|Title of Invention||
PROCESS FOR PURIFICATION OF SOLANESOL (95-%) FROM CRUDE/ENRICHED EXTRACTS OF TOBACCO GREEN LEAF/TOBACCO CURED LEAF/TOBACCO WASTE.
|Abstract||A process for purification of solanesol (95+%) from crude/ enriched extracts of tobacco green leaf/ tobacco cured leaf/ tobacco waste consisting of solvent extraction of raw material, enrichment of the extract containing solanesol by cooling at low temperature, further enrichment involving suitable adsorbent, isocratic elution with a solvent and recrystallisation from a solvent mixture at low temperature is described in this specification.|
|Full Text||The present invention relates to process for purification of solanesol (95+%) more particularly from crude/ enriched extracts of tobacco green leaf/ tobacco cured leaf/ tobacco waste.
Solanesol, a tri-sesquiterpenoid alcohol is present in many botanical and biological species. It is the starting material for synthesis of Vitamin K2, Vitamin E, Coenzyme Q9, Coenzyme Q10 (Colowick and Kaplan, 1975) and anti-cancer drug potentiating agents like N-solanesyl-N,N1-bis (3,4-dimethoxybenzyl) ethylenediamine (Suzuki et al., 1990 and Tomida et al., 1990). Tobacco is the richest source of solanesol, which has excellent prospects as drug intermediate. Rowland et al. (1956) first isolated solanesol by extracting tobacco with methanol and ether, removed the water soluble components and precipitated solanesol by addition of acetone to the concentrated extract and cooling at
-27°C. Grossman et al. (1963) employed a new technique which involved initial petroleum ether extraction, concentration of the neutral fraction after removing acids and
bases under vacuum. The waxes were removed from acetone solution at 4°C and the
dewaxed solution was subjected to molecular distillation at 300°C and 100-200 µ pressure. Solanesol was isolated from unsaponifiables of silk worm feces and mulberry leaves (Fukuwa et al., 1966; Toyoda et al, 1969, 1970) and potato leaves (Asahina et al., 1971) by adopting the techniques like solvent extraction, fractional crystallization, adsorption chromatography and molecular distillation. Narasimha Rao and Chakraborty (1979), developed a process involving selective extraction of solanesol with methanol followed by cooling the extract to precipitate solanesol and recrystallisation using methanol: chloroform (2:1) yielding a fraction containing approximately 75% solanesol. Takemura and Amano (1980) treated the unsaponifiable fraction of tobacco extract with mixture of acetone:methanol (2:1), insoluble matter was removed, solution was concentrated.dissolved in acetone:methanol (2:100), insoluble matter was removed, treated with charcoal and solanesol was precipitated at low temperature. The product was of high purity with 70% recovery. The patented process for recovery of solanesol involved centrifugal liquid partition chromatography using hexane as a stationary phase and a hydrous alcohol such as methanol, ethanol etc. having 5-10% water content as mobile phase. Subsequently, the liquid flow direction was reversed, followed by fractionation with hexane to obtain solanesol, which was further purified by HPLC (Jap. Patent No. 63190840). According to the Chin. Patent No. 1056486, n-hexane was used as extracting solvent and a pasty solanesol was obtained after two extractions followed by concentrations. In another process, solanesol was extracted from moldy or broken tobacco by extraction with solvents such as n-hexane, ultrafiltration and concentration by membrane evaporation (Chin. Pat. No. 1087076). As per the Chin. Pat. No. 1094705, extraction of unsaponifiable matter of solanesol involved addition of solvent and catalyst for saponification reaction, filtering, mixing, separation, ultrafiltration and concentration. In the technique of sprinkling extraction, pretreated tobacco was sent into a rotating grid, repeatedly and cyclically sprinkled and extracted by a solvent, finally the extraction liquid was purified and concentrated to obtain solanesol (Chin. Pat. No. 1108641). In the Chin. Pat. No. 1115751, tobacco after nicotine extraction was dried, crushed, extracted several times with C5 - C12 alkane as solvent. Extracts mixed and concentrated,
saponified with alcohol and alkali solution, after removing water and solvent, treated with mixed ketone and alcohol liquid I & II separately and cooled to separate pure solanesol crystal. Another Chin. Pat. No. 1118775 for solanesol recovery involved continuous extraction and twice settling process and tail-gas adsorption process. Recently, supercritical fluid extraction was adopted for extraction of solanesol (Chin. Pat. No. 1294111).
Summary of the invention
For extraction of solanesol on an industrial scale, there are two obvious routes viz., 1) hexane extraction followed by enrichment methods and 2) methanol extraction and fractional crystallisation. Solanesol and its esters can be extracted with hexane along with other lipids but enrichment of the hexane extract containing 10-15% solanesol, presents many problems and is cumbersome. Similarly, methanol extraction and cooling of the extracts, results in a fraction containing about 50% solanesol which can be enriched to about 75% by recrystallisation. As solanesol is utilised in the pharmaceutical industry for preparation of drug intermediates, much higher purity (95+%) is desirable. However, some of the techniques employed to attain higher purity of solanesol are cost intensive and time consuming.
The main object of the present invention is a process for purification of solanesol (95+%) from crude / enriched extracts of tobacco green leaf / tobacco cured leaf / tobacco waste, which obviates the draw backs as detailed above.
In an embodiment, the present invention provides a process for purification of a fraction containing about 50% to 75% solanesol obtained using a polar solvent like ethanol or methanol or isopropanol for extraction, may be by adsorbing the enriched extract on an adsorbent like activated bleaching earth or silica gel or alumina, leaching with non-polar solvent like benzene or toluene and recrystallisation of the solanesol rich fractions to yield solanesol (95+%).
In another embodiment, the present invention provides a process for enrichment of hexane / benzene / toluene extract containing about 10-15% solanesol may be by adsorbing on activated bleaching earth or diatomaceous earth, elution with hexane or benzene or toluene to yield extract containing 20-30% solanesol, further enrichment by removing waxy material by methyl ethyl ketone or isopropyl alcohol precipitation, adsorption on adsorbent like alumina or silica gel, elution with dichloroethane or benzene or toluene and recrystallisation.
In yet another embodiment, the present invention provides a process for enrichment of hexane extract containing about 10-15% may be by dissolving in 95:5 or 90:10 methanol:
chloroform, cooling the filtrate between 0°C to - 14°C, adsorbtion of the precipitate thus obtained on adsorbent like activated bleaching earth or alumina or silica gel, elution with dichloroethane or benzene and recrystalliation.
Importance of the process lies in the selection of a polar solvent for extraction of tobacco green leaf/ tobacco cured leaf/ tobacco waste and significant enrichment by cooling of the concentrated extract due to presence of lesser amount of non-polar substances which also makes further enrichment easy, compared to techniques reported in literature wherein fractional crystallisation, molecular distillation, ultrafiltration followed by membrane evaporation and column chromatography are employed. In column chromatography on alumina, silicic acid or florisil, the cost and time are high, as a selective gradient elution of the principal compound is involved. In the present process, cheaper adsorbents used, drastic reduction of adsorbent requirement, possibility of avoiding column, scope for regeneration of adsorbent and the single solvent employed to elute the compound contribute to the novelty. Another important feature is the adaptability of the process for extraction of solanesol (95+%) from the green leaf.
The following examples are given by way of illustration and therefore should not be construed to limit the scope of the present invention.
Tobacco powder (1kg) containing 1.8% solanesol was extracted with methanol in a Soxhelt extractor. Methanol extract was concentrated (133 g) and dissolved in 5-10 parts
of methanol: isopropanol (4:1) and cooled at -14°C. Precipitate obtained after cooling was dissolved in toluene to eliminate highly polar substances present yielding 27.1 g of enriched fraction 1 having 53.4% solanesol. This fraction was loaded on commercial grade silica gel (200 g) and eluted with toluene. Solanesol rich fractions were collected, pooled and solvent was removed by vacuum distillation to obtain enriched fraction 2 (16.0g) containing 90% solanesol. Pure solanesol (13.7g) was obtained by
recrystallisation of enriched fraction 2 from methanol: chloroform (9:1) at -14°C.
EXAMPLE - 2
Tobacco powder (1 kg ) containing 0.9% solanesol was extracted with 5 lit of isopropyl alcohol. Extract was concentrated to obtain 91 g of crude extract containing 9.0% solanesol. This crude extract was mixed with 900 g of activated bleaching earth and eluted with hexane. When solvent was removed by vacuum distillation, 41 g of enriched fraction 1 containing 50% solanesol was obtained. This fraction was loaded on alumina (300g) and eluted with benzene. Solanesol rich fractions were collected, pooled and solvent was removed by vacuum distillation, yielding enriched fraction 2(8.33 g) containing 90% solanesol. Pure solanesol (6.78 g) was obtained by recrystallising
enriched fraction 2 from methanol: dichloroethane (9:1) at -10°C.
EXAMPLE - 3
Crude hexane extract (940 g) of tobacco containing 11.2% solanesol was dissolved in 10 volumes of methanol: chloroform (9:1). Precipitate was removed by filtration and filtrate
was cooled at -15°C. The precipitate (enriched fraction 1, 105 g) having 80% solanesol
was dissolved in dichloroethane and loaded on silica gel (1kg) and eluted with dichloroethane. The enriched fraction 2 (90.0 g) containing 89.5% solanesol obtained after pooling the solanesol rich fractions and solvent removal by vacuum distillation was
recrystallised from acetone at 0°C to yield pure solanesol (79.5 g). The main advantages of the present invention are the following:
1. Use of polar solvent for extraction and subsequent enrichment by cooling, thus,
making attainment of higher purity easy.
2. Avoiding cost intensive and time consuming techniques like fractional crystallisation,
molecular distillation, ultrafiltration followed by membrane evaporation and column
chromatography, enhances the economic viability of the process, particularly with the
use of cheaper adsorbents, drastic reduction in the adsorbent requirement, possibility
of avoiding column, scope for regeneration of the adsorbent and isocratic elution of
the compound with a solvent.
3. Purity (95+%) of solanesol obtained in the process helps in utilization of the
compound in drug development.
4. It can be adopted for processing tobacco green leaf for extraction of pure solanesol,
thus enlarging the raw material base.
Asahina, M, H. Kato and H. Fukawa (1970) Chem. Abstr. 74:28733 t Colowick, S. P. and N. O. Kaplan (Eds.) (1975) Methods in Enzymology Vol. 18C Fukawa, H., M. Toyoda, T. Shimizu and M. Murohashi (1966) Tetrahedron Lett.
Grossman, J. D., R. M. Ikeda, E.. J. Deszyck and A. Bavley (1963) Nature 199:661-663 Narasimha Rao, C. V. and M. K. Chakraborty (1979) Research and Industry 24:83-86 Patent No. CN 1056486 (1991) Patent No. CN 1087076 (1994) Patent No. CN 1094705 (1994) Patent No. CN 1108641 (1995) Patent No. CN 1115751 (1996) Patent No. CN 1118775 (1996) Patent No. CN 1294111 (2001) Patent No. JP 63190840 (1988)
Rowland, R. L., P. H. Latimer and J. A. Giles (1956) J. Amer. Chem. Soc. 78:4680-4683 Suzuki, H., et al. (1990) Jpn. J. Cancer Res. 81(3):298-303 Tomida, A., et al. (1990) Jpn. J. Cancer Res. 81(11):1184-1190
Toyoda, M., H. Fukawa and T. Shimizu (1969) Nippon Nogei Kogaku Kaishi 43:688-693 Toyoda, H., H. Fukawa, H. Seo and T. Shimizu (1970) Chem. Abstr. 73:63143 b
1. A process for purification of solanesol from crude/ enriched extracts of tobacco green
leaf/ tobacco cured leaf/ tobacco waste which comprises use of organic solvent for
extraction of tobacco green leaf/ tobacco cured leaf/ tobacco waste, enrichment of
solanesol in the extract by low temperature cooling, further enrichment using a
suitable adsorbent, isocratic elution with a solvent and recrystallisation from a solvent
mixture at low temperature to yield pure solanesol (95+%) and herein described with
reference to the examples
2. A process as claimed in claim 1, that employs organic solvents like hexane or toluene
or dichloroethane or chloroform or isopropanol or methanol
3. A process as claimed in claims 1 and 2 which uses adsorbents like activated bleaching
earth or silica gel or alumina and subsequent isocratic elution with organic solvents
like hexane or benzene or dichloroethane or toluene
4. A process as claimed in claims, 1, 2 and 3 where recrystallisation of the final product
was accomplished using solvent mixtures like methanol : chloroform or methanol :
isopropanol or methanol : dichloroethane or methyl ethyl ketone in varying
proportions of 2:1 to 9:1 at a low temperature ranging from 0°C to -15°C
|Indian Patent Application Number||1071/DEL/2002|
|PG Journal Number||51/2007|
|Date of Filing||25-Oct-2002|
|Name of Patentee||INDIAN COUNCIL OF AGRICULTURAL RESEARCH|
|Applicant Address||KRISHI BHAWAN, DR. RAJENDRA PRASAD ROAD, NEW DELHI-110001, INDIA.|
|PCT International Classification Number||C07C 33/02|
|PCT International Application Number||N/A|
|PCT International Filing date|