DE-A-27 11 493, as well as in Drugs Exptl. 
Clin. Res., 6 (1), 53-64/1980.

The following Examples are given for the purpose of illustrating the 
present invention. 
EXAMPLE 1 
Obtaining the Sennoside Mixture Used as Starting Material 
In each case, 40 kg of senna drug (sennoside content about 1.5%), are 
introduced into two percolators, connected in series, with a volume of 250 
liters and covered with a perforated steel plate. As solvent for the 
extraction, there is used 70% methanol which is passed to the drug in the 
first percolator. The solution formed in the first percolator is passed to 
the drug which is present in the second percolator. The solvent is thereby 
allowed to flow freely through the first percolator. 
For the extraction of 40 kg of senna drug, there is used, in all, 160 
liters of solvent. After this volume of 70% methanol has been passed 
through the two percolators and the corresponding amount of percolate has 
been collected, the emptying pipe of the percolator is coupled with a 
post-percolate container and an additional 60 liters of 70% methanol are 
passed through the percolators. Thereafter, the remaining free solvent 
from the first percolator is passed into the upper part of the second 
percolator and the post-percolate is collected until it amounts to 120 
liters. The first percolator is then emptied, again filled with 40 kg of 
senna drug and the post-percolate is pumped on to the drug, the 120 liters 
of post-percolate thereby sufficing in order to cover the drug in the 
percolator. 
Subsequently, the temperature of the solution is brought to +30.degree. C. 
This percolator is connected with the one previously extracted and the 
extraction is carried out as described above. 
For each 40 kg of drug, there are collected 160 liters of percolate from 
which the methanol is removed in a vacuum rotary evaporator which is 
equipped with a packed column. There are obtained 30 liters of bottom 
product. This concentrate is extracted with an equal volume of butan-2-ol 
saturated with water. 
Step a) 
Reduction of the Sennosides to Rhein-9-Anthrone-8-Glucosides 
1.0 liter of the extracted concentrate is brought to pH 7.5 with 48% 
aqueous sodium hydroxide solution. The solution is heated to 60.degree. C. 
and, while stirring, 90 g of sodium dithionite in solid form are added to 
the solution over the course of half an hour. When the addition is 
completed, stirring is continued for a further hour. Subsequently, 
concentrated sulphuric acid is added thereto up to a pH of 2. The solution 
is cooled to ambient temperature in the course of 2 hours and the 
crystalline precipitate obtained is filtered off and washed with water 
containing sulphur dioxide. 
If desired, the crude rhein-9-anthrone-8-glucoside is reprecipitated. The 
still moist filter cake is dissolved in a mixture of 15 parts by volume of 
butan-2-ol, 85 parts by volume of water which contains 0.5% by weight of 
sodium pyrosulphite in such a manner that, by means of the addition of a 
48% aqueous solution of sodium hydroxide to pH 7, a 10% solution (w/v) is 
obtained. The solution is acidified with concentrated hydrochloric acid to 
a pH of 2.8 or below and left to stand for 2 hours. The precipitate 
obtained is filtered off, washed with water containing sulphur dioxide or 
sodium pyrosulphite and dried. Yield 90%. 
A renewed reduction (post-reduction) is carried out as follows with the 
product obtained in this manner: 
3.0 g of the crude dried rhein-9-anthrone-8-glucoside or the corresponding 
amount of the moist product are dissolved in 15 ml of water, together with 
1.4 g sodium dithionite and 2.3 ml 5 N aqueous sodium hydroxide solution. 
Subsequently, it is made up with water to 24 ml and the solution is heated 
to 55.degree. C. for 20 minutes. Thereafter, a further 1.5 g of sodium 
dithionite are added to the solution and heated for 20 minutes at 
55.degree. C. Subsequently, 0.9 ml of 5 N aqueous sodium hydroxide 
solution and 1.5 g of sodium dithionite are added thereto. After heating 
for 20 minutes to 55.degree. C., 0.9 ml of 5 N aqueous sodium hydroxide 
solution are again added thereto. The solution obtained is introduced 
directly into the following liquid-liquid extraction. 
Step b) 
Separation of the Aloe-Emodin Components 
The separation of the aloe-emodin components takes place by liquid-liquid 
partitioning of the anthrone-8-glucosides in countercurrent with the help 
of an apparatus containing 60 mixing-settler units. As aqueous heavier 
phase, there is used a solution of 3.0 g of sodium dithionite in 3.5 ml of 
5 N aqueous sodium hydroxide solution and 96 ml of water. As organic 
lighter phase, there is used butan-2-ol or acetone saturated with water. 
The two phases are supplied to the apparatus in such a manner that the 
volume ratio of heavier phase to lighter phase is 1:10. 
The mixture to be separated is supplied to the apparatus in the form of the 
freshly produced solution or in the form of a solution of corresponding pH 
value and of corresponding concentration which contains the 
9-anthrone-8-glucosides obtained from step a) in such a manner that 30 
parts by volume of the organic phase are used per part by volume of the 
mixture to be separated. 
The pH of the solution containing the mixture is maintained at 9 to 9.5 
with the help of a glycine buffer. The buffer comprising 3 parts by volume 
of a 7.5% glycine solution and 1 part by volume of 1 N aqueous sodium 
hydroxide solution is added in an amount of 240 ml of buffer solution per 
150 g of crude rhein-9-anthrone-9-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 acidified with sulphuric acid to pH 2.8 and the precipitate 
formed is filtered off and washed with water and acetone and dried in air 
at ambient temperature. In this way, rhein-9-anthrone-8-glucoside is 
obtained with a content of aloe-emodin components of 41 ppm, determined as 
aloe-emodin according to the method which is described at the end of this 
description. Yield 97%, referred to the rhein-9-anthrone-8-glucoside. 
Step c) 
Oxidation to Rhein-8-Glucoside 
The product from step b) (referred to a content of 3.0 kg of sennosides A, 
Al and B) is suspended in a solution of 185 liters of demineralised water 
and 75.5 kg of ferric sulphate hydrate (22% Fe.sup.3+). The suspension is 
heated to 55.degree. to 62.degree. C. and oxidised for 14 hours with the 
use of a rapidly running disperser. When the oxidation is complete, the 
rhein-8-glucoside formed is filtered off and washed with 50 liters of 
demineralised water which has been adjusted to pH 2 with sulphuric acid. 
Step d) 
Hydrolysis to Rhein 
The moist filter residue from step c) is suspended in 200 kg of 20% by 
weight sulphuric acid and stirred for 8 hours at 88.degree. to 92.degree. 
C. The rhein formed is filtered off and, for storage, can be dried at 1 
mbar vacuum for 48 hours at 40.degree. C. or can be used immediately in a 
moist state for the acetylation in step e). 
The total yield for steps a) to d) is 79%, referred to the sennosides A, Al 
and B used in step a). 
Step e) 
Acetylation to Give Diacetylrhein 
6.5 kg of rhein from step d) are suspended in 100 liters of acetic 
anhydride for 10 minutes, mixed with 2 kg of potassium acetate, heated to 
95.degree. C. while stirring, mixed with 0.65 kg of activated carbon and 
stirred for 30 minutes at 90.degree. to 95.degree. C. The activated carbon 
is filtered from the hot solution and the filtrate is mixed at 90.degree. 
C. with 2.1 kg of 96 to 98% by weight sulphuric acid. Subsequently, while 
stirring, it is cooled as quickly as possible to 20.degree. C. and the 
resulting suspension is filtered. The residue is washed free of sulphate 
with demineralised water. The yield is 83%. 
Step f) 
Recrystallisation, Drying and Grinding 
With rapid stirring, 7.5 kg of diacetylrhein from step e) (referred to the 
dry substance) are suspended in 375 liters of 90% by volume ethanol. The 
suspension is heated to 70.degree. C. and then mixed with 3.75 kg of 
potassium acetate. Upon cooling to 0.degree. to 2.degree. C., the pure 
potassium salt of diacetylrhein crystallises out from the clear solution 
which has, in the meantime, formed. The potassium salt is filtered off and 
dissolved in 300 liters of 40% by volume ethanol at 20.degree. to 
30.degree. C. with the addition of 3 kg potassium acetate. The clear 
solution is adjusted with 10% by weight sulphuric acid to pH 3.0. The 
diacetylrhein which crystallises out is filtered off and washed free of 
sulphate with demineralised water. 
The product is first dried in a vacuum at 1 mbar and 40.degree. C. within 
the course of 24 hours. When the residual water content has decreased to 
below 3%, the material is coarsely comminuted and further dried at 1 mbar 
vacuum and 70.degree. C. for 24 hours. Subsequently, it is ground to a 
sieving size of 0.5 mm and again dried at 1 mbar vacuum and 70.degree. C. 
for the removal of solvent residues. The yield from step f) is 95%. 
EXAMPLE 2 
The extraction of the senna drug and the reduction of the sennosides 
described in Example 1 is repeated. The subsequent reduction is then 
carried out as follows: 
140 g saccharose, 4.5 g 85% sodium dithionite and 13.3 g potassium acetate 
are dissolved in 133 ml of water and 1.3 ml of 48% sodium hydroxide 
solution and 17.3 g potassium carbonate are added thereto. Subsequently, 
the reaction mixture is mixed with 293 ml acetone and 50 ml of water. The 
mixture is shaken in a separating funnel and the phases are separated, 375 
ml of upper phase (acetone phase) and 130 ml of lower phase thereby being 
obtained. 
In 98 ml of the lower phase are dissolved 1.4 ml of a 48% sodium hydroxide 
solution and 10 g of crude rhein-9-anthrone-8-glucoside. The solution is 
warmed to 45.degree. to 50.degree. C. and maintained at this temperature 
for 20 C to 30 minutes. Subsequently, 1.0 ml of a 48% sodium hydroxide 
solution and 3.4 g of sodium dithionite are added thereto and heated for a 
further 20 to 30 minutes to 45.degree. to 50.degree. C. Subsequently, 
there are again added thereto 1.0 ml of 48% sodium hydroxide solution and 
3.4 g of sodium dithionite, followed by heating to 45.degree. to 
50.degree. C. for 20 to 30 minutes. 
The separation of the aloe-emodin components takes place by liquid-liquid 
partitioning of the reduced solution in countercurrent against the 
above-mentioned upper phase (acetone phase). The raffinate phase flowing 
off and containing the rhein-9-anthrone-8-glucoside is concentrated to 400 
ml and mixed with 20 ml butan-2-ol. 
Hydrochloric acid or sulphuric acid is added thereto up to a pH value of 
4.0 to 4.2. 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 in the manner described in Example 1. 
EXAMPLE 3 
The concentrate obtained after extraction of the senna drug is mixed with 
about 2 liters of butan-2-ol. The reduction of the mixture of senna fruit 
concentrate and butan-2-ol is then carried out in 7 steps under nitrogen 
as protective gas. After reduction step I, there follows a precipitation 
of the crude rhein-9-anthrone-8-glucoside. 
Reduction Step I 
100 liters of a mixture of senna fruit concentrate and butan-2-ol 
containing about 4 kg of sennosides are placed in a stirrer container and 
covered with nitrogen. While stirring, 6 liters of a 20% by weight aqueous 
solution of sodium hydroxide and thereafter 350 liters of water-saturated 
butan-2-ol, for example from step II, are successively added thereto and 
stirred for 15 minutes. The batch is heated to 42 to 50.degree. C., mixed 
with 7 kg sodium dithionite and further stirred for 45 minutes. The pH 
value is maintained at 7.5 to 8 with 20% by weight aqueous sodium 
hydroxide solution. The reduction potential (against an Ag/AgCl electrode) 
is, if necessary, maintained below -630 mV by the addition of sodium 
dithionite. After cooling to 30.degree. to 35.degree. C., precipitation is 
carried out within 1.5 hours with 10% by weight of sulphuric acid to pH&lt;4. 
The resultant suspension is stirred for about 10 hours at&lt;25.degree. C. 
with a slow speed of stirring and the resultant precipitate is filtered 
off. The precipitate is suspended in 60 liters of 15% by weight 
butan-2-ol, stirred for 30 minutes at 50.degree. to 60.degree. C. and 
subsequently filtered. The residue is washed with 100 liters of 
demineralised water. The crude yield of rhein-9-anthrone-8-glucoside is 
more than 82%, referred to the sennosides used. 
Reduction Step II 
3.3 kg crude rhein-9-anthrone-8-glucoside from step I are suspended in a 
mixture of 42 liters of demineralised water and 7.4 liters of butan-2-ol. 
The suspension is brought into solution with 2 liters of 20% by weight 
aqueous sodium hydroxide solution and 9.9 kg trisodium citrate and 
thereafter mixed with 3.3 kg sodium dithionite and 350 liters of 
water-saturated butan-2-ol, for example from step III. The batch is heated 
to 42.degree. to 45.degree. C., the pH value being maintained at 8.5 to 9 
with 20% by weight aqueous sodium hydroxide solution. The reduction 
potential (against Ag/AgCl electrode) is, if necessary, maintained below 
-750 mV by the addition of sodium dithionite. After standing for 30 
minutes, the upper phase is removed and the lower phase further worked up 
in step III. 
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 
0.8 liters 20% by weight sodium hydroxide solution and 
350 liters of water-saturated butan-2-ol, for example from step IV. 
Reduction Steps IV and VII 
The reduction/extraction process described in step II is repeated with the 
lower phase from the preceding step in question with the addition of the 
following chemicals: 
0.825 kg sodium dithionite 
0.4 liters 20% by weight aqueous sodium hydroxide solution and 
350 liters of water-saturated butan-2-ol, for example from the following 
steps in question using the countercurrent principle. 
The lower phase separated off in step VII is cooled to 30.degree. to 
35.degree. C. and the rhein-9-anthrone-8-glucoside is precipitated out as 
described in step I. The resultant precipitate is filtered off and washed 
with 100 liters of demineralised water. Subsequently, it is covered with 
10 liters of ferric sulphate solution (preparation see step b, Example 1). 
The rhein-9-anthrone-8-glucoside is then converted into the sennosides in 
the manner described in Example 1 or 2. 
Pharmacological investigations 
The effectiveness of diacetylrhein was determined in chronic inflammation 
models after oral administration. 
The following experimental models were used: cotton pellet granuloma in 
rats and arthrosis in rabbits induced by the intraarticular administration 
of vitamin A. 
A) Cotton Pellet Granuloma In Rats 
Young sexually mature rats (n=10) were given 25, 50 or 100 mg 
diacetylrhein/kg or 5 mg indomethacin/kg or 100 mg acetylsalicylic acid/kg 
daily for 5 days. A control group only treated with water was also used. 
Implantation of the pellets took place on the first day of treatment. 
Fresh and dry weights of the granuloma prepared at the end of the 
experiment showed a significant and clearly dosage-dependent reduction in 
comparison with the control group. The action of 100 mg diacetylrhein/kg 
thereby corresponded to about the action of 5 mg indomethacin or of 100 mg 
acetylsalicylic acid. The weights of the thymus and adrenals did not 
change during the treatment. 
B) Vitamin A Arthrosis 
An arthrosis-like joint change was initiated in two groups each of 10 
rabbits (white New Zealanders) by means of three intraarticular injections 
of 30,000 IU of vitamin A over the course of 9 days. 56 days later, 10 
animals were treated with 3 mg of diacetylrhein/kg/day for 8 weeks. In 
comparison with the control group, the macroscopically and microscopically 
recognisable joint changes in the treatment group were significantly 
reduced. 
Furthermore, the curative action of diacetylrhein was compared with that of 
acetylsalicylic acid on each of 7 rabbits which, after 6 days 
pre-treatment with three times 10,000 IU vitamin A and a 26 day 
treatment-free interval for 8 weeks, received either 5 mg of 
diacetylrhein/kg/day (experimental group), 15 mg of acetylsalicylic 
acid/kg/day (positive control group) or remained untreated (negative 
control group). In all three groups, 24 days after the last vitamin A 
injection, comparable disturbances of movement occurred in the form of 
dragging of the rear legs. In the negative control group, during the 
following 8 weeks, the clinical signs of a manifest arthrosis increased. 
In the experimental group and the positive control group, these symptoms 
improved significantly during the 8 weeks of treatment. 
Gastric Mucosa Changes 
Whereas a single administration of 400 mg of diacetylrhein/kg or of the 
solvent did not give rise to any erosions of the gastric mucosa in the 
case of the rat, after the administration of ibuprofen (200 mg/kg) or of 
indomethacin (20 mg/kg), there were found distinct mucosal damages in the 
form of punctiform (1 mm diameter) to large (3 mm diameter) erosions. Also 
the twice daily administration of 100 mg of diacetylrhein/kg over the 
course of 3 days also did not initiate any mucosal damage, whereas the 
corresponding use of 10 mg of indomethacin/kg certainly did: the erosions 
thereby having a diameter of 1 to 3 mm. 
Toxicology 
The acute toxicity LD.sub.50 was, depending upon the species investigated 
(rat, mouse, cat), after the oral administration 1.9 to 7.9 g/kg. The rat 
thereby proved to be the least sensitive. After parenteral administration 
(i.v. or i.p.), the LD.sub.50 values in the case of these species was from 
119 to 339 mg/kg. 
Clinical Investigations 
1. The action of diacetylrhein was investigated in coxarthrosis and 
gonarthrosis in 95 (49/46) patients in a double-blind study against 
naproxen and subsequent placebo after-treatment. The dosage administered 
was 50 mg of diacetylrhein twice daily or 750 mg of naproxen daily. The 
period of treatment was 60 days after a 7 day wash-out phase. The 
subsequent placebo treatment extended over 60 days. 
Test parameters were the pain and movement symptoms according to a score 
scale, function limitation and compatibility. 
In both treatment groups (diacetylrhein/naproxen), with regard to all test 
parameters there was ascertained a statistically significant rate of 
improvement (P&lt;0.01 and P&lt;0.05, respectively) in comparison with the 
initial values. After discontinuation of the treatment and subsequent 
administration of placebo, there was shown, however, on days 90 and 120 
with regard to the parameters of spontaneous pain and active and passive 
movement pain, a statistically significant superiority (P&lt;0.01) in 
comparison with the naproxen/placebo collective. On the 5% level, this 
difference was also verified for the variable night pain and pressure pain 
30 days after discontinuation of diacetylrhein. 
2. In an open running study with control, there was investigated the action 
of diacetylrhein against osteoarthrosis of the spine and of the knee in 70 
patients (35/35). The dosage administered was 100 mg of diacetylrhein per 
day. The period of treatment was 60 days and the period of observation was 
75 days. The test parameters were pain and movement limitation. The 
parameters were evaluated according to a score system. 
The control group comprised 35 patients in the case of which exclusively 
physiotherapeutic measures were carried out. Physiotherapy was also 
carried out in the diacetylrhein treatment group. 
With regard to all parameters, the evaluation of the results showed a 
statistically significant superiority of the treatment group with regard 
to the control group. Also after discontinuation of the treatment, a 
continuing therapeutic effect (hang-over effect) could be ascertained for 
the diacetylrhein group. 
3. The action of diacetylrhein in the case of localised arthrosis in 20 
patients was investigated in a single blind crossover study against 
naproxen. The patients were divided up into two groups: in the first 
group, initially 50 mg of diacetylrhein was administered twice daily for 
20 days. Subsequently, there followed a three day wash-out phase and a 
further treatment with 250 mg of naproxen twice daily for a further 20 
days. In the second group, the reverse sequence was used. The period of 
treatment was, in all, 43 days. The test parameters of pain, compression 
pain, passive movement pain, function limitation and swelling were 
determined according to a score system. 
The evaluation of the results showed a superiority of the treatment with 
diacetylrhein in comparison with the treatment with naproxen. No 
noteworthy side effects were observed and also no changes of the clinical 
laboratory parameters. 
4. The action of diacetylrhein was investigated in 23 patients (12/11) with 
osteoarthrosis in a randomised double blind study using the "double dummy 
technique" (compatibility study). The dosage administered was 50 mg of 
diacetylrhein twice daily and 250 mg of naproxen three times daily. The 
period of treatment was 4 weeks. The test parameters were the 
oesophagogastroduodenoscopic findings before and after the therapy. Only 
patients with normal mucosal findings or with slight mucosal lesions 
(grade 1) were used in the study. 
After 4 weeks, the endoscopic findings showed, in one case (10%) in the 
diacetylrhein group, mucosal lesions of grade 2, whereas in the naproxen 
treatment group 5 patients (50%) showed mucosal lesions of grade 2, 3 and 
4. In all cases, a normal take-up finding was present. 
Analytical Determination of Aloe-Emodin 
50 mg of diacetylrhein are dissolved in 25.3 ml of 0.5 M aqueous sodium 
hydroxide solution in a separating funnel and shaken for 10 minutes. 
Subsequently, 74.6 ml of a solution are added thereto which contains 0.5 M 
glycine and 0.5 M sodium chloride, a pH value of 9.5 thereby being 
obtained. 
This solution is extracted three times with 25 ml of chloroform. The 
combined organic phases are extracted once with 10 ml 0.5 M of a buffer of 
pH 9.5 (glycine, sodium hydroxide and sodium chloride) and once with 10 ml 
0.01 M sulphuric acid. The solvent is removed from the organic phase and 
the residue is dissolved in 1 ml methanol. 
For a standard solution, 2 mg of aloe-emodin are dissolved in 20 ml of 
N,N-dimethylacetamide and diluted with methanol to a concentration of 2 
.mu.g/ml, corresponding to 40 ppm. 
The content of the solutions is investigated by HPLC. The linearity of the 
HPLC method was demonstrated with aloe-emodin standard solution in the 
range of from 0.11 .mu.g/ml (corresponding to 2.2 ppm) to 53.6 .mu.g/ml 
(corresponding to 1072 ppm). The content determination takes place with a 
Merck HPLC column Lichrocart 250-4, packed with LiChrospher-100 RP-18, 5 
.mu.m, at 40.degree. C. with a mobile phase of 1% acetic acid in methanol 
(v/v), 1% acetic acid in water (v/v) and acetonitrile in a ratio of 
49:46:5. 
Analytical Determination of the Product of Step B), Namely, 
Rhein-9-Anthrone-8-Glucoside With a Content of Aloe-Emodin Components of 
41 PPM, Determined as Aloe-Emodin 
The substance to be investigated is converted into rhein and aloe-emodin by 
oxidation with ferric chloride with simultaneous hydrolysis with 
hydrochloric acid in a two-phase mixture of aqueous solution and carbon 
tetrachloride. The rhein is converted into a salt so that it can be 
separated from the aloe-emodin by liquid-liquid partitioning. The 
aloe-emodin present in the organic phase is determined by HPLC.