JPS6341396B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to
This invention relates to a method for obtaining pure Silymarin by extracting the fruits of Marianum Gaertn.

According to the known literature, silymarin refers to the silymarin group, i.e. polyhydroxyphenylchromanone (silymarin ~ silymarin), which has already been isolated with a purity of 70-80% (depending on the drug) from the berries of thistle. (West German Patent Application Publication No. 1923082).

Silymarin ~ Silymarin is a comprehensive formula
It is an isomer of C ₂₅ H ₂₂ O ₁₀ , and the literature describes silybin,
Silidianin and silycristin are characterized as structural chemistry [West German Patent Application Publication No.
Specification No. 1923082; "Tetrahedron Letters." by PELTER A and HANSEL.R., London.
In, vol. 25, p. 2911, 1968; Wagner (WAGNER.H.), Halehammer (HO¨
RHAMMER.L.), SELIGMANN.
O.) and FARNSWORTH.
NR), “Tetrahedron Letters” No. 31
Volume, pp. 2675-2678, 1970; WAGNER.H., SELIGMANN.
Tetrahedron Letters, Vol. 22, pp. 1895-1899, 1971]. Silymarin is taxifolin (West German Patent Application No. 1923082).

Silymarin is a useful liver treatment agent, and silymarin mixtures are more effective due to their synergistic action than the sum of the effects of the individual isomers (German Patent Application No. 1923082).

A method for obtaining silymarin (silymarin) is known from German Patent Application No. 192 3 082, in which the dried berries of thistle are mechanically squeezed to remove most of the fatty oil therefrom. Extract the pomace thoroughly with ethyl acetate, evaporate the ethyl acetate solution, take up the dry residue in aqueous methanol, and defatte in countercurrent by dividing into homogeneous shapes to increase potency, free from fat. A 70-80% polyhydroxyphenylchromanone mixture (silymarin) is obtained by evaporating the methanolic solution to dryness in vacuo. The ratio of silymarin is about 3:1:1, and the content of silymarin (taxifolin) is about 2-3% of the total amount of silymarin.

Another known state of the art is West German Patent Application No. 2020407. According to the method of this specification, without prior defatting, total extraction is performed directly using a solvent mixture, alcohol, water, lower fatty acid ester, or hydrocarbon, and then 10 to 40% by volume of the resulting extract is distilled. The silymarin is isolated by separating the fat content, then allowing it to stand, and removing the fat that floats to the top of the solution. This method can only extract 40% of silymarin, and further recrystallization with methanol is recommended, but no other processing means are described. This process is non-renewable if carried out later, and the final product contains approximately 13% original fat.
remained.

Another method is known from German Patent Application No. 2017789. The final product of this process contains 40-70% silymarin, with a final product yield of 0.8% up to 2.4% (at 40% silymarin). When carried out later, this method has shown to be regenerative as well.

Furthermore, as a state of the art, French patent No.
Mention may be made of specification 7214042, which relates to obtaining an extract from trumpet thistle without prior defatting. The specification indicates that the final product has approximately 82% polyhydroxyphenylchromone, of which 50% is silymarin. That is, this silymarin exists as other compounds, namely polyhydroxyphenylchromanone rather than polyhydroxyphenylchromone. This method is also non-reproducible.

However, no means for essentially improving the method described in West German Patent Application No. 1923082 has yet been proposed. It is difficult to separate the active ingredient from the drug given the silymarin mixture present, and it is also difficult to separate from it the approximately 30% fatty oil and accompanying substances that are difficult to remove.
This difficulty is only partly overcome by German Patent Application No. 1923082.

Since silymarin is a useful liver treatment agent, there is a need for silymarin in concentrations as high as possible, exceeding those previously known from the state of the art.

The fundamental task of the present invention is to develop a new method for obtaining pure silymarin (silymarin-), which is almost free of concomitant substances and includes all natural isomers. The new process should simply lead to particularly pure products and higher yields.

Unexpectedly, silymarin (silymarin) (purity
90-96%).

Silymarin (Silymarin The method of the present invention for obtaining silymarin consists of: a) Extracting the pomace with anhydrous ethyl acetate several times,
The extract is separated from the residue, the combined ethyl acetate extracts are evaporated, b the resulting evaporation residue a is defatted by multiple solid-liquid extractions with petroleum ether, or b′ the evaporation residue a is dissolved in methanol, the solution is degreased by liquid-liquid extraction several times with petroleum ether, c) the defatted residue b is sufficiently dissolved in methanol and insoluble components are separated from the solution, or c' the defatted methanol Separate petroleum ether from the aqueous solution b', d. Add a chlorine-substituted lower alkane and water as solvents to the methanolic solution c or c', shake it out, and separate the water-methanolic solution,
e the chlorinated solvent phase is mixed with methanol and water several times under stirring and decanted; f the combined water-methanolic phase is evaporated; g the evaporation residue is suspended in a small amount of methanol. Make it muddy,
Subsequently, the suspension is stirred into water, centrifuged, the precipitate is washed several times with water, h the precipitate is dried in vacuo, ground and dried again in vacuo. can be attached.

An advantageous embodiment of the process according to the invention comprises: a. The pomace is treated with anhydrous ethyl acetate for 2 to 4 times, preferably 3 times, each time for 1/2 to 11/2 hours, preferably for 1 hour. Extract with vigorous stirring at a temperature of 1:8 to 1:12, preferably 1:10, at 75°C, advantageously 75°C, separate the extracted drug and mix the combined ethyl acetate extracts to 40 to 55°C. °C, preferably 50 °C, in vacuum at 15 mbar, b evaporation residue a of 1:15 to 1:25, preferably 1:
Degreasing is carried out by solid-liquid extraction three times for 10-20 minutes, preferably for 15 minutes, with petroleum ether (60-70° C.) in a proportion of 20 or b' Dissolve in methanol, preferably 8 times its weight, in methanol, separate off the undissolved residual components, and add petroleum ether (boiling range 60-70°C) in a ratio of 1:1.5 to 1:2.5, preferably 1:2. defatted by liquid-liquid extraction three times for 10 to 20 minutes, preferably for 15 minutes using c′ Separate the petroleum ether from the defatted methanolic solution b′; d Add equal volumes of chloroform and water to the methanolic solution c or c′ and stir the mixture vigorously for about 15 minutes. Stir and decant, e Mix the chloroform phase with methanol and water in a volume ratio of 1:1:1 five times for about 15 minutes each time with vigorous stirring, then separate the chloroform phase, f Combined water-methanol. The sexual phase is evaporated at 30-50 °C, preferably 40 °C, under vacuum at 15 mbar and the evaporation residue is diluted with methanol 1:4-1:1.
6. Suspend preferably in a ratio of 1:5, stir the suspension into 8 to 12 times the volume of water, preferably 10 times the volume, centrifuge and rinse the precipitate with water several times. Wash and precipitate for 20-30 hours, advantageously 24 hours 30-50 hours.
Dry under vacuum at 10 mbar at 40 °C, advantageously at 40 °C, then grind and dry the ground product simultaneously at 30 to 50 °C, preferably at 40 °C under vacuum at 10 mbar.
Characterized by post-drying at mbar.

The product thus isolated contains 90-96% of silymarin (silymarin~) based on the anhydrous material.

The processing technique consists of individual steps, the special measures being selected in such a way that the active substance mixture present in the drug is virtually completely isolated. Deviation from these process steps will result in the loss of all or part of one or the other compound. This difficulty arises in the enrichment process of active substance mixtures, especially in the previous processing steps, since the desired active substance can be obtained partly as individual compounds or as a total mixture with accompanying substances to be separated. This is because it will be done.

The method of the present invention takes these points into account and aims to obtain the pure product with the highest yield while maintaining a sufficient proportion of each isomer present in the drug.

It is surprising that advantageous products can be obtained simply by appropriate combinations of these processing means, solvents or solvent systems and physical or chemical conditions.

Cooling and squeezing of the thistle seeds prevents chemical breakdown or polymerization of silymarin and also avoids tight emulsification of silymarin and accompanying substances in the fatty oil. This is because they have an undesirable effect on the subsequent separation process. The drug is partially degreased by this squeezing process. Also, about 25-30% of the oil is squeezed out.

Micronization makes it possible to speed up the subsequent extraction process, since the extractant can easily penetrate into the drug particles.

In this method, it is important to perform the ethyl acetate extraction treatment with anhydrous ethyl acetate. The water content may be up to 0.5%. If the water content exceeds 0.5%, the accompanying substances can no longer be separated in the next process step. This unexpected effect is decisive for the success of the method in conjunction with other processing means, ie for producing pure silymarin.

The drug/solvent ratio during the extraction with ethyl acetate from 1:8 to 1:12, preferably 1:10, is selected in such a way that the dissolution of the silymarin is uniquely and carefully ensured without unnecessary excess. 2 to 4 times, preferably 3
Silymarin can be completely obtained from the drug by multiple extractions. Temperatures of 60 DEG to 80 DEG C., preferably 75 DEG C., are preferred in order to achieve good dissolution of the active ingredient, without decomposition phenomena of the compounds present.

Stirring with a powerful stirrer is carried out in order to mechanically relax the drug and thereby favor extraction. A short time of 1/2 to 11/2 hours, preferably 1 hour, is selected for each of the several extraction treatments, but nevertheless sufficient extraction is carried out.

The combined ethyl acetate extracts are carefully evaporated in vacuo at low temperature.

A subsequent solid-liquid degreasing of the residue with petroleum ether (the ratio of residue:petroleum ether is very high, for example about 1:20), carried out several times, sufficiently separates the fatty oils in a short time and at little cost. have the advantage of obtaining Selectively dissolve the evaporation residue in about 8 times its weight of methanol, separate the insoluble components, and then liquefy it several times with petroleum ether in a ratio of about 1:2.
Perform liquid-extraction. The result is actually the same.

Methanol, as a very inexpensive solvent, is particularly suitable for dissolving defatted residues due to its large solvation potential. Silymarin (Silymarin)
) is particularly well dissolved in methanol. Residue/
A high proportion of solvent is advantageous for complete removal of silymarin. No silymarin remains in the residue.

Furthermore, in order to separate the remaining accompanying substances from silymarin (silymarin) (in this case, silymarin is not lost as a whole and the isomers are not lost), a chlorine-containing organic solvent, especially a chlorine-substituted one, is used. The use of lower alkanes such as chloroform, methylene chloride, 1,2-dichloroethane, preferably chloroform, is novel. In this case, lipophilic concomitant substances which were not precipitated out during the degreasing step with oil and petroleum ether, as well as slightly polar concomitant substances which are soluble in methanol but are appropriately retained in the partitioned phase system, are separated. The selected solvent system (in this case methanol/water/chloroform 1:1:
The solubility equilibrium (phase diagram) with respect to type and amount of 1) is particularly suitable in the presence of silymarin (silymarin) and, if necessary, accompanying substances still present. This is because a good separation into the two phases is achieved with this solvent system. The demixing range of the phase scheme encompasses the solvent ratios described above.

The distribution coefficient of silymarin (silymarin to silymarin) between both phases is extremely high. Ratio of silymarin in the combined aqueous-methanolic phase to silymarin remaining in chloroform after six shake-outs in a methanol/chloroform/water 1:1:1 (v/v/v) system. is 13:1.

This allows virtually complete recovery of silymarin (silymarin) from the methanolic phase.

According to the present invention, it was surprisingly possible for silymarin to remain in the aqueous/methanolic phase in a balanced state as an individual compound, thus maintaining the isomer ratio.

The aqueous-methanolic phase is carefully evaporated to dryness at low temperature and vacuum, and the residue is suspended in water several times for washing. This water washing step occurs in a normal filtration process. It is advantageous to prevent inclusion of impurities in the product.

Compared to the known method, the method of the present invention has 90-96% pure silymarin (silymarin) and approximately 1.7% pure silymarin.
It has the advantage of being easily obtained with a yield of . By means of the process of the present invention, which carries out the treatment procedure described above using anhydrous ethyl acetate and chlorine-substituted alkanes, such as chloroform, it is unexpectedly possible to obtain sufficiently pure silymarin from the berries of trumpet. .

The effect of extracting the drug with anhydrous ethyl acetate is particularly remarkable compared to the known state of the art. In this case the lipophilic substance of the drug remains in the residue. In subsequent processing steps, such as partitioning into a solvent, this lipophilic concomitant usually cannot be separated satisfactorily from silymarin.

What was also particularly unexpected was that the solvent partitioning effect of chlorinated lower alkanes, especially chloroform, occurs in the second stage of the process due to its special selectivity and is still present in this process step. All concomitant substances present are actually removed, so that silymarin remains in sufficient purity in the methanolic solution.

Example for obtaining silymarin (silymarin~) 1 Cooled and squeezed finely powdered thistle fruit
Add 3000 kg of anhydrous ethyl acetate to 300 kg, and
Heat to 75°C. Mix vigorously for 1 hour at this temperature using a high speed stirrer. It is then filtered and the crude drug residue is extracted twice more with ethyl acetate as described above.

Combine the cooled ethyl acetate solution at 50 °C under vacuum for 15 min.
Evaporate to dryness at mbar. To degrease the residue, add petroleum ether (boiling point 60/70) three times at room temperature each time.
Stir for 15 min at 300 °C and then remove from the solvent in vacuo. The crude product thus obtained is taken up in four times the volume of methanol, the undissolved solid material being centrifuged off and washed twice with half the volume of methanol each time, until it is confirmed that it actually contains no silymarin. Separate and remove it.

To the combined methanolic solution first add chloroform and then water in a volume ratio of 1:1:1 and transfer to a decant vessel.

Mixing is carried out vigorously for 15 minutes and after the phases have been separated, the lower phase (chloroform phase) is removed and extracted five more times with methanol-water in a volume ratio of 1:1:1 for 15 minutes each. The chloroform phase is then removed.

The combined aqueous-methanolic phase was heated at 50 °C under vacuum for 15 min.
Evaporate at mbar and suspend the residue in 5 times the volume of methanol. The suspension is added as a thin spray to 10 times the volume of water with stirring and then stirred vigorously for 15 minutes. It is then centrifuged and the centrifuged product is washed several times with water under suspension. Store the product at 40℃ for 24 hours
dried in a vacuum cabinet, ground and post-dried for a further 48 hours at 40°C.

A light tan powder is obtained, which is 90 to 96
% of silymarin [measured in the same manner as in the measurement of silymarin in the seeds of trumpet thistle by the dinitrophenylhydrazine method (DNPH-method), DAB, 1968, 2nd Supplement, 1975, No.
198 and quantitative evaluation of the diffuse reflectance curve). Confirmation was performed by thin layer chromatography.

The mutual proportions of the individual isomers were determined by quantitative evaluation of the diffuse reflectance curve (see Figure 1).The yield was 4.9 kg or about 1.7% based on the dry herbal medicine.

Example 2 300 pieces of fine powdered thistle fruit squeezed out when cold
Add 3,000 ml of ethyl acetate anhydride to the kg and mix at 75°C.
Heat to. Mix vigorously for 1 hour at this temperature using a high speed stirrer. It is then filtered and the crude drug residue is extracted twice more with ethyl acetate as described above.

Combine the cooled ethyl acetate solution at 50 °C under vacuum for 15 min.
Evaporate to dryness at mbar.

The evaporation residue is dissolved with methanol 120 ml, and the undissolved solids are centrifuged. The methanolic solution is liquid-liquid extracted three times each with petroleum ether 135 for 15 minutes.

To the methanolic solution first add chloroform and then water in a volume ratio of 1:1:1 and transfer to a decant vessel.

After vigorous mixing for 15 minutes and separation of the phases,
The lower phase (chloroform phase) is removed, and extracted in the same manner five more times with methanol and water at a volume ratio of 1:1:1 for 15 minutes each. The chloroform phase is then removed.

The combined aqueous/methanolic phase was heated at 50 °C under vacuum for 15 min.
Evaporate at mbar and suspend the residue in 5 times the volume of methanol. The suspension is added as a thin spray to 10 times the volume of water with stirring and then stirred vigorously for 15 minutes. It is then centrifuged and the centrifuged product is washed several times with water under suspension. The product is dried in a vacuum cabinet at 40° C. for 24 hours, ground and post-dried for a further 48 hours at 40° C.

This process yields 90-96% silymarin with properties similar to Example 1. Yield is 1.7%.

Example 3 A similar treatment using methylene chloride instead of chloroform yields 90-94% silymarin.

Yield is 1.7%.

Example 4 A similar treatment using 1,2-dichloroethane instead of chloroform yields 90-93% silymarin.

Yield is 1.8%.

In all examples, slight variations in the purity range and yield usually occur in connection with herbal medicines.

[Brief explanation of drawings]

The drawing is a diffuse reflection curve diagram that defines the mutual ratio of individual isomers by quantitative evaluation. 1...Silybin, 2...Silydianin, 3...
Silicristin, 4...taxifolin.

Claims (1)

[Claims] 1. Squeezing most of the fatty oil from the fruit of Silybum marianum while cold, pulverizing the squeezed substance, and extracting the pomace with ethyl acetate,
In the method for obtaining silymarin (silymarin) by evaporating the ethyl acetate extract and post-processing, a) extracting the pomace with anhydrous ethyl acetate many times,
The extract was separated from the residue, the combined ethyl acetate extracts were evaporated, and the evaporation residue a was filtrated several times with petroleum ether.
Degreasing by liquid-extraction, c. Sufficiently dissolve the degreased residue b in methanol and separate insoluble components from the solution, d. Add a chlorine-substituted lower alkane and water as solvents to the methanolic solution c, respectively, and shake it out. the water-methanolic solution is separated and stored; e the chlorinated solvent phase is mixed with methanol and water several times under stirring and decanted; f the combined water-methanolic phase is evaporated; g evaporated. Suspend the residue in a small amount of methanol,
Subsequently, the suspension is stirred into water, centrifuged, the precipitate is washed several times with water, h) the precipitate is dried in a vacuum, pulverized, and dried again in a vacuum. How to get silymarin from plants. 2 a Pomace 2 to 4 times 1/2 to 11/2 each
Extract with anhydrous ethyl acetate in a ratio of 1:8 to 1:12 at 65-76°C for hours with vigorous stirring, separate the extracted drug, and combine the ethyl acetate extracts at 40-55°C. Evaporate in vacuo at 15 mbar and evaporate the evaporation residue a three times using a petroleum ether boiling range (60-70 °C) in a ratio of 1:15-1:25.
Degrease by solid-liquid extraction for 20 minutes, c) Dissolve the defatted residue b in 6 to 10 times its weight of methanol to separate the insoluble residual components, d Add chloroform and water to the methanolic solution c, respectively. Equal volumes are added, the mixture is stirred vigorously for about 15 minutes, decanted, and the chloroform phase is mixed with methanol and water in a volume ratio of 1:1:1 five times with vigorous stirring for about 15 minutes each time, followed by addition of chloroform. Separate the phases, f evaporate the combined aqueous-methanolic phase under vacuum at 15 mbar at 30-50 °C, g evaporate residue with methanol 1:4-1:
The suspension was stirred and introduced into 8 to 12 times the volume of water, centrifuged, and the precipitate was washed with water many times. in vacuum at °C
2. A process as claimed in claim 1, which comprises drying at 10 mbar, subsequent comminution and simultaneous after-drying of the milled product at 30 DEG -50 DEG C. under vacuum at 10 mbar. 3. Squeeze most of the fatty oil from the fruit of Silybum marianum while cold, crush the squeezed material, extract the pomace with ethyl acetate,
In the method for obtaining silymarin (silymarin) by evaporating the ethyl acetate extract and post-processing, a) extracting the pomace with anhydrous ethyl acetate many times,
The extract is separated from the residue, the combined ethyl acetate extracts are evaporated, b′ this evaporation residue a is dissolved in methanol, the solution is defatted by multiple liquid-liquid extractions with petroleum ether, and c′ defatted. Separate petroleum ether from the methanolic solution b', d. Add a chlorine-substituted lower alkane and water as solvents to the methanolic solution c', shake it out, separate and store the water-methanolic solution, e. the oxidized solvent phase is mixed with methanol and water several times under stirring and decanted; f the combined aqueous-methanolic phase is evaporated; g the evaporation residue is suspended in a small amount of methanol;
Subsequently, the suspension is stirred into water, centrifuged, the precipitate is washed several times with water, h) the precipitate is dried in a vacuum, pulverized, and dried again in a vacuum. How to get silymarin from plants. 4 a 1/2 to 11/2 of the pomace 2 to 4 times each
Extract with anhydrous ethyl acetate in a ratio of 1:8 to 1:12 at 65-76°C for hours with vigorous stirring, separate the extracted drug, and combine the ethyl acetate extracts at 40-55°C. Evaporate in vacuo at 15 mbar, b' Dissolve the evaporation residue a in 6 to 10 times its weight of methanol, separate the undissolved residual components, and add 1:1.5 to
Petroleum ether (boiling range 60~
70℃) for 10 to 20 minutes three times.
Degrease by extraction, c′ separate the petroleum ether from the defatted methanolic solution b′, and d add equal volumes of chloroform and water to the methanolic solution c′ and stir the mixture vigorously for about 15 minutes. , decanting, e mixing the chloroform phase with methanol and water in a volume ratio of 1:1:1 five times with vigorous stirring for about 15 minutes each time, subsequently separating the chloroform phase, f combining water-methanolic phase; was evaporated under vacuum at 15 mbar at 30-50 °C and the evaporation residue was diluted with methanol 1:4-1:
The suspension was stirred and introduced into 8 to 12 times the volume of water, centrifuged, and the precipitate was washed with water many times. in vacuum at °C
4. The process as claimed in claim 3, comprising drying at 10 mbar, subsequent comminution and simultaneous after-drying of the milled product at 30 DEG -50 DEG C. under vacuum at 10 mbar.