Title of Invention	PROCESS OF OBTAINING SOLASODINE
Abstract	1.A process for obtaining Solasodine comprising: i)providing a selective source obtained by transformation of the Solarium species to cultures of said Solatium species having high content of Solasonine .; and ii)subjecting the glycoalkaloid Solasonine from said culture medium to enzymatic hydrolysis to convert into Solasodine.

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FORM2 THE PATENTS ACT, 1970 (39 of 1970) COMPLETE SPECIFICATION (See section 10; rule 13) PROCESS FOR OBTAINING SOLASODINE 2. (a) Registrar, University of Pune (b) Pune 411 007, State of Maharashtra, India, (c) established under the Poona University Act, 1948. The following specification (particularly) describes the nature of this invention (and the manner in which it is to be performed) ORIGINAL GRANTED 27-4-2004 Field of the invention The present invention relates to process for obtaining of Solasodine from Solanum Species especially ransformed Solarium species having high content of Solasonine glycoalkaloid and involving simple enzymatic hydrolysis of the Solasonine glycoalkaloid to release the desired Solasodine. In particular, the above process of simple and cost effective increased production of Solasodine from Solanum species involves genetically transformed root cultures of 5". Khasianum and Aspergillus niger mediated cleavage of its glycoalkaloid Solasonine for the release of Solasodine and its extracts. Background of the invention It has been observed that there is increased application of cortisones and other steroidal drugs in the treatment of Addison's disease, rheumatic arthritis, asthma, leukemia and psorias.is while progesterone and estrone are used in HRT and in the preparation of contraceptives. Since 1947, diosgenin was used commercially for the production of corticosteroid hormones (Marker R.E et al., 1947). Diosgenin can be readily converted to 16-dehydropregnenolone acetate a key intermediate in the synthesis of steroid drugs, e.g., cortisone, and sex hormones such as progesterone, estrone and testosterone (Sree et al., 1988). Dehydropregnenolone acetate (16-DPA) Diosgenin occurs as rhamno-rhamno-glucoside attached to dioscin, in the rhizome of several species of Dioscorea. However, the commercial cultivation of Dioscorea plant is not encouraging due to its stringent climatic conditions slow growth and the delayed maturity of tubers. This has created an acute shortage of raw material and now it has become an important mcentive to select a commercially profitable source for 16-DPA and increase its present production. Solasodine, a steroidal alkaloid has been considered as a potential alternative to diosgenin for commercial steroid drug synthesis (Glanes et al., 1984). These two compounds possess similar structure and share the characteristic that they can be readily converted to 16- dehydropregnenolone acetate (Sree et al., 1988). Solasodine occurs in various genera among these Solanum is the largest. Many species of this genus produce Solasodine (Jaggi et al., 1994) but only a few of these Solanum species are considered for commercial production. In plant, Solasodine is present as its glycoalkaloid viz. Solasonine and it contains as sugar component a trisaccharide, L-rhamnosido-D-galactosido-D-glucose (Schreiber, 1968). Table 1. Solanum species considered for commercial production: S. surattense Burm.f. (ripe berries) 1.10-2.08 ■ Gadwall977 S. trilobatum Linn (ripe berries) 3.50 Kaul and Zutshi 1977 S. verbascifoliwn auct. non. Linn.(ripe berries) 0.01-3.82 Kaul and Zutshi 1977 Therefore it is clear that S. khasianum is one of the species reported to produce maximum Solasodine. The conventional method of Solasodine production requires berries of Solanum khasianum Clarke and certain high performance equipments, chemicals and solvents. The conventional method of production of Solasodine requires large-scale cultivation of S.khasianum, harvesting the berries and extraction of Solasodine. Cultivation of Solanum khasianum Clarke plants requires large areas of land and extensive labor starting from planting, handpicking of berries, drying of berries and extraction of Solasodine. From the time of sowing to the harvest it takes about six months, which is a considerable amount of time. In practice there is considerable variation in the management of individual farms and that is reflected in the cost of cultivation and the profit. The seeds are known to loose their viability within two months of harvest; besides this they are also known to have dormancy. The steroidal alkaloid obtained does not show consistency owing to cross-pollination and the presence of chromosome B that leads to genetic instability. Newer pests (fruit borer, root eating grubs, leaf eating caterpillars and leaf hoppers) and diseases leaf spot disease, wilting caused by Fusarium oxysporum have been reported on this crop. Drying and attack of berries by fungus causes loss of crop. There is lack of information on several aspects of agronomy like optimum plant population, levels of nutrients, and effect of fertilizers on Solasodine content etc. Synchronous maturity of fruits for maximum Solasodine production is far from reality (Asolkar and Chadha, 1979). In the conventional method, conversion of Solasonine to Solasodine involves acid hydrolysis of the crude extract of the berries for hours together, which consumes excessive energy, time and money and requires large tanks and high temperature. The cooled reaction mixture deposits the sparingly soluble hydrochloride from which the free base may be regenerated. Therefore the known technology for extraction of Solasodine using conventional method requires among other equipments glassed steel reactors, high pressure stainless steel hydrogenation reactors, stainless steel fermentors, stainless steel solvent .distillation units and range of engineering services including compressed air, cryogenic systems, steam, electricity, tower water and auxiliary equipment and installations. (Kunjithapadam, 1982). Keeping these in view, today prospects for Solasodine from berries is bleak as a result of which majorities of the pharmaceutical companies are aborting the production of the same. It would be highly advantageous if the difficulties of conventional farming and extraction were overcome by some technique and would therefore be seen as a welcome replacement. Cell culture has been one such widely used technique. As parameters such as nutrition, plant growth regulators, light precursors, explant source, growth phase, age of culture and use of elicitors can be manipulated to increase the production of Solasodine in cell cultures hey are not discussed here. One obvious feature of cell culture is its ability for immobilization in a matrix and therefore special reference is made to immobilization of cell suspension cultures and its effect on Solasodine production in some Solarium species. Table 2. The callus and cell suspension cultures raised in some of the Solanum plants are listed here under with their alkaloid content: Table 3: hereunder shows explant sources reported to affect Solasodine production. E.g. in S. khasianum (Uddin and Chaturvedi 1979). Explant source Solasodine mg/gm DW Seedling 0.67 Root 0.41 Radicle 0.54 Stem 0.49 Leaf 0.48 Immobilized plant cells are similar in many respects to callus tissues (Dainty et al., 1985) and resemble the situation in vivo in that the cells grow together in a particularly organized environment and are maintained in a "stationary" condition with increased stability. Immobilized cells show the advantage of scaling up in a bioreactor as well. Immobilized cells accumulated two-fold higher alkaloid content than the free suspended cells. S. aviculare cells immobilized within Ca-alginate gel beads enhanced production of Scopolin (Roisin et al., 1997). Solasodine content in immobilized cells of 5". suratense was around two-fold that of in free cells at the same culture stage (Barnabas and David, 1988). Unfortunately cell cultures often differ qualitatively in their spectrum of secondary products from the parent plant and as is seen here quantitative changes occur as well. They are also accompanied by an apparent loss of the ability to accumulate secondary compounds due to the lack of organization. The reasons may be listed as below: i)The lack of expression in non-specialized cells of genes that control the essential steps in the biosynthetic pathway. ii)The diversion of substrate away from secondary product formation. iii)The non-operation of transport system by which potentially toxic end products may be removed from the biosynthetic site. iv)The non-availability of storage sites in which secondary metabolites would be normally be sequestered. v)The unregulated catabolism of synthesized product (Charlwood et al., 1990). As seen above the cell cultures of Solarium species; both free and immobilized do not show secondary metabolism comparable to the plant. The use of organized cultures to study the biosynthesis, transport, storage and turnover of natural products is an attractive approach. Plant tissue culture techniques offer cultivation root cultures devoid of plant. Therefore roots are now considered to contain the major sites of synthesis of many secondary metabolites. Roots derived from callus tissue of S.khasianum accumulated up to 5% steroidal alkaloids including Solasonine, Solanidine, which were also found in the intact plants (Kokate and Radwan 1979). However, the basic disadvantage with organized root culture was the growth limiting factor due to' which sufficient biomass could not be generated for extraction of Solasodine. Moreover, the present technology for extraction of Solasodine from hairy roots using conventional method requires, as discussed earlier, among other equipments glassed steel reactors, high pressure stainless steel hydrogenation reactors, stainless steel fermentors, stainless steel solvent distillation units and range of engineering services including compressed air, cryogenic systems, steam, electricity, tower water and auxiliary equipment and installations. Besides this the multi-step procedure is cumbersome and also involves processes like acid hydrolysis for conversion of Solasonine to Solasodine, which are not commercially preferred due to the involvement of high temperature operating conditions. It is also seen that although hairy root cultures are a very feasible source for Solasodine extraction, the extraction following the known processes could not be effected entirely and the product required to be obtained by crushing and damaging the cells of the hairy root cultures. Objects of the invention An object of the present invention is to provide an extraction grocess for obtainiing Solasodine from Solanum species achieving good yields and making it a continuous process. Yet further object is directed to effectuate the process for obtaining Solasodine without destroying the hairy root cultures of Solanum species thus making the extraction a continuous process. A further object of the present invention to economize the process for the release of Solasodine from feasible selective source of Solasodine that does away the use of high temperature and acid hydrolysis as adopted for field grown plants. Yet further object of the present invention to economize the process for the release of Solasodine that would avoid complex and cost extensive defatation, alcohol extraction, acid hydrolysis and basification and thereby achieve the desired isolation of Solasodine with reduced chemicals, equipments, time and energy involvement. A further object of the present invention is to provide a selective feasible source for Solasodine for increased, continuous and rapid production of Solasodine. A further object of the invention is directed to consistently provide the Solasodine of high content from a readily available source. Another object of the present invention is to provide an improved process for the increased, continuous and rapid production of Solasodine from a selective Solarium species having good content of Solasodine. Yet another object of the present invention is to provide a process for extraction Solasodine that is industrially feasible and economically attractive. Further object of the present invention is to overcome the drawbacks of prior art by providing an easy and effective two-phase extraction of Solasodine for continuous production in a controlled environment. Yet another object is to provide for a commercially viable source of Solasodine for use in steroidal drug synthesis. Summary of the invention Thus according to an aspect of the present invention, there is provided a process for extraction Solasodine from selective Solarium species comprising: ained byransformation of the Solarium species to cultures of said Solarium species having high content of Solasonine .; and ii) subjecting the glycoalkaloid Solasonine from said culture medium to enzymatic hydrolysis toconvert into Solasodine. Preferably, in the above process of the invention glycoalkaloid Solasonine is cleaved by way of an Aspergillus njger mediated cleavage. Such a process clearly avoids the complex and cost extensive production of defatation, alcohol extraction, acid hydrolysis and basification followed under the conventional art of producing Solasodine. Preferably, the above selective source of Solasodine used in the process comprise hairy root cultures of Solarium species grown in nutrient medium. The above selective culture is preferably obtained by Agrobacterium rhizogenes mediated transformation and hairy root production in Solarium khasianum. In accordance with a preferred aspect of the present invention, there is provided a process for obtaining Solasonine from Solanum species comprising: i) providing a selective source obtained by MTV transformation) of the Solanum species to cultures of said Solanum species having high content of Solasonine.; and ii) subjecting the glycoalkaloid Solasonine from said culture medium to enzymatic hydrolysis to convert into Solasodine. iii) extracting Solasodine from the medium by solvent extraction. Preferably, the solvent extraction is carried out with chloroform using a two-phase extraction system wherein the chloroform is evaporated to dryness to give pure Solasodine. In accordance with yet further preferred aspect of the present invention, there is provided a process for extraction of Solasodine from Solanum species in particular hairy root cultures comprising: a. genetic transformation of the Solanum species. b. growing the culture in plant tissue culture medium. c. permeabilizing the cells to leach glycoalkaloid Solasonine into the medium. d. enzymatically hydroiyzing the glycoalkaloid to convent IND Solasodine. e. extracting Solasodine from the medium using solvent system. Importantly, in the above process the step of permeabilization of the hairy roots is attended without causing any loss in their viability to facilitate the leaching of glycoalkaloid Solasonine into the culture medium. ' In accordance with another aspect of the present invention it is found that the presence of a combination of elicitor and precursor can synergistically further improve the Solasonine content in the hairy root cultures in accordance with the present invention. In accordance with yet further aspect of the present invention there is provided a process for the manufacture of steroidal drugs having Solasodine comprising using Solasodine obtained from extraction of Solasodine from selective Solarium species comprising: a) providing a selective source obtained by genetic transformation of the Solarium species to cultures of said Solarium species having high content of Solasonine .; and b) subjecting the glycoalkaloid Solasonine from said culture medium to enzymatic hydrolysis to convert into Solasodine. Detailed description The present invention thus provides an improved process for the increased, continuous and rapid production of Solasodine from hairy root cultures of Solarium khasianum preferably by using permeabilizing agents for the extraction of the glycoalkaloid Solasonine and an Aspergillus niger mediated cleavage of the glycoalkaloid Solasonine. This invention provides production of Solasodine without the use of field grown plants with an increased and continuous recovery of Solasodine mediated by Aspergillus niger. Following the above process of the invention, the amount of Solasodine produced by the roots is comparable to that present in the ripe berries. Solasodine can be obtained every 6-8 weeks as compared to 6-7 months required by the field grown plants. Suitable bioreactors can be used to further ensure a rapid and continuous process. The process involves obtaining Agrobacterium rhizogenes mediated transformation and hairy root production in S. khasianum and culture of hairy roots in nutrient medium free from phytohormones and subjecting the culture medium to Aspergillus niger lechate in order to obtain increased yields of Solasodine. The hairy roots for extraction of Solasodine can be initiated from any part of the Solanum species plant. In a preferred feature the hairy roots can be initiated from leaf tissues of S. khasianum using Agrobacterium rhizogenes; A4 strain. Agrobactenum rhizogenes are soil-borne bacterial pathogens of plants and these bacteria enter into any wounded part of plant cells. The Ri plasmid of A. rhizogenes induces the development on inoculated plant cells. Considerable increase in biomass and alkaloid accumulation in root cultures by R i-TDNA can be achieved. Agrobacterium rhizogenes with the T-DNA in the R i- plasmid when inoculated on the cut part —■ '■ -■■ ■ of the plant cells of Solanum species preferably the surface of the leaves of Solanum species, and when co-cultured with the same, leads to the production of hairy roots. The roots so obtained from transformation can be grown in a nutrient medium without hormones to produce root biomass that can be used for the extraction of Solasodine. Any plant tissue culture medium can be used for the purpose of this invention. In a preferred feature, elicitors can be added to the culture medium that can induce a pathogenic stress in the root, which enhances Solasodine production. Usually the pathogen, which attacks a specific plant, is useful as elicitor because such specificity of host pathogen interactions leads to a biotic stress that improves the production of metabolite (a defense mechanism or stress induced enhancement). Any pathogenic fungal organism like Colletotrichum, Botrytis, Pythium, Phytopthom, Alternaria etc. can be used as an elicitor for enhancing Solasodine production. The preferred elicitor used can be mycelial mat of pathogenic fungus Fusarium oxysporum. Similarly the precursor to Solasodine can be preferably added to increase the production of Solasodine. Some known intermediates like cholesterol, squalene, cycloartenol or a primary precursor acetyl CoA can be added as a precursor. Preferably, cholesterol, one of the precursors to Solasodine, can be added to the medium to enhance the production of Solasodine. As mentioned above, the presence of the elicitor and the precursor is found to also synergistically increase the Solasonine content and thereby the Solasodine content in the hairy root cultures. In a further aspect of the invention, permeabilizing agents are added to the culture medium to permeabilize the hairy roots without causing a loss in their viability and help in leaching of glycoalkaioid Solasonine into the culture medium. Permeabilization is a process where substances that can change / relax the membrane permeability to some extent are used so as the product can leach out from the cells. Permeabilizing agents like Tween-20, DMSO, copper sulphate, chitosan, organic solvents like chloroform, synthetic substances like pluronic F-68 etc. can be used for this purpose. Preferably permeabilizing agents such as DMSO (dimethyl sulfoxide) and Tween-20 are added to the culture medium. These can be synergistically used in combination to enable a higher extraction of the glycoalkaioid Solasonine into the culture medium. The culture medium thus containing the extracted glycoalkaloid is then subjected to Aspergillus niger lechate in order to enzymatically cleave the sugar moiety from the aglycone of the glycoalkaloid Solasonine to obtain Solasodine. Other organisms like mucor, Rhizopus, Endomyces, Aerobactor or Clostridium also exhibit such glucosidase/ galactosidase activity however Aspergillus is used as a commercial source for the enzyme amyloglycosidases that is responsible for the cleavage. Subsequently the medium rich in Solasodine can be analyzed for the content of Solasodine. For the purpose of the present invention, Aspergillus niger is preferably grown for a period of 5-7 days in czapek dox medium and its lechate is added to the spent medium of hairy roots grown for five-six weeks. The released Solasodine is extracted in a solvent preferably chloroform using a two-phase extraction system and the chloroform is evaporated to dryness to give pure Solasodine. Other solvents like petroleum ether, which can be used for two-phase extraction systems, can also be used for this purpose however chloroform is the preferred solvent. The parameters for the Aspergillus niger cleavage of the glycoalkaloid were selectively identified as further illustrated under the illustrative examples. The operating conditions for this conversion of Solasonine to Solasodine were also likewise identified. The details of the invention, its objects and advantages are explained hereunder in greater detail in relation to non-limiting exemplary illustrations hereunder: Examples: A comparative study of the Solasodine content of Normal roots (untransformed) with that of transformed undifferentiated hairy roots, transformed plants (shoots only) and transformed plants (roots only) was carried out as detailed hereunder: EXAMPLE 1 In vitro micropropagation of S. khasianum was established on MS medium (Murashige and Skoog, 1962) supplemented with 0.4 mg/1 of lAA, 0.2 mg/1 of Kn and lmg/1 of BAP. The leaves of these were cut into 1 cm2, pre-cultured for 2 days on MS basal with its adaxial surface facing up and used for infection with A. rhizogenes wild strain having the plasmid pRi A4 (agropine strain). Single colonies of pRiA4 were isolated from bacterial cultures grown on YMB medium (Hooykas 1988) containing 50mg/l of rifampicin. These were grovra in liquid media for 48 hrs at 220 rpm and 37° C or till it read an OD 1 at 610nm. The liquid cultures were then transferred to sterile nalgene tubes and centrifuged at 3000 rpm for 5 min. The supernatant was discarded and the pellet was resuspended in 5 ml of MS basal. 100 Μ.1 of this was used for infecting the S. khasianum leaves. The bacteria were co-cultured with the leaves for two days and transferred to liquid MS basal medium containing 300 mg /I of cefotaxime and kept on a shaker at 100 rpm and 25°C for one day. These were then transferred to solid MS basal medium containing 250mg/l of cefotaxime; this step was repeated till there was no further growth of bacteria. After 8-10 days hairy roots start to appear. This was grown on MS medium with no phytohormones. The Solasodine content of the above hairy roots was estimated by any of the following methods: Rapid estimation of Solasodine fspectrophotometric) Solasodine was extracted and estimated by a modified method of Bhatt and Bhatt, 1983. Solasodine extracted in the lower chloroform layer, is passed through anhydrous sodium sulphate (to remove moisture) and analysed spectrophotoraetrically at 400 nm in a UV-spectrophotometer (Solasodine is seen to show absorption maxima of 400nm). The concentration of Solasodine is calculated from a standard curve obtained by using solasodine standard (Source: Sigma). Estimation of Solasodine using HPLC: Solasodine was extracted and estimated as per the method of Kittipongpatana et al, 1999. HPLC Conditions: HLPC system: Merck (Germany) Column; C-8 normal phase column Mobile Phase: lOmM Tris buffer (pH 7): Methanol (25% : 75%) Flow rate: 0.1 ml/sec Wavelength: 205 nm Injection volume: 20 ul Solvent system: lOmM Tris buffer (pH 7): Methanol (25% : 75%) Sample Preparation: Solasodine was extracted in the lower chloroform layer and the chloroform was evaporated and residue was dissolved in 10 ml of 30% methanol. This prepared sample (3 ml) was injected in C-18 cartridge and passed through it at flow rate of 0.1 ml/sec followed by final extraction in ml of 100% methanol at 0.1 ml/ sec flow rate. This sample in 100% methanol was used for injection. The transformed older roots growing on MS basal medium, after 5-6 subcultures give rise to shoot regeneration. These shoots are separated and then rooted on MS basal medium without growth regulators. The shoots which are mentioned above when rooted and grown in sand-vermiculite (1: 1) mixture result in 2 types of roots viz normal roots which grow in the soil and hairy roots which are slightly negatively geotropic which are on the surface. The Solasodine content of all the above transformed species-were compared with normal roots (untransformed) and the results are provided in following Table I Table I: Comparison of Solasodine production Source of Solasodine Solasodine mg/g dry wt Normal roots (untransformed) 2.1197 + 0.220 Example lA-Transformed undifferentiated hairy roots Example IB-Transformed plants (shoots only) Example IC-Transformed plants (roots only). 7.9862 + 0.415 3.6114 + 0.480 8.6797+ 0.623 A1I the results are mean of three replicates + S.E. It would be clearly apparent from the above Table I that genetic modifications especially transformed undifferentiated hairy roots and transformed plants (roots) achieve higher content of Solasodine than the untransformed normal roots. EXAMPLE - 2 Preparation of Aspergillus niger culture used in the process of the extraction of Solasodine for the hairy root culture: Aspergillus niger culture is grown in 20 ml CEPEK DOX medium (a common medium used for microorganisms) in 100 ml flasks. Incubated on a shaker (100 rpm) at 30 °C. The fungal lechate of 6 ■©'old cultures having fresh wt. 1 gm (+ 0.362) and protein content of SOimg/g fresh wt. (+ 0.442) is used. This lechate is filter sterilized through bacterial filter (Sartorius) with pore size 0.2 microns. It is added to the spent medium of hairy root cultures at the end of 4 weeks at different concentrations and incubated at 30°C. EXAMPLE-3 Use of the Aspergillus niger culture of Example 2 in identification of preferred selective operating conditions for conversion of Solasonine to Solasodine: Preferred operating conditions like substrate concentration, reaction time, pH and temperature were identified to obtain the quantity of Solasodine. Example 3A To ascertain the effect of substrate concentration varying concentrations of standard Solasonine (Sigma) such as 2ffii^, 5ra(§:, Sra^., lOrag. and 13mg/15ml of medium, 2ml of A.nigerwas added and incubated for 30 min at room temperature. Solasodine released was extracted in chlorofonn . and estimated using HPLC. The results obtained are provided hereunder in Table II Table-II: Effect of different substrate concentrations on Solasodine content: Results are mean of 3 replicates + S.E. Example 3B To ascertain the effect of time of incubation to 15 ml spent medium 2ml of A. niger leachate was added and incubated for different time like 10, 30, 50, 70 arid 90 min at room temperature. The chlorfrom : sanple volume is 1:10, MTV Solasodine released was extracted using cnloroformvand estimated using HPLC as mentioned above. The result obtained are provided in Table III hereunder: Table-Ill: Effect of different incubation time on Solasodine content: Results are mean of 3 replicates + S.E. Example 3C To ascertain the effect of temperature 15 ml of spent medium and 2 ml of A. niger leachatg was added and incubated at different temperatures like 10, 20, 30 40, 50 and 60 °C for 30 min. The resultsobtained are shown in Table IV hereunder: Table-IV: Effect of different temperatures on Solasodine content: All the results are mean of 3 replicates + SE Example 3D To ascertain the effect of pH, 15 ml of spent medium was taken and pH set to 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5. To this medium 2 ml of A. niger leachate was added and incubated for 30 min. at room temperature. Solasodine obtained was extracted and estimated as mentioned above. The result obtained are shown in Table V hereunder: Table-V: Effect of different pH on Solasodine content: All the results are mean of 3 replicates + SE As would be apparent from the results above, for effective conversion of Solasonine to Solasodine substrate concentration of 5 10 mg/I5 ml (most preferably 8mg/15 ml), incubation for a period of 25 to 35 mins (most preferably 30 minutes), temperature of 25 to 35 °C (most preferably 30°C) and pH range of 4 to 5.5 (most preferably 4.5) is preferred. The above selective identification of conditions like substrate concentration, reaction time, pH and temperature for the extraction. of Solasodine as demonstrated by this example can be further exemplified by the accompanying Figure 1A to ID. EXAMPLE -4 The process of increased production of Solasodine by selective addition of elicitor and precursor: The Agrobacterium rhizogenes mediated hairy root cultures of S. khasianum obtained as mentioned above were grown in hormone free MS medium for a period of five to six weeks. Seven to ten day old mycelial mat of pathogenic fungus Fusarium oxysporum is grown on Cepex dox medium. Then it is weighed autoclaved in 50 ml distilled water and filtered using 0.2-micron filter under vacuum and sterile conditions. The reducing sugars of the filtrate were estimated using DNS method and 2.5 ml of filtrate (equivalent to 30 mg/1 of reducing sugars) is added to the culture medium of 4-6 week old hairy root cultures. This is called as day -14. As represented in Fig 2A the favorable Solasodine production from hairy root cultures using elicitor was found to be about 7.9 mg/gm dry wt. To ascertain the effect of precursor, 0.5mg/l cholesterol was added to the culture medium on day 10. Hairy root cultures were incubated for 10 days in cholesterol containing medium for Solasodine production. Fig 2B shows the favorable effect of cholesterol in generation of Solasodine which was found to be about 6.8 mg/gm dry wt. The selective use of both elicitor and precursor cholesterol showed synergistic effect on Solasodine production vis-a-vis use of only one of the Elicitor or Precursor as evident from the results provided in Table VI hereunder. In this case elicitor is added on day 14 and cholesterol on day 10. This content of Solasodine is seen in the first cycle starting from the day of inoculation of the hairy root cultures. 1 able VI : Synergistic effect of elicitor and precursor on. Solasodine production. The Solasodine content in use of both elicitor and precursor in the hairy root cultures increased to 8.7 + 0.1763 mg/gm dry weight of hairy roots on day 1. EXAMPLE-5 Under the Example the increased extraction of Solasodine achieved by addition of permeabiiizing agents is demonstrated as here below: On day 0, 0.5% (v/v) DMSO and 1% (v/v) Tween-20 is added to the medium. The next day culture medium is drained and fresh MS medium containing 30mg/l of elicitor is added to the hairy root cultures. This is counted as day 14. After this on day 10 0.5 mg/1 cholesterol is added. The addition of 0.5% DMSO and 1% Tween-20 synergistically extracted 80-85% of the glycoalkaloid present in the roots. This is clearly revealed by the results provided under Table VII hereunder: Table-VII: % total Solasodine extracted into the medium (Results are mean of 3 repticates) The effect of permeabiiizing agents and their synergy and on Solasodine production in hairy root cultures is further demonstrated by means of the accompanying Figure-3A to 3C. EXAMPLE-6 The advantages in the selective extraction 6f Solasodine using a two-phase extraction system is detailed hereunder: A two-phase extraction system is provided wherein the Solasodine released in the medium was extracted in chloroform and evaporated to dryness. The Solasodine obtained was dissolved in 1ml of methanol and quantified by HPLC (Kittipongpatana, 1999). 15 ml of hairy root culture spent medium containing 1 mg of Solasonine on being treated with 2 ml of Aspergillus niger leachate at 30°C temperature, reaction time 30 minutes and pH 4.0 gave 158.942 + 2.31 μg of Solasodine. EXAMPLE 7: This Example illustrates the proposed process of extraction of Solasodine from hairy roots involving preferably the elicitor, precursor and permeabilizing agent in accordance with a preferred aspect of the invention. Hairy root cultures are used for scale up in the biomass. To the growing hairy root cultures 30 mg/1 of reducing sugar equivalent of elicitor prepared from Fusarium oxysporum culture was added. After 5 days, precursor 0.5mg/l of cholesterol was added and this was allowed to grow for 6-8 weeks. Subsequently the permeabilizing substances as a pulse of one day (0.5% DMSO and 1% Tween 20) were added. At the end of day one, the medium was removed and replaced with the fresh MS medium with elicitor as stated above starting the cycle again. The drained medium was used for bioconversion using 30mg/l reducing sugar equivalent from filtrate of Aspergillus niger. This was allowed to react for 30 min at 30 °C at pH 4.3. The medium thus treated was then extracted with chloroform to get the residue of Solasodine. Solasodine yield was found to be 8.701+ 1.672 mg/gm dry weight. It would be clearly apparent from the above that the present process of production of Solasodine is directed to provide a plant free system, which is neither dependent on climatic conditions nor is it susceptible to pests or diseases thus making it possible to get a continuous supply of Solasodine at high yield through out the year under controlled conditions. The process clearly avoids the problems of hand picking and drying of berries and provides a simple procedure. Importantly, the process of the invention is directed to consistently provide the Solasodine of high content by using hairy root cultures, as they are biochemically and genetically stable and can produce the same content of Solasodine generation after generation. The process of the invention is simple in terms of providing a simple Aspergillus niger mediated conversion of Solasonine to Solasodine thus eliminating use of processes like defatation, alcohol extraction, acid hydrolysis and basification and therefore also affording a reduction in number of chemicals, equipments, time and energy required. We claim; 1. A process for obtaining Solasodine comprising: i) providing a selective source obtained by transformation of the Solarium species to cultures of said Solatium species having high content of Solasonine .; and ii) subjecting the glycoalkaloid Solasonine from said culture medium to enzymatic hydrolysis to convert into Solasodine. 2. A process for obtaining Solasodine as claimed in claim 1 wherein said selective source of Solasodine used in the process comprise hairy root cultures of Solarium species grown in nutrient medium. 3. A process for obtaining Solasodine as claimed in anyone of claims 1 or 2 wherein the selective culture is preferably obtained by Agrobacterium rhizogenes mediated transformation and hairy root production in Solarium khasianum. 4. A process for obtaining Solasodine as claimed in claim 1 wherein the said glycoalkaloid Solasonine is cleaved by way of a leachate of Aspergillus niger. 5. A process for obtaining Solasodine as claimed in anyone of claims 1 to 4 comprising: i) providing a selective source obtained by genetic transformation of the Solarium species to cultures of said Solarium species having high content of Solasonine; and ii) subjecting the glycoalkaloid Solasonine from said culture medium to enzymatic hydrolysis to convert into Solasodine and iii) extracting Solasodine from the medium by solvent extraction. 6. A process for obtaining Solasodine as claimed in claim 5 wherein said organic solvent extraction is carried out with chloroform using a two-phase extraction system wherein the chloroform is evaporated to dryness to give pure Solasodine. 7. A process for obtaining Solasodine as claimed in anyone of claims 1 to 6 comprising: -a. genetic transformation of the Solarium species. b. - growing the culture in plant tissue culture medium. c. permeabilizing the cells for leaching glycoalkaloid Solasonine into the medium, d.enzymatically hydrolyzing the glycoalkaloid to convert into Solasodine. e. extracting Solasodine from the medium using organic solvent system. 8. A process for obtaining Solasodine as claimed in anyone of claims 1 to 7 wherein the Solasodine content in the hairy root cultures is increased in the presence of a combination of elicitor and/or precursor. 9. A process for obtaining Solasodine as claimed in claim 8 wherein the elicitor is selected from any pathogenic fungal organism preferably Fusarium oxysporum or Colletotrichum or Botrytis or Pythium or Phytopthora or Altemaria. 10. A process for obtaining Solasodine as claimed in claim 8 wherein the precursors are preferably selected from cholesterol, squalene, cycloartenol or a primary precursor acetyl CoA. 11. A process according to anyone of claims 1 to 10, wherein the genetic transformation of Solanum species is brought about by inoculating Agrobacterium rhizogenes with the T-DNA in the Ri- plasmid on the cut part of the plant cells to produce hairy root cultures. 12. A process according to anyone of claims 1 to 11, wherein the culture medium is MS medium. 13. A process according to anyone of claims 7 to 12 wherein the permeabilizing agent used is selected from Tween-20, DMSO, copper sulphate, chitosan, organic solvents like chloroform, synthetic substances like pluronic F-68 preferably Tween 20. 14. A process according to anyone of claims 7 to 12 wherein the permeabilizing agent used is selected from Tween-20, DMSO, copper sulphate, chitosan, organic solvents like chloroform, synthetic substances like pluronic F-68 preferably dimethyl sulfoxide. 15. A process according to anyone of claims 1 to 14 wherein the permeabilizing agents Tween 20 and dimethyl sulfoxide are preferably used in combination. 16. A process according to anyone of claims 8 to 15 wherein elicitors are added to the medium to increase the production of Solasodine. 17. A process according to claim 16 wherein the elicitor used is reducing sugar obtained from the processed filtrate of Fusarium oxysporum fungi. 18. A process according to anyone of claims 8 to 17 wherein precursors of Solasodine are added to the medium to increase the production of Solasodine. 19. A process according to claim 18 wherein precursor of Solasodine used is cholesterol. 20. A process for obtaining Solasodine according to anyone of claims 1 to 19 wherein operating parameters like substrate concentration, reaction time, pH, and temperature are selectively identified comprising preferred ranges for substrate concentration of 5 to 10 mg/15 ml, incubation for a period of 25 to 35 minutes, temperature of 25 to 35°C and pH range of 4 to 5.5. 21. A process for obtaining Solasodine substantially herein described and illustrated in relation to the accompanying examples. Dated this 15th day of September 2003. Meghna S. Vaidya Of S. MAJUMDAR & CO. Applicants' Agent

Documents:

955-mum-2003-cancelled pages(27-04-2004).pdf

955-mum-2003-claims(granted)-(27-04-2004).doc

955-mum-2003-claims(granted)-(27-04-2004).pdf

955-mum-2003-correspondence(08-02-2007).pdf

955-mum-2003-correspondence(ipo)-(24-04-2007).pdf

955-mum-2003-drawing(27-04-2004).pdf

955-mum-2003-form 1(02-12-2003).pdf

955-mum-2003-form 1(15-09-2003).pdf

955-mum-2003-form 19(15-09-2003).pdf

955-mum-2003-form 2(granted)-(27-04-2004).doc

955-mum-2003-form 2(granted)-(27-04-2004).pdf

955-mum-2003-form 3(15-09-2003).pdf

955-mum-2003-power of attorney(13-04-2004).pdf

955-mum-2003-power of attorney(27-04-2004).pdf

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