Process for isolation of hepatoprotective agent silymarin...

Details Process for isolation of hepatoprotective agent silymarin... Process for isolation of hepatoprotective agent silymarin...

2000-03-31

2001-10-30

Witz, Jean C. (Department: 1651)

Drug, bio-affecting and body treating compositions

Plant material or plant extract of undetermined constitution...

Containing or obtained from a seed or nut

C424S725000

Reexamination Certificate

active

06309678

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to a novel process for the isolation of a hepatoprotective agent silymarin from the plant
Silybum marianum
(L) Gaertn.
1. Background of the Invention
Silymarin is the name given to a mixture of flavanolignans found in
Silybum marianum
which have exhibited clinically proven hepatoprotective action. Positive results obtained in numerous pharmacological, biochemical and toxicological studies have made silymarin a drug of choice in the treatment of diseases of the liver (Morazzoni, P. and Bombardelli, E.,
Fitoterapia,
116(1), p 1005, 1995). Major constituents of silymarin have been identified as the flavanolignans silybin, silydianin and silychristin and their chemical structures have been elucidated (Wagner, H., Diesel, P., and Seitz, M., Arzneim Forsch. 24, p 466, 1974; Pelter, A., Hansel, R.,
Tetrahedron Letters
, No.25, p 2911-2916, 1968; Abraham D J, Takagi S, Wagner H, Farnsworth N R, et al,
Tetrahedron Letters
, No.31, p. 2675-2678, 1970; Wagner H, Cahri, M. V, Seitz M; Riess-Muarer, I,
Tertrahedron Letters
, No.4, p 381-384, 1978). The mechanism of hepatoprotective action of silymarin has been attributed to its ability to act as a powerful scavenger of free radicals capable of interrupting the peroxidative processes involved in liver damage induced by hepatotoxic agents like alcohol, paracetamol and carbon tetrachloride (Leng, P. E., Strange, H. A., Z. Phytotherapie 12, 162, 1991). Considering the widespread use of alcohol causing liver problems in humans and the general prevalence of liver diseases caused by impure drinking water, the demand for silymarin is huge. Silymarin is extracted from the mature dry seeds of
Silybum marianum
. A few methods for isolation of silymarin have been reported.
2. Prior Art References
According to one process, the powdered drug was extracted in soxhlet extractor with methanol or ethanol, the extract, concentrated under reduced pressure, concentrate made into a slurry with silica gel and loaded onto a silica gel column, eluted with benzene-ethyl acetate mixtures of increasing polarity, silymarin containing fractions combined and solvent removed, residue crystallized from MeOH gave 86% pure silybin (mp 167° C.-180° C.), mother liquor completely dried under vacuum gave additional yellow mixture of flavanoids having 58% purity as silybin (Gupta, G. K., Raj, S., Rao, P. R.,
Research and Industry
27, p 37-42, 1982). The highest yield of Silymarin reported by these authors was 2.46% based on the weight of seeds. This process involves the costly step of silica gel chromatography which is not economical when used on industrial scale. Another process presently being used consists of removing part of the fatty oil by pressing the seeds in a screw press, breaking up the compressed seed mass, extracting the pressed seed mass with ethyl acetate at 75° C., evaporating the solvent to obtain a semisolid residue, defatting the residue by repeated solid liquid extraction with light petroleum, dissolving defatted residue in methanol, mixing methanol solution with chloroform and water, separating and retaining methanolic layer, evaporating aqueous methanol, suspending residue in a little methanol, precipitating the silymarin rich solid by mixing with excess water, centrifuging, washing and drying the precipitate in vacuo. Yield of Silymarin is 1.7% based on dry seed weight. (Madaus, R. Klaus, G., Werner, M., U.S. Pat. No. 4,368,195 Jan. 11, 1983).
OBJECT OF THE INVENTION
The main objective of the present invention is to provide a simple process to isolate the hepatoprotective agent silymarin from the seeds of
Silybum marianum.
Another object is to provide a novel, cheap and economic process for isolation of silymarin on commercial scale.
SUMMARY OF THE INVENTION
Accordingly, the invention provides a novel process for the isolation of Silymarin, a hepatoprotective agent, from the seeds of
Silybum marianum.
DETAILED DESCRIPTION
Accordingly, the invention provides a novel process for the isolation of Silymarin, a hepatoprotective agent, from the seeds of
Silybum marianum
, said process comprising the steps of:
i) chilling the seeds of
Silybum marianum,
ii) grinding the chilled seeds to a fine powder,
iii) defatting the pulverized seeds by extracting it with a hydrocarbon solvent,
iv) extracting the defatted seed powder with acetonitrile at 20-30° C. to obtain silymarin-containing fraction as an extract,
v) concentration of the sensitive silymarin fraction,
vi) stirring the silymarin rich dry powder with cold dichloromethane at 5° C. followed by filtration and drying with a slow purge of nitrogen gas,
vii) purification of silymarin by suspending it in acetonitrile in an amount 5 times its weight and precipitating it by adding distilled water in an amount 8-12 times its weight, at 20-30° C.,
viii) filtering the precipitated silymarin in a closed vacuum filter having 1 to 2 &mgr;m screw, washing the cake three times with distilled water, and
ix) drying the silymarin cake in vacuum oven to obtain substantially pure silymarin.
In an embodiment, the seeds of the plant
Silybum marianum
are cooled for 24 hrs. at −20° C. in a deep freezer or cold room.
In an embodiment, the hydrocarbon solvent for removal of fatty acids is hexane.
In an embodiment, the chilled seeds are pulverized in a hammer mill fitted with about 40 mesh discharge screen.
Still another embodiment, the fatty oil in the seed powder is removed by extraction in a soxhlet type extractor without using screw/press.
In yet another embodiment, the extract containing sensitive silymarin obtained in step (iv) is concentrated using an agitated wiped thin film evaporator.
In yet another embodiment. In another embodiment of invention the filtration of the silymarin containing fractions is carried out in closed vacuum filter under the inert environment of nitrogen gas to prevent the degradation of the product.
In still another embodiment the silymarin precipitate is dried under vacuum at 40-45° C., maintaining oven pressure at 5-15 torr for 15 hours.
The invention is described in detail herein below with reference to the following examples which are provided merely to illustrate the invention and should not be construed to limit the scope of the invention in any manner.
Primarily, the improved process consists of following operations
1. Chilling the seeds of
Silybum marianum
at (−)20° C. by keeping overnight in a deep freezer/cold room.
2. Grinding the chilled seeds in a hammer mill fitted with 40 mesh discharge screen.
3. Extracting the powdered seeds in a soxhlet apparatus with a hydrocarbon solvent such as hexane to remove all the fatty oil.
4. Percolating the defatted seed powder 34 times with acetonitrile at (20° C.-30° C.) for separating the silymarin containing fraction, as extract.
5. Removing the acetonitrile under vacuum in a wiped film evaporator to obtain a concentrate rich in silymarin, recovered acetonitrile being recycled to the process.
6. Drying the concentrate in a batch type stirred vacuum concentrator to obtain a product of semi solid consistency and its further drying in a vacuum oven to obtain a dry residue.
7. Stirring the powdered residue in cold dichloromethane (5° C.) and filtering in a closed vacuum filter for removing the impurities from silymarin rich solid product.
8. Dissolving the above solid in small amount of acetonitrile and precipitating silymarin by adding cold distilled water.
9. Filtering the light yellow precipitate and washing it three times with distilled water.
10. Transferring the cake to a vacuum oven and drying it at 40-45° C., maintaining the oven pressure at 5-15 torr for 15 hours.
11. Checking the purity of product obtained by DNPH method using a UV spectrophotometer at 490 nm (Wagner, V. H., Diesel, P., Saity, M., Ahzneim.—Forsch, 24 (4), p 466-471, 1974) and by HPLC (Tittel and Wagner,
Journal of Chromatography,
153, p 227-232, 1978).
Salient Features of the Invention
1. The prior art process depends on a screw press to remove part of fatty oil present in the seeds. The remaining oil is

Affiliated with

Also associated with

Rating

Process for isolation of hepatoprotective agent silymarin... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Process for isolation of hepatoprotective agent silymarin..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Process for isolation of hepatoprotective agent silymarin... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2609586