FIELD OF INVENTION
The present invention relates to an improved method for isolation and purification of Lysergol from Ipomoea hederacea seeds. The invention particularly describes a method for the isolation and purification of upto 99.2% pure lysergol from the seeds of Kaladana {Ipomoea hederacea) .The present invention provides a method which consist of drying and grinding the seeds and extracting them by primary alcohol at a temperature in the range of 45°C -75°C with less than 0.5% impurities.
The present invention also provides an eco-friendly, hygienic and simplified method for commercial manufacturing of Lysergol. Lysergol is a compound that has been identified as a source of therapeutic value and commercial high value compound, used in the treatment of schizophrenia and motor disorders. It is also used in the synthesis of Lumilysergol and Nicergoline. The drug consisting of seeds is locally used for its aperient action.
BACKGROUND OF THE INVENTION
Lysergol is a compound that has been identified as a source of therapeutic value and commercial high value compound, used in the treatment of schizophrenia and motor disorders. It is also used in the synthesis of Lumilysergol and Nicergoline. It is an alkaloid, of ergolic structure. Its use in traditional medicine dates back to the modern medicines of 19th century. Some less known but promising traditional formulations of Chhatisgarh, India, are based on the kaladana as main ingredient. The drug consisting of seeds, is locally used for its aperient action whereas the powder is used as an antipyretic.
Kaladana is the native name by which it is indicated a spontaneous plant of the subtropical mountainous territories, more particularly the pre-Himalayan belt of India and Pakistan, upto altitude of 1500 meters. As a matter of fact in the publication Chopra's Indigenous Drugs of India, U.N. Dhur and Sons Private
Limited, Calcutta, 1958, page 51, Kaladana is indicated as corresponding to the scientific term Ipomoea hederacea (I.P).
Reference may be made to US patent No. 3920663, "method for extraction of Lysergol and ergot alkaloids from plants of the ipomoea genus" which describes a method where the seeds of the plant Kaladana are ground to a flour and fat stripped and then extracted with a halogen - substituted aliphatic hydrocarbonaceous solvent selected from the group consisting of chloroform, carbon tetrachloride, and methylene chloride, and the extraction step being repeated from 3 to 5 times.
Reference may be made to US patent No. 2809920, which describes a process for the preparation of ergotamine,ergotamine and ergometrine by saprophytic culture of ergot invitro and isolation of the alkaloids thus produced .The mycelium and the culture medium are made alkaline,extracted with ether and the ethereal solution with a 1% of tartaric acid.
Reference may be made to US patent No. 3219545, which relates to microbiological production of lysergic acid derivatives.The lysergic acid derivatives are isolated and hydrolysed by dissolving the crude mixture of lysergic acid derivatives formed in the nutrient medium in ethanol solution .Barium hydroxide and sodium dithionate are used as alkaline solution.
Reference may be made to US patent No.3224945, wherein a method has been described for the production of lysergic acid by fermentation process. Here ethylene chloride is used for the separation of alkaloids.
Reference may be made to the article entitled "Alkaloids of an Ipomoea Seed commonly known as Kaladana in Pakistan" of Charles I., Abou-Chaar and George A. Digenis (1966) in Nature vol: 212, page: 618 where in the alkaloids were identified by paper and thin-layer chromatography in seven different solvent
systems, by behaviour in ultra-violet light arid coloration by van Urk's reagent and by determination of the melting point.
Reference may be made to the article entitled "Ergot alkaloid transport across ruminant gastric tissues" of N. S. Hill, F. N. Thompson et al (2001) in Journal of Animal Science, Vol 79, Issue 2 542-549, where they have described relative and potential transport of ergoline and ergopeptine alkaloids across isolated gastric tissues in vitro.
Reference may be made to the article entitled "Improved extraction and HPLC methods for ergovaline from plant material and rumen fluid" of Rottinghaus, GE, Schultz LM et al (1993) in J Vet Diagn Invest 6:348-352, which describes an improvement in extraction and cleanup and the high-pressure liquid chromatographic. It also describe a 24-hour extraction time, the use of smaller solvent volumes during sample preparation, and fast analysis on the polymeric reverse-phase column.
DRAWBACK OF THE PRIOR ART
1) It does not provide eco-friendly and hygienic method for manufacturing of Lysergol.
2) The processes involved are tedious and multi-step.
3) The use of corrosive acid for extraction as well as purification leads to high cost.
4) The final product obtained with less purity.
To overcome all the aforementioned barriers there is a need to develop a simple and fast method for the recovery of Lysergol from extraction of seeds of Kaladana which can reduce the long operation span and can provide eco-friendly and economically beneficial manufacturing process.
OBJECTIVES OF THE INVENTION
The main object of the present invention is to provide an eco-friendly and hygienic method for commercial manufacturing of Lysergol.
Another object of the present invention is to develop economically beneficial manufacturing process by cost cutting.
Another object of the present invention is to avoid direct human exposure with hazardous chemicals solvents.
Yet another object of this invention is to reduce the long operation span.
Still another object of the present invention is to provide a method for purification of Lysergol with purity upto 99.2%.
Another object of this invention is to provide final product with impurity less than 0.5%.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides A method for the isolation and purification of Lysergol from the seeds of Kaladana (Ipomoea hederacea) plant comprising the following steps:
a) drying the seed of the plant at room temperature to the exclusion of light;
b) grinding the dried seeds obtained in step (a) to 20 to 80 mesh size to obtain a free flowing powder;
c) extracting the free flowing powder obtained in step (b) with primary alcohol at a temperature in the range of 45-75° C along with their TLC analysis;
d) filtering the extract obtained in step (c);
e) distilling out the filtrate so obtained in step (d) at a temperature in the range of 45-75° C under vacuum to obtain a soft mass containing Lysergol and derivatives;
f) mixing the soft mass containing Lysergol and derivatives obtained in step (e) with aqueous solution of a weak base such as NH4OH (basified water), and subsequently with an
organochlorine solvent, to facilitate exchange of alkaloids
(Lysergol and derivatives) from the basified water into the
organochlorine solvent; g) distilling out the organochlorine solvent completely to obtain
Lysergol containing soft mass; h) dissolving the Lysergol containing soft mass obtained in (g) in an
aqueous solution of weak organic acid; i) treating the above acidic solution obtained in step (h) with
activated charcoal for decolorization; j) filtering the decolorized solution obtained in step (i) at pH 4.0-
4.5; k) adding NH4OH to the filtered solution obtained in step (j) to
make the water phase alkaline to a pH of 9-9.5 and allowing
Lysergol rich fraction to settle down; 1) dissolving the Lysergol rich fraction obtained in step (k) in an
organochlorine solvent; m) extracting organochlorine solvent phase with a solution of glacial
acetic acid in water; n) mixing the acid phase with mineral acid such as hydrochloric
acid to prepare Lysergol hydrochloride; o) treating the Lysergol hydrochloride obtained in step (n) with
NH4OH solution to obtain free Lysergol; p) drying the free Lysergol obtained in step (o) at a temperature in
the range of 55-65° C under vacuum for 9 hours to obtain the
final product. In one embodiment of the present invention, the primary alcohol used for extraction of powdered seeds is selected from ethanol, methanol and n-propanol, preferably ethanol.
In another embodiment of the present invention, the organochlorine solvent is selected from ethylene dichloride (EDC), dichloromethane and chloroform, preferably ethylene dichloride (EDC).
In another embodiment of the present invention, the weak organic acid solution used is selected from acetic acid, tartaric acid, ascorbic acid and formic acid, preferably acetic acid.
In another embodiment of the present invention, the final product obtained has more than 99.2% purity.
In another embodiment of the present invention, the final product obtained has impurity less than 0.5%.
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses a method for the isolation of high purity Lysergol, purity deciphered either TLC, HPTLC or HPLC as per specification, from the seeds of the Kaladana (Ipomoea hederacea). Kaladana is the native name by which is indicated a spontaneous plant of the subtropical mountainous territories, more particularly the pre-Himalayan belt of India and Pakistan, upto altitude of 1500 meters.
As a matter of fact in the publication Chopra's Indigenous Drugs of India, U.N. Dhur and Sons Private Limited, Calcutta, 1958, page 51, Kaladana is indicated as corresponding to the scientific term Ipomoea hederacea (LP).
Lysergol is a compound that has been identified as a source of therapeutic value and commercial high value compound, used in the treatment of schizophrenia and motor disorders. It is also used in the synthesis of Lumilysergol and Nicergoline. It is an alkaloid, of ergolic structure. Its use in traditional medicine dates back to the modern medicines of 19th century. Some less known but promising traditional formulations of Chhatisgarh, India, are based on the kaladana as main ingredient. The drug consisting of seeds, is locally used for its aperient action whereas the powder is used as an antipyretic.
According to the invention, the seeds of the Kaladana are dried at room temperature to the exclusion of light and ground to a powedered. The free flowing powder of the seeds was extracted with primary alcohol at a temperature in the range of 45-75° C, with 5 times solvent (w/v) at each. Extraction continued till the Thin Layer Chromatographic finger prints exhibit traces in extract. The extract was analyzed by TLC (mobile phase Chloroform: Methanol: ammonia solution:: 95: 5:0.5,Merck: Silica gel 6OF254, at 254 nm as well as 365 nm) to quantify Lysergol content. It was established that following aforesaid operating parameters after third extraction the herb completely exhaust for Lysergol content, if present, only in ppm level. The extract was filtered and mixed .Then filtrate was distilled out at a temperature in the range of 45-75° C under vacuum. The resulting mass left behind after distillation of alcohol, was mixed with basic water i.e. NH4OH in water and the water phase was extracted with organochlorine solvent. The Organochlorine solvent was completely distilled out.
The resulting softmass was dissolved in aqueous solution of weak organic acid and filtered, using filter aid, at a pH in the range of 4.0-4.5 after treating with activated charcoal for decolorisation.
The aqueous acidic phase was basified with NH4OH.The Lysergol rich fraction settle-down. Lysergol rich fraction dissolved in Organochlorine solvent again. Organochlorine solvent phase was extracted with glacial acetic acid in water.
The organochlorine phase was mixed with mineral acid to prepare its chloride derivative. The chloride derivative was treated with NH4OH solution and free Lysergol was obtained.
The final product so obtained was dried at a temperature in the range of 55-65 C under vacuum for 9 hours.
The purity of the product is defined by Thin layer chromatography (mobile phase Chloroform: Methanol: ammonia solution:: 95: 5:0.5,Merck: Silica gel 60F254, at 254 nm as well as 365 nm) as well as High performance liquid chromatographic
[(Silica column 250x 4.6mm,I.D.,5u., mobile phase: (n-Heptane: THF: MeOH: Diethyl amine:: 55: 35: 6: 0.02), wave length: 230nm, Flow rate 1.5 ml/min)] techniques with Lysergol more than 98% purity and Elymoclavin less than 0.5%. Elymoclavin is a geometrical isomer of Lysergol, which is considered as an associated impurity. According global market specification it should be less than 0.5%) (wt./wt) by High Performance Liquid Chromatographic (HPLC) analysis, as a high value product.
Thus, the novelty of our invention lies in the fact that the Lysergol is extracted from the seeds of Kaladana ((Ipomoea hederacea), which consist of grinding and drying the seeds and extracting them by a primary alcohol, which on further re-extracted by an Organochlorine solvent recovers Lysergol selectively at basic medium.
The extraction and purification of Lysergol is described hereinafter with examples, which are given by way of illustration of the present invention and should not be construed to limit the scope of the present invention.
EXAMPLE 1:
10.0 kg dried Kaladana seeds were powdered 20 mesh size. The free flowing powder of the seeds was extracted with primary alcohol at 45° C temperature, with 5 times solvent, at each [i.e. for 10 kg mass 50L solvent, at once, (three extractions were done so total solvent used 50L x 3=150L)]. Extraction continued till the Thin Layer Chromatographic finger prints have traces of Lysergol in extract. The extract was analyzed by TLC (mobile phase Chloroform: Methanol: ammonia solution:: 95: 5:0.5,Merck: Silica gel 6OF254, at 254 run as well as 365 nm) to quantify Lysergol content. The extract was filtered and mixed, then the filtrate was distilled out at 45° C under vacuum. As alcohol was distilled-out, the mass left on vessels was mixed 6L water (having 300ml of NH4OH, pH 9 and the water phase was extracted with 5L ethylene dichloride (EDC). EDC was completely distilled out. The resulting softmass was dissolved in 5L water having 14% glacial acetic acid, pH=4 and filtered, using filter aid, at pH 4.0, after treating with activated charcoal

for decolorisation. The aqueous solution of weak organic acid of pH 4.0 was basified with NH4OH, 300ml of NH4OH used and Lysergol rich fraction precipitated out which is again dissolved in organochlorine solvent. EDC phase was then extracted with 3.0L of 14% acetic acid solution. The acidic phase was mixed with 75 ml of HC1, LR grade to prepare its hydrochloride derivative. The hydrochloride derivative was treated with 120ml of NH4OH solution to give free Lysergol. The product is then dried at 65° C temperature under vacuum for 9 hours, having 7.87 gm, of 99.14% Lysergol and Elymoclavin 0.35% as determined by HPLC analysis.
EXAMPLE 2:
10.0 kg dried Kaladana seeds were powdered 40 mesh size. The free flowing powder of the seeds was extracted with primary alcohol at 55° C temperature, with 5 times solvent, (wt/V) at each [i.e. for 10 kg mass 50L solvent, at once, ( three extractions were done so total solvent used 50L x 3=150L)]. Extraction continued till the Thin Layer Chromatographic finger prints have traces of Lysergol in extract. The extract was analyzed by TLC (mobile phase Chloroform: Methanol: ammonia solution:: 95: 5:0.5,Merck: Silica gel 6OF254, at 254 nm as well as 365 run) to quantify Lysergol content. The extract was filtered and mixed, and then the filtrate was distilled out at 55° C under vacuum. As alcohol was distilled-out, the mass left on vessels was mixed with 6.0L water (having 300ml of NH4OH, pH=9.0) and the water phase was extracted with 5.0L of EDC. EDC was completely distilled out. The resulting soft mass was dissolved in 5.0L water having 14% glacial acetic acid, and filtered, using filter aid, after treating with activated charcoal for decolorisation. The aqueous solution of weak organic acid of pH 4.0 was basified with 300ml of NH4OH and Lysergol rich fraction precipitate out which is again dissolved in 3.0L EDC. EDC phase was then extracted with 3.0L water having 14% glacial acetic acid. The acidic phase was mixed with 75ml HC1, LR grade, to prepare hydrochloride derivative. The hydrochloride derivative was treated with 120 ml NH4OH solution to give free Lysergol. The product is then dried at 65° C temperature under vacuum for 9 hours, having 7.94 gm, of 99.2 % Lysergol and Elymoclavin 0.33% as determined by HPLC analysis.
EXAMPLE 3:
10.0 kg dried Kaladana seeds were powdered 60 mesh size. The free flowing powder of the seeds was extracted with primary alcohol at 55° C temperature, with 5 times solvent,(Wt/ V) at each [i.e. for 10 kg mass SOL solvent, at once, (three extractions were done so total solvent used 50L x 3=150L)]. Extraction continued till the Thin Layer Chromatographic finger prints have traces of Lysergol in extract. The extract was analyzed by TLC (mobile phase Chloroform: Methanol: ammonia solution:: 95: 5:0.5,Merck: Silica gel 6OF254, at 254 nm as well as 365 nm) to quantify Lysergol content. The extract was filtered and mixed, and then the filtrate was distilled out at 65° C under vacuum. As alcohol was distilled-out, the mass left on vessels was mixed with 6.0L water (having 300ml NH4OH, pH=9.0) and the water phase was extracted with 5.0L EDC. EDC was completely distilled out. The resulting soft mass was dissolved in 5.0L water having 14% glacial acetic acid and filtered, using filter aid, at pH 4.0, after treating with activated charcoal for decolorisation. The aqueous solution of weak organic acid of pH 4.0 was basified with 300ml NH4OH and Lysergol rich fraction precipitate out which is again dissolved in 3.0L EDC. EDC phase was then extracted with 14% acetic acid solution, total volume=3.0L. The acidic phase was mixed with 75 ml of HC1, LR grade, to prepare its hydrochloride derivative. The hydrochloride derivative was treated with NH4OH solution to give free Lysergol. The product is then dried at 65° C temperature under vacuum for 9 hours, having 7.82 gm, of 98.82 % Lysergol and Elymoclavin 0.36% as determined by HPLC analysis.
EXAMPLE 4:
10.0 kg dried Kaladana seeds were powdered 80 mesh size. The free flowing powder of the seeds was extracted with primary alcohol at 75° C temperature, with 5 times solvent,(Wt/ V) at each [i.e. for 10 kg mass 50L solvent, at once, (three extractions were done so total solvent used 50L x 3=150L)]. Extraction continued till the Thin Layer Chromatographic finger prints have traces of Lysergol in extract. The extract was analyzed by TLC (mobile phase Chloroform: Methanol: ammonia solution:: 95: 5:0.5,Merck: Silica gel 6OF254, at 254 nm as well as 365 nm) to quantify Lysergol content. The extract was filtered and mixed, and then the filtrate
was distilled out at 75° C under vacuum. As alcohol was distilled-out, the mass left on vessels was mixed with 6.0L basic water (water having 300ml NH4OH, pH=9.0) and the water phase was extracted with 5.0L EDC. EDC was completely distilled out. The resulting soft mass was dissolved in 14% glacial acetic acid solution, total volume5.0L and filter, using filter aid, after treating with activated charcoal for decolorisation. The aqueous solution of weak organic acid of pH 4.0 was basified with300ml of NH4OH and Lysergol rich fraction precipitate out which is again dissolved in 3.0L EDC. EDC phase was then extracted with 3.0L of 14% glacial acetic acid solution. The acidic phase was phase was mixed with 75ml of HC1, LR grade, to prepare hydrochloride derivative. The hydrochloride derivative was treated with 120 ml of NH4OH solution to give free Lysergol. The product is then dried at 65° C temperature under vacuum for 9 hours, having 8.00 gm, of 98.54 % Lysergol and Elymoclavin 0.30% as determined by HPLC analysis.
THE MAIN ADVANTAGES OF THE PRESENT INVENTION:
1.) Fat/oils extracted during extraction are separated by solvent- solvent exchange
method during purification, by adjusting pH of the phase. 2.) No use of corrosive highly acids as phosphoric acid in the whole process. 3.) Mild alkali and weak organic acids are used in the whole process. However
mineral acid was used but it is diluted. 4.) It is an eco-friendly and hygienic method for commercial manufacturing of
Lysergol. 5.) Economically beneficial manufacturing process by cost cutting. 6.) Reduces the long operation span.
7.) Use of latest analytical techniques e.g. TLC, HPTLC and HPLC for assay. 8.) Establish a standardized specification for the final product along purity and
associated impurity.

We Claim:
1. A method for the isolation and purification of Lysergol from the seeds of Kaladana (Ipomoea hederacea) plant comprising the following steps:
a) drying the seed of the plant at room temperature to the exclusion of light;
b) grinding the dried seeds obtained in step (a) to 20 to 80 mesh size to obtain a free flowing powder;
c) extracting the free flowing powder obtained in step (b) with primary alcohol at a temperature in the range of 45-75° C along with their TLC analysis;
d) filtering the extract obtained in step (c);
e) distilling out the filtrate so obtained in step (d) at a temperature in the range of 45-75 C under vacuum to obtain a soft mass containing Lysergol and derivatives;
f) mixing the soft mass containing Lysergol and derivatives obtained in step (e) with aqueous solution of a weak base such as NH4OH (basified water), and subsequently with an organochlorine solvent, to facilitate exchange of alkaloids (Lysergol and derivatives) from the basified water into the organochlorine solvent;
g) distilling out the organochlorine solvent completely to obtain Lysergol containing soft mass;
h) dissolving the Lysergol containing soft mass obtained in (g) in an
aqueous solution of weak organic acid; i) treating the above acidic solution obtained in step (h) with
activated charcoal for decolorization; j) filtering the decolorized solution obtained in step (i) at pH 4.0-
4.5; k) adding NH4OH to the filtered solution obtained in step (j) to
make the water phase alkaline to a pH of 9-9.5 and allowing
Lysergol rich fraction to settle down; 1) dissolving the Lysergol rich fraction obtained in step (k) in an
organochlorine solvent; m) extracting organochlorine solvent phase with a solution of glacial
acetic acid in water; n) mixing the acid phase with mineral acid such as hydrochloric
acid to prepare Lysergol hydrochloride; o) treating the Lysergol hydrochloride obtained in step (n) with
NH4OH solution to obtain free Lysergol; p) drying the free Lysergol obtained in step (o) at a temperature in
the range of 55-65° C under vacuum for 9 hours to obtain the
final product.
2. A method according to claim 1, wherein the primary alcohol used for extraction of powdered seeds is selected from ethanol, methanol and n-propanol, preferably ethanol.
3. A method according to claim 1, wherein the organochlorine solvent is selected from ethylene dichloride (EDC), dichloromethane and chloroform, preferably ethylene dichloride (EDC).
4. A method according to claim 1, wherein the weak organic acid solution used is selected from acetic acid, tartaric acid, ascorbic acid and formic acid, preferably acetic acid.
5. A method according to claim 1, wherein the final product obtained has more than 99.2% purity.
6. A method according to claim 1, wherein the final product obtained has impurity less than 0.5%.
7. A method for the isolation and purification Lysergol from the seeds of Kaladana (Ipomoea hederacea) substantially as herein described with reference to the examples.