FIELD OF INVENTION
The present invention relates to a standardized method for extraction and purification of Forskolin from Coleus forskohlii roots. The invention particularly describes a method for the extraction and purification of upto 99.6% pure Forskolin from the roots of Coleus forskohlii (Hindi Name: Coleus, English Name: Coleus). The present invention provides a method consist of drying and grinding the roots and extracting them by organochlorine solvent at a temperature in the range of 45°C -50°C with assay not less than 99.6%, by HPLC analysis.
The present invention also provides an eco-friendly, hygienic and simplified method for commercial manufacturing of Forskolin from natural source, in-spite of synthetic route. Forskolin is a compound that has been identified as a source of therapeutic potential and high commercial value compound, used as anti-glucoma, anti-platelet, bronchospasmolytic, cardiotonic, arterial relaxant, anti-histamatic, anti-aging, anti-allergic and anti-hypotensive
BACKGROUND OF THE INVENTION
Forskolin (Colforsin, Boforsin, Molecula formula: C22H34O7, Molecular weight: 410.15) [α ]D25= -26.190, is a compound that has been identified as a source of therapeutic potential and high commercial value compound, used in lowering blood pressure and producing positive inotropic activity, without affecting myocardial oxygen consumption.. It is libdane diterpene, founding roots of C. forskohlii, growing naturally in dry and barren hills of subtropical Himalayas including Kumaun and Nepal ascending to 2700 Meters, and in the Deccan Peninsula, Gujarat and Bihar; cultivated in Baroda and Maharashtra. Coleus ( C. forskohlii, C. barbaratus, C. aromaticus) is perennial herb with fleshy fibrous roots. Coleus has been used since ancient times in the Indian folk medicine known as Ayurveda traditional medicine. A large scale screening of
medicinal plants by the Central Drug Research Institute, Lucknow, India have shown its unique biological activity like anti-hypotensive, anti-sposmaltic and management of cardiovascular because of Forskolin is used as cardiotonic in the treatment of congestive heart failure, glaucoma therapy. Presently as a source of Forskolin, C. forskohlii is now being cultivated in different part of the world.
Development of Coleus forskohlii as a medicinal crop"of Virbala Saha and Kalakoti B.S., Research Centre, Hoechst Marion Roussel Limited, Mulund, Bombay 400 080, India (http;www.foa.org/docrep/w3735e/w3735e27.hmt) for the development studies to increased yield of root tubers and Forskolin content. The development research entailed studies on natural species populations, evaluation of infra-specific variation, identification of suitable agro-climatic regions, standardization of growth conditions, and genetic improvement of elite genotypes. Analytical results for Forskolin content varies from 0.07 to 0.58% (on dry bio-mass basis) within and between the populations.
Reference may be made to US Patent No. 4734513, which describes a process for synthesizing Forskolin from 9-deoxyforskolin for manufacturing of Forskolin through synthetic route only, not from natural source.
Reference may be made to the article entitled "A total synthesis of (±)-Forskolin " of Shun-ichi Hasimoto Shinji sakata, Motoharu sonegawa and Shiro Lkegami (1998) in J. Am. Chem.. Soc. vol: 110, page: 3670-3672, where the synthetic Forskolin is compared to the authentic sample of natural Forskolin by 400 MHz'H NMR, l3C NMR, IR, MS and Thin-Layer Chromatography.
Reference may be made to the article entitled "Total synthesis of (±)-Forskolin" of E.C. Corey., Paul da silva Jardine and John C. Rohn C. Rohloff (1988) in J.Am. Chem. Soc,Vol 110, pages 3672-3673, where they have described attempts for its synthesis, problems in sterio-selectivity and progress in synthetic root to have Forskolin as identical as from natural source.
Reference may be made to the article entitled "The synthesis of the Labdane Diterpenoid Erigerol and of analogous compounds" of van Frank Kienzle et. al (1990) in the HELVETICA CHIMICA ACTA, Vol. 73, pages 1108-1138, where they have described the attempts to find a suitable root to have Forskolin as from natural source.
Reference may be made to the article entitled "Quantitative Determination of Forskolin by TLc and HPLC" of Inamdar PK, Kanitkar PV et al (1984) in Planta Med. 50 (1) 30-34, which describes Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC) method for quantitative determination of Forskolin.
Reference may be made to the article entitled "Evaluation of Coleus forskohlii accessions for Tuber and Forskolin yield" of Hegde L, Kumar TV and Himabindu K.(http://www.actahort.org/members/showpdf?booknrarnr=675 32),which describes the evaluation of 13 accessions of C forskohlii , from family Lamiaceae, for Forskolin content. This study also reflect the value of the forskolin in modern society.
DRAWBACK OF THE PRIOR ART
1) There is no document for extraction and purification from natural source.
2) As synthetic routes are known but the processes involves toxic chemicals as raw material for synthesis.
3) The steps in the synthetic processes are tedious and multi-step.
4) The end product is mostly a racemic mixture.
5) Attempts to purify the racemic mixture leads to high cost.
To overcome all the aforementioned barriers there is a need to develop a simple and fast method for the recovery of Forskolin from extraction of roots of Coleus forskohlii, a natural source, 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 a method for purification of Forskolin from natural source, with purity upto 99.6%.
Another object of the present invention is to develop economically beneficial manufacturing process by standardization.
Another object of the present invention is to avoid direct human exposure with hazardous chemicals solvents such as pyridine.
Yet another object of this invention is to reduce the long operation span.
Still another object of the present invention is to provide an eco-friendly and hygienic method for commercial manufacturing of Forskolin.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides "A method for the extraction and purification of Forskolin from the roots of Coleus forskohlii" plant comprising the following steps:
a) drying the root of the plant at room temperature to the exclusion of light;
b) grinding the dried roots obtained in step (a) to 10 to 40 mesh size to obtain a free flowing powder;
c) extracting the free flowing powder obtained in step (b) with organochlorine solvent at a temperature in the range of 30-50° 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 35-50° C under vacuum to obtain a soft mass containing Forskolin and other terpenoids;
f)mixing the soft mass containing Forskolin and derivatives obtained in step (e) with n-hexane with stirring to facilitate exchange of resinous material from the soft mass,
g) softmass was chromatographed on silica gel(60-120 mesh) column, for purification of Forskolin, using n-hexane. n-hexane continued to elute till it become colorless; h) elute the column obtained in (g) with 1.0% ethyl acetate in n-hexane and monitor with TLC the elution of less polar components than Forskolin; i) elute column with 3% ethyl acetate in n-hexane after removal of less polar components in step (h), elutes Forskolin rich fraction collected separately and elution monitored with TLC and continued with 4%, 5% and 6% ethyl acetate in n-hexane for elution of Forskolin rich fraction; j) Forskolin rich crop obtained in step (i) was mixed together and distilled to remove solvent, at 30-50°C, under vacuum, soft mass left behind; k) softmass obtained in step (j) dissolved in acetone and refluxed with
activated charcoal for ½ an hour ; 1) filter the charcoalised solution obtained in step (k) using celite as filter aid, the and distill-out the solvent till crystallizations appears; m) the solution obtained in step (1) was kept at 5°C for 2hours and filter,
dry the ppt. at 40 c for 5 hours under vacuum; n) on drying the ppt. obtained in step (m), off-white crystals of Forskolin of more than 99.6% purity, with HPLC analysis, obtains.
In one 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 final product obtained has more than 99.6% purity by HPLC.
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses a method for the isolation of high purity Forskolin. Purity was deciphered either TLC, HPTLC or HPLC as per specification, from the roots of the Coleus forskohlii. Coleus forskohlii is the botanical name of Coleus, the Trade as well as English name of the species used. C. forskohlii grows wild on sun-exposed arid and semi-arid hill slopes of the Himalayas from Simla eastward to Sikkim and Bhutan, Deccan Plateau, Eastern Ghats, Eastern Plateau and rainshadow regions of the Western Ghats in India. Forskolin is a compound that has been identified as a source of therapeutic value and high commercial value compound, used in the treatment of congestive heart failure, glaucoma therapy; antihypertensive, remedy for metastatic condition, in thrombosis , as bronchoditator as well as cardiotonic
According to the invention, the roots of the Coleus forskohlii are dried at room temperature to the exclusion of light and ground to a powedered. The free flowing powder of the roots was extracted with organochlorine solvent at a temperature in the range of 45-50° 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, Cyclohexane: Ethyl acetate:: 65: 35, Merck: Silica gel 6OF254, spots were developed with 7.5 % H2SO4, just after iodization and subsequent heating at 110 C for 10 minutes.) to quantify Forskolin content. It was established that following aforesaid operating parameters after third extraction the herb completely exhaust for Forskolin content. The extract was filtered and mixed .Then filtrate was distilled out at a temperature in the range of 30-50° C under vacuum. The resulting mass left behind after distillation of organochlorine solvent, was mixed with n-hexane with stirring to remove resinous material in n-hexane. n-hexane phase was separated and soft-mass was packed in silica gel (60-120 mesh), using n-hexane as elutent. n-hexane used till it become colorless and 1% ethyl acetate with n-hexane was used for further elution to remove less polar components than Forskolin, with TLC monitoring. Further elution was carried-out with 3% ethyl acetate in n-hexane and major spot of Forskolin appears in TLC. The Forskolin cone, remain in column dominating till 6% ethyl acetate as elutent.The
whole fraction was collected together and solvent was removed at 40 C temp.under vacuum. The resulting softmass was dissolved in acetone and charcoalized, filtered with filter aid. The filtrate was distilled-out at 40°C under vacuum, till crystallization appears. The mass was kept at 5 C for 2 hours and filtered, dry the ppt at 40°C temp, under vacuum for 5 hours, off-white crystals of Forskolin, of more than 99.6% [using HPLC (mobile phase; methanol: water::60:40) Column: C-18 (250 x 4.6mm, 5pm) wave length: 210 nra, flow rate; l.Oml/min] obtains.
The examples below are only illustrative in nature and should not be constructed to limit the scope of the present investigation. EXAMPLE 1:
2.0 kg dried Coleus forskohlii roots were powdered 10 mesh size. The free flowing powder of the roots was extracted with ethylene dichloride (EDC) at 45° C temperature, with 5 times solvent, at each [i.e. for 2.0 kg mass 10 L solvent, at once, (three extractions were done so total solvent used 10L x 3=30L)]. Extraction continued till the Thin Layer Chromatographic finger prints have traces of Forskolin in extract. The extract was analyzed by TLC (mobile phase; Cyclohexane: Ethyl acetate :: 65: 35, Merck: Silica gel 6OF254, spots were developed with 7.5% H2SO4 just after iodization and subsequent heating at 110°C for 10 minutes) to quantify Forskolin content. The extract was filtered and mixed, then the filtrate was distilled out at 45° C under vacuum. As EDC was distilled-out, the mass left on vessels was mixed 100 ml n-hexane, with stirring to remove resinous material in n-hexane. n-hexane phase was separated and soft-mass was chromatographed on column (stationary phase height 6", width 3 ") packed with silica gel (60-120 mesh), using n-hexane as elutent. n-hexane used till it become colorless and 1% ethyl acetate with n-hexane was used for further elution to remove less polar components than Forskolin, with TLC monitoring. Further elution was carried-out with 3% ethyl acetate in n-hexane and major spot of Forskolin appears in TLC. The Forskolin cone, remain in column dominating till 6% ethyl acetate as elutent. The whole fraction was collected together and solvent was removed at 40 C temp, under vacuum. The resulting softmass was dissolved in 50ml acetone and 1.0

gm activated charcoalized was added. The solution was refluxed for ½ hour at 60°C, filtered with filter aid. The filtrate was distilled-out at 40 °C under vacuum, till crystallization appears. The mass was kept at 5 °C for 2 hours then filtered and. ppt dried at 40°C temp, under vacuum for 5 hours. Off-white crystals of Forskolin, 4.02 gm, of purity not less than 99.6% [using HPLC (mobile phase; methanol: water: :60:40) Column; C-18 (250 x 4.6mm, 5µm) wave length; 210 nm, flow rate;1.0ml/min] obtains. EXAMPLE 2:
2.0 kg dried Coleus forskohlii roots were powdered 20 mesh size. The free flowing powder of the roots was extracted with ethylene dichloride (EDC) at 45° C temperature, with 5 times solvent, at each [i.e. for 2.0 kg mass 10 L solvent, at once, (three extractions were done so total solvent used 10L x 3= 30L)]. Extraction continued till the Thin Layer Chromatographic finger prints have traces of Forskolin in extract. The extract was analyzed by TLC (mobile phase; Cyclohexane:Ethyl acetate :: 65: 35, Merck: Silica gel 6OF254, spots were developed with 7.5% H2SO4 just after iodization and subsequent heating at 110 C for 10 minutes) to quantify Forskolin content. The extract was filtered and mixed, then the filtrate was distilled out at 45° C under vacuum. As EDC was distilled-out, the mass left on vessels was mixed 100 ml n-hexane, with stirring to remove resinous material in n-hexane. n-hexane phase was separated and soft-mass was chromatographed on column (stationary phase height 6", width 3 ") packed with silica gel (60-120 mesh), using n-hexane as elutent. n-hexane used till it become colorless and 1 % ethyl acetate with n-hexane was used for further elution to remove less polar components than Forskolin, with TLC monitoring. Further elution was carried-out with 3% ethyl acetate in n-hexane and major spot of Forskolin appears in TLC. The Forskolin cone, remain in column dominating till 6% ethyl acetate as elutent. The whole fraction was collected together and solvent was removed at 40 C temp, under vacuum. The resulting softmass was dissolved in 50ml acetone and 1.0 gm activated charcoalized was added. The solution was refluxed for ½ hour at 60°C, filtered with filter aid. The filtrate was distilled-out at 40 °C under vacuum, till crystallization appears. The mass was kept at 5 °C for 2 hours then filtered and.
ppt dried at 40 C temp, under vacuum for 5 hours. Off-white crystals of Forskolin, 4.11 gm, of purity not less than 99.66% [using HPLC (mobile phase; methanol: water::60:40) Column; C-18 (250 x 4.6mm, 5pm) wave length; 210 nm, flow rate;1.0ml/min] obtains.
EXAMPLE 3:
2.0 kg dried Coleus forskohlii roots were powdered 30 mesh size. The free flowing powder of the roots was extracted with ethylene dichloride (EDC) at 45° C temperature, with 5 times solvent, at each [i.e. for 2.0 kg mass 10 L solvent, at once, (three extractions were done so total solvent used 10L x 3=30L)]. Extraction continued till the Thin Layer Chromatographic finger prints have traces of Forskolin in extract. The extract was analyzed by TLC (mobile phase; Cyclohexane: Ethyl acetate :: 65: 35, Merck: Silica gel 60F254, spots were developed with 7.5% H2SO4 just after iodization and subsequent heating at 110°C for 10 minutes) to quantify Forskolin content. The extract was filtered and mixed, then the filtrate was distilled out at 45° C under vacuum. As EDC was distilled-out, the mass left on vessels was mixed 100 ml n-hexane, with stirring to remove resinous material in n-hexane. n-hexane phase was separated and soft-mass was chromatographed on column (stationary phase height 6", width 3 ") packed with silica gel (60-120 mesh), using n-hexane as elutent. n-hexane used till it become colorless and 1% ethyl acetate with n-hexane was used for further elution to remove less polar components than Forskolin, with TLC monitoring. Further elution was carried-out with 3% ethyl acetate in n-hexane and major spot of Forskolin appears in TLC. The Forskolin cone, remain in column dominating till 6% ethyl acetate as elutent. The whole fraction was collected together and solvent was removed at 40 C temp, under vacuum. The resulting softmass was dissolved in 50ml acetone and 1.0 gm activated charcoalized was added. The solution was refluxed for ½ hour at 60°C, filtered with filter aid. The filtrate was distilled-out at 40 C under vacuum, till crystallization appears. The mass was kept at 5 °C for 2 hours then filtered and. ppt dried at 40°C temp, under vacuum for 5 hours. Off-white crystals of Forskolin, 4.12 gm, of purity not less than 99.61% [using HPLC (mobile phase; methanol:
water::60:40) Column; C-18 (250 x 4.6mm, 5]am) wave length; 210 nm, flow rate;1.0ml/min] obtains.
EXAMPLE 4:
2.0 kg dried Coleus forskohlii roots were powdered 40 mesh size. The free flowing powder of the roots was extracted with ethylene dichloride (EDC) at 45° C temperature, with 5 times solvent, at each [i.e. for 2.0 kg mass 10 L solvent, at once, (three extractions were done so total solvent used 10L x 3=30L)]. Extraction continued till the Thin Layer Chromatographic finger prints have traces of Forskolin in extract. The extract was analyzed by TLC (mobile phase; Cyclohexane:Ethyl acetate :: 65: 35, Merck: Silica gel 6OF254, spots were developed with 7.5% H2SO4 Just after iodization and subsequent heating at 110°C for 10 minutes) to quantify Forskolin content. The extract was filtered and mixed, then the filtrate was distilled out at 45° C under vacuum. As EDC was distilled-out, the mass left on vessels was mixed 100 ml n-hexane, with stirring to remove resinous material in n-hexane. n-hexane phase was separated and soft-mass was chromatographed on column (stationary phase height 6", width 3 ") packed with silica gel (60-120 mesh), using n-hexane as elutent. n-hexane used till it become colorless and 1 % ethyl acetate with n-hexane was used for further elution to remove less polar components than Forskolin, with TLC monitoring. Further elution was carried-out with 3% ethyl acetate in n-hexane and major spot of Forskolin appears in TLC. The Forskolin cone, remain in column dominating till 6% ethyl acetate as elutent. The whole fraction was collected together and solvent was removed at 40 C temp, under vacuum. The resulting softmass was dissolved in 50ml acetone and 1.0 gm activated charcoalized was added. The solution was refluxed for ½ hour at 60°C, filtered with filter aid. The filtrate was distilled-out at 40 °C under vacuum, till crystallization appears. The mass was kept at 5 °C for 2 hours then filtered and. ppt dried at 40°C temp, under vacuum for 5 hours. Off-white crystals of Forskolin, 4.22 gm, of purity not less than 99.6% [using HPLC (mobile phase; methanol: water::60:40) Column; C-18 (250 x 4.6mm, 5µm) wave length; 210 nm, flow rate;1.0ml/min] obtains.
THE MAIN ADVANTAGES OF THE PRESENT INVENTION:
1.) The product is from natural sources ,
2.) The whole process of extraction and purification is eco-friendly than synthetic route.
3). Expensive and hazardous chemicals are not in use any where in the process.
4) As the product is from herb used in traditional system of medicines, it is
non- toxic. The product (i.e. Forskolin) isolated from C. forskohlii does not
have any unknown/ foreign toxic compound /impurity, while in case of
synthetic Forskohn, traces of unknown impurity may lead to a fatal threat.
5) A standardized extraction and purification methodology available.

We Claim:
1. A method for the extraction and purification of Forskolin from the roots of Coleus forskohlii plant comprising the following steps:
a) drying the roots of the plant at room temperature to the exclusion of light;
b) grinding the dried roots obtained in step (a) to 10 to 40 mesh size to obtain a free flowing powder;
c) extracting the free flowing powder obtained in step (b) with organochlorine solvent at a temperature in the range of 35-50° 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 35-50° C under vacuum to obtain a soft mass containing Forskolin and derivatives;
f) mixing the soft mass containing Forskolin and derivatives obtained
in step (e) with n-hexane under stirring to remove resinous
material in n-hexane and settled mass containing Forskolin rich
fraction separately.
g) purifying the settled mass containing Forskolin rich fraction obtained
in step (f)above by column chromatography using n-hexane with
ethyl acetate(l-6%) as elutant, to obtain Forskolin rich fraction,
which on distillation gives soft mass of Forskolin rich fraction;
h) dissolving the forskolin containing soft mass obtained in (g) in
acetone and treating it with activated charcoal for decolorization; i) filtering the decolorized solution obtained in step (h) above with a filter aid. j.) distilling the of solution obtained in step (i) to crystallize the
Forskolin; k.) drying of Forskolin obtained in step (j) at a temperature up-to 40°C, under vacuum. The dried Forskolin having more than 99.6% purity by HPLC analysis.
2. A method according to claim 1, wherein the organochlorine solvent used, is selected from ethylene dichloride (EDC), dichloromethane and chloroform, preferably ethylene dichloride (EDC).
3. A method for the isolation and purification of Forskolin from the roots of Coleus forskohlii substantially as herein described with reference to the examples.